WO2022082183A1

WO2022082183A1 - Compounds and methods for the treatment of inflammatory and metabolic diseases

Info

Publication number: WO2022082183A1
Application number: PCT/US2021/071844
Authority: WO
Inventors: Kaoru Saijo; Greg TIMBLIN
Original assignee: The Regents Of The University Of California
Priority date: 2020-10-13
Filing date: 2021-10-13
Publication date: 2022-04-21

Abstract

The present disclosure relates to hydroxyestrogen derivatives that activate mitohormesis by mediating mitochondrial stress in microphages. The hydroxyestrogen derivatives repress inflammation and are useful in treating, e.g., acute and chronic inflammation and metabolic diseases.

Description

COMPOUNDS AND METHODS FOR THE TREATMENT OF INFLAMMATORY

AND METABOLIC DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Application No. 63/091,217, filed October 13, 2020, which is incorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] Provided in certain instances here are hydroxyestrogens that activate mitohormesis by mediating mitochondrial stress in macrophages. The hydroxyestrogens repress inflammation and are useful in treating, e.g., acute and chronic inflammation and metabolic diseases.

BACKGROUND

[0003] Acute and chronic diseases, as well as age-dependent diseases, are serious medical issues in the US. For example, approximately 1.7 million adults in the US develop sepsis and about 270,000 people die as a result of this condition. In addition, about 5% of Covid-19- infected patients die in the US, and it has been speculated that this high mortality rate is due to the immune system becoming hyperactive and inducing a condition called a cytokine storm. Controlling severe inflammation may be a good strategy for rescuing patients.

Cytokine storms are also a cause of death of patients with sepsis. Thus, controlling proper immune responses is important for the treatment of patients with acute and severe inflammation.

[0004] Moreover, macrophage-mediated chronic inflammation triggers metabolic syndrome, which includes type 2 diabetes and obesity. With more than 30 million people in the US having type 2 diabetes and one third of Americans being obese, these chronic inflammatory diseases have a profound impact on human health as well as medical costs in the US.

[0005] To control hyperactive immunity or cytokine storms, some drugs that suppress the immune system have been used. One class of these drugs is a group of steroid hormones called glucocorticoids. Glucocorticoids efficiently repress inflammation; however, they have serious adverse effects, including hypertension, diabetes, and osteoporosis. Another example is cyclosporine, an immunosuppressant. However, cyclosporine is known to damage kidney as well as cause hypertension, and infections are known adverse effects of it. [0006] Therefore, more efficient and safer strategies for controlling the immune system are urgently needed for treatment of both acute and chronic inflammatory diseases.

SUMMARY

[0007] Described herein are pharmaceutical compositions comprising hydroxylated estrogens (also referred to herein interchangeably as hydroxy estrogens). Further described herein are uses of hydroxy estrogens to activate mitohormesis, an adaptation process for controlling the immune response in macrophages. In some embodiments, hydroxyestrogens are useful at targeting mitochondrial function and activating mitohormesis in subjects. Some exemplary hydroxyestrogens are hydroxyestrones, e.g., 4-hydroxyestrone (4-OHE1) and 2- hydroxyestrone (2-OHE1). Other exemplary hydroxyestrogens are hydroxy estradiols, e.g., 2- hydroxyestradiol (2-OHE2).

[0008] The present disclosure is based on the unexpected discovery that hydroxyestrogens (e.g., 4-OHE1, 2-OHE1, 2-OHE2) are mediators of mitochondrial stress in macrophages. In some embodiments, hydroxyestrogens decrease the mitochondrial acetyl-Coenzyme A (acetyl-CoA) production required for histone acetylation and lipopolysaccharide (LPS)- induced proinflammatory gene transcription in macrophages. In some embodiments, hydroxyestrogens induce a mild mitochondrial stress that triggers adaptations in macrophages that appear to render them tolerant to subsequent stress perturbations, and less able to produce inflammatory mediators. This response is termed mitohormesis. The induction of tolerance in macrophages can also result in immunosuppression, which may lead to increased susceptibility to infections and may increase mortality. It has now been discovered that mitohormesis leads to macrophage tolerance via a reduction in acetyl-CoA production by aerobic respiration, and that administration of acetyl Coenzyme A (acetyl-CoA) or CoA restores both the acetylation of histones and proper immune reactions in macrophages. Thus, compounds that control mitohormesis, such as hydroxyestrogens and acetyl-CoA/CoA, may have a broad range of applications for treating diseases that require suppressing inflammation or reactivating immune responses.

[0009] In some embodiments, targeting mitochondrial function and mitohormesis in macrophages may have various clinical applications for treating subjects with overactive or underactive immune systems. Thus, in some embodiments, hydroxyestrogens that target mitohormesis can be used to treat acute and chronic inflammatory disorders. In some embodiments, hydroxyestrogens are useful in treating metabolic diseases, e.g., diabetes. In some embodiments, hydroxyestrogens are useful at treating age-dependent diseases. For example, as demonstrated herein, hydroxyestrogens are effective at treating mouse model of metabolic syndrome. Furthermore, the injection of hydroxylated estrogens for 12-14 weeks did not cause any adverse effects in the mice.

[0010] In one aspect, the disclosure provides a pharmaceutical composition comprising a unit dose of a hydroxyestrogen or a salt thereof and a pharmaceutically acceptable excipient, wherein the unit dose of the hydroxyestrogen comprises an amount of the hydroxyestrogen sufficient to induce mitohormesis.

[0011] In some embodiments, provided herein is a pharmaceutical composition comprising a hydroxyestrogen or a salt thereof and a pharmaceutically acceptable excipient.

[0012] In some embodiments, provided herein is a pharmaceutical composition comprising a hydroxyestrone or a salt thereof and a pharmaceutically acceptable excipient.

[0013] In some embodiments, provided herein is a pharmaceutical composition comprising a hydroxyestradiol or a salt thereof and a pharmaceutically acceptable excipient.

[0014] In some embodiments, the hydroxyestrogen comprises 4-hydroxyestrone (4-OHE1) or a salt thereof.

[0015] In some embodiments, the hydroxyestrogen comprises 4-hydroxyestradiol (4-OHE2) or a salt thereof.

[0016] In some embodiments, the hydroxyestrogen comprises 2-hydroxyestrone (2-OHE1) or a salt thereof.

[0017] In some embodiments, the hydroxyestrogen comprises 2-hydroxyestradiol (2-OHE2) or a salt thereof.

[0018] In some embodiments, the hydroxyestrogen comprises 4-hydroxyestrone (4-OHE1). [0019] In some embodiments, the hydroxyestrogen comprises 4-hydroxyestradiol (4-OHE2). [0020] In some embodiments, the hydroxyestrogen comprises 2-hydroxyestrone (2-OHE1). [0021] In some embodiments, the hydroxyestrogen comprises 2-hydroxyestradiol (2-OHE2). [0022] In some embodiments, the hydroxyestrogen is 4-hydroxyestrone (4-OHE1), 4- hydroxyestradiol (4-OHE2), or 2-hydroxyestrone (2-OHE1), and the amount of the hydroxyestrogen sufficient to induce mitohormesis is at least about 0.2 mg/kg based on mass of a subject administered the pharmaceutical composition. In some embodiments, the amount of the hydroxyestrogen sufficient to induce mitohormesis is about 1 mg/kg to about 20 mg/kg. In some embodiments, the amount of the hydroxyestrogen sufficient to induce mitohormesis is about 1 mg/kg to about 10 mg/kg. [0023] In some embodiments, the hydroxyestrogen is 4-hydroxyestrone (4-OHE1), 4- hydroxyestradiol (4-OHE2), 2-hydroxyestrone (2-OHE1), or 2-hydroxyestradiol (2-OHE2), and the amount of the hydroxyestrogen sufficient to induce mitohormesis is greater than 2 mg/kg based on mass of a subject administered the pharmaceutical composition.

[0024] In some embodiments, the amount of the hydroxy estrogen sufficient to induce mitohormesis is at least about 10 mg. In some embodiments, the amount of the hydroxyestrogen sufficient to induce mitohormesis is between about 10 mg and about 100 mg. In some embodiments, the amount of the hydroxyestrogen sufficient to induce mitohormesis is between about 50 mg and about 60 mg.

[0025] In some embodiments, the amount of the hydroxy estrogen sufficient to induce mitohormesis is at least about 0.5 uM. In some embodiments, the amount of the hydroxyestrogen sufficient to induce mitohormesis is between about 0.5 uM and about 10 uM. In some embodiments, the amount of the hydroxyestrogen sufficient to induce mitohormesis is between about 1 uM and about 5 uM.

[0026] Provided herein is a method of mediating mitochondrial stress in a cell comprising contacting the cell with an effective amount of a hydroxyestrogen or a salt thereof to thereby mediate mitochondrial stress. In some embodiments, the effective amount results in reduction in mitochondrial acetyl-CoA production in the cell. In some embodiments, the effective amount further results in a reduction in total intracellular acetyl-CoA levels.

[0027] Provided herein is a method of activating or inducing mitohormesis in a cell comprising contacting the cell with an effective amount of a hydroxyestrogen or salt thereof. In some embodiments, mitochondrial oxidative stress resistance increases in the cell. In some embodiments, the ratio of aerobic glycolysis to mitochondrial oxidative metabolism increases in the cell.

[0028] In some embodiments, the cell is a macrophage.

[0029] In some embodiments, the hydroxy estrogen is provided at a concentration of at least about 1 pM. In some embodiments, the hydroxyestrogen is provided at a concentration of about 5 pM.

[0030] In some embodiments, a method of mediating mitochondrial stress in a cell or activating or inducing mitohormesis comprises administering or causing to be administered an effective amount of 4-hydroxyestrone (4-OHE1) or a salt thereof. In some embodiments, exogenous 4-hydroxyestrone (4-OHE1) or a salt thereof is provided.

[0031] In some embodiments, a method of mediating mitochondrial stress in a cell or activating or inducing mitohormesis comprises administering or causing to be administered an effective amount of 2-hydroxyestrone (2-OHE1) or a salt thereof. In some embodiments, exogenous 2-hydroxyestrone (2-OHE1) or a salt thereof is provided.

[0032] In some embodiments, a method of mediating mitochondrial stress in a cell or activating or inducing mitohormesis comprises administering or causing to be administered an effective amount of 2-hydroxyestradiol (2-OHE2) or a salt thereof. In some embodiments, exogenous 2-hydroxyestradiol (2-OHE2) or a salt thereof is provided. In some embodiments, exogenous 2-hydroxyestradiol (2-OHE2) or a salt thereof is provided at a concentration of greater than 2 mg/ml.

[0033] Provided herein is a method of reducing an inflammatory response in a subject comprising administering or causing to be administered an effective amount of an hydroxyestrogen to the subject to thereby inhibit an inflammatory response. In some embodiments, induction of II lb expression by an inflammatory stimulus in a macrophage of the subject is reduced. In some embodiments, the inflammatory response is induced by a lipopolysaccharide (LPS). In some embodiments, Illb expression in a visceral white adipose tissue macrophage of the subject is reduced.

[0034] In some embodiments, the inflammatory response is an acute inflammatory response. In some embodiments, the inflammatory response is a chronic inflammatory response. In some embodiments, the subject is afflicted by an inflammatory condition. In other embodiments, the subject is afflicted by an acute inflammatory conditions.

[0035] Provided herein is a method of treating an acute inflammatory condition in a subject comprising administering or causing to be administered an effective amount of a hydroxyestrogen. In some embodiments, the acute inflammatory condition comprises a cytokine storm. In some embodiments, a subject is treated with the hydroxyestrogen.

[0036] In some embodiments, a method of inhibiting an inflammatory response in a subject or treating an acute inflammatory condition in a subject comprises administering or causing to be administered an effective amount of 4-hydroxyestrone (4-OHE1) or a salt thereof. In some embodiments, exogenous 4-hydroxyestrone (4-OHE1) or a salt thereof is administered. [0037] In some embodiments, a method of inhibiting an inflammatory response in a subject or treating an acute inflammatory condition in a subject comprises administering or causing to be administered an effective amount of 2-hydroxyestrone (2-OHE1) or a salt thereof. In some embodiments, 2-hydroxyestradiol (2-OHE2) or a salt thereof is administered.

[0038] In some embodiments, a method of inhibiting an inflammatory response in a subject or treating an acute inflammatory condition in a subject comprises administering or causing to be administered an effective amount of 2-hydroxyestradiol (2-OHE2) or a salt thereof. In some embodiments, 2-hydroxyestradiol (2-OHE2) or a salt thereof is administered.

[0039] Provided herein is a method of mitigating effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce a body weight gain of the subject. In some embodiments, the body weight gain of the subject reduced by at least 10% compared to a body weight gain of a control subject not administered a hydroxyestrogen.

[0040] Provided herein is a method of mitigating the effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce a fasting blood glucose of the subject.

[0041] In some embodiments, the subject is obese. In some embodiments, the subject is morbidly obese. In some embodiments, the subject is overweight. In some embodiments, the subject is not obese. In some embodiments, the subject is not overweight.

[0042] Provided herein is a method of mitigating the effects of a high fat diet comprising administering or causing to be administered an effective amount of a hydroxyestrogen to a subject, wherein glucose tolerance is improved. In some embodiments, a blood glucose level 60 minutes after a meal is reduced by at least 10% compared to a blood glucose level of a control subject not administered a hydroxy estrogen.

[0043] Provided herein is a method of mitigating the effects of a high fat diet comprising administering or causing to be administered an effective amount of a hydroxyestrogen to a subject, to thereby reduce liver triglyceride content of the subject. In some embodiments, a liver triglyceride content is reduced by at least 20 nmol per mg of liver tissue compared to a liver triglyceride content of a control subject not administered a hydroxyestrogen.

[0044] Provided herein is a method of mitigating the effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of a hydroxyestrogen to thereby reduce liver fibrosis of the subject. In some embodiments, a liver collagen content is reduced by at least 0.5 pg per mg of liver tissue compared to a liver collagen content of a control subject not administered a hydroxyestrogen.

[0045] Provided herein is a method of treating obesity in a subject comprising administering or causing to be administered an effective amount of a hydroxyestrogen to the subject to thereby reduce body weight of the subject. In some embodiments, the body weight of the subject is reduced by at least about 10%.

[0046] Provided herein is a method of mitigating effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of a hydroxyestrogen to thereby increase insulin sensitivity in the subject. In some embodiments, the amount of insulin required to clear glucose from blood of the subject is lower than a control subject not administered the hydroxyestrogen.

[0047] Provided herein is a method of increasing insulin sensitivity in a subject consuming a high fat diet comprising administering or causing to be administered to the subject an effective amount of a hydroxyestrogen.

[0048] Provided herein is a method of increasing glucose tolerance comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen.

[0049] In some embodiments, a fed blood glucose is reduced. In some embodiments, the subject has ingested a high fat diet. In some embodiments, the high fat diet comprises at least 20% fat.

[0050] In some embodiments, a fasting blood glucose of the subject is reduced compared to a fasting blood glucose of the subject prior to administration of the hydroxyestrogen, or a control subject not administered a hydroxy estrogen. In some embodiments, infiltration of immune cells into visceral white adipose tissue is reduced. In some embodiments, infiltration of CD45+ leukocytes into visceral white adipose tissue is reduced. In some embodiments, infiltration of CDllb+ macrophages into visceral white adipose tissue is reduced. In some embodiments, oxygen consumption is increased. In some embodiments, energy expenditure of the subject is increased.

[0051] In some embodiments, fat mass is reduced. In other embodiments, visceral white adipose tissue mass is reduced. In other embodiments, subcutaneous white adipose tissue mass is reduced.

[0052] In some embodiments, the hydroxyestrogen is an exogenous hydroxy estrogen. In some embodiments, the hydroxyestrogen is an isolated hydroxyestrogen.

[0053] In some embodiments, the hydroxyestrogen is a synthetic or semi-synthetic hydroxyestrogen.

[0054] In some embodiments, the hydroxyestrogen is 4-hydroxyestrone (4-OHE1), 4- hydroxyestradiol (4-OHE2), or 2-hydroxyestrone (2-OHE1), and the effective amount of the hydroxyestrogen is at least about 0.2 mg/kg based on mass of a subject administered the pharmaceutical composition.

[0055] In some embodiments, the hydroxyestrogen is 4-hydroxyestrone (4-OHE1), 4- hydroxyestradiol (4-OHE2), 2-hydroxyestrone (2-OHE1), or 2-hydroxyestradiol (2-OHE2), and the effective amount is greater 2 mg/kg based on mass of a subject administered the pharmaceutical composition.

[0056] In some embodiments, a method of mitigating the effects of a high fat diet, or a method of treating obesity, comprises administering or causing to be administered 4- hydroxyestrone (4-OHE1) or a salt thereof. In some embodiments, exogenous 4- hydroxyestrone (4-OHE1) or a salt thereof is administered.

[0057] In some embodiments, a method of mitigating the effects of a high fat diet, or treating obesity, comprises administering or causing to be administered 2-hydroxyestrone (2-OHE1) or a salt thereof. In some embodiments, 2-hydroxyestrone (2-OHE1) or a salt thereof is administered.

[0058] In some embodiments, a method of mitigating the effects of a high fat diet, or a method of treating obesity, comprises administering or causing to be administered 2- hydroxyestradiol (2-OHE2) or a salt thereof. In some embodiments, 2-hydroxyestradiol (2- OHE2) or a salt thereof is administered.

[0059] In some embodiments applicable to all methods of inhibiting an inflammatory response, methods of treating an acute inflammatory condition, methods of mitigating effects of a high fat diet, or methods of treating obesity as described herein, the subject being treated is a male. In some embodiments applicable to all methods of inhibiting an inflammatory response, methods of treating an acute inflammatory condition, methods of mitigating effects of a high fat diet, or methods of treating obesity as described herein, the subject being treated is a female.

[0060] In some embodiments applicable to all methods of inhibiting an inflammatory response, methods of treating an acute inflammatory condition, methods of mitigating effects of a high fat diet, or methods of treating obesity as described herein, the exogenous hydroxyestrogen is administered subcutaneously. In some embodiments applicable to all methods of inhibiting an inflammatory response, methods of treating an acute inflammatory condition, methods of mitigating effects of a high fat diet, or methods of treating obesity as described herein, the exogenous hydroxyestrogen is administered intraperitoneally.

[0061] Provided herein is a method of enhancing an inflammatory response in a subject comprising administering or causing to be administered an effective amount of CoA or Acetyl-CoA or a salt thereof to the subject to thereby enhance an inflammatory response of the subject.

[0062] In some embodiments, the method further comprises administering or causing to be administered a Toll-like receptor ligand. A non-limiting example of a Toll-like receptor ligand is monophosphoryl lipid A (MPLA). Other non- limiting examples of Toll-like receptor ligands are rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD- 101, Bacillus Calmette- Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

[0063] Provided herein is a method of enhancing an inflammatory response in a subject comprising administering or causing to be administered an effective amount of 4'- phosphopantetheine or salt thereof to the subject to thereby enhance an inflammatory response in the subject.

[0064] In some embodiments, induction of Illb expression by an inflammatory stimulus in a macrophage of the subject is increased.

[0065] Provided herein is a method of treating a cancer in a subject in need thereof comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby inhibit growth of a transformed cell in the subject.

[0066] Provided herein is a method of treating or preventing a cancer metastases in a subject in need thereof comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby reduce cancer metastases in the subject.

[0067] In some embodiments, the method of treating cancer or treating or preventing a cancer metastases further comprises administering or causing to be administered a proinflammatory signaling pathway agonist. In some embodiments, the proinflammatory signaling pathway agonist is monophosphoryl lipid A (MPLA). In some embodiments, the proinflammatory signaling pathway agonist is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

[0068] In some embodiments, the method of treating cancer or treating or preventing a cancer metastases further comprises administering or causing to be administered an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a fragment thereof.

[0069] In some embodiments, the cancer comprises a solid tumor, or metastases thereof. In some embodiments, the solid tumor is a melanoma, colon adenocarcinoma, bladder cancer, hepatoma, or breast cancer, or metastases thereof.

[0070] In some embodiments, the cancer comprises a hematologic malignancy, or metastases thereof. In some embodiments, the hematologic malignancy is lymphoma or acute myeloid leukemia, or metastases thereof.

[0071] In some embodiments, the cancer is lung cancer, or metastases thereof. [0072] In some embodiments, the coenzyme A derivative is acetyl-CoA. In some embodiments, the coenzyme A derivative is 4-phosphopanthetheine.

[0073] In some embodiments, the coenzyme A (Co A) or derivative thereof is an exogenous coenzyme A (Co A) or derivative thereof.

[0074] In some embodiments, the coenzyme A (Co A) or derivative thereof is an isolated coenzyme A (Co A) or derivative thereof.

[0075] In some embodiments, the coenzyme A (Co A) or derivative thereof is a synthetic or semi-synthetic coenzyme A (CoA) or derivative thereof.

[0076] Provided herein is a method of enhancing macrophage anti-tumor immunity, comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to a subject in need thereof.

[0077] Provided herein is a method of restoring macrophage innate immunity in an aging subject, comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to the subject.

[0078] In some embodiments, the proinflammatory signaling pathway agonist is monophosphoryl lipid A (MPLA). In some embodiments, the proinflammatory signaling pathway agonist is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

[0079] In some embodiments applicable to all methods of treatment described herein, the subject is a mammal. In some embodiments applicable to all methods of treatment described herein, the subject is a human.

[0080] Various embodiments provided herein include, but are not limited to one or more of the following:

[0081] Embodiment 1 : A pharmaceutical composition comprising a unit dose of a hydroxyestrogen or a salt thereof and a pharmaceutically acceptable excipient, wherein the unit dose of the hydroxyestrogen comprises an amount of the hydroxyestrogen sufficient to induce mitohormesis.

[0082] Embodiment 2: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen is a hydroxyestrone or a salt thereof.

[0083] Embodiment 3: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen is a hydroxyestradiol or a salt thereof. [0084] Embodiment 4: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen is a synthetic or semi-synthetic hydroxyestrogen or a salt thereof.

[0085] Embodiment 5: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen comprises 4-hydroxyestrone (4-OHE1) or a salt thereof.

[0086] Embodiment 6: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen comprises 4-hydroxyestradiol (4-OHE2) or a salt thereof.

[0087] Embodiment 7: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen comprises 2-hydroxyestrone (2-OHE1) or a salt thereof.

[0088] Embodiment 8: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen comprises 2-hydroxyestradiol (2-OHE2) or a salt thereof.

[0089] Embodiment 9: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen is 4-hydroxyestrone (4-OHE1).

[0090] Embodiment 10: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen is 4-hydroxyestradiol (4-OHE2).

[0091] Embodiment 11 : The pharmaceutical composition of embodiment 1 , wherein the hydroxyestrogen is 2-hydroxyestrone (2-OHE1).

[0092] Embodiment 12: The pharmaceutical composition of embodiment 1, wherein the hydroxyestrogen is 2-hydroxyestradiol (2-OHE2).

[0093] Embodiment 13: The pharmaceutical composition of any one of embodiments 1-7 and 9-11, wherein the amount of the hydroxyestrogen sufficient to induce mitohormesis is at least about 0.2 mg/kg based on mass of a subject administered the pharmaceutical composition.

[0094] Embodiment 14: The pharmaceutical composition of any one of embodiments 1-13, wherein the amount of the hydroxyestrogen sufficient to induce mitohormesis is greater than 2 mg/kg based on mass of a subject administered the pharmaceutical composition.

[0095] Embodiment 15: The pharmaceutical composition of any one of embodiments 1-14, wherein the pharmaceutical composition does not include carboxymethylcellulose.

[0096] Embodiment 16: The pharmaceutical composition of any one of embodiments 1-14, wherein the pharmaceutical composition does not include 1% carboxymethylcellulose.

[0097] Embodiment 17: A method of mediating mitochondrial stress in a cell, said method comprising contacting the cell with an effective amount of a hydroxyestrogen or a salt thereof to thereby mediate mitochondrial stress.

[0098] Embodiment 18: The method of embodiment 17, wherein the effective amount results in reduction in mitochondrial acetyl-CoA production in the cell. [0099] Embodiment 19: The method of embodiment 18, wherein the effective amount results in reduction of total intracellular acetyl-CoA levels.

[0100] Embodiment 20: A method of activating or inducing mitohormesis in a cell, said method comprising contacting the cell with an effective amount of a hydroxyestrogen or salt thereof.

[0101] Embodiment 21: The method of embodiment 20, wherein the effective amount results in an increase in mitochondrial chaperone activity.

[0102] Embodiment 22: The method of embodiment 20, wherein the effective amount results in an increase in mitochondrial oxidative stress resistance in the cell.

[0103] Embodiment 23 : The method of embodiment 20, wherein the effective amount results in an increase of a ratio of aerobic glycolysis to mitochondrial oxidative metabolism in the cell.

[0104] Embodiment 24: The method of any one of embodiments 17-23, wherein the cell is a macrophage.

[0105] Embodiment 25: The method of any one of embodiments 17-23, wherein the hydroxyestrogen is provided at a concentration of at least 1 pM.

[0106] Embodiment 26: The method of embodiment 25, wherein the hydroxyestrogen is provided at a concentration of about 5 pM.

[0107] Embodiment 27: The method of any one of embodiments 17-26, wherein the hydroxyestrogen comprises 4-hydroxyestrone (4-OHE1) or a salt thereof.

[0108] Embodiment 28: The method of any one of embodiments 17-26, wherein the hydroxyestrogen comprises 2-hydroxyestrone (2-OHE1) or a salt thereof.

[0109] Embodiment 29: The method of any one of embodiments 17-26, wherein the hydroxyestrogen comprises 2-hydroxyestradiol (2-OHE2) or a salt thereof.

[0110] Embodiment 30: A method of reducing an inflammatory response in a subject in need thereof, said method comprising administering or causing to be administered an effective amount of an hydroxyestrogen to the subject to thereby inhibit an inflammatory response.

[0111] Embodiment 31: The method of embodiment 30, wherein induction of Illb expression by an inflammatory stimulus in a macrophage of the subject is reduced.

