CN112274535B - Application of spermidine modified macrophage in development of immunotherapy medicaments - Google Patents

Abstract

The invention discloses application of spermidine modified macrophages in development of immunotherapeutic drugs, and belongs to the technical field of biomedicine. The invention can change from glycolysis to oxidative phosphorylation in vivo and in vitro reprogramming of macrophage sugar metabolism mode through spermidine, so as to improve macrophage anti-inflammatory capability in vivo and in vitro, and spermidine can deactivate macrophage response to LPS, maintain anti-inflammatory phenotype, thus providing spermidine or use of spermidine in vitro modified macrophage immunotherapeutic agent in treating inflammatory bowel disease; the invention can improve the expression and secretion of macrophage liver growth factor (HGF) through spermidine, and can improve the expression of macrophage and Matrix Metalloproteinase (MMP) MMP8, MMP9, MMP12 and MMP13, thereby providing the application of spermidine or spermidine in vitro modified macrophage immunotherapeutic agent in treating hepatic fibrosis.

Description

Application of spermidine modified macrophage in development of immunotherapy medicaments

Technical Field

The invention relates to application of spermidine modified macrophages in development of immunotherapeutic drugs, and belongs to the technical field of biomedicine.

Background

Today's society, autoimmune diseases and degenerative and chronic inflammatory diseases such as: inflammatory bowel disease Crohn's disease, obesity, type II diabetes, liver and kidney fibrosis, and the like have become public health problems that seriously jeopardize human health. Because of the lack of curable treatments, treatment of patients often depends on the use of immunosuppressive drugs such as steroids, but most of these treatments are accompanied by various side effects. In recent years, preclinical and clinical studies of immunotherapy with adoptive transplantation of stem cells have demonstrated an important role in regulating the tissue microenvironment in the treatment of inflammatory diseases. The tissue microenvironment serves as a complex ecosystem, consists of tissue parenchymal cells, interstitial cells and various types of immune cells, and generates complex interactions between them, and orderly dynamic regulation is an important guarantee for maintaining body homeostasis. Among them, macrophages account for a large proportion of tissue microenvironments, and excessive accumulation and secretion of inflammatory factors by activated macrophages are important causes of inflammatory diseases. Although clinical treatment studies of antibodies against inflammatory factors such as TNFα and IL-6 have been conducted, the effect has been limited.

In fact, macrophages have a very strong plasticity and, in different microenvironments, exhibit different phenotypes by performing different metabolic pathways. Currently, macrophages can be simply classified into classical activated macrophages (M1 macrophages, pro-inflammatory macrophage phenotype) and alternative activated macrophages (M2 macrophages, anti-inflammatory macrophage phenotype). Macrophages initiate an energy-consuming, biosynthetic, aerobic glycolysis under stimulation of LPS and inflammatory factors such as ifnγ, presenting a pro-inflammatory M1 phenotype, secreting multiple types of pro-inflammatory cytokines and chemokines, and by expressing a large number of mhc ii and B7 molecules, enhancing antigen presentation, participating in Th 1-type immune responses, killing infectious pathogens and tumor cells. In addition, macrophages undergo oxidative catabolism-oxidative phosphorylation under treatment with IL4, IL13, IL10, and the like to produce a large amount of ATP, mediate Th 2-type immune responses, and are important participants in suppressing immune responses and performing tissue repair. Considering that excessive proinflammatory macrophage accumulation is often the most leading cause of inflammatory diseases, the latest clinical data further demonstrate the safety of autologous macrophage infusion therapy, so that the selective targeting of proinflammatory macrophages in the microenvironment, or the infusion of autologous macrophages of patients with anti-inflammatory phenotype after in vitro reprogramming, will become an important strategy for new inflammatory immunotherapy. There is a need in the art for intensive research aimed at developing macrophage-targeted immunotherapy.

Disclosure of Invention

In order to solve the problems, the invention provides an agent which can realize polarization of anti-inflammatory macrophages in vivo and in vitro through metabolic reprogramming and improve the anti-inflammatory capacity of the anti-inflammatory macrophages to enhance treatment of inflammatory diseases and application thereof.

A first object of the present invention is to provide the use of spermidine for the preparation of a medicament for the treatment of a disease based on metabolic reprogramming macrophage immunotherapy.

Further, the metabolic reprogramming macrophage immunotherapy-based disease is an autoimmune disease.

Further, the metabolic reprogramming macrophage immunotherapy-based disease is inflammatory bowel disease.

Further, the metabolic reprogramming macrophage immunotherapy-based disease is liver fibrosis.

Further, the metabolic reprogramming macrophage is an in vitro reprogramming macrophage or an in vivo reprogramming macrophage.