[0112] Embodiment 32: The method of embodiment 31, wherein the inflammatory response is induced by a lipopolysaccharide (LPS).

[0113] Embodiment 33: The method of any one of embodiments 20-31, wherein Illb expression in a visceral white adipose tissue macrophage of the subject is reduced. [0114] Embodiment 34: The method of any one of embodiments 30-33, wherein the inflammatory response is an acute inflammatory response.

[0115] Embodiment 35: The method of any one of embodiments 30-33, wherein the inflammatory response is a chronic inflammatory response.

[0116] Embodiment 36: The method of any one of embodiments 30-33, wherein the subject is afflicted with an inflammatory condition.

[0117] Embodiment 37: The method of embodiment 36, wherein the acute inflammatory condition comprises a cytokine storm.

[0118] Embodiment 38: The method of any one of embodiments 30-37, wherein the hydroxyestrogen comprises 4-hydroxyestrone (4-OHE1) or a salt thereof.

[0119] Embodiment 39: The method of any one of embodiments 30-37, wherein the hydroxyestrogen comprises 4-hydroxyestradiol (4-OHE2) or a salt thereof.

[0120] Embodiment 40: The method of any one of embodiments 30-37, wherein the hydroxyestrogen comprises 2-hydroxyestrone (2-OHE1) or a salt thereof.

[0121] Embodiment 41: The method of any one of embodiments 30-37, wherein the hydroxyestrogen comprises 2-hydroxyestradiol (2-OHE2) or a salt thereof.

[0122] Embodiment 42: The method of any one of embodiments 30-41, wherein the hydroxyestrogen is administered subcutaneously.

[0123] Embodiment 43 : A method of mitigating effects of a high fat diet, said method comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce a body weight gain of the subject. [0124] Embodiment 44: The method of embodiment 43, wherein the body weight gain of the subject is reduced by at least 10% compared to a body weight gain of a control subject not administered a hydroxyestrogen.

[0125] Embodiment 45 : A method of mitigating effects of a high fat diet, said method comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce a fasting blood glucose of the subject.

[0126] Embodiment 46: The method of embodiment 45, wherein the subject is obese.

[0127] Embodiment 47: The method of embodiment 45, wherein the subject is not obese. [0128] Embodiment 48: A method of mitigating the effects of a high fat diet, said method comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby improve a glucose tolerance of the subject. [0129] Embodiment 49: The method of embodiment 46 or embodiment 48, wherein a blood glucose level 60 minutes after a meal is reduced by at least 10% compared to a blood glucose level of a control subject not administered a hydroxyestrogen.

[0130] Embodiment 50: A method of mitigating the effects of a high fat diet, said method comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce liver triglyceride content of the subject.

[0131] Embodiment 51: The method of embodiment 50, wherein a liver triglyceride content is reduced by at least 20 nmol per mg of liver tissue compared to a liver triglyceride content of a control subject not administered a hydroxyestrogen.

[0132] Embodiment 52: A method of mitigating the effects of a high fat diet, said method comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce liver fibrosis of the subject.

[0133] Embodiment 53: The method of embodiment 50 or embodiment 52, wherein a liver collagen content is reduced by at least 0.5 pg per mg of liver tissue compared to a liver collagen content of a control subject not administered a hydroxyestrogen.

[0134] Embodiment 54: A method of treating obesity in a subject, said method comprising administering or causing to be administered an hydroxyestrogen to the subject to thereby reduce body weight of the subject.

[0135] Embodiment 55: The method of embodiment 54, wherein the body weight of the subject is reduced by at least about 10%.

[0136] Embodiment 56: A method of mitigating effects of a high fat diet, said method comprising administering or causing to be administered to a subject in need thereof an effective amount of a hydroxyestrogen to thereby increase insulin sensitivity in the subject. [0137] Embodiment 57: The method of embodiment 56, wherein the amount of insulin required to clear glucose from blood of the subject is lower than a control subject not administered the hydroxyestrogen.

[0138] Embodiment 58: A method of increasing insulin sensitivity in a subject consuming a high fat diet, said method comprising administering or causing to be administered to the subject an effective amount of a hydroxyestrogen.

[0139] Embodiment 59: A method of increasing glucose tolerance, said method comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen. [0140] Embodiment 60: The method of any one of embodiments 43-59, wherein a fed blood glucose is reduced.

[0141] Embodiment 61: The method of any one of embodiments 43-60, wherein the subject has ingested a high fat diet.

[0142] Embodiment 62: The method of embodiment 61, wherein the high fat diet comprises at least 20% fat.

[0143] Embodiment 63: The method of any one of embodiments 43-62, wherein the subject is a male.

[0144] Embodiment 64: The method of any one of embodiments 43-62, wherein the subject is a female.

[0145] Embodiment 65: The method of any one of embodiments 43-64, wherein a fasting blood glucose of the subject is reduced compared to a fasting blood glucose of the subject prior to administration of the hydroxyestrogen, or a control subject not administered a hydroxyestrogen.

[0146] Embodiment 66: The method of any one of embodiments 43-65, wherein infiltration of immune cells into visceral white adipose tissue is reduced.

[0147] Embodiment 67: The method of any one of embodiments 43-66, wherein infiltration of CD45+ leukocytes into visceral white adipose tissue is reduced.

[0148] Embodiment 68: The method of any one of embodiments 43-67, wherein infiltration of CDllb-i- macrophages into visceral white adipose tissue is reduced.

[0149] Embodiment 69: The method of any one of embodiments 43-68, wherein oxygen consumption is increased.

[0150] Embodiment 70: The method of any one of embodiments 43-69, wherein energy expenditure is increased.

[0151] Embodiment 71: The method of any one of embodiments 43-70, wherein the hydroxyestrogen comprises 4-hydroxyestrone (4-OHE1).

[0152] Embodiment 72: The method of any one of embodiments 43-70, wherein the hydroxyestrogen comprises 2-hydroxyestrone (2-OHE1).

[0153] Embodiment 73: The method of any one of embodiments 43-70, wherein the hydroxyestrogen comprises 2-hydroxyestradiol (2-OHE2).

[0154] Embodiment 74: The method of any one of embodiments 43-73, wherein the hydroxyestrogen is administered subcutaneously.

[0155] Embodiment 75: The method of any one of embodiments 43-74, wherein fat mass is reduced. [0156] Embodiment 76: The method of embodiment 75, wherein visceral white adipose tissue mass is reduced.

[0157] Embodiment 77: The method of embodiment 75, wherein subcutaneous white adipose tissue mass is reduced.

[0158] Embodiment 78: The method of any one of embodiments 17-77, wherein the hydroxyestrogen is an exogenous hydroxyestrogen.

[0159] Embodiment 79: The method of any one of embodiments 17-77, wherein the hydroxyestrogen is an isolated hydroxyestrogen.

[0160] Embodiment 80: The method of any one of embodiments 17-77, wherein the hydroxyestrogen is a synthetic or semi-synthetic hydroxyestrogen.

[0161] Embodiment 81: The method of any one of embodiments 17-80, wherein the effective amount of the hydroxyestrogen is at least about 0.2 mg/kg based on mass of a subject administered the pharmaceutical composition.

[0162] Embodiment 82: The method of any one of embodiments 17-80, wherein the hydroxyestrogen comprises 2-hydroxyestrone (2-OHE1) and wherein the effective amount of the 2-OHE1 greater than 2 mg/kg based on mass of a subject administered the pharmaceutical composition.

[0163] Embodiment 83: The method of any one of embodiments 17-82, wherein said contacting, said administering, or said causing to be administered does not comprise contacting, administering, causing to be administered the hydroxyestrogen with carboxymethylcellulose.

[0164] Embodiment 84: The method of any one of embodiments 17-82, wherein said contacting, said administering, or said causing to be administered does not comprise contacting, administering, or causing to be administered the hydroxy estrogen with 1 % carboxymethylcellulose.

[0165] Embodiment 85: A method of enhancing an inflammatory response in a subject, said method comprising administering or causing to be administered an effective amount of CoA or Acetyl-CoA or a salt thereof to the subject to thereby enhance an inflammatory response of the subject.

[0166] Embodiment 86: The method of embodiment 85, further comprising administering or causing to be administered a Toll-like receptor ligand.

[0167] Embodiment 87: The method of embodiment 86, wherein the Toll-like receptor ligand is monophosphoryl lipid A (MPLA). [0168] Embodiment 88: The method of embodiment 86, wherein the Toll-like receptor ligand is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD- 101, Bacillus Calmette- Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

[0169] Embodiment 89: A method of enhancing an inflammatory response in a subject, said method comprising administering or causing to be administered an effective amount of 4'- phosphopantetheine or salt thereof to the subject to thereby enhance an inflammatory response in the subject.

[0170] Embodiment 90: The method of embodiment 85 or embodiment 89, wherein induction of II lb expression by an inflammatory stimulus in a macrophage of the subject is increased.

[0171] Embodiment 91: A method of treating a cancer in a subject in need thereof, said method comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby inhibit growth of a transformed cell in the subject. [0172] Embodiment 92: A method of treating or preventing a cancer metastases in a subject in need thereof, said method comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby reduce cancer metastases in the subject.

[0173] Embodiment 93: The method of embodiment 91 or embodiment 92, wherein the coenzyme A derivative is acetyl-CoA.

[0174] Embodiment 94: The method of embodiment 91 or embodiment 92, wherein the coenzyme A derivative is 4-phosphopanthetheine.

[0175] Embodiment 95: The method of any one of embodiments 91-94, further comprising administering or causing to be administered a proinflammatory signaling pathway agonist. [0176] Embodiment 96: The method of embodiment 95, wherein the proinflammatory signaling pathway agonist is monophosphoryl lipid A (MPLA).

[0177] Embodiment 97: The method of embodiment 95, wherein the proinflammatory signaling pathway agonist is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

[0178] Embodiment 98: The method of any one of embodiments 91-96, further comprising administering or causing to be administered an immune checkpoint inhibitor.

[0179] Embodiment 99: The method of embodiment 98, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a fragment thereof. [0180] Embodiment 100: The method of any one of embodiments 91-99, wherein the cancer comprises a solid tumor, or metastases thereof.

[0181] Embodiment 101: The method of embodiments 100, wherein the solid tumor is a melanoma, colon adenocarcinoma, bladder cancer, hepatoma, or breast cancer, or metastases thereof.

[0182] Embodiment 102: The method of any one of embodiments 91-99, wherein the cancer comprises a hematologic malignancy, or metastases thereof.

[0183] Embodiment 103: The method of embodiment 102, wherein the hematologic malignancy is lymphoma or acute myeloid leukemia, or metastases thereof.

[0184] Embodiment 104: The method of any one of embodiments 91-103, wherein the coenzyme A (CoA) or derivative thereof is an exogenous coenzyme A (CoA) or derivative thereof.

[0185] Embodiment 105: The method of any one of embodiments 91-103, wherein the coenzyme A (CoA) or derivative thereof is an isolated coenzyme A (CoA) or derivative thereof.

[0186] Embodiment 106: The method of any one of embodiments 91-103, wherein the coenzyme A (CoA) or derivative thereof is a synthetic or semi- synthetic coenzyme A (CoA) or derivative thereof.

[0187] Embodiment 107: The method of any one of embodiments 91-106, wherein the cancer is breast cancer or metastases thereof.

[0188] Embodiment 108: The method of any one of embodiments 91-106, wherein the cancer is lung cancer or metastases thereof.

[0189] Embodiment 109: A method of enhancing macrophage anti-tumor immunity, said method comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to a subject in need thereof.

[0190] Embodiment 110: A method of restoring macrophage immunity in sepsis-associated immunosuppression, said method comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to a subject in need thereof.

[0191] Embodiment 111: A method of restoring macrophage innate immunity in an aging subject, said method comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to the subject. [0192] Embodiment 112: The method of embodiment any one of embodiments 109-111, wherein the proinflammatory signaling pathway agonist is monophosphoryl lipid A (MPLA). [0193] Embodiment 113: The method of embodiment any one of embodiments 109-111, wherein the proinflammatory signaling pathway agonist is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

[0194] Embodiment 114: The method of any one of embodiments 17-113, wherein the subject is a mammal.

[0195] Embodiment 115: The method of any one of embodiments 17-114, wherein the subject is a human.

[0196] The present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

DESCRIPTION OF THE DRAWINGS

[0197] The disclosed embodiments and other features, advantages, and aspects contained herein, and the matter of attaining them, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure. Such detailed description will be better understood when taken in conjunction with the accompanying drawings, wherein:

[0198] FIG. 1 : schematic representation of acute and chronic effects of hydroxyestrogens on LPS-induced transcription, LPS tolerance and mitohormesis.

[0199] FIGs. 2 and 3: depict anti-inflammatory activity of hydroxyestrogens in vitro.

[0200] FIG. 2A. BMDMs pretreated with EtOH vehicle control or 1 pM estrogens for 1 hour, followed by 6-hour LPS stimulation (lOOng/mL) and Nos2 qPCR.

[0201] FIG. 2B. Gene ontology (GO) analysis of 253 genes repressed by 1 hour hydroxyestrogen pretreatment (IpM) in 6-hour LPS -stimulated BMDMs (lOOng/mL) identified by RNAseq. [0202] FIG. 2C. RNA-seq hierarchical clustering dendrogram, with heatmap highlighting genes with reduced relative expression- in hydroxyestrogen-pretreated, LPS-stimulated BMDMs (*log2-transformed RPKM centered on the mean of each gene).

[0203] FIG. 2D. BMDMs pretreated with EtOH or indicated concentrations of 4-OHE1 for 1 hour, followed by 6-hour LPS stimulation (lOOng/mL) and II lb qPCR. Percentages indicate induction relative to max (100%) in “EtOH +LPS” control BMDMs.

[0204] FIG. 2E. RAW macrophages pretreated with EtOH or 5pM 4-OHE1 for 1 hour, followed by 6-hour LPS stimulation (lOOng/mL) before pro-IL-lb measurement by western blot.

[0205] FIG. 2F. RAW macrophages pretreated with EtOH or 5pM 4-OHE1 for 1 hour, followed by 4-hour LPS stimulation (lOOng/mL) before pro-IL-lb measurement by intracellular staining and flow cytometry.

[0206] FIG. 2G. RAW macrophages pretreated with EtOH or 2.5 pM estrogens for 1 hour, followed by 3 hour stimulation with LPS (TLR4, lOOng/mL), Pam3CSK4 (PAM, TLR2, lOOng/mL), polyinosinic-polycytidylic acid (pIC, TLR3, 25ug/mL), or CpG oligodeoxynucleotides (CpG, TLR9, IpM), and Illb qPCR. Percentages indicate induction relative to max (100%) in the “EtOH +TLR ligand” control BMDMs for each ligand.

[0207] FIG. 2H. BMDMs from ERafl/fl LysM-Cre mice pretreated with EtOH or IpM estrogens for 1 hour, followed by 6-hour LPS stimulation (lOOng/mL) and Illb qPCR.

[0208] FIG. 21. BMDMs pretreated with EtOH or 1 pM hydroxyestrogens in the absence (left) or presence (right) of lOpM ICI 182780 for 1 hour, followed by 6-hour LPS stimulation (lOOng/mL) and Illb qPCR.

[0209] FIG. 3A. BMDMs pretreated for 1 hour with EtOH vehicle control or IpM estrogens before 6-hour LPS stimulation (lOOng/mL) and qPCR to validate cytokine and chemokine targets repressed by hydroxyestrogens from RNA-seq data.

[0210] FIG. 3B. RAW macrophages pretreated for 1 hour with EtOH vehicle control or 5pM estrogens before 6-hour LPS stimulation (lOOng/mL) and Illb qPCR.

[0211] FIG. 3C. RAW macrophages pretreated for 1 hour with EtOH vehicle control or indicated concentrations of 4-OHE2 before 6-hour LPS stimulation (lOOng/mL) and Illb qPCR. [0212] All qPCR data for FIG. 2 and 3 represented as mean ± SEM. Each data point is an independent biological replicate (n=2 or 3 for each condition). *P<0.05, **P<0.01, ***P<0.001, ****P<0.001 by Student’s T Test versus appropriate “EtOH +LPS” control sample. All qPCR data representative of at least 2 independent experiments.

[0213] FIGs. 4 and 5: depict anti-inflammatory activity of hydroxyestrogens in vivo.

[0214] FIG. 4A. Experimental setup (top), and serum IL- lb levels (bottom) in 8 week old C57BL/6 male mice injected IP with EtOH vehicle control or estrogens (lOmg/kg) prior to IP LPS injection (2mg/kg). n=2,4,5, and 4 mice for the EtOH, EtOH +LPS, 4-OHE1 +LPS, and E2 +LPS groups, respectively.

[0215] FIG. 4B. qPCR for proinflammatory gene expression in spleenocytes isolated from mice in FIG. 4A.

[0216] FIG. 4C. Experimental setup (top), and hierarchical clustering of vWAT macrophage RNA-seq data (bottom) from 8 week old C57BL/6 male mice fed a high-fat diet (HFD) and injected subcutaneously every 6 days with EtOH, E2, or 4-OHE1 (lOmg/kg). Relative expression heatmap displays the log2-transformed RPKM centered on the mean of each gene. n=5 mice for all groups.

[0217] FIG. 4D. Venn Diagram displaying overlap between genes significantly repressed by E2 or 4-OHE1 versus EtOH control in vWAT macrophages from HFD-fed mice.

[0218] FIG. 4E. GO analysis of 666 and 152 genes uniquely repressed by 4-OHE1 (right) or E2 (left), respectively, versus EtOH control vWAT macrophages.

[0219] FIG. 4F. Glucose tolerance test (GTT) in 12-week-old male C57BL/6 mice after 30 days of normal chow (NC) or high-fat diet (HFD) feeding and s.c. injection with either ethanol or 4-OHE1 (10 mg kg^-1) every 6 days.

[0220] FIG. 4G. Serum insulin ELISA measuring glucose-stimulated insulin secretion in fasted mice from FIG. 4F. at 15 min after i.p. glucose injection.

[0221] FIG. 4H. Serum IL-ip levels in a subset of mice from f, 4.5 h after i.p. LPS injection (3 mg kg^-1), which was done 48h after the last s.c. EtOH or 4-OHE1 dose.

[0222] FIG. 5A. Hydroxyestrogens are anti-inflammatory in vivo. a. Representative gating strategy for identifying forward scatter/side scatter (FS/SS) live gate+, DAPI-, CD45+, F4/80+CDllb-i- visceral white adipose tissue (vWAT) macrophages for sorting and flow cytometry analysis. [0223] FIG. 5B. vWAT mass in mice after 30 days HFD feeding and EtOH control or estrogen injections, n = 5 mice per group.

[0224] FIG. 5C. vWAT macrophage cellularity in mice after 30 days HFD feeding and EtOH control or estrogen injections, n = 5 mice per group.

[0225] FIG. 5D. Relative expression* of select pro-inflammatory genes in vWAT macrophages from HFD-fed mice injected with EtOH, 4-OHE1, or E2 (*log2-transformed RPKM centered on the mean of each gene), n = 5 mice per group.

[0226] FIG. 5E. Blood glucose levels at 30min and Ih post-glucose injection in mice from FIG. 2F. n = 10, 10, and 11 mice per condition.

[0227] FIG. 5F. qPCR for Nos2 expression in splenocytes isolated from mice in FIG. 2H. n = 3, 5, 5, and 5 mice per condition.

[0228] For FIG. 5 bar graphs, each data point is an independent biological replicate, and data are represented as mean ± SEM. All P values from unpaired, two-sided Student’s T Test (planned comparisons). HFD chronic inflammation model was performed once for transcriptional profiling (a-d), and a second time for metabolic studies (e,f).

[0229] FIGs. 6 and 7: Hydroxyestrogens activate NRF2, but NRF2 is not required for their anti-inflammatory effects.

[0230] FIG. 6A. Natural production and metabolism of 4-OHE1, highlighting detoxification via methylation and glutathione (GSH) conjugation, and mechanisms by which 4-OHE1 can cause oxidative and electrophilic stress.

[0231] FIG. 6B. BMDMs pretreated with EtOH vehicle control or 5pM estrogens for 1 hour, followed by 4 hour LPS stimulation (lOOng/mL) and Illb qPCR. Black box highlights Illb repression by hydroxy estrogens, but not their precursors or methylated metabolites.

*P>0.0001 for all hydroxyestrogen + LPS samples vs. EtOH + LPS control sample by Student’s T Test.

[0232] FIG. 6C. HOMER promoter motif analysis showing NRF2 as a top transcription factor binding motif enriched in promoters of the 341 genes upregulated by Ih hydroxyestrogen pretreatment (IpM) in 6h LPS -stimulated BMDMs (lOOng/mL).

[0233] FIG. 6D. Relative expression* of putative NRF2 target genes in estrogen pretreated, LPS-stimulated BMDM RNA-seq dataset (*log2-transformed RPKM centered on the mean of each gene). [0234] FIG. 6E. RAW macrophages treated with 5pM E2, 5pM 4-OHE1, or lOOpM DEM, for indicated times before NRF2 stabilization was assessed by western blot. *non-specific band

[0235] FIG. 6F. Wild type and NRF2 KO BMDMs were pretreated Ih with EtOH or indicated concentrations of 4-OHE1 before 6h LPS stimulation (lOOng/mL) and Illb qPCR. Percentages indicate induction relative to max (100%) in “EtOH +LPS” control BMDMs for each genotype. WT BMDM data are from FIG. 2D.

[0236] FIG. 7A. GO analysis of 341 genes significantly upregulated in hydroxyestrogen- pretreated, LPS-stimulated BMDMs relative to control pretreatments. Gray highlights GO categories involved in oxidative stress resistance (OSR) and detoxification of reactive oxygen species (ROS).

[0237] FIG. 7B. Wild type and Nrf2 KO BMDMs were pretreated Ih with EtOH or indicated concentrations of 4-OHE1 before 6h LPS stimulation (lOOng/mL) and qPCR for the NRF2 target gene Hmoxl . Data represented as mean ± SEM. n=3 per condition. qPCR data are represented as mean ± SEM. Each data point is an independent biological replicate (n=2 or 3 for each condition). qPCR and western blot data representative of two independent experiments.

[0238] FIGs. 8 and 9: Hydroxyestrogens cause mitochondrial stress.

[0239] FIGs. 8A, 8B, and 8C. Relative expression* of genes indicative of mitochondrial stress in Ih estrogen pretreated, 6h LPS-stimulated BMDM RNA-seq dataset (*log2- transformed RPKM centered on the mean of each gene).

[0240] FIG. 8D. IsoTOP- ABBP strategy to identify covalent targets of 4-OHE1 acting through reactive cysteines. BMDMs were treated with EtOH vehicle control or IpM 4-OHE1 for 1 hour.

[0241] FIGs. 8E and 8F. All targets (left) and mitochondrial targets (right) of 4-OHE1 identified by isoTOPABPP. Cysteine-containing peptides above dashed line have light/heavy ratio>2.0, indicating at least a 50% reduction in cysteine-reactive probe targeting of these cysteines in 4-OHE1 -treated BMDMs relative to control BMDMs. 18 of 20 mitochondrial targets are in MitoCarta 2.0. FKBP4 and GCLC mitochondrial localization prediction from Uniprot. [0242] FIGs. 9A, 9B, 9C. GO analysis of 341 genes significantly upregulated in hydroxyestrogen-pretreated, LPS-stimulated BMDMs relative to control pretreatments. Gray highlights GO categories indicative of HSF1 and ATF4 activity, and upregulation of glycolysis/pentose phosphate pathway (PPP) genes.

[0243] FIG. 9D. Experimental setup for steroid extraction and liquid chromatography /mass spectrometry (LC/MS) to measure 4-OHE1 extracted from cell culture media (top, control), or from whole cell and mitochondrial fractions prepared from RAW macrophages treated with 5pM 4-OHE1 for 1 hour.

[0244] FIG. 9E. Western blot showing enrichment of mitochondrial marker VDAC, and depletion of cytoplasmic marker vinculin, in mitochondrial fractions verses whole cell lysates prepared from RAW macrophages.

[0245] FIG. 9F top - Mass spectrum (positive ion mode) of Img/mL 4-OHE1 standard (2pL injection volume) showing 4-OHE1 peak at m/z = 287.1642. Mass range - m/z=287.1624- 287.1660; FIG. 9F bottom - Extracted ion chromatogram over the mass range above for indicated samples. Peaks at retention time RT=13.14 and RT=13.23 in the 4-OHE1 standard and media +4-OHE1 sample (gray arrows) indicates ability to detect 4-OHE1 with our LC/MS method, and validates our 4-OHE1 steroid extraction method. Lack of an abundant chromatographic peak at RT=13.1-13.2 in whole cell or mitochondrial extracts prepared from RAW macrophages treated with 4-OHE1 indicates lack of free 4-OHE1 in these samples.

[0246] FIG. 9G. Chi-square test comparing the observed frequency of mitochondrial targets (18) in isoTOP-ABPP target list (118 total targets) versus the expected frequency of mitochondrial targets from MitoCarta 2.0.

[0247] FIGs. 10 and 11: Hydroxyestrogens impair mitochondria acetyl-CoA production and histone acetylation required for LPS -induced proinflammatory gene transcription.

[0248] FIG. 10A. RAW macrophages pretreated for Ih with indicated concentrations 4- OHE1 before LPS stimulation (lOOng/mL) for 6 hours and Illb, 116, and Tn/ PCR.

[0249] FIG. 10B. BMDM metabolomics and ¹³Ce glucose labeling strategy.

[0250] FIG. 10C. Normalized metabolite abundance in 4- OHE1 -treated BMDMs (5pM) relative to EtOH control BMDMs (where metabolite level was set to 1.0). Gray highlighted metabolites above dashed line showed significant change (P<0.05, one-way ANOVA). [0251] FIGs. 10D and 10E. Absolute abundance of acetyl-CoA (left) and CoA (right) in control versus 4- OHEl-treated BMDMs. *P<0.05, **P<0.01 by Student’s T test.

[0252] FIG. 10F. ChlP-seq for p65 (left) and H3K27ac (right) in BMDMs pretreated for 1 hour with IpM of the indicated hydroxy estrogen, followed by 30 minute LPS stimulation (lOOng/mL). Histograms represent read density at LPS-inducible p65 peaks/H3K27ac regions in each of the 3 conditions. H3K27ac regions were centered on the nucleosome-free region (NRF) of associated promoters/enhancers, yielding a classic “2 peak” histogram.

[0253] FIG. 10G. Model for how mitochondrial stress caused by 4-OHE1 impairs metabolic/epigenetic control of proinflammatory gene transcription.

[0254] FIG. 10H. BMDMs cultured in absence or presence of CoA (250pM) or acetyl-CoA (Ac-CoA, 200pM) (red bars) for 3 hours prior to 1 hour 5pM 4-OHE1 pretreatment, 1.5 hour LPS stimulation (lOOng/mL), and Illb qPCR.

[0255] FIG. 101. RAW macrophages cultured in absence or presence of acetyl-CoA (Ac- CoA, 200pM, red bars) for 2 hours prior to 1 hour electrophile pretreatment, 1.5 hour LPS stimulation, and Illb qPCR. Electrophile concentrations: 250nM Celastrol, lOOpM DEM, lOpM FCCP.

[0256] FIG. 11A. BMDMs were pretreated Ih with EtOH or indicated concentrations of 4- OHE1 before 6h LPS stimulation (lOOng/mL) and qPCR for III b, 116, and Tnf. The percent induction of at each 4-OHE1 concentration ((induction in presence of 4-OHEl/EtOH control induction)xl00)) was calculated and plotted for each gene. n=3 for each data point.

[0257] FIG. 11B. Absolute abundance (area under the curve, AUC) of citrate, aconitate, and alpha-ketoglutarate in EtOH versus 4- OHEl-treated BMDMs. P values from Student’s T Test.

[0258] FIG. 11C. Fractional contribution (FC) of ¹³Ce glucose-derived carbons to total carbons for TCA cycle metabolites, and amino acids derived from these metabolites, in EtOH versus 4- OHEl-treated BMDMs. Metabolites with significantly enhanced ¹³C labeling in bold (identified by one-way ANOVA). Question marks indicate possible alternative entry routes for ¹³Ce glucose-derived carbons into the TCA cycle other than pyruvate conversion to acetyl-CoA and citrate. *P<0.05, ***P<0.001, ****P<0.0001 by Student’s T Test versus EtOH. [0259] FIG. HD. RAW macrophages cultured in absence or presence of CoA (500pM, left) or acetyl-CoA (Ac-CoA, 500pM, right) (bars in the far right for each graph) for 3 hours prior to 1 hour EtOH or 5pM 4-OHE1 pretreatment, 6 hour LPS stimulation (lOOng/mL), and Illb qPCR. *P<0.05, **P<0.01 by Student’s T Test.

[0260] FIG. HE. RAW macrophages cultured in absence or presence of sodium acetate (5mM, labeled “acetate”) or CoA (500pM, labeled “CoA”) for 15 minutes prior to 1 hour EtOH or 5pM 4-OHE1 pretreatment, 1 hour LPS stimulation (lOOng/mL), and Illb qPCR. ***P<0.001 by Student’s T Test versus “EtOH +LPS” control sample. All bar graph data represented as mean ± SEM. n=2 or 3 biological replicates per condition.

[0261] FIG. HF. Data from reference 38. Acetyl-CoA levels as measured by mass spectrometry in HeLa cells treated for 24 hours with DMSO vehicle control or lOpM FCCP. Dat represented as mean ± SEM, n=7 for both groups.

[0262] For FIGs. 10 and 11, all qPCR data represented as mean ± SEM. Each data point is an independent biological replicate (n=2 or 3 for each condition). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by Student’s T Test versus appropriate “EtOH +LPS” control sample. All qPCR data are representative of at least 2 independent experiments.

[0263] FIGs. 12 and 13: Hydroxyestrogen-driven mitochondrial stress triggers mitohormesis.

[0264] FIG. 12A. RAW matrix-oxGFP macrophages were treated with EtOH or 5pM estrogens for indicated times and matrix-oxGFP florescence quantified by flow cytometry.

[0265] FIG. 12B. Basal (left) and LPS-induced (right, lOOng/mL LPS) mitochondrial H2O2 levels were measured in EtOH control and estrogen (pre)treated (5pM) RAW macrophages using MitoPyl staining and flow cytometry. Menadione (50pM) and H2O2 (500pM) serve as positive controls.

[0266] FIG. 12C. Left - Schematic describing RAW macrophages expressing roGFP proteins targeted to cytosol, mitochondrial inner membrane space (IMS), and mitochondrial matrix. roGFP oxidation favors excitation by 405nm violet laser relative to 488nm blue laser. Right - 405nm/488nm excitation ratio measured by flow cytometry (510nM emission) in roGFP RAW macrophages untreated (NT) or treated with LPS (lOOng/mL) for 6 hours.

[0267] FIG. 12D. IMS-roGFP macrophages pretreated with EtOH or estrogens (5pM) for 1 hour and stimulated with LPS (lOOng/mL) for 6 hours before 405/488 excitation ratio was measured by flow cytometry (510nM emission). [0268] FIG. 12E. Left - Schematic describing menadione mitochondrial superoxide resistance assay. RAW macrophages were treated overnight (18-24 hours) with EtOH or 4- OHE1 (5pM) before next day treatment with DMSO vehicle control or menadione (50pM, 4 hours) and viability assessment by flow cytometry. Right - Flow cytometry forward scatter/side scatter plots (FS/SS) of treated RAW macrophages. DAPI staining demonstrates cells in “live” FS/SS gate are viable and exclude DAPI, whereas “dead” FS/SS gate cells have increased cell membrane permeability and take up DAPI. Percentages represent events in “live” and “dead” FS/SS gates relative to total events.

[0269] FIG. 12F. RAW macrophages treated overnight with EtOH or 5pM estrogens were treated the following day with DMSO vehicle control (left) or 50pM menadione (right) for 4 hours before viability was assessed the next day by flow cytometry. Viability is represented as percentage of “live” FS/SS gate positive, DAPI negative cells per total events collected for each sample.

[0270] FIG. 13A. Seahorse respirometry measurement of oxygen consumption in RAW macrophages treated with EtOH vehicle control or indicated concentrations of 4-OHE1. Cells were treated immediately before loading into Seahorse XF24 analyzer, after which 5 measurements per condition (n=4) were taken over 45 minutes and averaged.

[0271] FIG. 13B. TMRE measurement of mitochondrial membrane potential in RAW macrophages treated with EtOH vehicle control, Oligomycin (5pM), FCCP (5pM), or 4- OHE1 (5pM) for 20 minutes before TMRE staining and flow cytometry.

[0272] FIG. 13C. RAW matrix-oxGFP macrophages were treated with EtOH vehicle control or indicated concentrations of 4-OHE1 for 8 hours and matrix-oxGFP fluorescence quantified by flow cytometry.

[0273] FIG. 13D. Mitochondrial DNA/genomic DNA (mtDNA/gDNA) ratio in RAW macrophages treated with EtOH vehicle control or 5pM 4-OHE1 for indicated times (n=3 per condition).

[0274] FIG. 13E. RAW matrix-oxGFP macrophages were pretreated with DMSO vehicle control or indicated concentrations of KRIBB11 for 1 hour before treatment with 5pM 4- OHE1 for 8 hours. Matrix-oxGFP fluorescence was then quantified by flow cytometry.

[0275] FIGs. 13F and 13G. RAW macrophages (F) and BMDMs (G) were treated with EtOH vehicle control or estrogens (5pM) for 7 hours before MitoTracker Green staining and flow cytometry. [0276] FIG. 13H. BMDMs pretreated for 1 hour with EtOH vehicle control or estrogens (5pM) before LPS stimulation (lOOng/mL) for 6 hours and mitochondrial H2O2 measurement with MitoPY 1 staining and flow cytometry.

[0277] FIG. 131. Left - RAW matrix-roGFP emission at 510nM with 405nm UV laser (y axis) versus 488nM blue laser (x axis) excitation. Shift of untreated matrix-roGFP cells (black population) following H2O2 treatment (ImM, purple population) for 10 minutes demonstrates ability to detect matrix-roGFP oxidation with flow cytometry. Right - RAW matrix-roGFP 405nm laser excitation/488nM laser excitation ratio (405ex/488ex) after 10 minutes of H2O2 (ImM) and DTT (lOrnM) treatment.

[0278] FIG. 13J. BMDMs treated overnight with EtOH or 5pM 4-OHE1 and treated the following day with DMSO vehicle control (4 hours) or 50pM menadione for (2,4 hours) before viability was assessed by flow cytometry.

[0279] All flow cytometry data in FIGs. 12 and 13 represented as mean ± SEM. Each data point is an independent biological replicate (n=2 or 3 for each condition). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student’s T Test versus appropriate control samples. All flow cytometry data are representative of at least 2 independent experiments.

[0280] FIGs. 14 and 15: LPS-driven mitochondrial stress triggers mitohormesis identical to that observed in hydroxyestrogen-treated macrophages.

[0281] FIG. 14A. BMDMs were treated with 4-OHE1 (5pM) or LPS (lOOng/mL) alone for 6 and 24 hours, and RNA-seq was performed. Venn diagrams show overlap between genes significantly upregulated or downregulated by either treatment relative to control BMDMs at each timepoint.

[0282] FIG. 14B. GO analysis of 1622 genes upregulated by both 4-OHE1 and LPS at 24 hours.

[0283] FIG. 14C. Heatmap showing relative expression* of select genes upregulated by both 4-OHE1 and LPS at 24 hours. (*DESeq2 counts centered on the mean of each gene).

[0284] FIG. 14D. Expression of Prdxl and Hsp90abl (RPKM) at 24 hours from RNA-seq.

[0285] FIG. 14E. Matrix-oxGFP RAW macrophages treated with EtOH, 4-OHE1 (5pM), LPS (lOOng/mL), or both, for indicated times and matrix-oxGFP florescence quantified by flow cytometry. ****P<0.0001 by Student’s T Test versus time-matched EtOH control sample. [0286] FIG. 14F. Menadione resistance assay in BMDMs treated overnight (18-24 hours) with EtOH, 4-OHE1 (5pM), LPS (lOOng/mL), or both, before treatment with DMSO control or menadione (50pM, 4 hours) and viability assessment by flow cytometry. ****P<0.0001 by Student’s T Test for EtOH +DMSO versus EtOH +Menadione samples, and EtOH +DMSO versus 4-OHE1/LPS +Menadione samples.

[0287] FIG. 15A. GO analysis of 1622 genes upregulated by both 4-OHE1 and LPS at 6 hours in BMDMs.

[0288] FIG. 15B. Heatmap showing relative expression* of select genes upregulated by both 4-OHE1 and LPS at 6 hours in BMDMs. (*DESeq2 counts centered on the mean of each gene).

[0289] FIG. 15C. RAW matrix-oxGFP macrophages pretreated with DMSO vehicle control or lOpM KRIBB11 for 1 hour before treatment with 4-OHE1 (5pM, left) or LPS (lOOng/mL, right) for 8 hours. Matrix-oxGFP fluorescence was then quantified by flow cytometry. ***P<0.001 by Student’s T Test. n=3 per condition.

[0290] FIG. 15D.. MitoTracker Green signal in RAW macrophages and BMDMs measured by flow cytometry after 24h LPS simulation.

[0291] FIG. 15E. Mitochondrial DNA/genomic DNA (mtDNA/gDNA) ratio in RAW macrophages treated with PBS vehicle control or LPS for indicated times.

[0292] FIG. 15F. WT (left) and Nrf2 KO (right) BMDMs were treated with EtOH vehicle control, or 4- OHE1/LPS (5pM/100ng/mL) for 7 hours before Prdx6 qPCR. ***P<0.001 by Student’s T Test. n=2 per condition.

[0293] For FIGs. 14 and 15, RNA-seq RPKM and flow cytometry data represented as mean ± SEM. Each data point is an independent biological replicate (n=3 for each condition). Flow cytometry data representative of 2 independent experiments. All bar graph data represented as mean ± SEM.

[0294] FIGs. 16 and 17. Mitohormesis in macrophages involves metabolic reprogramming that enforces an LPS-tolerant state.

[0295] FIG. 16A. Schematic drawing describing how mitochondrial oxidative metabolism (dark gray line, labeled “mito OXPHOS”) supports pro-inflammatory gene expression (light gray line, labeled “pro-inflammatory gene expression”) after LPS treatment, but is suppressed as macrophages transition to an LPS-tolerant state where proinflammatory genes are refractory to upregulation by secondary LPS exposure. Mitohormetic adaptations (gray line) occur in parallel with this process, but whether metabolic reprogramming and suppression of mitochondrial oxidative metabolism is a coincident mitohormetic adaptation is unknown.

[0296] FIG. 16B. Seahorse energy map plotting basal oxygen consumption rate (OCR) versus basal extracellular acidification rate (ECAR) in RAW macrophages treated overnight (18-24 hours) with EtOH, 4-OHE1 (5pM), LPS (lOOng/mL), or both.

[0297] FIG. 16C. Seahorse mitochondrial stress test in RAW macrophages treated overnight (18-24 hours) with EtOH, 4-OHE1 (5pM), LPS (lOOng/mL), or both.

[0298] FIG. 16D. Illb qPCR in RAW macrophages treated overnight (18-24 hours) with EtOH, 4-OHE1 (5pM), LPS (lOOng/mL), or both before treatments were washed out and cells allowed to recover (1-2 hours) before secondary LPS stimulation (lOOng/mL) for 6 hours.

[0299] FIG. 16E. Same as FIG. 16D, with CoA (2.5mM) provided to cells during the washout/recovery period before secondary LPS stimulation.

[0300] FIG. 16F. Relative expression* of select genes in BMDMs treated with EtOH, 4- OHE1 (5pM), or LPS (lOOng/mL) for 24 hours (*log2-transformed RPKM centered on the mean of each gene).

[0301] FIG. 17A. Basal and maximal OCR from RAW macrophage Seahorse Mito Stress Test in FIG. 16C. Data is average of 3 measurements taken prior to oligomycin injection (basal) and following FCCP injection (maximal), respectively. n=5 per condition.

[0302] FIG. 17B. Seahorse mitochondrial stress test in BMDMs treated overnight (18-24 hours) with EtOH, 4-OHE1 (5pM), LPS (lOOng/mL), or both.

[0303] FIG. 17C. Illb qPCR in BMDMs treated overnight (18-24 hours) with EtOH, 4- OHE1 (5pM), LPS (lOOng/mL), or both, before treatments were washed out and cells allowed to recover (1-2 hours) before secondary LPS stimulation (lOOng/mL) for 6 hours. Data is plotted as +/- LPS fold induction for cells with the same primary overnight treatment. n=2 per condition.

[0304] All qPCR data in FIG. 16 and 17 are represented as mean ± SEM. Each data point is an independent biological replicate (n=2 for each condition). *P<0.05, **P<0.01 by Student’s T Test versus the indicated condition. All qPCR data representative of 2 independent experiments except for CoA rescue. All bar graph data represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 by Student’s T Test versus EtOH control. RAW macrophage Seahorse data representative of 2 independent experiments.

[0305] FIGs. 18 and 19. 4-OHE1 prevents HFD-driven obesity and metabolic dysfunction in male mice.

[0306] FIG. 18A. Body weight of normal chow (NC) and (HFD)-fed male C57BL/6N mice injected weekly with EtOH or 4-OHE1 (10 mg/kg) for 13 weeks [n=4,4, 8,7 for NC EtOH, NC 4- OHEl(not shown), HFD EtOH, and HFD 4-OHE1 groups, respectively].

[0307] FIGs. 18B and 18C. Fasting blood glucose (B) and intraperitoneal glucose tolerance test (GTT, C) after 13 weeks.

[0308] FIG. 18D. EchoMRI measurement of fat mass (left) and lean mass (right) after 13 weeks.

[0309] FIG. 18E. Weight of visceral (vWAT, left) subcutaneous (scWAT, right) white adipose tissue after 13 weeks.

[0310] FIG. 18F. Representative mice from HFD EtOH and HFD 4-OHE1 groups, with arrows highlighting the reductions in both vWAT and scWAT in 4-OHEl-treated mice.

[0311] FIG. 18G. vWAT cellularity measurements for leukocytes (left) and macrophages (right).

[0312] FIG. 18H. vWAT macrophage CD301 and CDllc mean fluorescence intensity (MFI).

[0313] FIG. 19A. Body weight (left), fasting blood glucose (middle), and GTT (right) for NC and HFD-fed male C57BL/6N mice injected weekly with EtOH or 2-OHE1 (10 mg/kg) for 8 weeks [n=4,4, 8,7 for NC EtOH, NC 2-OHE1 (not shown), HFD EtOH, and HFD 2- OHE1 groups, respectively].

[0314] FIG. 19B. Body weight (left), fasting blood glucose (middle), and GTT (right) for NC and HFD-fed male C57BL/6N mice injected weekly with EtOH or 2-OHE2 (10 mg/kg) for 8 weeks [n=3 ,4,7,6 for NC EtOH, NC 2-OHE2(not shown), HFD EtOH, and HFD 2-OHE2 groups, respectively].

[0315] FIGs. 19C, 19D, 19E, and 19F. Food consumption, activity, oxygen consumption, and respiratory exchange ratio (RER) in individually-housed HFD EtOH and HFD 4-OHE1 mice in metabolic cages at 4 weeks (n=4,4) and 13 weeks (n=8,7) following initiation of HFD feeding and injections. After an overnight acclimation to metabolic cages, data was collected for a 24 hour period (i.e., one dark- light cycle) and averaged.

[0316] All data points and error bars for FIGs. 18 and 19 represent mean ± SEM. *P<0.01, **P<0.01, ***P<0.001, ****P<0.0001 by Student’s T Test.

[0317] FIGs. 20 and 21. 2 -OHE2 ameliorates HFD-driven metabolic dysfunction in OVX female mice.

[0318] FIG. 20A. Body weight of normal chow (NC) and (HFD)-fed OVX female C57BL/6N mice injected weekly with EtOH or 2-OHE2 (10 mg/kg) for 14 weeks [n=3,3, 6,6 for NC EtOH, NC 2-OHE2 (not shown), HFD EtOH, and HFD 2-OHE2 groups, respectively].

[0319] FIGs. 20B and 20C. Fasting blood glucose (FIG. 20B) and intraperitoneal glucose tolerance test (GTT, FIG. 20C) after 14 weeks.

[0320] FIG. 20D. EchoMRI measurement of fat mass (left) and lean mass (right) after 14 weeks.

[0321] FIG. 20E. Weight of visceral (vWAT, left) subcutaneous (scWAT, right) white adipose tissue after 14 weeks.

[0322] FIG. 20F. vWAT cellularity measurements for leukocytes (left) and macrophages (right).

[0323] FIGs. 21A, 21B, 21C, and 21D: Food consumption, activity, oxygen consumption, and respiratory exchange ratio (RER) in individually-housed HFD EtOH and HFD 2-OHE2 mice in metabolic cages after 14 weeks (n=5,6). After an overnight acclimation to metabolic cages, data was collected for a 24 hour period (i.e., one dark-light cycle) and averaged.

[0324] FIG. 21E: Body weight of normal chow (NC) and (HFD)-fed OVX female C57BL/6N mice injected weekly with EtOH or 4-OHE1 (10 mg/kg) for 14 weeks [n=7,7,2 for NC EtOH, HFD EtOH, and HFD 2-OHE2 groups, respectively].

[0325] FIG. 21F: Fasting intraperitoneal GTT after 14 weeks.

[0326] All data points and error bars for FIGs. 20 and 21 represent mean ± SEM. *P<0.01, **P<0.01, ***P<0.001, by Student’s T Test.

[0327] FIGs. 22 and 23. 4-OHE1 alters adipose tissue gene expression.

[0328] FIG. 22A. Hierarchical clustering of whole vWAT RNAseq data from NC EtOH, HFD EtOH, and HFD 4-OHE1 mice (n=4 per group). Heatmap represents relative gene expression, centered on the log2 transformed mean, for each gene.

[0329] FIG. 22B. Normalized expression of select genes in vWAT from RNAseq data. [0330] FIGs. 22C and 22D. Summary of differential gene expression analysis comparing genes significantly (log2 fold change > 1.5, FDR <0.05) upregulated or downregulated in vWAT, scWAT, and BAT. Lists represent genes with significantly altered expression in all three adipose tissues (AT) of HFD 4-OHE1 mice versus HFD EtOH control tissues. The number of total genes significantly altered in each tissue is also indicated.

[0331] FIG. 22E. Cfd, Irf4, and Irsl expression in vWAT.

[0332] FIG. 22F. Sppl expression in vWAT.

[0333] FIG. 23A. Cfd, Irf4, and Irsl expression in primary bone-marrow derived macrophages (BMDMs) treated with 5|xM 4-OHE1 for 24 hours.

[0334] FIG. 23B. Sppl expression in (BMDMs) treated with 5|xM 4-OHE1 for 24 hours.

[0335] FIG. 23C. Thermogenic gene expression in BAT (left) and scWAT (right) from HFD EtOH and HFD 4-OHE1 mice.

[0336] All data points and error bars in FIGs. 22 and 23 represent mean ± SEM.

[0337] FIGs. 24 and 25. 4-OHE1 promotes weight loss and improves glucose tolerance in male mice with existing diet-induced obesity.

[0338] FIG. 24A. Body weight of male C57BL/6N mice fed HFD chow for 16 weeks, and then injected SQ with EtOH, E2 (lOmg/kg), or 4-OHE1 (lOmg/kg) for 8 weeks (n=5 for each group) while continuing HFD feeding.

[0339] FIG. 24B. Food consumption tracked at the cage in grouped housed animals from FIG. 13A. Each treatment group of 5 mice was housed in 2 cages with 2 or 3 animals each.

[0340] FIG. 24C. EchoMRI measurement of fat mass (left) and lean mass (right) after 8 weeks of treatment.

[0341] FIG. 24D. Blood glucose levels in ad libitum fed mice measured at 1 and 8 weeks of treatment.

[0342] FIG. 24E and 24F. Fasting blood glucose (E) and intraperitoneal glucose tolerance test (GTT, F) after 8 weeks.

[0343] FIG. 24G. Weight of visceral (vWAT, left) subcutaneous (scWAT, right) white adipose tissue after 8 weeks.

[0344] FIG. 24H. Representative tissue from EtOH- and 4-OHEl-treated mice groups.

[0345] FIG. 241. Triglyceride (TG) levels in liver tissue from age matched normal chow (NC)- fed control mice versus HFD-fed mice treated with EtOH or 4-OHE1 (lOmg/kg).

[0346] FIG. 24J. Collage content of liver tissue from age matched NC-fed control mice versus HFD-fed mice treated with EtOH or 4-OHE1 (lOmg/kg). [0347] FIG. 24K. vWAT macrophage CDllc and CD301 mean fluorescence intensity (MFI). [0348] FIG. 25A. Body weight of male C57BL/6N mice fed HFD chow for 16 weeks, and then injected SQ with EtOH or 4-OHE1 (Img/kg) for 8 weeks (n=5 for each group) while continuing HFD feeding.

[0349] FIG. 25B. Fasting intraperitoneal glucose tolerance test (GTT) on mice from A. after 8 weeks.

[0350] FIG. 25C. Food consumption tracked at the cage in grouped housed animals from FIG. 13A. Each treatment group of 5 mice was housed in 2 cages with 2 or 3 animals each.

[0351] FIGs. 25D, 25E, 25F, and 25G. Food consumption, activity, oxygen consumption, and respiratory exchange ratio (RER) in individually-housed EtOH control, 4-OHE1 (lOmg/kg), and E2 (lOmg/kg) -treated mice in metabolic cages after 8 weeks of treatment (n=4,3,4). After an overnight acclimation to metabolic cages, data was collected for a 24 hour period (i.e. one dark-light cycle) and averaged.

[0352] FIG. 25H. vWAT cellularity measurements for leukocytes (left) and macrophages (right).

[0353] All data points and error bars in FIGs. 24 and 25 represent mean ± SEM. *P<0.01, **P<0.01, ***P<0.001, ****P<0.0001 by Student’s T Test.

[0354] FIG. 26. CoA and Acetyl-CoA enhance TLR-dependent inflammatory responses in murine and human macrophages. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student’s T Test vs. indicated condition.

[0355] FIG. 26a. Murine RAW macrophages were pretreated with 500uM CoA for 15min, followed by 7.5 LPS stimulation (lOOng/mL) before harvest and Illb qPCR.

[0356] FIG. 26b. Murine BMDMs were pretreated with 250uM CoA for 3h, followed by 1.5h LPS stimulation (lOOng/mL) before harvest and Illb qPCR.

[0357] FIG. 26c. Human THP-1 cells were pretreated with 500uM CoA for 15min, followed by 6h LPS stimulation (lOOng/mL) before harvest and Illb qPCR.

[0358] FIG. 26d. Murine BMDMs were pretreated with 500uM CoA for 15min, followed by 6h MPLA stimulation (lOOng/mL) before harvest and III b qPCR.

[0359] FIG. 27. In vivo Coenzyme A + LPS administration. FIG. 27a - study design. FIG. 27b-d: Serum concentrations of cytokines (FIG. 27b), chemokines (FIG. 27c), and growth factors following administration of the test articles (FIG. 27d). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student’s t test. [0360] FIG. 28. Combination of TLR4 ligand MPLA and CoA significantly slows tumor growth and weight as compared with MPLA alone in PyMPT breast cancer cells. FIG. 28a: tumor growth curve; FIG. 28b: Day 35 tumor weights.

[0361] FIG. 29. CoA/Acetyl-CoA enhance mitochondrial glucose oxidation to support proinflammatory gene expression. FIG. 29a. Murine bone-marrow derived macrophages (BMDMs) were provided with either vehicle or 250uM CoA or Ac-CoA. After 2h, cells were pretreated with EtOH or 5uM 4-OHE1, which causes mitochondrial oxidative stress that suppresses LPS-induced gene expression. After Ih, cells were stimulated with lOOng/mL LPS. 1.5h later (with cells switched to isotopically labeled C13-glucose containing media the last 30min), cells were harvested to assess gene expression and C13-glucose flux. LPS- induced Illb expression is suppressed by 4-OHE1, but enhanced in presence of CoA r Ac- CoA. FIG. 29b. LPS-induced C13-glucose flux into the TCA cycle metabolites acetyl-CoA and citrate is suppressed by 4-OHE1, but enhanced in presence of CoA or Ac-CoA. FIG.

29c. Model for how CoA supplementation promotes/enhances mitochondrial glucose utilization and oxidation, especially in the face of mitochondrial oxidative stress when CoA/ Ac-CoA is used as an anti-oxidant to protect the mitochondrial proteome.

DETAILED DESCRIPTION OF THE INVENTION

[0362] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details.

Certain Definitions

[0363] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0364] The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range.

[0365] The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, may "consist of" or "consist essentially of" the described features.

Compounds

[0366] Provided herein are hydroxyestrogen compounds that target mitohormesis and are useful in the treatment in various conditions. Hydroxy estrogens are known as estrogen metabolites that may retain some biological activities of the parent molecule estrogen. The present disclosure is based in part on the unexpected finding that hydroxyestrogens have a unique regulatory role and biological activities as compared with estrogens in that they target mitohormesis and therefore have unique applications in inflammation and metabolic diseases. [0367] In some embodiments, the hydroxyestrogen is a hydroxy estrone. As used herein, the term “hydroxyestrone” means a hydroxylated estrone. As used herein, the term “estrone” means the compound having the chemical designation (8R,9S,13S,14S)-3-hydroxy-13-methyl- 7,8,9,ll,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-17-one. As used, herein, the term “estrone” means the compound having the chemical structure:

[0368] In some embodiments, the hydroxyestrone is 4-hydroxyestrone (4-OHE1). As used herein, the term “4-hydroxyestrone” means the compound having the chemical designation (8R,9S,13S,14S)-3,4-dihydroxy-13-methyl-7,8,9,ll,12,14,15,16-octahydro-6H- cyclopenta[a]phenanthren- 17-one. [0369] In some embodiments, the hydroxyestrone is 2-hydroxyestrone (2-OHE2). As used herein, the term “2-hydroxyestrone” means the compound having the chemical designation (8R,9S,13S,14S)-2,3-dihydroxy-13-methyl-7,8,9,ll,12,14,15,16-octahydro-6H- cyclopenta[a]phenanthren- 17-one.

[0370] In some embodiments, the hydroxyestrogen is a hydroxyestradiol. As used herein, the term “hydroxyestradiol” means a hydroxylated estradiol. As used herein, the term “estradiol” means the compound having the chemical designation (8R,9S,13S,14S,17S)-13-Methyl- 6,7,8,9,ll,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol. As used, herein, the term “estradiol” means the compound having the chemical structure:

[0371] In some embodiments, the hydroxyestradiol is 4-hydroxyestradiol (4-OHE2). As used herein, the term “4- hydroxyestradiol” means the compound having the chemical designation (8R,9S,13S,14S,17S)-13-methyl-6,7,8,9,ll,12,14,15,16,17-decahydrocyclopenta [a] phenanthrene-3 ,4, 17-triol.

[0372] In some embodiments, the hydroxyestradiol is 2-hydroxyestradiol (2-OHE2). As used herein, the term “2 -hydroxyestrone” means the compound having the chemical designation (8R,9S,13S,14S,17S)-13-methyl-6,7,8,9,ll,12,14,15,16,17-decahydrocyclopenta [a]phenanthrene-2 , 3 , 17 -triol.

[0373] The structures of 4-hydroxyestrone (4-OH-E1), 2-hydroxyestrone (2-OHE1), 4- hydroxyestradiol (4-OH-E2) and 2-hydroxyestradiol (2-OH-E2) are presented in Scheme 1.

4-hydroxyestradiol

= 4-OHE2

Scheme 1

[0374] The compounds of the present disclosure may be present in the form of a salt. The compounds of the present invention may be present in the form of a pharmaceutically acceptable salt. Pharmaceutically-acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically- acceptable salt is an ammonium salt.

[0375] Metal salts can arise from the addition of an inorganic base to a compound of the invention. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[0376] In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.

[0377] Ammonium salts can arise from the addition of ammonia or an organic amine to a compound of the invention. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N- methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine. In some embodiments, an ammonium salt is a triethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N- methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrazole salt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or a pipyrazine salt.

[0378] Acid addition salts can arise from the addition of an acid to a compound of the invention. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.

[0379] In some embodiments, the salt is a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a glucaronate salt, a saccharate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a propionate salt, a butyrate salt, a fumarate salt, a succinate salt, a methanesulfonate (mesylate) salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt, or a maleate salt. [0380] The hydroxyestrogens can be obtained by any manner known in the art. In some embodiments, the hydroxyestrogens can be isolated from a natural source (herein “isolated hydroxyestrogen”). In some embodiments, the hydroxyestrogen can be synthetically obtained (herein “synthetic hydroxy estrogen”). The term “synthetic” as used herein encompasses total synthesis or semi-synthesis.

[0381] In some embodiments, the hydroxyestrogen is an isolated hydroxy estrogen. In some embodiments, the hydroxyestrone is an isolated hydroxyestrone. In some embodiments, the hydroxyestradiol is an isolated hydroxyestradiol.

[0382] In some embodiments, the hydroxyestrogen is a synthetic hydroxyestrogen. In some embodiments, the hydroxyestrone is a synthetic hydroxyestrone. In some embodiments, the hydroxyestradiol is a synthetic hydroxyestradiol.

[0383] In some embodiments, the hydroxyestrogen is a semi- synthetic hydroxyestrogen. In some embodiments, the hydroxyestrone is a semi-synthetic hydroxyestrone. In some embodiments, the hydroxyestradiol is a semi-synthetic hydroxyestradiol.

[0384] In some embodiments, the hydroxyestrogen is an exogenous hydroxy estrogen. As used herein, the term “exogenous” means growing or originating from outside an organism. An exogenous hydroxyestrogen also means non-endogenous hydroxy estrogen, i.e., it is produced, located, or isolated outside of a living organism, e.g., a human body.

[0385] Any compound herein can be purified. A compound herein can be least 1% pure, at least 2% pure, at least 3% pure, at least 4% pure, at least 5% pure, at least 6% pure, at least 7% pure, at least 8% pure, at least 9% pure, at least 10% pure, at least 11% pure, at least 12% pure, at least 13% pure, at least 14% pure, at least 15% pure, at least 16% pure, at least 17% pure, at least 18% pure, at least 19% pure, at least 20% pure, at least 21% pure, at least 22% pure, at least 23% pure, at least 24% pure, at least 25% pure, at least 26% pure, at least 27% pure, at least 28% pure, at least 29% pure, at least 30% pure, at least 31% pure, at least 32% pure, at least 33% pure, at least 34% pure, at least 35% pure, at least 36% pure, at least 37% pure, at least 38% pure, at least 39% pure, at least 40% pure, at least 41% pure, at least 42% pure, at least 43% pure, at least 44% pure, at least 45% pure, at least 46% pure, at least 47% pure, at least 48% pure, at least 49% pure, at least 50% pure, at least 51% pure, at least 52% pure, at least 53% pure, at least 54% pure, at least 55% pure, at least 56% pure, at least 57% pure, at least 58% pure, at least 59% pure, at least 60% pure, at least 61% pure, at least 62% pure, at least 63% pure, at least 64% pure, at least 65% pure, at least 66% pure, at least 67% pure, at least 68% pure, at least 69% pure, at least 70% pure, at least 71% pure, at least 72% pure, at least 73% pure, at least 74% pure, at least 75% pure, at least 76% pure, at least 77% pure, at least 78% pure, at least 79% pure, at least 80% pure, at least 81% pure, at least 82% pure, at least 83% pure, at least 84% pure, at least 85% pure, at least 86% pure, at least 87% pure, at least 88% pure, at least 89% pure, at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.1% pure, at least 99.2% pure, at least 99.3% pure, at least 99.4% pure, at least 99.5% pure, at least 99.6% pure, at least 99.7% pure, at least 99.8% pure, or at least 99.9% pure.

[0386] Compounds herein can include all stereoisomers, enantiomers, diastereomers, mixtures, racemates, atropisomers, and tautomers thereof. The compounds disclosed herein, in some embodiments, contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R)- or (S)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the compounds disclosed herein are contemplated by this disclosure.

Biological Activity

[0387] Macrophage inflammatory responses are important for host defense, but if not tightly controlled, can be detrimental to the host in acute and chronic inflammatory disease. Macrophage tolerance evolved to protect the host from overproduction of inflammatory mediators; however, this immunoparalyzed state impairs the ability of macrophages to clear pathogens, tumors, and perform tissue homeostatic function. Thus, understanding how the balance between responsiveness versus tolerance is regulated has far-reaching implications in heath and disease.

[0388] Metabolism transforms nutrients into substrates utilized by enzymes catalyzing epigenetic modifications controlling cellular gene expression, and in turn, functional states. The metabolic state of responsive versus tolerant macrophages is divergent. Mitochondrial oxidative metabolism and acetyl-Coenzyme A (acetyl-CoA) production from oxidized glucose regulate histone acetylation and pro-inflammatory gene transcription in macrophages acutely stimulated with the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS). In contrast, a shift away from oxidative mitochondrial metabolism and towards aerobic glycolysis occurs during the development of LPS tolerance. LPS is the endotoxin portion of the cell wall of a gram-negative bacteria. It is one of the most abundant proinflammatory stimuli in the gastrointestinal tract. LPS is a non-limiting example of a broad range of natural immune stimulators that can be used in the present disclosure. [0389] Mitochondrial integrity is closely monitored by quality control systems. This includes nuclear-encoded transcriptional factors that detect signs of mitochondrial stress, including increased mitochondrial reactive oxygen species (mtROS) and mitochondrial protein misfolding. In model organisms such as Caenorhabditis elegans and Saccharomyces cerevisiae, stress-induced activation of these transcription factors promotes persistent cyto- /mito-protective adaptations and stress resistance in a process known as mitohormesis, which influences organismal metabolism, health, and longevity. LPS- stimulation induces mitochondrial stress in macrophages. Mitohormetic adaptations to LPS-driven mitochondrial stress control the transition to an LPS-tolerant state via metabolic reprogramming and suppression of mitochondrial oxidative metabolism.

[0390] Macrophages induce tolerance to avoid the hyperactivation of inflammation that results in a cytokine storm. Tolerance may prevent death in hyperactive conditions, but it may also cause immunoparalysis. As contemplated herein, it has now been discovered that administration of acetyl-CoA or CoA breaks tolerance in macrophages and restores responsive immune reactions. This could be useful for treating sepsis, where macrophage immunoparalysis leaves patients susceptible to secondary infections. It could also be useful for treating cancer and/or reducing cancer metastases, where suppression of macrophage immune reactions in the tumor microenvironment helps cancer cells evade killing by the immune system.

[0391] Proinflammatory cytokines have been implicated as playing a causative role in insulin resistance in metabolic disease. Accordingly, genetic and pharmacological manipulations that antagonize myeloid inflammatory responses have been shown to improve glucose tolerance in murine models of high-fat diet (HFD)-driven obesity and type 2 diabetes (T2D).

[0392] In some embodiments, the present disclosure is based on the discovery of hydroxyestrogens with anti-inflammatory activity in macrophages. In some embodiments, targeting mitochondrial function may repress or promote macrophage inflammatory responses for host benefit.

[0393] In some embodiments, hydroxyestrogens are repressors of LPS-induced proinflammatory gene transcription. Unexpectedly, these effects were estrogen receptor (ER) -independent.

[0394] In some embodiments, the present disclosure provides hydroxyestrogens as lipophilic mediators of oxidative and electrophilic mitochondrial stress in macrophages. Upon acute treatment, hydroxyestrogens including 4-hydroxyestrone (4-OHE1) impair mitochondrial acetyl-CoA production required for histone acetylation and lipopolysaccharide (LPS)-induced proinflammatory gene transcription (FIG. 1).

[0395] In some embodiments, hydroxyestrogen (e.g., 4-OHEl)-driven mitochondrial stress triggers mitohormetic adaptations in macrophages, including increased mitochondrial chaperone activity, and mitochondrial oxidative stress resistance. Similar mitohormetic adaptations were induced by LPS-driven mitochondrial stress as macrophages transition from a responsive to LPS-tolerant state, suggesting mitochondrial stress triggers transition. As demonstrated herein, similar to LPS, hydroxyestrogens (e.g., 4-OHEl)-induced mitohormesis reprogrammed macrophage metabolism away from mitochondrial oxidative metabolism and towards aerobic glycolysis, enforcing an immunoparalyzed state of diminished LPS responsiveness. Targeting mitochondrial production of metabolites utilized for epigenetic modifications supporting pro-inflammatory gene transcription with lipophilic electrophiles such as hydroxyestrogens represents an attractive anti-inflammatory strategy (FIG. 1).

[0396] In some embodiments, mitohormetic adaptations to LPS-driven mitochondrial stress control the transition to an LPS-tolerant state via metabolic reprogramming and suppression of mitochondrial oxidative metabolism (FIG. 1).

[0397] Provided herein is a method of activating or inducing mitohormesis in a cell comprising administering or causing to be administered a hydroxyestrogen or salt thereof. In some embodiments, chaperone activity is increased. In some embodiments, mitochondrial oxidative stress resistance increases in the cell. In some embodiments, the ratio of aerobic glycolysis to mitochondrial oxidative metabolism increases in the cell. In some embodiments, the cell is a macrophage.

[0398] In some embodiments, a method of mediating mitochondrial stress in a cell or activating or inducing mitohormesis comprises administering or causing to be administered 4-hydroxyestrone (4-OHE1) or a salt thereof.

[0399] In some embodiments, a method of mediating mitochondrial stress in a cell or activating or inducing mitohormesis comprises administering or causing to be administered 2- hydroxyestrone (2-OHE1) or a salt thereof.

[0400] In some embodiments, a method of mediating mitochondrial stress in a cell or activating or inducing mitohormesis comprises administering or causing to be administered 2-hydroxyestradiol (2-OHE2) or a salt thereof. Therapeutic Uses

[0401] In some embodiments, the present disclosure relates to therapeutic methods for treating conditions and diseases. In some embodiments, the present disclosure relates to therapeutic methods for treating inflammatory conditions. In some embodiments, the present disclosure relates to methods to activate mitohormesis and repress inflammation with clinical applications to treat various conditions and disorders including acute inflammation, chronic inflammation, and age-dependent diseases. In other embodiments, the present disclosure relates to therapeutic methods of mitigating effects of a high fat diet. In other embodiments, the present disclosure relates to therapeutic methods for reducing body weight. In some embodiments, the present disclosure relates to methods of reducing fasting blood glucose levels. In some embodiments, the present disclosure relates to method of treating obesity. In some embodiments, the present disclosure relates to method of treating metabolic disorders. Types of metabolic disorders include, but are not limited to, acid-base imbalance, metabolic brain diseases, disorders of calcium metabolism, DNA repair-deficiency disorders, glucose metabolism disorders, hyperlactatemia, iron metabolism disorders, lipid metabolism disorders, malabsorption syndromes, and the like.

[0402] In some embodiments, the present disclosure relates to methods of treating cancer, which can refer to any malignant disease. As used herein, the term “treating cancer” also includes reducing, inhibiting or preventing the formation or progression of cancer metastases.

[0403] The compounds and compositions of the present invention can be administered to patients or subjects in need thereof. The term “patient” or “subject” refers to a mammal, such as a human, bovine, rat, mouse, dog, monkey, ape, goat, sheep, cow, or deer. Generally, a patient as described herein is human.

[0404] The term “effective amount” refers to the amount of a therapy which is sufficient to accomplish a stated purpose or otherwise achieve the effect for which it is administered. An effective amount can be sufficient to reduce and/or ameliorate the progression, development, recurrence, severity and/or duration of a given disease, disorder or condition and/or a symptom related thereto. An effective amount can be a “therapeutically effective amount” which refers to an amount sufficient to provide a therapeutic benefit such as, for example, the reduction or amelioration of the advancement or progression of a given disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a given disease, disorder or condition, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy. A therapeutically effective amount of a composition described herein can enhance the therapeutic efficacy of another therapeutic agent.

[0405] The terms “therapies” and “therapy” refer to any protocol(s), method(s), and/or agent(s) that can be used in the prevention, treatment, management, and/or amelioration of a disease, disorder, or condition or one or more symptoms thereof. In certain instances the term refers to active agents such as a hydroxyestrogen described herein. The terms “therapy” and “therapies” can refer to anti-inflammatory, anti-obesity and/or other therapies useful in treatment, management, prevention, or amelioration of a disease, disorder, or condition or one or more symptoms thereof known to one skilled in the art, for example, a medical professional such as a physician.

[0406] Provided herein is a method of inhibiting an inflammatory response in a subject comprising administering or causing to be administered a hydroxyestrogen to the subject. In some embodiments, induction of Illb expression by an inflammatory stimulus in a macrophage of the subject is reduced. In some embodiments, the inflammatory stimulus is a lipopolysaccharide (LPS). In some embodiments, Illb expression in a visceral white adipose tissue macrophage of the subject is reduced.

[0407] In some embodiments, the inflammatory response is an acute inflammatory response. In some embodiments, the inflammatory response is a chronic inflammatory response.

[0408] Provided herein is a method of treating an acute inflammatory condition in a subject comprising administering or causing to be administered a pharmaceutical composition comprising a hydroxyestrogen.

[0409] In some embodiments, the acute inflammatory condition comprises a cytokine storm. In some embodiments, a subject is treated with the hydroxy estrogen.

[0410] In one embodiment, the subject is infected with a coronavirus. In some embodiments, the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (beta coronavirus that causes Middle East Respiratory Syndrome, or MERS), SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS) SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or CO VID- 19, also referred to herein as SARS-Covid-19). In one embodiment, the coronavirus is SARS-Covid-19.

[0411] In some embodiments, the inflammation is associated with an inflammatory disease or disorder. In some embodiments, the inflammatory disease or disorder is selected from inflammatory bowel disease, arthritis, obesity, radiation-induced inflammation, psoriasis, T cell-mediated hypersensitivity diseases, allergic diseases, atopic dermatitis, non-alcoholic steatohepatitis (NASH), systemic lupus erythematosus (SLE), autoimmune thyroiditis (Grave's disease), multiple sclerosis, ankylosing spondylitis and bullous diseases due to overproduction of pro-inflammatory cytokines, Crohn's disease, or asthma.

[0412] In some embodiments, use of hydroxy estrogens to activate mitohormesis and repress inflammation can be used as a treatment for various pathological conditions including acute inflammation (e.g., severe infection, early sepsis, injury), chronic inflammation (e.g., obesity, type 2 diabetes, atherosclerosis, NASH), and age-dependent diseases (e.g., Parkinson’s disease, Alzheimer’s disease, arthritis, inflam- aging).

[0413] In some embodiments, administration of acetyl-CoA or Co A may be useful as a treatment for patients during late-stage sepsis and other immune-compromised conditions to reverse the effects of mitohormesis and rescue patients from an immunosuppressed status (e.g. cancer, including metastases thereof, age- and obesity-related immunosuppression). [0414] In some embodiments, a method of inhibiting an inflammatory response in a subject or treating an acute inflammatory condition in a subject comprises administering or causing to be administered 4-hydroxyestrone (4-OHE1) or a salt thereof.

[0415] In some embodiments, a method of inhibiting an inflammatory response in a subject or treating an acute inflammatory condition in a subject comprises administering or causing to be administered 2-hydroxyestrone (2-OHE1) or a salt thereof.

[0416] In some embodiments, a method of inhibiting an inflammatory response in a subject or treating an acute inflammatory condition in a subject comprises administering or causing to be administered 2-hydroxyestradiol (2-OHE2) or a salt thereof.

[0417] Provided herein is a method of mitigating effects of a cause, parameter or condition that can lead to the development of diabetes (e.g., type-2 diabetes) or obesity, such as sedentary lifestyle, being overweight, or consuming an unhealthy or a high fat diet comprising administering or causing to be administered a hydroxyestrogen to a subject, wherein a body weight gain of the subject is reduced. In some embodiments, the body weight gain of the subject reduced by at least 10% compared to a body weight gain of a control subject not administered a hydroxyestrogen.

[0418] Provided herein is a method of mitigating the effects of a high fat diet comprising administering or causing to be administered a hydroxyestrogen to a subject, wherein a fasting blood glucose level of the subject is reduced.

[0419] Provided herein is a method of mitigating the effects of a high fat diet comprising administering or causing to be administered a hydroxyestrogen to a subject, wherein glucose tolerance is improved. In some embodiments, a blood glucose level 60 minutes after a meal is reduced by at least 10% compared to a blood glucose level of a control subject not administered a hydroxyestrogen. In some embodiments, a blood glucose level 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes after a meal is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27% 28%, 29% or 30% compared to a blood glucose level of a control subject not administered a hydroxy estrogen.

[0420] Provided herein is a method of treating obesity in a subject comprising administering or causing to be administered a hydroxyestrogen to the subject, thereby reducing body weight of the subject.

[0421] In some embodiments, the body weight of the subject is reduced by at least about 5%. In some embodiments, the body weight of the subject is reduced by at least about 10%. In some embodiments, the body weight of the subject is reduced by at least about 15%. In some embodiments, the body weight of the subject is reduced by at least about 20%. In some embodiments, the body weight of the subject is reduced by at least about 25%. In some embodiments, the body weight of the subject is reduced by at least about 30%.

[0422] In some embodiments, a fed blood glucose is reduced. In some embodiments, the subject has ingested a high fat diet. In some embodiments, the high fat diet comprises at least 10% fat. In some embodiments, the high fat diet comprises at least 20% fat. In some embodiments, the high fat diet comprises at least 30% fat.

[0423] In some embodiments, a fasting blood glucose of the subject is reduced compared to a fasting blood glucose of the subject prior to administration of the hydroxyestrogen, or a control subject not administered a hydroxy estrogen. In some embodiments, infiltration of immune cells into visceral white adipose tissue is reduced. In some embodiments, infiltration of CD45+ leukocytes into visceral white adipose tissue is reduced. In some embodiments, infiltration of CDllb+ macrophages into visceral white adipose tissue is reduced. In some embodiments, oxygen consumption and energy expenditure of the subject is increased.

[0424] In some embodiments, fat mass is reduced. In other embodiments, visceral white adipose tissue mass is reduced. In other embodiments, subcutaneous white adipose tissue mass is reduced.

[0425] Provided herein is a method of treating a cancer comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby inhibit growth of a transformed cell in the subject. In some embodiments, the coenzyme A derivative is acetyl-CoA. In some embodiments, the coenzyme A derivative is 4-phosphopanthetheine. [0426] Provided herein is a method of treating, or preventing the formation of, a cancer metastases comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby inhibit growth of a transformed cell in the subject. In some embodiments, the coenzyme A derivative is acetyl-CoA. In some embodiments, the coenzyme A derivative is 4-phosphopanthetheine.

[0427] In some embodiments, the coenzyme A (CoA) or derivative thereof is coadministered with a proinflammatory signaling pathway agonist. In some embodiments, the proinflammatory signaling pathway agonist is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette- Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

[0428] In some embodiments, the coenzyme A (CoA) or derivative thereof is coadministered with an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises an anti-PD- 1 antibody, an anti-CTLA-4 antibody, or a fragment thereof.

[0429] In some embodiments, the coenzyme A (CoA) or derivative thereof is coadministered with a proinflammatory signaling pathway agonist and an immune checkpoint inhibitor.

[0430] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the solid tumor is a melanoma, colon adenocarcinoma, bladder cancer, hepatoma, or breast cancer. In some embodiments, the cancer comprises a hematologic malignancy. In some embodiments, the hematologic malignancy is lymphoma or acute myeloid leukemia.

[0431] In some embodiments, the coenzyme A (CoA) or derivative thereof is an exogenous coenzyme A (CoA) or derivative thereof. In some embodiments, the coenzyme A (CoA) or derivative thereof is an isolated coenzyme A (CoA) or derivative thereof. In some embodiments, the coenzyme A (CoA) or derivative thereof is a synthetic or semi-synthetic coenzyme A (CoA) or derivative thereof.

Dosages and Dosing Regimens

[0432] As used herein, the term “therapeutically effective dose” means (unless specifically stated otherwise) a quantity of a compound which, when administered either one time or over the course of a treatment cycle affects the health, wellbeing or mortality of a subject (e.g., and without limitation, delays the onset of and/or reduces the severity of one or more of the symptoms associated with a fibrotic disease or condition and/or a cancer, as applicable).

Useful dosages of the compounds of the present disclosure can be determined by comparing their in vitro activity, and the in vivo activity in animal models. Methods of the extrapolation of effective dosages in mice and other animals to human subjects are known in the art. Indeed, the dosage of the compound can vary significantly depending on the condition of the host subject, the cancer or fibrotic disease being treated, how advanced the pathology is, the route of administration of the compound and tissue distribution, and the possibility of cousage of other therapeutic treatments (such as radiation therapy or additional drugs in combination therapies). The amount of the composition required for use in treatment (e.g., the therapeutically or diagnostically effective amount or dose) will vary not only with the particular application, but also with the salt selected (if applicable) and the characteristics of the subject (such as, for example, age, condition, sex, the subject’s body surface area and/or mass, tolerance to drugs) and will ultimately be at the discretion of the attendant physician, clinician, or otherwise.

[0433] In some embodiments, a therapeutically-effective amount of a hydroxy estrogen sufficient to induce mitochondrial stress and/or mitohormesis is greater than an amount endogenously produced in a subject. In some embodiments, a therapeutically-effective amount of a hydroxyestrogen sufficient to induce mitochondrial stress and/or mitohormesis can range from about 1 mg to about 1000 mg; from about 1 mg to about 500 mg; from about 1 mg to about 250 mg; from about 1 mg to about 200 mg; from about 1 mg to about 100 mg; from about 1 mg to about 50 mg; from about 5 mg to about 1000 mg; from about 2 mg to about 1000 mg; from about 2 mg to about 500 mg; from about 2 mg to about 250 mg; from about 2 mg to about 200 mg; from about 2 mg to about 100 mg; from about 2 mg to about 50 mg; from about 5 mg to about 1000 mg; from about 5 mg to about 500 mg; from about 5 mg to about 250 mg; from about 5 mg to about 200 mg; from about 5 mg to about 100 mg; from about 5 mg to about 50 mg; from about 10 mg to about 1000 mg; from about 10 mg to about 500 mg; from about 10 mg to about 250 mg; from about 10 mg to about 200 mg; from about 10 mg to about 100 mg; from about 10 mg to about 50 mg; from about 20 mg to about 500 mg; from about 20 mg to about 250 mg; from about 20 mg to about 200 mg; from about 20 mg to about 100 mg; from about 20 mg to about 50 mg; from about 25 mg to about 500 mg; from about 25 mg to about 250 mg; from about 25 mg to about 200 mg; from about 25 mg to about 100 mg; from about 25 mg to about 50 mg; from about 50 mg to about 500 mg; from about 50 mg to about 250 mg; from about 50 mg to about 200 mg; from about 50 mg to about 100 mg; from about 100 mg to about 150 mg; from about 150 mg to about 200 mg; from about 200 mg to about 250 mg; from about 250 mg to about 300 mg; from about 300 mg to about 350 mg; from about 350 mg to about 400 mg; from about 400 mg to about 450 mg; from about 450 mg to about 500 mg; from about 500 mg to about 550 mg; from about 550 mg to about 600 mg; from about 600 mg to about 650 mg; from about 650 mg to about 700 mg; from about 700 mg to about 750 mg; from about 750 mg to about 800 mg; from about 800 mg to about 850 mg; from about 850 mg to about 900 mg; from about 900 mg to about 950 mg; or from about 950 mg to about 1000 mg.

[0434] In some embodiments, a therapeutically-effective amount of a hydroxy estrogen sufficient to induce mitochondrial stress and/or mitohormesis can be about 10 mg per day, about 20 mg per day, about 30 mg per day, about 40 mg per day, about 50 mg per day, about 60 mg per day, about 70 mg per day, about 80 mg per day, about 90 mg per day, about 100 mg per day, about 110 mg per day, about 120 mg per day, about 130 mg per day, about 140 mg per day, about 150 mg per day, about 160 mg per day, about 170 mg per day, about 180 mg per day, about 190 mg per day, about 200 mg per day, about 210 mg per day, about 220 mg per day, about 230 mg per day, about 240 mg per day, about 250 mg per day, about 260 mg per day, about 270 mg per day, about 280 mg per day, about 290 mg per day, about 300 mg per day, about 310 mg per day, about 320 mg per day, about 330 mg per day, about 340 mg per day, about 350 mg per day, about 360 mg per day, about 370 mg per day, about 380 mg per day, about 390 mg per day, about 400 mg per day, about 410 mg per day, about 420 mg per day, about 430 mg per day, about 440 mg per day, about 450 mg per day, about 460 mg per day, about 470 mg per day, about 480 mg per day, about 450 mg per day, or about 500 mg per day.

[0435] I In some embodiments, a therapeutically-effective amount of a hydroxyestrogen sufficient to induce mitochondrial stress and/or mitohormesis can be about 1 mg, about 2 mg, about 3 mg, , about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 120 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1000 mg.

[0436] In some embodiments, a dose can be expressed in terms of an amount of the drug divided by the mass of the subject, for example, milligrams of drug per kilograms of subject body mass. In some embodiments, a compound is administered in an amount ranging from about 0.1 mg/kg to about 100 mg/kg, for example about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 25 mg/kg, about 3 mg/kg to about 25 mg/kg, about 3 mg/kg to about 50 mg/kg, about 3 mg/kg to about 75 mg/kg, about 3 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50 mg/kg, about 10 mg/kg to about 75 mg/kg, or about 10 mg/kg to about 100 mg/kg. In some embodiments, a dose (e.g., a unit dose) is about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg, about 0.005 mg/kg, about 0.006 mg/kg, about 0.007 mg/kg, about 0.008 mg/kg, about 0.009 mg/kg, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, about 1.9 mg/kg, about 2.0 mg/kg, about 2.1 mg/kg, about 2.2 mg/kg, about 2.3 mg/kg, about 2.4 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, about 5.0 mg/kg, about 5.5 mg/kg, about 6.0 mg/kg, about 6.5 mg/kg, 7.0 mg/kg, about 7.5 mg/kg, 8.0 mg/kg, about 8.5 mg/kg, 9.0 mg/kg, about 9.5 mg/kg, 10.0 mg/kg, about 15 mg/kg, about 20 mg/kg, about 55 mg/kg, about 30 mg/kg, about 55 mg/kg, about 40 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg. In some embodiments, a compound is administered to a human. In some embodiments, the compound is 4-hydroxyestrone (4-OHE1). In some embodiments, the compound is 2-hydroxyestrone (2- OHE1). In some embodiments, the compound is 4-hydroxyestradiol (4-OHE2).

[0437] In some embodiments, the compound is 2-hydroxyestradiol (2-OHE2) or a salt thereof, and is administered in an amount ranging from greater than 2 mg/kg to about 100 mg/kg, for example, greater than 2 mg/kg to about 5 mg/kg, greater than 2 mg/kg to about 10 mg/kg, greater than 2 mg/kg to about 5 mg/kg, greater than 2 mg/kg about 10 mg/kg, greater than 2 mg/kg to about 5 mg/kg, greater than 2 mg/kg to about 25 mg/kg, about 10 mg/kg to about 50 mg/kg, about 10 mg/kg to about 75 mg/kg, or about 10 mg/kg to about 100 mg/kg. In some embodiments, a dose (e.g., a unit dose) is about 2.1 mg/kg, about 2.2 mg/kg, about 2.3 mg/kg, about 2.4 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, about 5.0 mg/kg, about 5.5 mg/kg, about 6.0 mg/kg, about 6.5 mg/kg, 7.0 mg/kg, about 7.5 mg/kg, 8.0 mg/kg, about 8.5 mg/kg, 9.0 mg/kg, about 9.5 mg/kg, 10.0 mg/kg, about 15 mg/kg, about 20 mg/kg, about 55 mg/kg, about 30 mg/kg, about 55 mg/kg, about 40 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg.

[0438] A compound described herein can be present in a composition or a unit dose in a range of from about 100 nM to about 1000 nM, from about 200 nM to about 1000 nM, from about 300 nM to about 1000 nM, from about 400 nM to about 1000 nM, from about 500 nM to about 1000 nM, from about 600 nM to about 1000 nM, from about 700 nM to about 1000 nM, from about 800 nM to about 1000 nM, from about 900 nM to about 1000 nM, from about 1 pM to about 1000 pM, from about 1 pM to about 500 pM, from about 1 pM to about 200 pM, from about 1 pM to about 100 pM, from about 1 pM to about 50 pM, from about 1 pM to about 25 pM, from about 1 pM to about 20 pM, from about 1 pM to about 15 pM, from about 1 pM to about 10 pM, from about 1 pM to about 5 pM, from about 10 pM to about 25 pM, from about 50 pM to about 250 pM, from about 100 pM to about 200 pM, from about 1 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 150 pM, from about 150 pM to about 200 pM, from about 200 pM to about 250 pM, from about 250 pM to about 300 pM, from about 300 pM to about 350 pM, from about 350 pM to about 400 pM, from about 400 pM to about 450 pM, from about 450 pM to about 500 pM, from about 500 pM to about 550 pM, from about 550 pM to about 600 pM, from about 600 pM to about 650 pM, from about 650 pM to about 700 pM, from about 700 pM to about 750 pM, from about 750 pM to about 800 pM, from about 800 pg to about 850 Mg, from about 850 Mg to about 900 Mg, from about 900 Mg to about 950 Mg, from about 950 Mg to about 1000 Mg, or from about 950 Mg to about 1000 pM.

Pharmaceutical Compositions

[0439] The compounds described herein can be administered alone or in a pharmaceutical composition comprising the compound or compounds and one or more pharmaceutically acceptable excipients.

[0440] The compositions of the present invention are in biologically compatible form suitable for administration in vivo for subjects. The pharmaceutical compositions further comprise a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the VLP is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water may be a carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose may be carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried slim milk, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0441] As used herein, the term “administering” generally refers to any and all means of introducing compounds described herein to the host subject including, but not limited to, by oral, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and like routes of administration. Compounds described herein may be administered in unit dosage forms and/or compositions containing one or more pharmaceutically-acceptable carriers, adjuvants, diluents, excipients, and/or vehicles, and combinations thereof.

[0442] The phrase “causing to be administered” or “cause to be administered” refers to the actions taken by a medical professional (e.g., a physician), or a person prescribing and/or controlling medical care of a subject, that control and/or determine, and/or permit the administration of the agent(s)/compound(s) at issue to the subject. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular agent(s)/compounds for a subject. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like. [0443] As used herein, the term “composition” generally refers to any product comprising more than one ingredient, including the compounds described herein. It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein.

[0444] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredients that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example and without limitation, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol(s), and the like), vegetable oils, nontoxic glyceryl esters, and/or suitable mixtures thereof.

[0445] Pharmaceutical compositions described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Nonlimiting examples are liquids in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

EXAMPLES

Example 1: Materials and Methods

[0446] Animals. All experiments were approved by the UC Berkeley Animal Care and Use Committee (ACUC) (AUP-2017-02-9539-1 to K.S.). Nrf2 KO mice (Stock No. 017009) were purchased from Jackson Labs.

[0447] Cell culture. Bone marrow-derived macrophages (BMDMs) were prepared by flushing leg and hip bones from 6-12 week old mice, filtering cells through a 40|xm filter, RBC lysis, and plating in DMEM supplemented with 20ng/mL murine M-CSF (Shenandoah) in 15cm petri dishes (non-TC treated). Media/M-CSF was changed on days 3 and 6. Cells were harvested by scraping and plated in appropriate TC-treated multiwell plates/dishes for experiments on days 7-10. For initial estrogen metabolite experiments, phenol red-free DMEM (Corning 17-205-CV) and 10% charcoal-stripped FBS (HyClone SH30068.03) were used to eliminate estrogenic effects of phenol red and serum estrogens. All experiments after determining hydroxyestrogen effects were ER-independent were performed in DMEM with phenol red (Corning 10-013-CV) supplemented with 10% regular FBS (HyClone SH30071.03). Both DMEM/FBS formulations were supplemented with Pen/Strep (Gibco). All BMDM data presented are from cells derived from female mice, though the inventors confirmed the hydroxyestrogens are anti-inflammatory in BMDMs derived from male mice. RAW 264.7 macrophages and HEK293T cells (both ATCC) were also cultured in DMEM with 10% FBS and Pen/Strep. The latter were used to produce lentivirus with psPAX2 (Addgene 12260), pMD2.G (Addgene 12259), and various lentiviral constructs described hereafter.

[0448] Chemicals. Estrogens (estrone (El), 17P-estradiol (E2), estriol (E3), 2- hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone, 4-hydroxyestradiol, 2- methoxyestrone, 2-methoxyestradiol, 4-methoxyestrone, 4-methoxyestradiol,16a- hydroxyestrone, 16-keto-17P-estradiol, and 16-epiestriol) were from Steraloids Inc. Pam3CSK4 (tlrl-pms) ODN (tlrl-1826-1) were from InvivoGen. LPS (L3024), poly IC (P0913), Diethyl maleate (D97703), sodium acetate (S5636), and Celastrol (C0869) were from Sigma. Coenzyme A (CoA) and acetyl-CoA (Ac-CoA) from both Sigma (C4780, A2056) and Cayman (16147, 16160) were tested behaved similarly in their rescue of proinflammatory gene expression in hydroxyestrogen-pretreated macrophages.

[0449] Quantitative real-time PCR (qPCR). Cells in multiwell plates were directly lysed in Trizol (Invitrogen) and total RNA isolated using Direct- zol kit (Zymo). cDNA was prepared with Superscript III (Invitrogen) and diluted in H2O for qPCR using ROX low SYBR FAST 2X mastermix (KAPA/Roche) and QuantStudio6 qPCR machine (Thermo Fisher) in 96 well plate fast run mode. Primers were designed to span exon-exon junctions in Primer-BLAST (Ye, J. et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134-134 (2012)) and determined to have one product via melt curve analysis. All data presented is normalized to Hprt expression using the delta-delta Ct method.

[0450] RNA-seq. Total RNA was converted into sequencing libraries using mRNA HyperPrep kit (KAPA/Roche) and custom Illumina-compatible unique dual index (UDI) adaptors (IDT). Libraries were quantified with Illumina Library qPCR quantification kit (KAPA/Roche) and pooled for sequencing on HiSeq4000 (Illumina). Sequencing reads were aligned to mm9 or mmlO using STAR (Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 (2013)), and reads counted (raw counts and RPKM) using HOMER (Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576-589 (2010)). EdgeR (Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010)) and DESeq2 (Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.

Genome Biol. 15, 550-8 (2014)) were used for differential expression analysis using raw read counts and specific cutoff parameters. Hierarchical clustering was performed using Cluster (Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. U. S. A. 95, 14863-14868 (1998)) and visualized with Java TreeView. Heatmaps were produced from normalized expression data in Prism. Gene ontology (GO) analysis was performed by inputting gene lists into Metascape⁷. Promoter motif finding from gene lists was performed using HOMER.

[0451] Western blotting. Cells/mitochondrial fractions were lysed in RIPA buffer plus protease inhibitor cocktail (Roche) 20 minutes on ice, followed by centrifugation 10,000g x 10 minutes at 4°C. Supernatant was quantified with DC Protein Assay (BioRad), and 15-30ug of soluble protein mixed with 5x NuPAGE loading dye and 2x NuPAGE reducing reagent (Life Technologies) and heated 10 minutes 70°C. Samples were loaded on NuPAGE 4-12% Bis-Tris gels (Life Technologies) against SeeBlue Plus2 prestained protein ladder (Thermo Fisher) and ran in NuPAGE MOPs buffer (Life Technologies). Size-separated proteins were transferred to PVDF membrane (GE Healthcare) by semidry transfer, and membrane blocked with SuperBlock (Thermo Fisher) for 1 hour. Membranes were probed with primary antibodies overnight at 4°C, followed by washes with 0.1% TBS-T, and fluorophore- conjugated secondary antibody probing at room temperature for 1 hour. After 0.1% TBS-T washes, membrane fluorescence was visualized using Licor Odyssey imaging system.

Primary antibodies: Tubulin (CP06, Cal Biochem), pro-IL-ip (AF-401-NA, R&D Systems), NRF2 (MABE1799, EMD Millipore), vinculin (sc-73614, Santa Cruz), VDAC (abl54856, Abeam). Secondary antibodies: AlexaFluor 680 conjugates were from Invitrogen. IRDye 800CW conjugates were from Rockland. All antibodies were diluted in 0.1% TBS-T with 5% BSA and 0.02% sodium azide for probing.

[0452] Flow cytometry and cell sorting. All sample analysis was done on LSR II, LSR Fortessa, or LSR Fortessa X20 analyzers (BD). All cell sorting was performed on Influx or Aria Fusion sorters (BD). Data was analyzed using FlowJo (Tree Star).

[0453] Pro-IL-1 P ICS for flow cytometry. RAW macrophages were treated, harvested, and processed with FIX & PERM Cell Fixation & Permeabilization Kit (Thermo Fisher) in 96 well round-bottom plate. Primary antibody: pro-IL-ip (AF-401-NA, R&D Systems) diluted 1:50 in Solution B. Secondary antibody: anti-goat AlexaFluor 647 (Invitrogen) diluted 1:500 in Solution B. All staining done in 50|xL volume. All washes performed with PBS. [0454] Acute in vivo inflammation Male C57BL/6J mice (Jackson Labs) mice were injected intraperitoneally (IP) with EtOH vehicle control or estrogens (lOmg/kg). 1 hour later, mice received IP injection of PBS or LPS (2mg/kg). After 3 hours, submandibular bleeding was performed to collect blood for measurement of serum IL-ip levels by ELISA (Invitrogen 88- 7013-22). At 4 hours, mice were sacrificed and spleenocytes isolated by crushing spleen through 40|xm filter, performing RBC lysis, and lysing total spleenocyte cell pellet in Trizol for RNA extraction, cDNA preparation, and qPCR for proinflammatory genes.

[0455] Chronic in vivo inflammation. Male C57BL/6N mice (Charles River) mice were placed on high-fat diet (Research Diets Inc, D 12492, 60 kcal% fat) and given subcutaneous EtOH vehicle control or estrogen injections (lOmg/kg) every 6 days in rear flank. After 30 days, mice were sacrificed and visceral white adipose tissue (vWAT) isolated and weighed. Stromal vascular fraction (SVF) was then prepared from vWAT. Tissue was minced with scissors and incubated in DMEM with 0.1% collagenase (Sigma C6885) and 5% BSA for 1 hour at room temperature with gentle shaking. Digested tissue was filtered through a 70|xm filter, RBCs lysed, and SVF stained for analysis and cell sorting on ice in FACs buffer (PBS with lOmM HEPES and 5% BSA) using standard techniques. After incubation in Fc Block (BD Biosciences), cells were stained with anti-CD45-PerCP-Cy5.5 (clone 30-F11, BioLegend), anti-F4/80-PE (clone BM8, eBioscience), and anti-CDllb-APC (clone MI/70, eBioscience). After washes, cells were resuspended in FACs buffer with DAPI (Thermo Fisher D1306, reconstituted according to manufactures instructions, used at 1:100,000) for exclusion of dead cells. F4/80+CDllb+ vWAT macrophages were sorted directly into Trizol- LS (Invitrogen) for RNA isolation. Total live SVF cell counts were also determined using hemocytometer and Trypan Blue exclusion so that macrophage cellularity could be calculated from flow cytometry percentages.

[0456] Mitochondrial fractions for steroid extraction and LC/MS. RAW macrophages (15-30 million total) were treated with EtOH vehicle control or 5|xM 4-OHE1 for 1 hour before lx PBS wash and harvest by scraping. For whole cell steroid extraction, 1 million macrophages were pelleted (800g x 5 minutes, 4°C) in a 1.5mL Eppendorf tube, flash frozen, and stored at -80°C. From remaining cells, mitochondrial fractions were isolated using a previously described differential centrifugation method⁸ with modification. Briefly, cells were resuspended in 2mL cell isolation buffer (IBc) in a 15mL conical and disrupted using a Bioruptor sonicator (Diagenode) with metal probe adaptor and short 1 second pulses on “HI” setting. Cell disruption was tracked by flow cytometry scatter, with 10-15 pulses getting cells from 70% to <10% “live scatter” gate-positive. Mitochondrial fractions were then isolated by differential centrifugation, flash frozen, and stored at -80°C. Fractions were checked for mitochondrial protein enrichment versus whole cell lysates by western blot.

[0457] For steroid extraction, whole cell and mitochondrial fraction pellets were thawed at room temperature and resuspended in 1 mL of acetonitrile by pipetting and vortexing. The resulting homogenate was stored for 30 min at -20°C and then centrifuged for 5 min at 12000 x g and at 4°C. The supernatant was transferred to a glass round-bottom tube and evaporated under a N2 stream. The residue was resuspended in 2mL of 0.2M sodium acetate buffer (pH 5.0) and extracted with lOmL of hexane. The mixture was centrifuged for 2 min at 1200 x g, and the upper hexane layer was transferred to a new glass tube and evaporated under nitrogen with gentle heating at 25-30°C in a water bath (Model N-EVAP 112, Organomation Associates). The residue was reconstituted in 50pL of MeOH with vortexing. As a positive control to confirm this method could extract 4-OHE1, an extraction was performed on 20 .L of cell culture media spiked with 2 .L of 5mM 4-OHE1.

[0458] Extracts and 4-OHE1 standard were analyzed using a liquid chromatography system (LC; 1200 series, Agilent Technologies, Santa Clara, CA) that was equipped with a reversed- phase analytical column (length: 150 mm, inner diameter: 1.0 mm, particle size: 5 pm, Viva Cl 8, Restek, Bellefonte, PA). The LC system was connected in line with an LTQ-Orbitrap- XL mass spectrometer that was equipped with an electrospray ionization (ESI) source and operated in the positive ion mode (Thermo Fisher Scientific, Waltham, MA). Mass spectrometry data acquisition and processing were performed using Xcalibur software (version 2.0.7, Thermo). Injection volumes were as follows: 2pL for Img/mL 4-OHE1 standard (Steraloids) in MeOH, 5pL for whole cell extracts, 2pL for mitochondrial extracts. This instrumentation is located in the QB3/Chemistry Mass Spectrometry Facility, on the campus of the University of California, Berkeley.

[0459] isoTOP-ABPP. IsoTOP- ABPP analysis was performed as previously described⁹. Briefly, proteomes (prepared from BMDMs treated with EtOH or I M 4-OHE1 for 1 hour) were labeled with lAyne (lOOpM) for 1 hour at room temperature, and subsequently treated with lOOpM isotopically light (control) or heavy (treated) TEV-biotin and click chemistry was performed as previously described. Proteins were precipitated, washed, resolubilized and insoluble components were precipitated. Soluble proteome was diluted and labeled proteins were bound to avidin-agarose beads while rotating overnight at 4°C. Bead-linked proteins were enriched, then resuspended, alkylated with iodoacetamide, then washed and resuspended with sequencing grade trypsin overnight. Non-bead-linked tryptic peptides were washed away, and the TEV-biotin tag was digested overnight in TEV buffer containing and Ac-TEV protease at 29°C. Liberated peptides were diluted in water, acidified and stored at - 80°C.

[0460] Analysis was performed as previously described using Multidimensional Protein Identification Technology (MudPIT) with an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher)⁹. Data was extracted in the form of MSI and MS2 files using Raw Extractor 1.9.9.2 (Scripps Research Institute) and searched against the Uniprot mouse database using ProLuCID search methodology in IP2 v.3 (Integrated Proteomics Applications, Inc)¹¹. ProLUCID data was filtered through DTASelect to achieve a peptide false-positive rate below 1 % and cysteine residues were searched with a static modification for carboxyaminomethylation (+57.02146) and up to two differential modifications for the light or heavy TEV tags (+464.28596 or +470.29977, respectively).

[0461] Metabolomics. BMDMs were cultured in DMEM (Corning 17-207-CV) supplemented with One Shot dialyzed FBS (Thermo Fisher), ImM sodium pyruvate, 4mM L- glutamine, and 25mM glucose (all from Gibco). Cells were seeded into 6-well plates at 500,000 cells per well, and the next day treated with EtOH vehicle control or 5pM 4-OHE1. After two hours, media was aspirated and cells washed lx with unsupplemented DMEM. Media was then replaced with DMEM described above except with 25mM ¹³Ce-glucose (Cambridge Isotope Laboratories, CLM-1396-PK). After 30 minutes, media was aspirated, cells washed lx with ImL PBS, and moved to dry ice for addition of ImL ice cold 80% MeOH. Cells were moved to -80°C for 15 minutes, after which cells were scraped on dry ice and moved to Safe Lock 1.5mL Eppendorf tube (cat.022363204). After centrifugation 20,000g x 10 minutes at 4°C, supernatant was transferred to a new tube and evaporated overnight using a Speed Vac (no heating). Dried metabolite extracts were stored at -80°C before shipping on dry ice. MeOH extraction pellet was saved for protein quantification to be used in metabolite normalization.

[0462] Dried metabolites were resuspended in 50% ACN:water and 1/10* was loaded onto a Luna 3um NH2 100A (150 x 2.0 mm) column (Phenomenex). The chromatographic separation was performed on a Vanquish Flex (Thermo Scientific) with mobile phases A (5 mM NH4AcO pH 9.9) and B (ACN) and a flow rate of 200pL/min. A linear gradient from 15% A to 95% A over 18 min was followed by 9 min isocratic flow at 95% A and reequilibration to 15% A. Metabolites were detection with a Thermo Scientific Q Exactive mass spectrometer run with polarity switching (+3.5 kV7- 3.5 kV) in full scan mode with an m/z range of 65-975. TraceFinder 4.1 (Thermo Scientific) was used to quantify the targeted metabolites by area under the curve using expected retention time and accurate mass measurements (< 5 ppm). Values were normalized to sample protein concentration. Relative amounts of metabolites were calculated by summing up the values for all isotopologues of a given metabolite. Fraction contribution (FC) of ¹³C carbons to total carbon for each metabolite was calculated as previously described¹². Data analysis, including principal component analysis and hierarchical clustering, was performed using in-house R scripts.

[0463] ChlP-seq. Approximately 30 million BMDMs were treated with EtOH vehicle control or lp.M hydroxyestrogen for 1 hour, followed by PBS or EPS (lOOng/mL) stimulation for 30 minutes. Cells were then washed 2x with PBS and fixed with 0.67mg/mL DSG (Thermo Fisher) in PBS for 30 minutes at room temperature with shaking, followed by addition of paraformaldehyde (Electron Microscopy Sciences) to 1% and an additional 15 minutes of room temperature fixation. Fixation was quenched by adding glycine to 125mM and shaking 10 minutes, after which fixed cells were scraped and washed 2x with PBS. Cell pellet was resuspended in 1.5mL ice cold ChIP RIPA buffer (20mM Tris HC1 pH 8.0, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100) with protease inhibitors (Roche) and sonicated with metal probe adaptor using Bioruptor (Diagenode) for 60 minutes (continuous cycles of 20 seconds ON/40 seconds OFF, “medium” setting). Samples were spun 20 minutes max speed at 4°C in benchtop centrifuge to remove insoluble material, and supernatant containing soluble sheared chromatin transferred to new tube, saving 1% volume for input library preparation. Immunoprecipitation was performed overnight at 4°C with 2p.g of primary antibody (p65: Santa Cruz sc-372, H3K27ac: Abeam ab4729), or corresponding IgG control antibody (GenScript), conjugated to Protein A Dynabeads (Invitrogen). The next day, beads were captured with magnet on ice and washed with ice cold wash buffer II (20mM Tris HC1 pH 8.0, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.5% NaDOC) 3 times, wash buffer III (WmM Tris HC1 pH 8.0, 250mM LiCl, 2mM EDTA, 1% NP-40, 0.5% NaDOC) 3 times, and TE with 50mM NaCl two times, all supplemented with protease inhibitors. Beads with antibody-target-DNA complexes were then resuspended in 200|xL elution buffer (50mM Tris HC1 pH 8.0, lOmM EDTA, 1% SDS) for 30 minutes at 37°C to elute immunoprecipitated complexes. 200|xL elute was collected, 10|xL of 5M NaCl added, and DNA-protein crosslinks reversed overnight at 65 °C. Next day, samples were treated with RNAse A (Thermo Fisher) 1 hour at 37°C, Proteinase K (NEB) for 1 hour at 50°C, and DNA fragments recovered with Zymo ChIP DNA Clean & Concentrator kit.

[0464] Sequencing libraries were prepared from recovered DNA using in-house protocol. Briefly, DNA was blunted, A-tailed, and ligated to Illumina-compatible NEXTFlex sequencing adaptors (Bioo). Libraries were PCR amplified, gel size selected, quantified, pooled, and sequenced on an Illumina HiSeq2500. Sequencing reads were aligned to mm9 using STAR. HOMER findPeaks was used to call peaks/regions in each sample relative to input. HOMER getDifferentialPeaks was used to identify peaks/regions with significantly increased read density induced by LPS. HOMER getDifferentialPeaks was then used to quantify the percentage of LPS-induced peaks/regions that were significantly reduced in read density in hydroxyestrogen-pretreated, LPS stimulated samples. HOMER annotatePeaks.pl was used to make histograms showing read density at LPS-induced peaks/regions across the 3 conditions for both p65 and H3K27ac ChlP-seqs.

[0465] TMRE staining. TMRE (Sigma 87917) was prepared as a ImM stock in DMSO. TMRE was added directly to cell culture media to macrophages in multiwell tissue culture plates at 37°C (lOnM final concentration). 10 minutes after TMRE addition, cells were treated with EtOH, 4-OHE1, oligomycin (EMD Millipore 495455), or FCCP (Sigma C2920) at indicated concentrations for 20 minutes. Cell were then placed on ice and washed lx ice cold PBS, followed by scraping into PBS supplemented with DAPI for flow cytometry analysis.

[0466] RAW matrix-oxGFP macrophages. Cells were transduced with matrix-oxGFP lentivirus encoding oxGFP¹³ with N-terminally fused COX4L mitochondrial matrix targeting sequence and selected with 10|Xg/mL blasticidin (Thermo Fisher). For experimental use, treatments of RAW matrix-oxGFP macrophages were performed as described, after which cells were put on ice, media aspirated, and cells scraped into PBS supplemented with DAPI for flow cytometry analysis. HSF1 transcriptional inhibitor KRIBB11 (EMD Millipore 385570) was prepared as a lOmM stock in DMSO and used at indicated concentrations.

[0467] mtDNA/gDNA ratio. RAW macrophages in 24-well plates were treated with EtOH or 5pM 4-OHE1 for indicated times, then lysed directly in lOOpL total DNA isolation buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 6.25 mM MgCh, 0.045% NP-40, 0.45% Tween-20). Lysate was moved to PCR tube, supplemented with Proteinase K (NEB), and incubated 1 h at 56°C. Proteinase K was inactivated by incubation at 95 °C for 15 minutes, and 5pL of lysate was used in qPCR reactions for mitochondrial DNA (mtDNA, mt-Cytb gene locus) and genomic DNA (gDNA, Actb gene locus) amplicons using primers described in de Almeida, M. J., Luchsinger, L. L., Corrigan, D. J., Williams, L. J. & Snoeck, H. W. Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells. Cell. Stem Cell. 21, 725-729.e4 (2017).

[0468] MitoTracker Green staining. RAW macrophages and BMDMs were treated with EtOH or estrogens as described. MitoTracker Green (Invitrogen M7514, reconstituted in DMSO) was added directly to cell culture media at a final concentration of lOOnM, and mitochondria were labeled for 45 minutes at 37°C. Cells were then placed on ice and washed lx ice cold PBS, followed by scraping into PBS supplemented with DAPI for flow cytometry analysis.

[0469] mitoPyl staining. RAW macrophages and BMDMs in multiwell tissue culture plates were (pre)treated with estrogens and LPS as described. MitoPyl (Tocris 4428), prepared as a lOmM stock, was added to cell culture media at 1:1000 for direct staining (37°C for 1 hour). Menadione (Sigma M5615) or H2O2 (Fisher H325) were added the last 10-15 minutes of MitoPyl staining as positive controls. To harvest, cells were placed on ice and washed lx ice cold PBS, followed by scraping into PBS supplemented with DAPI for flow cytometry analysis.

[0470] roGFP RAW macrophages. RAW macrophages were transduced with lentiviral constructs encoding for N-terminal fusions of roGFP to COX4L targeting sequence (matrix- roGFP), LACTB targeting sequence (inner membrane space- or IMS-roGFP), or nuclear export sequence (cyto-roGFP), and selected with lOug/mL puromycin (Thermo Fisher). For experimental use, treatments were performed as described, after which cells were put on ice, media aspirated, and cells scraped into PBS supplemented with DAPI. Using an ESR Fortessa analyzer, roGFP emission after excitation with 405nm (violet) and 488nm (blue) lasers was collected using a 505nm longpass: 525/50 bandpass filter combo coupled to each respective laser line. H2O2 (Fisher H325) and DTT (Fisher BP172-5) treatment were used to confirm ability to detect roGFP oxidation and reduction.

[0471] Menadione toxicity/mitochondrial superoxide resistance assay. RAW macrophages or BMDMs were treated overnight (18-24 hours) with EtOH vehicle control or 5pM estrogens. The next day, cells were treated with DMSO or 50pM menadione for a short time period (2-6 hours) before harvest. Cells were moved to ice, media aspirated, and cells scraped into PBS supplemented with DAPI. Cell viability was assessed and quantified as a percentage of total events collected for each sample. Viable cells were defined as “live scatter” gate positive, DAPI negative events, with these gates defined in control EtOH/DMSO-treated samples.

[0472] Seahorse respirometry. For mitochondrial stress test, RAW macrophages and BMDMs were plated in XF24 microplates (50K and per well, respectively) in a small volume of media (100pL) to promote attachment. After attachment (5-6 hours), media volume was brought to 250pL, and cells were treated overnight (18-24 hours) with EtOH, 5pM 4-OHE1, lOOng/mL LPS, or both. The next day, fresh XF Assay media (supplemented with ImM sodium pyruvate and 25mM glucose) was prepared and brought to pH 7.4. Cells were washed lx with XF Assay media, 500|xL of XF assay media was added to each well, and plate incubated at 37°C (no CO2) for 45-60 minutes. During this time, XF24 sensor cartridge (in calibrant overnight 37°C) was loaded with Oligomycin, FCCP, and Antimycin A (Sigma A8674), and Rotenone (Cayman 13995), and loaded into Agilent Seahorse XF24 analyzer for calibration. After calibration, cells were loaded for mitochondrial stress test with sequential injections of mitochondrial inhibitors (all at 1.5pM final concentration). All data presented is raw data/unnormalized, as protein quantification after stress test reveal no significant differences between the various treatments. For measurement of 4-OHEl’s acute effects on basal oxygen consumption, cells were plated and moved to fresh XF assay media as described, and treated with EtOH or 4-OHE1 immediately prior to loading into Seahorse analyzer.

[0473] Statistical analysis All statistical analysis for qPCR and flow cytometry experiments was performed using GraphPad Prism 8 software. Metabolomics statistical analysis was performed using R scripts available upon request.

Example 2: Anti-inflammatory Activity of Hydroxyestrogens in Macrophages In Vitro

[0474] To screen for estrogens capable of repressing LPS-induced proinflammatory gene transcription, bone marrow-derived macrophages (BMDMs) were pretreated with endogenous estrogen metabolites (estrone (El), 17P-estradiol (E2), estriol (E3), 2- hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone, 4-hydroxyestradiol, 2- methoxyestrone, 2-methoxyestradiol, 4-methoxyestrone, 4-methoxyestradiol,16a- hydroxyestrone, 16-keto-17P-estradiol, and 16-epiestriol) for 1 hour, followed by LPS stimulation for 6 hours and Nos2 quantitative real-time PCR (qPCR). Hydroxyestrogens 2- hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), and 2-hydroxyestradiol (2-OHE2), significantly repressed Nos2 induction, while other estrogens including Estradiol (E2) and 16- Epiestriol (16-Epi) lacked this activity (FIG. 2A). RNA-seq identified 253 common genes repressed by individual hydroxyestrogen pretreatment in LPS- stimulated BMDMs. Gene Ontology (GO) analysis of these genes revealed enrichment for categories including “Inflammatory response” and “Cytokine production” (FIG. 2B). Hierarchical clustering of the RNA-seq data revealed a broad set of pro-inflammatory cytokines and chemokines repressed by hydroxyestrogens (FIG. 2C), which were validated by qPCR (FIG. 3A). Illb was chosen as hydroxyestrogen-repressed gene as its transcriptional induction in BMDMs was potently repressed in a dose-dependent manner by 4-OHE1 pretreatment (FIG. 2D). Strong Illb repression by hydroxyestrogens (including 4-OHE2, which was not included in the initial screen) occurred in RAW macrophages (FIG. 3B, C), making them suitable for mechanistic studies. In agreement with the transcriptional effects, 4-OHE1 pretreatment strongly repressed LPS-induced pro-IL-ip protein levels (FIG. 2E,F). 4-OHE1 pretreatment also repressed Illb induction by multiple TLR agonists (FIG. 2G), suggesting a common pathway downstream of all TLRs is targeted. To test whether these anti-inflammatory effects are dependent on ERa, the primary ER expressed in macrophages, the experiments were repeated using BMDMs from ERa™ LysM-Cre mice. Surprisingly, the anti-inflammatory activity of the hydroxyestrogens was still intact (FIG. 2H). Moreover, cotreatment with the high-affinity ERa antagonist ICI 182780 had no effect on the ability of the hydroxyestrogens to repress Illb (FIG. 21).

[0475] Together, these results demonstrate hydroxyestrogens are strong repressors of macrophage pro-inflammatory gene transcription in vitro that act in an ERa-independent manner.

Example 3: Hydroxy estrogens are anti-inflammatory in vivo.

[0476] To test whether hydroxyestrogens have anti-inflammatory activity in vivo, the effects of 4-OHE1 on acute LPS-induced inflammation were examined. Mice were intraperitoneally (IP) injected with EtOH vehicle control, E2, or 4-OHE1, followed by IP injection with LPS (FIG. 4A, top). 4-OHE1, but not E2, significantly repressed both the LPS-induced increase in serum IL-ip levels (FIG. 4A, bottom), and LPS-induced proinflammatory gene expression in spleenocytes (FIG. 4B). Thus, 4-OHE1, but not E2, repressed acute LPS-induced inflammation in vivo. [0477] To test if the hydroxyestrogens have anti-inflammatory effects in vivo in a chronic inflammatory setting, the inventors examined effects on gene expression in visceral white adipose tissue (vWAT) macrophages during the early stages of high-fat diet (HFD)-induced metabolic inflammation. Mice were placed on HFD and given subcutaneous injections of EtOH, E2, or 4-OHE1 every 6 days. After 30 days, vWAT macrophages were profiled by RNA-seq (FIG. 4C, top, FIG. 5A). While both E2 and 4-OHE1 reduced adiposity and macrophage cellularity in vWAT (FIG. 5B,C), vWAT macrophages from 4- OHE1 -treated mice displayed a distinct gene expression profile compared to macrophages from EtOH and E2-treated mice (FIG. 4C, bottom). 4-OHE1 repressed expression of a distinct set of genes compared to E2 (FIG. 4D). GO analysis of genes uniquely repressed by 4-OHE1 revealed enrichment for categories including “Inflammatory response” and “Leukocyte migration”, (FIG. 4E), while genes uniquely repressed by E2 showed no enrichment for inflammatory processes (FIG. 4F). Many hydroxy estrogen targets repressed in vitro were repressed by 4- OHE1, but not E2, in vWAT macrophages (FIG. 4G). Thus, 4-OHE1, but not E2, displays anti-inflammatory effects in vWAT macrophages during HFD-induced metabolic inflammation in vivo.

Example 4: Hydroxy estrogens activate NRF2, but NRF2 is not required for their antiinflammatory activity.

[0478] Given their ERa-independent anti-inflammatory effects, other mechanisms by which hydroxyestrogens might act given their production and metabolism (FIG. 6A) were explored. Hydroxylation of estrone (El) or E2 by CYP1 family cytochrome P450 monooxygenases creates a catechol moiety, giving hydroxyestrogens their “catechol estrogens” nickname Present in a variety of drugs and natural compounds, this catechol moiety can cause cellular stress in 2 ways. First, it can be oxidized to its quinone form, and redox cycling between these forms (which involves a semiquinone free radical intermediate) can produce reactive oxygen species (ROS). Second, because the quinone form possesses a,P-unsaturated carbonyls and is highly electrophilic, it can be attacked by nucleophiles, such as reactive cysteines on proteins, forming covalent adducts. Accordingly, cells have evolved in two ways to detoxify hydroxyestrogens: catechol methylation by COMT (which reduces redox cycling), and glutathione (GST) conjugation of the quinone. Given the hydroxy estrogens, but not their precursors or methylated metabolites, repressed LPS-induced proinflammatory gene transcription (FIG. 6B, FIG. 3B), it was hypothesized that their anti-inflammatory activity may be dependent on their ability to cause oxidative and electrophilic stress.

[0479] The Keapl-Nrf2 system regulates cytoprotection in response to both oxidative and electrophilic stress. LPS-treated BMDM RNA-seq dataset identified 341 genes significantly upregulated in hydroxyestrogen-pretreated cells versus control pretreatments. GO analysis revealed enrichment of categories including “Response to oxidative stress”, “Detoxification of ROS”, and “Glutathione metabolism” (FIG. 7A), and NRF2 was identified as a top transcription factor binding motif enriched in promoters of these genes (FIG. 6C). Expression of NRF2 targets Hmoxl, Nqol, and genes involved in GST biosynthesis and ROS detoxification, were significantly upregulated by hydroxyestrogen pretreatment (FIG. 6D). 4- OHE1 rapidly stabilized NRF2 in macrophages, similar to diethyl maleate (DEM), a known electrophilic NRF2 activator (FIG. 6E).

[0480] Next experiments tested whether NRF2 was required for 4-OHEl’s anti-inflammatory activity. BMDMs were prepared from WT and Nfe2l2~'~ (referred to herein as Nrf2 KO) mice. Impaired Hmoxl induction confirmed lack of NRF2 function in Nrf2 KO BMDMs (FIG. 7H). However, 4-OHE1 repressed LPS-induced Il lb in both WT and Nrf2 KO BMDMs (FIG. 6F), demonstrating that while hydroxyestrogens are NRF2 activators, NRF2 is not required for their anti-inflammatory activity.

Example 5: Hydroxy estrogens cause mitochondrial stress.

[0481] This example tested whether hydroxyestrogen cause mitochondria stress via NRF2 activation, given that these lipophilic compounds localize to mitochondria, produce ROS and covalently modify mitochondrial proteins. GO analysis of the 341 genes upregulated in hydroxyestrogen-pretreated LPS-stimulated BMDMs revealed 3 additional transcriptional signatures indicative of mitochondrial stress (FIG. 5A-C and FIG. 9A-C). The Heat Shock Factor 1 (HSF1) signature includes putative HSF1 target genes, suggesting HSF1 activation in response to mitochondrial dysfunction (FIG. 8A). The ATF4/mitochondrial damage signature includes Atf4, which coordinates cytoprotection in response to mitochondrial stress in mammalian cells, and the ATF4 target Gdfl5, encoding a mitokine indicative of mitochondrial dysfunction (FIG. 8B). Finally, the Glycolysis/Pentose Phosphate Pathway (PPP) signature includes enzymes in these pathways known to be upregulated in response to mitochondrial stress in other systems (FIG. 8C). [0482] Next, steroid extraction and liquid chromatography/mass spectrometry (LC/MS) were performed to determine if 4-OHE1 was enriched in mitochondrial fractions relative to whole cells (FIG. 9 D,E). Free 4-OHE1 was not detected in macrophages treated with 4-OHE1 for 1 hour (FIG. 9F), suggesting 4-OHE1 is rapidly covalently conjugated by GST and/or proteins (FIG. 6A).

[0483] To identify covalent 4-OHE1 protein targets acting through reactive cysteines, competitive isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) was performed in EtOH control and 4-OHE1 -treated BMDMs (FIG. 8D). This revealed 127 cysteines on 118 proteins targeted by 4-OHE1 (FIG. 8E). Crossreferencing this target list with MitoCarta 2.0 revealed 18 of the 118 targets are mitochondrial proteins (FIG. 8F). A chi-square test comparing the observed frequency of mitochondrial targets with the expected frequency of mitochondrial proteins from MitoCarta 2.0 confirmed significant enrichment for mitochondrial proteins in the 4-OHE1 target list (FIG. 9G).

[0484] Together, these transcriptional signatures and proteomic data suggest hydroxyestrogens localize to mitochondria and cause mitochondrial stress via ROS production and covalently targeting mitochondrial proteins.

Example 6: Hydroxy estrogens impair mitochondria acetyl-CoA production and histone acetylation required for LPS -induced proinflammatory gene transcription.

[0485] This experiment considered how oxidative and electrophilic mitochondrial stress caused by hydroxyestrogens might exert anti-inflammatory effects. Mitochondria utilize oxidized glucose for acetyl-CoA production, which in turn is used to acetylate histones, a process crucial for Illb upregulation in LPS-stimulated macrophages. Pharmacological inhibition of glucose utilization in LPS-stimulated macrophages has selective repressive effects on Illb, but not cytokine transcripts such as 116 and Tnf. suggesting the glucose>acetyl-CoA axis is more important for transcription of the former gene. Similarly, in both RAW macrophages (FIG. 10A) and BMDMs (FIG. 11A), 4-OHE1 pretreatment had much stronger repressive effects on LPS-induced Illb than 116 and Tnf, suggesting 4-OHE1 interferes with mitochondrial glucose utilization for acetyl-CoA production.

[0486] A metabolomics screen was performed to identify metabolites whose levels were acutely affected by 4-OHE1 treatment. BMDMs were treated with EtOH or 4-OHE1 for 2 hours, followed by 30 minute ¹³Ce glucose labeling before whole cell metabolite extraction (FIG. 10B). Of the 136 metabolites measured, 8 were significantly changed by 4-OHEf (FIG. 10C). Free GSH levels were reduced (FIG. IOC), supporting the notion that 4-OHE1 is rapidly glutathione-conjugated after treatment (FIG. 6A). The metabolite with the most significant statistical change was acetyl-CoA, as levels in 4- OHE1 -treated BMDMs decreased by 45% (FIG. 10C,D). Coenzyme A (CoA) levels were also significantly reduced (41%) by 4-OHE1 treatment (FIG. 10 C,E). As CoA biosynthesis occurs within mitochondria, and 80%-90% of intracellular CoA is intra-mitochondrial, this suggests mitochondrial stress caused by 4-OHE1 disrupts CoA homeostasis, and in turn, acetyl-CoA production. For TCA cycle metabolites quantified, total levels of citrate and aconitase (immediately downstream of acetyl-CoA) showed the largest decreases in abundance (FIG. 11B). Moreover, ¹³Ce glucose labeling revealed increased ¹³C fractional contribution to all TCA cycle metabolites (and amino acids derived from these metabolites) except acetyl-CoA, citrate, and aconitase, upon 4-OHE1 treatment (FIG. 11C), suggesting alternative entry routes of glucose-derived carbon into the TCA cycle to replenish intermediates in response to 4- OHE1 -induced mitochondrial stress.

[0487] Together, this data demonstrates mitochondrial stress caused by 4-OHE1 impairs mitochondrial acetyl-CoA production.

[0488] Since acetyl-CoA is required for histone acetylation and proinflammatory gene transcription, ChlP-seq was performed to undertake a genome-wide look at how LPS-induced transcription factor binding and histone acetylation were affected by hydroxyestrogen pretreatment. For the NFKB subunit p65, 6,149 peaks were absent in unstimulated BDMDs but present 30 minutes post-LPS stimulation (FIG. 10F, left). Pretreatment with the hydroxyestrogen 2-OHE2 had minimal effects on LPS-induced NFKB binding, as only 13% of LPS-induced p65 binding peaks were significantly reduced in read density. For histone acetylation, 10,999 regions of H3K27ac, a mark of active promoters and enhancers, were absent in naive BMDMs but present 30 minutes post-LPS stimulation (FIG. 10F, right). In contrast to NFKB, pretreatment with the hydroxy estrogen 2-OHE1 strongly impaired H2K27ac deposition, as nearly two-thirds (65%) of LPS-induced H3K27ac regions were significantly reduced in read density. Thus, while hydroxyestrogens largely leave TLR4 signaling and transcription factor nuclear translocation/DNA binding intact, they strongly impair histone acetylation required for pro-inflammatory gene transcription, further supporting the hypothesis that hydroxyestrogens impair mitochondrial acetyl-CoA production (FIG. 10G). [0489] Administration of exogenous CoA to cells with impaired CoA biosynthesis, or treated with CoA-depleting compounds, can rescue histone acetylation and gene expression defects caused by reduced acetyl-CoA levels. Indeed, supplying BMDMs with either exogenous CoA or acetyl-CoA fully rescued LPS-induced Illb in 4- OHE1 -pretreated macrophages at early timepoints (1-1.5h) post-LPS (FIG. 10H), and partially at 6 hours (FIG. 11D). Acetate supplementation was unable to rescue Illb (FIG. HE), suggesting nucleocytosolic acetyl- CoA generation by ASCC2 does not contribute to the acetyl-CoA pool in macrophages. These rescue experiments further support a model where impairment of mitochondrial acetyl- CoA production by hydroxyestrogens underlies their anti-inflammatory activity.

[0490] It was further investigated whether the anti-inflammatory mechanism extends to other electrophilic small molecules with immunomodulatory properties. For example, Celastrol and DEM are electrophiles that repress Illb transcription in myeloid cells, and activate the same stress response pathways as 4-OHE1. Mitochondrial uncouplers including carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) are known for their ability to dissipate mitochondrial membrane potential and reduce mtROS levels, an effect thought to be behind Illb repression in macrophages by uncouplers as mtROS is considered an inflammatory signal However, uncouplers are also electrophilic, and like 4-OHE1, FCCP treatment significantly reduces intracellular acetyl-CoA levels (FIG. HF). Thus, it was investigated whether acetyl-CoA supplementation could rescue LPS-induced Illb expression in macrophages pretreated with these electrophiles. Indeed, supplying acetyl-CoA prior to electrophile treatment restored Illb induction (FIG. 10H), suggesting the anti-inflammatory effects of these electrophiles may also lie in their ability to cause mitochondrial stress and impair acetyl-CoA production.

Example 7 : Hydroxy estrogen-driven mitochondrial stress triggers mitohormesis.

[0491] The long-term effect of mitochondrial stress caused by hydroxyestrogens on macrophage function was further investigated. High levels of mitochondrial stress trigger apoptosis; however, no changes were observed in mitochondrial oxygen consumption (FIG. 13A), mitochondrial membrane potential (FIG. 13B), or cell viability upon acute (1 hour) hydroxyestrogen treatment.

[0492] The ability of hydroxy estrogen-driven mitochondrial stress to trigger mitohormesis in macrophages was evaluated. Two hallmarks of mitohormesis are mitochondrial biogenesis, and increased mitochondrial chaperone activity. To determine if hydroxyestrogen-driven mitochondrial stress triggered these mitohormesis hallmarks, a mitochondrial matrix-targeted oxidation-resistant green fluorescent protein was expressed in RAW macrophages, creating the RAW matrix-oxGFP reporter cell line. Treatment with 4-OHE1, but not El or 4-MeOEl, induced a steady increase in RAW matrix-oxGFP fluorescence at 7 and 24 hours as measured by flow cytometry (FIG. 12A), an effect that was dose-dependent (FIG. 13C). Quantification of the mitochondrial DNA/genomic DNA (mtDNA/gDNA) ratio in RAW macrophages treated with 4-OHE1 revealed no significant increase (FIG. 13D). As matrix-oxGFP is a mitochondrial chaperone client that needs to be folded upon mitochondrial import, this suggests RAW matrix-oxGFP cells are primarily reporting mitochondrial chaperone activity. Pharmacologically blocking HSF1 transcriptional activity with KRIBB11 partially blunted increased matrix ox-GFP fluorescence in response to 4-OHE1, suggesting HSF1 -dependent chaperone expression contributes to this mitohormetic response (FIG. 13E). This increase in mitochondrial chaperone activity occurred concurrently with an increase in mitochondrial volume, as 4-OHE1, but not El or 4-MeOEl, increased MitoTracker Green signal in both RAW macrophages and BMDMs (FIG. 13F,G)).

[0493] Together, these results suggest hydroxyestrogen-driven mitochondrial stress triggers an adaptive increase in mitochondrial chaperone activity.

[0494] Another hallmark of mitohormesis is mitochondrial oxidative stress resistance (OSR), i.e., a transient increase in mitochondrial stress triggers adaptations that both lower steadystate levels of mtROS at later timepoints and provide defense against subsequent oxidative stress challenge. Eike all cells, macrophages produce mtROS from the mitochondrial electron transport chain (mtETC), and LPS stimulation enhances mtROS production for bactericidal purposes. The effect of hydroxyestrogen-driven mitochondrial stress affected mtROS levels in macrophages at later timepoints was investigated using the mitochondrial-targeted H2O2 sensor MitoPy. RAW macrophages treated for 7 hours with 4-OHE1, but not El or 4- MeOEl, showed reduced MitoPy 1 fluorescence by flow cytometry, suggesting decreased basal mtROS levels (FIG. 12B, left). In control 1-hour EtOH pretreated cells, 6-hour LPS stimulation enhanced mtROS levels as expected (FIG. 12B, right). However, in cells pretreated 1 hour with hydroxyestrogens, but not their precursor or methylated metabolites, there was a significant decrease in LPS -induced mtROS levels. This specific reduction in LPS-induced mtROS by hydroxyestrogen pretreatment also occurred in BMDMs (FIG.

13H). [0495] To corroborate these effects, sensitive-GFPs (roGFPs) was expressed and targeted to the cytosol (cyto-roGFP), mitochondrial inner membrane space (IMS-roGFP), and mitochondrial matrix (matrix-roGFP), in order to quantify subcellular redox status in RAW macrophages (FIG. 12C, left). Treatment of these cells with H2O2 and DTT confirmed the ability to detect changes in the redox state of roGFPs by flow cytometry (FIG. 131). LPS stimulation for 6 hours revealed oxidation of IMS-roGFP, but not matrix-roGFP or cyto- roGFP, the latter of which showed a shift to a reduced state (FIG. 12C, right). This demonstrates the ability of the roGFPs to monitor compartment-specific redox changes in LPS-stimulated macrophages, and supports the hypothesis that LPS-induced mtROS is produced by Complex III into the IMS. Consistently, pretreatment with hydroxy estrogens, but not their precursor or methylated metabolites, significantly reduced IMS-roGFP oxidation by LPS (FIG. 12D).

[0496] Together, these results suggest hydroxyestrogen-driven mitochondrial stress triggers mitohormetic mitochondrial OSR in macrophages, reducing endogenous mtROS levels.

[0497] Next, it was evaluated whether this mitohormetic mitochondrial OSR could provide macrophages defense against normally toxic levels of pharmacologically-generated mtROS (FIG. 12E, left schematic). Macrophages were treated overnight with EtOH or 4-OHE1, and viability was assessed the next day by flow cytometry. The majority of the EtOH and 4- OHEl-treated macrophages fell into a live forward scatter/side scatter (FS/SS) gate containing viable cells as assessed by DAPI exclusion (FIG. 12E, right, top row). These macrophages were then treated with DMSO or a high dose of menadione, a redox cycling quinone that produces mitochondrial superoxide. After 2 hours, an apoptotic cell population appeared in our EtOH control cultures, indicated by their FS/SS shift and inability to exclude DAPI; however, this population did not appear in the 4-OHE1 -treated macrophages (FIG. 12E, right, bottom row). This resistance to menadione toxicity was specifically conferred by 4-OHE1, and not El or 4-MeOEl (FIG. 12F), and it occurred in BMDMs (FIG. 13J). Thus, mitohormesis in response to hydroxyestrogen-driven mitochondrial stress confers macrophages increased mitochondrial OSR and lasting “vaccine-like” protection against subsequent oxidative stress challenge with toxic levels of pharmacologically-generated mtROS. Example 8: LPS-driven mitochondrial stress triggers mitohormesis identical to that observed in hydroxyestrogen-treated macrophages.

[0498] To test the similarity between the mitochondrial stress caused by 4-OHE1 and LPS at the transcriptional level, RNA-seq was performed on BMDMs treated for 6 and 24 hours with either 4-OHE1 or LPS. This revealed an extremely high degree of overlap for both activated and repressed genes at each timepoint (FIG. 14A). Chi-square tests confirmed such overlap would be extremely unlikely by chance (P values <0.0001), suggesting 4-OHE1 very closely mimics physiological oxidative and electrophilic mitochondrial stress signals induced by LPS. GO analysis of genes upregulated by both 4-OHE1 and LPS at 6 and 24 hours revealed enrichment for categories including “Regulation of cellular response to stress”, “Protein folding”, and “Detoxification of ROS” (FIG. 14B, FIG. 15A). Examination of genes in these categories revealed concerted upregulation of HSF1 -regulated molecular chaperones (HSPs = Heat Shock Proteins) that control mitochondrial protein folding, and enzymes of the peroxiredoxin/thioredoxin system that scavenge mitochondrial H2O2, by both 4-OHE1 and LPS (FIG. 14C, FIG. 15B). 4-OHE1/LPS co-treatment further enhanced upregulation of many of these genes (FIG. 14D).

[0499] The next experiment evaluated whether, like 4-OHE1, LPS-driven mitochondrial stress triggered mitohormetic adaptations in macrophages. With regards to mitochondrial chaperone activity, treatment of RAW matrix-oxGFP reporter cells with LPS drove a progressive increase in fluorescence in a manner identical to 4-OHE1 treatment (FIG. 14E). And like stress-induced gene expression, 4-OHE1/LPS co-treatment enhanced this effect. This suggests the increased expression of molecular chaperones in response to both 4-OHE1 and LPS identified in our transcriptional profiling contributes to increased mitochondrial chaperone activity, a hallmark of mitohormesis. HSF1 transcriptional inhibition with KRIB11 partially blunted increased matrix-oxGFP fluorescence in response to both 4-OHE1 and LPS, consistent with HSF1 regulating HSP expression (FIG. 15C).

[0500] To determine if like 4-OHE1, LPS-driven mitochondrial stress triggered mitohormetic mitochondrial OSR, menadione toxicity resistance experiments were performed. 24 hour treatment with either 4-OHE1 or LPS increased BMDM viability relative to EtOH control cultures, with 4-OHE1/LPS co-treatment enhancing this effect (FIG. 14F, left). Treatment of EtOH control BMDMs with menadione significantly decreased viability; however, both 4- OHE1- and LPS -treated macrophages were resistant to menadione-induced toxicity, with 4- OHE1/LPS co-treatment enhancing resistance (FIG. 14F, right). This suggests the increased expression of mtROS scavenging enzymes in response to both 4-OHE1 and LPS identified in transcriptional profiling contributes to mitohormetic mitochondrial OSR. Induction of mtROS scavengers including Prdx6 by 4-OHE1/LPS was impaired in NRF2 KO BMDMs, suggesting a role for NRF2 in mitohormetic OSR (FIG. 15D).

[0501] Taken together, these data demonstrate 4-OHE1 -driven mitochondrial stress closely mimics physiologically relevant LPS-driven mitochondrial stress, and that both trigger classic mitohormetic adaptations in macrophages.

Example 9: Mitohormesis in macrophages involves metabolic reprogramming that enforces an LPS-tolerant state.

[0502] Following primary EPS exposure, macrophages transition to LPS-tolerant state where pro-inflammatory gene induction is refractory to upregulation by secondary LPS treatment (FIG16A). Evidence suggests that suppression of mitochondrial oxidative metabolism following LPS exposure limits acetyl-CoA production required for pro-inflammatory gene transcription (FIG. 16A). mtETC inhibition by LPS-induced nitric oxide (NO) production has been proposed to drive this suppression; however, macrophages unable to produce NO still show significant suppression of mitochondrial oxidative metabolism following LPS treatment. Metabolic reprogramming is often a part of mitohormetic responses to mitochondrial stress, as a shift away from mitochondrial oxidative metabolism towards aerobic glycolysis provides a damaged mitochondrial network an opportunity to recover from stress, while simultaneously augmenting ATP and NADPH production for energy and antioxidant defense, respectively. This experiment evaluated whether the mitochondrial stress-induced mitohormesis observed in LPS-treated macrophages, which includes increased mitochondrial chaperone activity and OSR, also includes metabolic reprogramming that enforces tolerance via suppression mitochondrial oxidative metabolism (FIG. 16A). If so, mitochondrial stress may be a key signal in inducing the transition from an LPS-responsive to LPS-tolerant state via mitohormetic metabolic reprogramming.

[0503] The next experiment evaluated whether 4-OHE1 was sufficient to trigger mitohormetic metabolic reprogramming away from mitochondrial oxidative metabolism and towards aerobic glycolysis. In the absence of other TLR-4 dependent events, RAW macrophages were treated with EtOH vehicle control, 4-OHE1, LPS, or both 4-OHE1/LPS for a tolerizing duration (18-24h) before treatments were washed out and cells subjected to Seahorse respirometry. Plotting basal extracellular acidification rate (ECAR, a proxy for glycolysis) versus basal oxygen consumption rate (OCR, a proxy for mitochondrial oxidative metabolism) to create an energy map showed that while naive EtOH control macrophages are relatively aerobic, both 4-OHE1 and LPS treatment shifted the macrophages away from mitochondrial metabolism and towards aerobic glycolysis, with 4-OHE1/LPS cotreatment inducing an even stronger metabolic shift (FIG. 16B). Seahorse mitochondrial stress testing revealed treatment with either 4-OHE1 and LPS alone was sufficient to significantly reduce basal and maximal OCR, with 4-OHE1/LPS cotreatment causing an even stronger reduction (FIG. 16C, FIG. 17A). A similar response occurred in BMDMs (FIG. 17B). Thus, 4-OHE1- driven mitochondrial stress is sufficient to trigger mitohormetic metabolic reprogramming in a manner essentially identical to that observed in LPS -treated macrophages.

[0504] To test if the 4-OHE1 metabolically reprogrammed macrophages were LPS-tolerant, RAW macrophages were treated with EtOH, 4-OHE1, LPS, or both 4-OHE1/LPS for a tolerizing duration before treatments were washed out and cells allowed to recover. These macrophages were then either left untreated, or LPS -stimulated for 6 hours followed by Illb qPCR (FIG. 16D). While naive macrophages responded robustly to LPS, cells treated overnight with primary LPS showed classic tolerance and impaired Illb upregulation in response to secondary LPS. Similarly, cells treated overnight 4-OHE1 also displayed impaired Illb induction, demonstrating 4-OHEl-induced metabolic reprogramming coincides with transition to an LPS-tolerant state. Finally, in agreement with 4-OHE1/LPS cotreatment driving a stronger metabolic shift than either treatment alone, macrophages co-treated with 4- OHE1/LPS overnight displayed an even more severely impaired secondary LPS response.

CoA supplementation during the washout and recovery period boosted LPS responsiveness in both LPS- and 4-OHEl-tolerized RAW macrophages, suggesting impaired CoA/ acetyl-CoA homeostasis is a FIG. 17C).

[0505] Together, these data demonstrate that in addition to increased mitochondrial chaperone activity and OSR, 4-OHE1 -induced mitohormesis involves metabolic reprogramming that coincides with transition to an LPS-tolerant state essentially identical to that induced by LPS.

[0506] RNA-seq data revealed that unlike LPS, 4-OHE1 -induced LPS tolerance occurs in the absence of transcriptional upregulation of negative regulators of TLR4 signaling such as Tnfaip3, 1110, and without Nos2 induction (FIG. 16F). Thus, mitohormetic metabolic reprogramming to an LPS-tolerant state can be uncoupled from TLR4 signaling, and an LPS- tolerant state can be enforced in the absence of these TLR4-dependent events. Discussion - Mitohormesis Experiments

[0507] Described herein are hydroxyestrogens as lipophilic compounds capable of causing oxidative and electrophilic mitochondrial stress. Two primary conclusions are made from the above-referenced experiments. First, hydroxyestrogens target mitochondrial acetyl-CoA production and epigenetic support of pro-inflammatory gene transcription to antagonize macrophage inflammatory responses in vitro and in vivo in murine models of acute and chronic inflammation. The anti-inflammatory activities of other electrophiles may lie in their ability to cause mitochondrial stress that impairs mitochondrial acetyl-CoA production (FIG. 6H). Much lower doses (high nanomolar/low micromolar) of the more lipophilic electrophiles 4-OHE1, Celastrol, and FCCP, are sufficient to repress Illb to levels similar to I OOpM DEM, suggesting that the ability to localize to mitochondrial membranes controls anti-inflammatory potency of immunomodulatory electrophiles.

[0508] Second, in studying macrophage responses to hydroxyestrogen-driven mitochondrial stress, classic mitohormetic adaptations were discovered. These same mitohormetic adaptations are triggered by LPS-driven mitochondrial stress. This suggests mitochondrial- localized ROS production and protein conjugation by hydroxyestrogens very closely mimics the mitochondrial stress macrophages experience following LPS stimulation, which increases mtROS and production of electrophilic itaconate. In turn, this stress activates transcription factors monitoring mitochondrial integrity, and they coordinate mitohormetic adaptations via mito-nuclear communication. Without wishing to be bound by any theory or mechanism of action, the data indicates that metabolic reprogramming involves both transcriptional changes in expression of nuclear-encoded mitochondrial, glycolytic, and PPP genes, along with post- transcriptional alterations in mitochondrial composition by the ubiquitin-proteasome system, which can tune mitochondrial composition and function in response to stress.

Example 10: Effect of Hydroxyestrogens on High Fat Diet (HFD)-driven Obesity and Metabolic Dysfunction in Mice

[0509] As shown in Example 3, during the early stages (<1 month) HFD-induced metabolic dysfunction in male mice, hydroxyestrogens, but not E2, have unique anti-inflammatory effects on gene expression in visceral white adipose tissue (vWAT) macrophages. To determine if the repression of vWAT macrophage inflammation by 4-OHE1 improved glucose tolerance during more long-term HFD feeding, the duration of these experiments was extended. Surprisingly, 4-OHE1 administration via weekly subcutaneous injection completely abrogated weight gain in HFD-fed male mice (FIG. 18A). After 13 weeks of HFD feeding, 4- OHE1 -treated male mice had normal fasting blood glucose levels and glucose tolerance (FIG. 18B,C). EchoMRI analysis demonstrated that while 4-OHE1 had minimal effects on lean mass, 4-OHE1 reduced fat mass in both normal chow- and HFD-fed male mice (FIG. 18D). Similar results were obtained when the related hydroxyestrogens 2- OHE1 and 2-OHE2 were administered to male mice (FIG. 19A,B), suggesting that in addition to their anti-inflammatory effects on macrophages, hydroxyestrogens also have antiobesity and anti-diabetic properties in this mouse model of HFD-driven metabolic disease.

[0510] High Fat Diet (HFD)-fed EtOH- and 4- OHEl-treated male mice were housed in metabolic cages at 4 and 13 weeks after initiating HFD feeding and weekly subcutaneous injections. Surprisingly, at both timepoints, 4-OHE1 -treated mice ate more HFD chow than their EtOH control counterparts (FIG. 19C), suggesting the 4-OHE1 anti-obesity effect is independent of anorexia. 4-OHE1 -treated mice displayed normal levels of activity at 4 weeks, and elevated activity at 13 weeks, which could be due to their resistance to obesity (FIG. 19D). Differences in energy expenditure were clearly evident at 13 weeks (FIG. 19E). Interestingly, 4- OHEl-treated male mice displayed a significantly increased respiratory exchange ratio (RER) at both 4 and 13 weeks (FIG. 19F), suggesting they have enhanced ability to utilize glucose for fuel. As demonstrated above in Example 9, in macrophages, mitochondrial stress caused by 4-OHE1 results in metabolic reprogramming that suppresses oxidative metabolism and increases glucose utilization via enhanced aerobic glycolysis. Thus, this increased RER suggests similar metabolic reprogramming may occur in vivo in response to mitochondrial stress caused by 4-OHE1 in energy-consuming tissues.

[0511] Post-mortem analysis after 13 weeks demonstrated that the mass of both visceral and subcutaneous white fat (scWAT) pad size was significantly reduced in both normal chow- and HFDfed mice treated with 4-OHE1 (FIG. 18E,F). Flow cytometry of the visceral fat stromal vascular fraction (SVF) reveal that 4-OHE1 prevented HFD-driven infiltration of both CD45+ leukocytes and macrophages (FIG. 18G). Moreover, macrophages from 4- OHEl-treated mice showed significantly higher expression of the alternative/M2 macrophage marker CD301, and reduced expression of the classically activated/Ml macrophage marker CD 11c (FIG. 18H). Example 11: Hydroxyestrogens ameliorate HFD-driven metabolic dysfunction in OVX female mice

[0512] Estrogens are thought to provide females protection against metabolic disease. However, menopause increases the likelihood that females will develop metabolic dysfunction. To test if hydroxyestrogens can prevent metabolic disease in female mice, two hydroxyestrogens (2-OHE2 and 4-OHE1) were administered to ovariectomized (OVX) female mice, where the ovariectomy models the decrease in estrogen production experienced post menopause. 2-OHE2 reduced weight gain, lowered fasting blood glucose, and improved glucose tolerance in HFD-fed OVX female mice compared to HFD EtOH controls after 14 weeks (FIG. 20A,C).

[0513] EchoMRI showed that 2-OHE1 decreased fat mass while, leaving lean mass unchanged, in HFD 2-OHE2 OVX female mice (FIG. 21D). Metabolic cage measurements after 14 weeks showed that food consumption was not affected by 2-OHE1, nor was activity (FIG. 21A,B). Oxygen consumption was elevated, while RER was unchanged (FIG. 21C,D). Post-mortem analysis showed that both vWAT and scWAT accumulation were reduced by 2- OHE2 in HFD-fed mice (FIG. 20E). Accumulation of both leukocytes and macrophages in vWAT was also prevented (FIG. 20F). A similar trend was seen in 4-OHE1 -treated OVX female mice (FIG. 21E,F); however, post-surgery complications with 4-OHE1 -treated OVX female mice in this cohort led to a small cohort (n=2) for this experimental group.

Example 12: 4-OHE1 alters adipose tissue gene expression

[0514] Adipose tissue is an endocrine organ that plays a key role in energy storage and expenditure, and secretes various hormones, known as adipokines, that regulate systemic nutrient handling and energy balance through communication with other tissues. To evaluate whether 4-OHE1 might affect adipose tissue function, gene expression profiling was performed by RNA-seq of vWAT from the NC EtOH, HFD EtOH, and HFD 4-OHE1 male mice. Unbiased hierarchical clustering of the RNAseq data revealed that global gene expression in vWAT from HFD 4-OHE1 mice most closely resembled that of vWAT from NC EtOH control mice (FIG. 22A). While HFD suppressed expression of genes involved in glucose uptake, fatty acid (FA) mobilization, and FA oxidation, expression of these genes was mostly rescued, or even enhanced, in vWAT from HFD 4-OHE1 mice (FIG. 22B). This suggests 4-OHE1 helps maintain “healthy”, functional gene expression in vWAT, though whether this is a direct effect on adipocytes, or a secondary effect from 4-OHEl’s prevention of obesity, remain unclear. [0515] To evaluate how 4-OHE1 affects gene expression in adipose tissues, scWAT and brown adipose tissue (BAT) from HFD EtOH and HFD 4-OHE1 mice was profiled, and compared differentially expressed genes amongst the three adipose tissues. This revealed both common and distinct genes activated and repressed in each of these tissues (FIG. 22C,D), with 16 and 32 common genes upregulated and downregulated by 4-OHE1 treatment in all three adipose depots, respectively. Among the genes upregulated by 4-OHE1 was Cfd, which encodes the adipokine adipsin (FIG. 22C). Adipsin production by adipose tissue, which is suppressed during obesity, supports and protects insulin-producing beta cells in the pancreas, positively influencing glucose homeostasis. Expression of Irf4, which encodes the transcriptional regulator Interferon Regulatory Factor 4 (IRF4), was also increased in all three tissues by 4-OHE1 (FIG. 22C). In adipocytes, IRF4 is a key transcriptional regulator of lipolysis, and when induced by fasting, IRF4 upregulates the expression of genes that control the liberation of FA from triglycerides (TGs) for energy utilization. Finally, Irsl, which encodes the insulin receptor signaling adaptor IRS-1, was upregulated in all three depots by 4-OHE1. Signaling through IRS-1 is critical for glucose uptake in response to insulin, and defects in IRS-1 phosphorylation have been linked to insulin resistance in T2D. The expression of Cfd, Irf4, and Irsl in HFD 4-OHE1 vWAT was elevated beyond expression observed in NC EtOH control vWAT. (FIG. 22D). This raises the possibility that 4-OHE1 might not simply keep expression of these genes close to “normal” levels observed in vWAT of healthy mice, but rather might induce supraphysiologic levels of expression that provide protection against the development of obesity and glucose intolerance during HFD feeding, a hypothesis which will need testing. Furthermore, the expression of Cfd, Irf4, and Irsl in primary macrophages in vitro was not upregulated by 4-OHE1 treatment (FIG. 23A), suggesting that these in vivo changes in whole adipose tissue gene expression are not direct effects on macrophages, but rather on adipocytes.

[0516] Many of the 32 common genes repressed by 4-OHE1 in all adipose tissues were related to macrophage activation and immune function (e.g. Itgax, Cd68, Ccl2, Ccl9, Illrri) (FIG. 12D). This suggests that 4-OHE1 might act directly on macrophages in all of these adipose tissues to suppress proinflammatory gene expression as shown above for vWAT macrophages. However, since this is whole tissue gene expression, these changes could also be attributed to the prevention of macrophage infiltration into scWAT and BAT as was observed for vWAT (FIG. 20F).

[0517] One gene repressed by 4-OHE1 in all three adipose tissues was Sppl (a.k.a. Opr), which encodes the extracellular protein Osteopontin (OPN). OPN has been implicated in the recruitment of pro-inflammatory macrophages into adipose tissue during DIO, and HFD-fed OPN KO mice are protected from DIO, adipose tissue and liver inflammation, and hepatic steatosis. Notably, while Sppl expression was increased in response to HFD in vWAT, 4- OHE1 treatment completely prevented this increase (FIG. 22F). Again, whether this is a primary versus secondary effect related to obesity prevention by 4-OHE1 will require further investigation. In macrophages in vitro, Sppl expression is not repressed by 4-OHE1 treatment (FIG. 23B), suggesting the in vivo repression might be a direct effect on adipocytes, which are known to express the Sppl gene.

[0518] Finally, estrogens have been reported to influence energy expenditure by activating adaptive thermogenesis in BAT and so-called “beige fat” in scWAT. This could occur through direct action on adipocytes, or indirectly though activation of neurons in the hypothalamus that promote thermogenesis in BAT via the sympathetic nervous system (SNS). However, no increase in thermogenic gene expression, including Ucpl, in either BAT or scWAT was detected in HFD 4-OHE1- treated mice versus controls (FIG. 23C). This suggests that adaptive thermogenesis does not underlie the anti-obesity effects of 4-OHE1.

Example 13: 4-OHE1 promotes weight loss and improves glucose tolerance in male mice with existing diet-induced obesity.

[0519] This experiment evaluated whether 4-OHE1 could treat metabolic disease in animals with existing diet- induced obesity (DIO). Male mice were put on HFD for 16 weeks to induce DIO. Obese mice were then given weekly SQ injections of EtOH, 4-OHE1 (both at lOmg/kg) or an ethanol control for 8 weeks as the mice continued ad libitum HFD chow feeding. Interestingly, while E2 prevented further weight gain and caused a minor decrease in body weight, 4-OHE1 -treated mice rapidly lost nearly 20% of their body weight within 4 weeks of the first injection (FIG. 24A). To ascertain whether these effects were due to anorexia, food consumption was tracked at the whole cage level in these grouped-housed animals. While both E2 and 4-OHE1 caused significant reductions in food consumption following the initial injections, food consumption in both groups recovered and was not significantly different than EtOH control mice from 3 weeks of treatment onward (FIG. 24B). Thus, while both E2 and 4-OHE1 caused acute anorexia, only 4-OHE1 -treated mice continued to lose weight and maintained a significantly lower body weight than E2-treated mice despite consuming similar amounts of HFD chow. EchoMRI analysis after 8 weeks revealed that 4-OHE1 treatment significantly reduced fat mass compared to E2 treatment, while having only a minor impact on lean mass compared to EtOH control mice (FIG. 24C). Blood glucose levels in ad libitum fed mice decreased in 4-OHE1-, but not E2-treated animals despite our consuming similar amounts of chow (FIG. 24D). Fasting blood glucose levels (FIG. 24E) and glucose tolerance (FIG. 24F) were improved in both 4-OHE1- and E2-treated mice. This suggests that 4-OHE1 and E2 treatment accomplish similar improvements in glucose handling via different mechanisms, as 4-OHE1 had much larger effects on body weight.

[0520] A treatment group given a 10-fold lower dose of 4-OHE1 (Img/kg) had nearly identical effects on bodyweight (FIG. 25A) and glucose tolerance (FIG. 25B), with minimal effects on food consumption (FIG. 25C), as observed in lOmg/kg E2- treated mice, suggesting that the anti-obesity and glucose handling effects of 4-OHE1 are still present at this reduced dosage. Metabolic cage measurements of individually housed animals at 8 weeks confirmed that there were no differences in food consumption between the groups (FIG.

25D). Moreover, no differences in activity, oxygen consumption, or RER could be detected (FIG. 25 E,F,G).

[0521] Post-mortem analysis revealed significant reductions in the size of both visceral and subcutaneous fat pads in 4-OHE1 -treated mice (FIG. 24G and 24H), along with a visible improvement in liver lipid levels (FIG.24H). A reduction in liver triglyceride (TG) levels upon 4-OHE1 treatment was confirmed (FIG. 241), as was an improvement in liver fibrosis by 4- OHE1 as assayed by measuring tissue collagen content (FIG. 24J). The number of CD45+ leukocytes and CDllb-i- F4/80+ macrophages in visceral adipose tissue SVF was not changed by either 4-OHE1 or E2 treatment (FIG. 25H). However, but 4-OHE1 and E2 caused a coordinate increase in CD301 expression/decrease in CDllc expression, suggesting both estrogens might have effects on macrophage polarization (FIG. 24K).

Example 14: CoA and Acetyl-CoA synergize with LPS to enhance macrophage proinflammatory gene transcription in vitro.

[0522] In this example, CoA and Acetyl-CoA were tested for their ability to enhance proinflammatory gene expression in macrophages. RAW macrophages were pretreated with 500pM Coenzyme A (CoA) for 15 minutes, followed by stimulation with lOOng/mL LPS for 7.5 hours. Illb gene expression was measured by quantitative real time PCR (qPCR). Results are depicted in FIG. 26a.

[0523] Primary bone marrow-derived macrophages (BMDMs) were pretreated with 400pM Acetyl-Coenzyme A (Acetyl-CoA) for 2 hours, followed by stimulation with lOOng/mL LPS for 1.5 hours. Illb gene expression was measured by qPCR. The results are depicted in FIG. 26b. Human THP-1 cells were pretreated with 500uM CoA for 15min, followed by 6h LPS stimulation (lOOng/mL) before harvest and Illb qPCR. The results are depicted in FIG. 26c. [0524] Murine BMDMs were pretreated with 500uM CoA for 15min, followed by 6h MPLA stimulation (lOOng/mL) before harvest and Illb qPCR. The results are depicted in FIG. 26d. The results show that CoA and Acetyl-CoA alone can enhance proinflammatory gene expression in macrophages in vitro, and that when provided to macrophages in combination with TLR ligands such as LPS, show synergistic (not additive) enhancement of proinflammatory gene expression. Additionally, the data shows that MPLA enhances proinflammatory gene expression.

Example 15: Anti-cancer effect of CoA and CoA derivatives in in vivo xenograft tumor models (prophetic)

[0525] Exogenous CoA and acetyl-CoA bypass the mitochondrial stress produced by hydroxyestrogens and thereby enhances the activation potential of inflammatory cells, including macrophages. Moreover, CoA and acetyl-CoA restore the activation potential of macrophages tolerized with hydroxyestrogens or the TLR agonist LPS. Activating immune responses, including immune responses by macrophages, is useful for treating cancers, as demonstrated in well-established xenograft models where CoA and its derivatives (e.g. acetyl-CoA and 4-phosphopanthetheine) act synergistically with proinflammatory signaling pathway agonists (e.g. monophosphoryl lipid A (MPL), rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di- AMP, and anti-CD40 antibody) and immune checkpoint inhibitors (e.g. anti-PD-1, anti- CTLA-4) to inhibit the growth of solid tumors (melanoma, colon adenocarcinoma, bladder cancer, hepatoma, breast cancer) and hematologic malignancies (lymphoma and acute myeloid leukemia).

[0526] Bl 6- 10 murine melanoma cells are maintained in RPMI 1640 supplemented with 10% FCS, lx nonessential amino acids, 1 mM sodium pyruvate, 2 mM 1-glutamine, and penicillin with streptomycin. Tumors are initiated in 8-week-old male and female C57BL/6J mice (Jackson Labs) via subcutaneous (s.c.) injection of 5 x 10⁵ B16-F10 melanoma cells into both right and left rear flanks (day 0). Mice are randomized with equal numbers of males and females in each group, and then treated on days 4, 8, 12, and 16 in the right flank tumor (left flank tumor serves as untreated control). Mice will be divided into six treatment groups: 1) PBS/PBS/IgG, 2) MPL/PBS/IgG, 3) MLP/CoA/IgG, 4) PBS/PBS/anti-PD-1, 5) MPL/PBS/anti-PD-1, 6) MPL/CoA/anti-PD-1. The TLR4 agonist monophosphoryl lipid A (MPL, 5 pg) or PBS control are administered intratumorally (i.t). CoA (50pg) or PBS control are administered intratumorally (i.t). Checkpoint blockade inhibitor anti-PD-1 (RMP1-14) or corresponding IgG isotype controls (2A3) 2A3 are administered intraperitoneally (i.p.) twice weekly (250pg). Tumor growth is measured with calipers for the right (treated) and left (untreated) tumors and is plotted until the point at which any mice in the group died, or their tumors on either flank reached 1000mm.

[0527] MPL/CoA/IgG combination therapy reduces treated tumor growth and improves survival better than MPL/PBS/IgG monotherapy therapy or PBS/PBS/IgG control therapy. MPL/PBS/anti-PD-1 combination therapy also reduces treated tumor growth and improves survival better than either MPL/PBS/IgG or PBS/PBS/anti-PD-1 monotherapies alone. Furthermore, MPL/CoA/anti-PD-1 triple therapy reduces treated tumor growth and improves survival better than MPL/PBS/anti-PD-1 combination therapy. For untreated tumor growth, MPL/PBS/anti-PD-1 combination therapy reduces growth better than control or any monotherapy. However, MPL/CoA/anti-PD- 1 triple therapy is superior in reducing untreated tumor growth.

[0528] A safer and more effective compound is identified by comparing the efficacy of CoA with CoA derivatives including acetyl-CoA and 4-phosphopanthetheine. An effective dose for inhibiting tumor growth in mice is determined in a dose-response experiment testing the efficacy of different doses (Ipg, 5pg, 25 pg, 50pg, lOOpg) of CoA and its derivatives. CoA and its derivatives act synergistically with other proinflammatory signaling pathway agonists: TLR3 agonist (e.g. poly I:C, rintatolimod), TLR5 agonist (e.g. entolimod), TLR7 agonist (e.g. Imiquimod, R848, 1V720, Resiquimod), TLR9 agonist (e.g. ODN1826, SD-101), Bacillus Calmette- Guerin (BCG, TLR2/4/9 agonist), STING agonist (e.g. MIW815, ci-di- AMP, 25pg), or anti-CD40 antibody (FGK45, 20pg) and immune checkpoint inhibitors including anti-CTLA-4 (9D9 [Mouse IgG2a], lOOpg/dose). CoA combination therapy with proinflammatory signaling pathway agonists and immune checkpoint inhibitors has efficacy in multiple in vivo xenograft models including MC38 or CT26 (colon adenocarcinoma), MB49 (bladder cancer), or Hep-55.1c (hepatoma), RMA (lymphoma), 4T1 (breast cancer), and C1498 (acute myeloid leukemia) cells.

Example 16: CoA and Acetyl-CoA synergize with LPS to enhance macrophage proinflammatory gene transcription in vivo.

[0529] As shown in Example 14, Coenzyme A (CoA) and acetyl-CoA (Ac-CoA) can boost Toll-like receptor 4 (TLR4) ligand-dependent inflammatory responses in murine and human macrophages. To test if CoA has this effect in vivo, an intraperitoneal (i.p.) LPS injection model was used. The model causes a systemic, macrophage-dependent inflammatory response that can be quantified by the upregulation of proinflammatory cytokines, chemokines, and growth factors in circulation.

Experimental details & data description

[0530] a. Six-week-old male C57BL/6 mice were i.p. injected with 250mg/kg CoA, or vehicle control. 15 minutes later, mice were injected with 3mg/kg LPS, or vehicle control. 4 hours later, blood was collected for multiplex measurement of cytokines, chemokines, and growth factors to assess their upregulation by vehicle, LPS, LPS + CoA, or CoA alone treatments. The experiment is schematically shown in FIG. 27a.

[0531] FIG. 27 shows the serum concentrations of b. cytokines (FIG. 27b), c. chemokines (FIG. 27c), and d. growth factors following administration of the test articles (FIG. 27d) . *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Student’s t test.

[0532] The results show that, compared to LPS alone, CoA co-administration with LPS significantly enhanced the magnitude of the inflammatory response.

[0533] CoA co-administration with LPS, MPLA (a clinically-approved TLR4 ligand), or other TLR ligands, may be useful to (a) boost macrophage anti-tumor immunity; (b) restore macrophage immunity in sepsis-associated immunosuppression, where patients that survive a severe, systemic primary infection (sepsis) enter an immunosuppressed state where they are susceptible to secondary (often nosocomial) infections; and/or (c) restore macrophage innate immunity in aging. Aging is associated with increased susceptibility to infections and cancer, and decreased vaccination responses. This is in part due to decreased macrophage-dependent immunity, decreased macrophage-dependent tumor surveillance, and decreased macrophage inflammatory responses to adjuvants present in vaccine cocktails (resulting in decreased activation of the adaptive immune system by the innate immune system).

Example 17: Anti-cancer effect of CoA and CoA derivatives in PyMT Breast Cancer Model

[0534] Toll-like receptor (TLR) ligands have been proposed to be able to stimulate inflammatory responses and anti-tumor immunity in tumor-associated macrophages (TAMs). However, their success as monotherapies have been limited. This may be because TAMs are continually exposed to damage- associated molecular patterns (DAMPs) in the tumor microenvironment (TME) that signal through TLRs to induce tolerance. Moreover, chronic administration of the TLR ligands themselves can drive TAM tolerance. This experiment evaluated whether clinically-approved TLR4 ligand monophosphoryl lipid A (MPLA), combined with Coenzyme A (Co A), a factor which reverses macrophage tolerance and primes/boost TLR-dependent inflammatory responses in macrophages, would have greater efficacy at reducing tumor growth and metastasis than MPLA treatment alone. As a positive control, MPLA was combined with Interferon gamma (IFNy), a cytokine known to prime/enhance TLR4-dpendent inflammatory responses, reverse macrophage tolerance, and reduce breast cancer growth in preclinical models in combination with MPLA (but it is not clinically used to deleterious side effects).

Experimental details

[0535] On Day 0, 2.5 x 10^A5 PyMT breast cancer cells were injected into the mammary fat pad 6 week old female C57BL/6 mice (lOOuL volume in Matrigel). On Day 10 when all mice had palpable tumors, intratumor (i.t) injection of the TLR4 ligand MPLA (lug), MPLA (lug) + CoA (250mg/kg), or MPLA (lug) + IFNy (lug) was performed. Treatments (twice weekly) and tumor growth monitoring via caliper measurements continued until Day 35 when tumors were harvested and weighed.

Results

[0536] Compared to MPLA alone, MPLA + CoA significantly slows tumor growth (FIG. 28A) and tumor weights (FIG. 28B). Thus, combining CoA with MPLA (or other TLR ligand monotherapies) provides a superior therapeutic combination as CoA boosts TLR- dependent inflammatory responses, and prevents myeloid cell tolerance to TLR ligands in the tumor microenvironment, to potentiate anti-tumor immunity.

[0537] Moreover, compared to the combination of TLR ligands alone + checkpoint blockade (e.g. anti-PD-Ll antibodies), the triple therapy of TLR ligands + CoA + checkpoint blockade may provide a superior anti-tumor therapeutic combination.

Example 18.

[0538] The mechanism by which CoA/Acetyl-CoA enhances TLR-dependent inflammatory response was investigated. The experimental design is shown in FIG. 29a. Murine bone- marrow derived macrophages (BMDMs) were provided with either vehicle or 250uM CoA or Ac-Co A. After 2h, cells were pretreated with EtOH or 5uM 4-OHE1, which causes mitochondrial oxidative stress that suppresses LPS-induced gene expression. After Ih, cells were stimulated with lOOng/mL LPS. 1.5h later (with cells switched to isotopically labeled C13-glucose containing media the last 30min), cells were harvested to assess gene expression and C13-glucose flux. LPS-induced Illb expression is suppressed by 4-OHE1, but enhanced in presence of Co A r Ac-Co A. FIG. 29b shows LPS-induced C13-glucose flux into the TCA cycle metabolites acetyl-CoA and citrate is suppressed by 4-OHE1, but enhanced in presence of CoA or Ac-CoA. The results show that CoA/Ac-CoA enhances mitochondrial glucose oxidation to support proinflammatory gene expression.

[0539] FIG. 29c schematically summarizes ow CoA supplementation promotes/enhances mitochondrial glucose utilization and oxidation, especially in the face of mitochondrial oxidative stress when CoA/Ac-CoA is used as an anti-oxidant to protect the mitochondrial proteome.

[0540] While certain embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification.

[0541] While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is Claimed Is:

1. A pharmaceutical composition comprising a unit dose of a hydroxyestrogen or a salt thereof and a pharmaceutically acceptable excipient, wherein the unit dose of the hydroxyestrogen comprises an amount of the hydroxyestrogen sufficient to induce mitohormesis.

2. The pharmaceutical composition of claim 1 , wherein the hydroxy estrogen is a hydroxyestrone or a salt thereof.

3. The pharmaceutical composition of claim 1, wherein the hydroxy estrogen is a hydroxyestradiol or a salt thereof.

4. The pharmaceutical composition of claim 1 , wherein the hydroxy estrogen is a synthetic or semi-synthetic hydroxyestrogen or a salt thereof.

5. The pharmaceutical composition of claim 1, wherein the hydroxy estrogen comprises 4- hydroxyestrone (4-OHE1) or a salt thereof.

6. The pharmaceutical composition of claim 1 , wherein the hydroxy estrogen comprises 4- hydroxyestradiol (4-OHE2) or a salt thereof.

7. The pharmaceutical composition of claim 1, wherein the hydroxy estrogen comprises 2- hydroxyestrone (2-OHE1) or a salt thereof.

8. The pharmaceutical composition of claim 1, wherein the hydroxy estrogen comprises 2- hydroxyestradiol (2-OHE2) or a salt thereof.

9. The pharmaceutical composition of claim 1 , wherein the hydroxy estrogen is 4- hydroxyestrone (4-OHE1).

10. The pharmaceutical composition of claim 1, wherein the hydroxy estrogen is 4- hydroxyestradiol (4-OHE2).

11. The pharmaceutical composition of claim 1 , wherein the hydroxy estrogen is 2- hydroxyestrone (2-OHE1).

12. The pharmaceutical composition of claim 1, wherein the hydroxy estrogen is 2- hydroxyestradiol (2-OHE2).

86

13. The pharmaceutical composition of any one of claims 1-7 and 9-11, wherein the amount of the hydroxyestrogen sufficient to induce mitohormesis is at least about 0.2 mg/kg based on mass of a subject administered the pharmaceutical composition.

14. The pharmaceutical composition of any one of claims 1-13, wherein the amount of the hydroxyestrogen sufficient to induce mitohormesis is greater than 2 mg/kg based on mass of a subject administered the pharmaceutical composition.

15. The pharmaceutical composition of any one of claims 1-14, wherein the pharmaceutical composition does not include carboxymethylcellulose.

16. The pharmaceutical composition of any one of claims 1-14, wherein the pharmaceutical composition does not include 1 % carboxymethylcellulose.

17. A method of mediating mitochondrial stress in a cell comprising contacting the cell with an effective amount of a hydroxyestrogen or a salt thereof to thereby mediate mitochondrial stress.

18. The method of claim 17, wherein the effective amount results in reduction in mitochondrial acetyl-CoA production in the cell.

19. The method of claim 18, wherein the effective amount results in reduction of total intracellular acetyl-CoA levels.

20. A method of activating or inducing mitohormesis in a cell comprising contacting the cell with an effective amount of a hydroxyestrogen or salt thereof.

21. The method of claim 20, wherein the effective amount results in an increase in mitochondrial chaperone activity.

22. The method of claim 20, wherein the effective amount results in an increase in mitochondrial oxidative stress resistance in the cell.

23. The method of claim 20, wherein the effective amount results in an increase of a ratio of aerobic glycolysis to mitochondrial oxidative metabolism in the cell.

24. The method of any one of claims 17-23, wherein the cell is a macrophage.

25. The method of any one of claims 17-23, wherein the hydroxy estrogen is provided at a concentration of at least 1 pM.

26. The method of claim 25, wherein the hydroxyestrogen is provided at a concentration of about 5 pM.

87

27. The method of any one of claims 17-26, wherein the hydroxyestrogen comprises 4- hydroxyestrone (4-OHE1) or a salt thereof.

28. The method of any one of claims 17-26, wherein the hydroxyestrogen comprises 2- hydroxyestrone (2-OHE1) or a salt thereof.

29. The method of any one of claims 17-26, wherein the hydroxyestrogen comprises 2- hydroxyestradiol (2-OHE2) or a salt thereof.

30. A method of reducing an inflammatory response in a subject in need thereof comprising administering or causing to be administered an effective amount of an hydroxyestrogen to the subject to thereby inhibit an inflammatory response.

31. The method of claim 30, wherein induction of Illb expression by an inflammatory stimulus in a macrophage of the subject is reduced.

32. The method of claim 31, wherein the inflammatory response is induced by a lipopolysaccharide (LPS).

33. The method of claim 30 or claim 31, wherein Illb expression in a visceral white adipose tissue macrophage of the subject is reduced.

34. The method of any one of claims 30-33, wherein the inflammatory response is an acute inflammatory response.

35. The method of any one of claims 30-33, wherein the inflammatory response is a chronic inflammatory response.

36. The method of any one of claims 30-33, wherein the subject is afflicted with an inflammatory condition.

37. The method of claim 36, wherein the acute inflammatory condition comprises a cytokine storm.

38. The method of any one of claims 30-37, wherein the hydroxyestrogen comprises 4- hydroxyestrone (4-OHE1) or a salt thereof.

39. The method of any one of claims 30-37, wherein the hydroxyestrogen comprises 4- hydroxyestradiol (4-OHE2) or a salt thereof.

40. The method of any one of claims 30-37, wherein the hydroxyestrogen comprises 2- hydroxyestrone (2-OHE1) or a salt thereof.

88

41. The method of any one of claims 30-37, wherein the hydroxyestrogen comprises 2- hydroxyestradiol (2-OHE2) or a salt thereof.

42. The method of any one of claims 30-41, wherein the hydroxyestrogen is administered subcutaneously.

43. A method of mitigating effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce a body weight gain of the subject.

44. The method of claim 43, wherein the body weight gain of the subject is reduced by at least 10% compared to a body weight gain of a control subject not administered a hydroxyestrogen.

45. A method of mitigating effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce a fasting blood glucose of the subject.

46. The method of claim 45, wherein the subject is obese.

47. The method of claim 45, wherein the subject is not obese.

48. A method of mitigating the effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby improve a glucose tolerance of the subject.

49. The method of claim 48, wherein a blood glucose level 60 minutes after a meal is reduced by at least 10% compared to a blood glucose level of a control subject not administered a hydroxyestrogen.

50. A method of mitigating the effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce liver triglyceride content of the subject.

51. The method of claim 50, wherein a liver triglyceride content is reduced by at least 20 nmol per mg of liver tissue compared to a liver triglyceride content of a control subject not administered a hydroxyestrogen.

52. A method of mitigating the effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen to thereby reduce liver fibrosis of the subject.

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53. The method of claim 52, wherein a liver collagen content is reduced by at least 0.5 |ig per mg of liver tissue compared to a liver collagen content of a control subject not administered a hydroxyestrogen.

54. A method of treating obesity in a subject comprising administering or causing to be administered an hydroxy estrogen to the subject to thereby reduce body weight of the subject.

55. The method of claim 54, wherein the body weight of the subject is reduced by at least about 10%.

56. A method of mitigating effects of a high fat diet comprising administering or causing to be administered to a subject in need thereof an effective amount of a hydroxyestrogen to thereby increase insulin sensitivity in the subject.

57. The method of claim 56, wherein the amount of insulin required to clear glucose from blood of the subject is lower than a control subject not administered the hydroxyestrogen.

58. A method of increasing insulin sensitivity in a subject consuming a high fat diet comprising administering or causing to be administered to the subject an effective amount of a hydroxyestrogen.

59. A method of increasing glucose tolerance comprising administering or causing to be administered to a subject in need thereof an effective amount of an hydroxyestrogen.

60. The method of any one of claims 43-59, wherein a fed blood glucose is reduced.

61. The method of any one of claims 43-60, wherein the subject has ingested a high fat diet.

62. The method of claim 61, wherein the high fat diet comprises at least 20% fat.

63. The method of any one of claims 43-62, wherein the subject is a male.

64. The method of any one of claims 43-62, wherein the subject is a female.

65. The method of any one of claims 43-64, wherein a fasting blood glucose of the subject is reduced compared to a fasting blood glucose of the subject prior to administration of the hydroxy estrogen, or a control subject not administered a hydroxyestrogen.

66. The method of any one of claims 43-65, wherein infiltration of immune cells into visceral white adipose tissue is reduced.

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67. The method of any one of claims 43-66, wherein infiltration of CD45+ leukocytes into visceral white adipose tissue is reduced.

68. The method of any one of claims 43-67, wherein infiltration of CDllb-i- macrophages into visceral white adipose tissue is reduced.

69. The method of any one of claims 43-68, wherein oxygen consumption is increased.

70. The method of any one of claims 43-69, wherein energy expenditure is increased.

71. The method of any one of claims 43-70, wherein the hydroxy estrogen comprises 4- hydroxyestrone (4-OHE1).

72. The method of any one of claims 43-70, wherein the hydroxyestrogen comprises 2- hydroxyestrone (2-OHE1).

73. The method of any one of claims 43-70, wherein the hydroxy estrogen comprises 2- hydroxyestradiol (2-OHE2).

74. The method of any one of claims 43-73, wherein the hydroxy estrogen is administered subcutaneously.

75. The method of any one of claims 43-74, wherein fat mass is reduced.

76. The method of claim 75, wherein visceral white adipose tissue mass is reduced

77. The method of claim 75, wherein subcutaneous white adipose tissue mass is reduced.

78. The method of any one of claims 17-77, wherein the hydroxy estrogen is an exogenous hydroxyestrogen.

79. The method of any one of claims 17-77, wherein the hydroxyestrogen is an isolated hydroxyestrogen.

80. The method of any one of claims 17-77, wherein the hydroxyestrogen is a synthetic or semi-synthetic hydroxyestrogen.

81. The method of any one of claims 17-80, wherein the effective amount of the hydroxyestrogen is at least about 0.2 mg/kg based on mass of a subject administered the pharmaceutical composition.

82. The method of any one of claims 17-80, wherein the hydroxyestrogen comprises 2- hydroxyestrone (2-OHE1) and wherein the effective amount of the 2-OHE1 greater than 2 mg/kg based on mass of a subject administered the pharmaceutical composition.

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83. The method of any one of claims 17-82, wherein said contacting, said administering, or said causing to be administered does not comprise contacting, administering, or causing to be administered the hydroxyestrogen with carboxymethylcellulose.

84. The method of any one of claims 17-82, wherein said contacting, said administering, or said causing to be administered does not comprise contacting, administering, or causing to be administered the hydroxyestrogen with 1% carboxymethylcellulose.

85. A method of enhancing an inflammatory response in a subject comprising administering or causing to be administered an effective amount of CoA or Acetyl-CoA or a salt thereof to the subject to thereby enhance an inflammatory response of the subject.

86. The method of claim 85, further comprising administering or causing to be administered a Toll-like receptor ligand.

87. The method of claim 86, wherein the Toll-like receptor ligand is monophosphoryl lipid A (MPLA).

88. The method of claim 86, wherein the Toll-like receptor ligand is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

89. A method of enhancing an inflammatory response in a subject comprising administering or causing to be administered an effective amount of 4'-phosphopantetheine or salt thereof to the subject to thereby enhance an inflammatory response in the subject.

90. The method of claim 85 or claim 89, wherein induction of II lb expression by an inflammatory stimulus in a macrophage of the subject is increased.

91. A method of treating a cancer in a subject in need thereof comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby inhibit growth of a transformed cell in the subject.

92. A method of treating or preventing a cancer metastases in a subject in need thereof comprising administering or causing to be administered coenzyme A (CoA) or a derivative thereof to the subject to thereby reduce cancer metastases in the subject.

93. The method of claim 91, wherein the coenzyme A derivative is acetyl-CoA.

94. The method of claim 91, wherein the coenzyme A derivative is 4-phosphopanthetheine.

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95. The method of any one of claims 91-94, further comprising administering or causing to be administered a proinflammatory signaling pathway agonist.

96. The method of claim 95, wherein the proinflammatory signaling pathway agonist is monophosphoryl lipid A (MPLA).

97. The method of claim 95, wherein the proinflammatory signaling pathway agonist is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody.

98. The method of any one of claims 91-96, further comprising administering or causing to be administered an immune checkpoint inhibitor.

99. The method of claim 98, wherein the immune checkpoint inhibitor comprises an anti-PD- 1 antibody, an anti-CTLA-4 antibody, or a fragment thereof.

100. The method of any one of claims 91-99, wherein the cancer comprises a solid tumor, or metastases thereof.

101. The method of claims 100, wherein the solid tumor is a melanoma, colon adenocarcinoma, bladder cancer, hepatoma, or breast cancer, or metastases thereof.

102. The method of any one of claims 91-99, wherein the cancer comprises a hematologic malignancy, or metastases thereof.

103. The method of claim 102, wherein the hematologic malignancy is lymphoma or acute myeloid leukemia, or metastases thereof.

104. The method of any one of claims 91-103, wherein the coenzyme A (CoA) or derivative thereof is an exogenous coenzyme A (CoA) or derivative thereof.

105. The method of any one of claims 91-103, wherein the coenzyme A (CoA) or derivative thereof is an isolated coenzyme A (CoA) or derivative thereof.

106. The method of any one of claims 91-103, wherein the coenzyme A (CoA) or derivative thereof is a synthetic or semi- synthetic coenzyme A (CoA) or derivative thereof.

107. The method of any one of claims 91-106, wherein the cancer is breast cancer or metastases thereof.

108. The method of any one of claims 91-106, wherein the cancer is lung cancer or metastases thereof.

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. A method of enhancing macrophage anti-tumor immunity, comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to a subject in need thereof. . A method of restoring macrophage immunity in sepsis-associated immunosuppression, comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to a subject in need thereof. . A method of restoring macrophage innate immunity in an aging subject, comprising administering or causing to be administered a coenzyme A (CoA) or a derivative thereof in combination with a proinflammatory signaling pathway agonist to the subject. . The method of claim any one of claims 109-111, wherein the proinflammatory signaling pathway agonist is monophosphoryl lipid A (MPLA). . The method of claim any one of claims 109-111, wherein the proinflammatory signaling pathway agonist is rintatolimod, entolimod, Imiquimod, R848, 1V720, Resiquimod, ODN1826, SD-101, Bacillus Calmette-Guerin, MIW815, ci-di-AMP, or an anti-CD40 antibody. . The method of any one of claims 17-113, wherein the subject is a mammal. . The method of any one of claims 17-114, wherein the subject is a human.