Further, the drug is a preparation containing spermidine or a macrophage preparation treated with spermidine.

It is a second object of the present invention to provide a pharmaceutical formulation for treating metabolic reprogramming macrophage based immunotherapy, said pharmaceutical formulation comprising spermidine treated macrophages.

Further, the pharmaceutical formulation further comprises a carrier compatible with the macrophages.

Further, the carrier comprises one or more of saline, buffer solution, glucose, water, glycerol and ethanol.

Further, the carrier also comprises an auxiliary agent, wherein the auxiliary agent is one or more of a lubricant, a retention aid, a wetting agent, an emulsifying agent and a pH buffering agent.

The invention has the beneficial effects that:

the invention can change from glycolysis to oxidative phosphorylation in vivo and in vitro reprogramming of macrophage sugar metabolism mode through spermidine, so as to improve macrophage anti-inflammatory capability in vivo and in vitro, and spermidine can deactivate macrophage response to LPS, maintain anti-inflammatory phenotype, thus providing spermidine or use of spermidine in vitro modified macrophage immunotherapeutic agent in treating inflammatory bowel disease; the invention can improve the expression and secretion of macrophage liver growth factor (HGF) through spermidine, and can improve the expression of macrophage and Matrix Metalloproteinase (MMP) MMP8, MMP9, MMP12 and MMP13, thereby providing the application of spermidine or spermidine in vitro modified macrophage immunotherapeutic agent in treating hepatic fibrosis.

Drawings

Figure 1, spermidine is effective in alleviating dextran sulfate sodium salt (DSS) induced inflammatory bowel disease.

(A-B) oral administration of 4% DSS to wild type C57 mice induced inflammatory bowel disease (control 5, spermidine treated 6). Daily intraperitoneal injections of 50mg/kg of spermidine, PBS as a control group, and daily monitoring and recording of changes in body weight of the mice. On day 7, mice were sacrificed and colon tissue was subsequently isolated.

(C) Statistical analysis of isolated mouse colon tissue photographed and measured

(D) Cleaning up the feces in colon tissues by using PBS, sucking up the feces by using water-absorbing paper, weighing the feces, and counting the ratio of the weight to the length of the cecum tissues;

(E) The colon tissue of the mice was paraffin-embedded into paraffin sections after fixation with 4% pfa, stained for H & E tissue and photographed for analysis. * P <0.05, < P <0.01, < P <0.001.

Figure 2, spermidine, is capable of reprogramming macrophages to a stable anti-inflammatory phenotype both in vitro and in vivo.

(A-D) isolation of mouse bone marrow-derived macrophages, washing out a suspension of bone marrow cells in DMEM/F12 medium containing 20% L929 supernatant, culturing for 7 days with 20. Mu.M spermidine, (A) extraction of total cellular RNA, and detection of the expression level of anti-inflammatory macrophage-related genes using Q-PCR. (B) RIPA lyses cells, high-speed centrifugation extracts protein, and Western blot detects protein expression of anti-inflammatory macrophage Arg-1. (C) Confocal microscopy was used to observe Arg-1 expression and photograph. (D) The proportion of macrophages of different phenotypes was examined using flow cytometry. (E) Bone marrow-derived macrophages were incubated in a medium containing spermidine (20. Mu.M) for 7 days, the supernatant was discarded, washed 3 times with PBS and replaced with fresh complete medium without spermidine for further incubation for different times, and cellular proteins were extracted and Western blot examined for protein expression by anti-inflammatory macrophages Arg-1. (F-G) intraperitoneal injection of 1ml of starch broth and 50mg/kg of spermidine per day, DMSO as a control. On the third day, mice were sacrificed and peritoneal macrophages were harvested, (F) RNA was extracted and Q-PCR was performed to detect gene expression from anti-inflammatory macrophages. (G) The phenotype of peritoneal macrophages was examined by flow cytometry. P <0.05, < P <0.01, < P <0.001.

FIG. 3, transition of spermidine metabolism reprogrammed macrophages from glycolysis to oxidative phosphorylation.

(A) The starch broth was intraperitoneally injected into wild type mice, 50mg/kg spermidine was intraperitoneally injected daily, PBS was used as a control group, the mice were sacrificed on the third day, macrophages in the abdominal cavity of the mice were isolated or bone marrow cell suspensions of the wild type mice were washed out, and cultured in DMEM/F12 medium containing 20% L929 supernatant, 20. Mu.M spermidine was added for 7 days to obtain macrophages derived from bone marrow of the mice, and 2-NBDG probe was incubated for 1 hour, and the amount of glucose absorbed by the macrophages was examined by flow cytometry.

(B-C) isolating and culturing to obtain mouse bone marrow-derived macrophages, marking mitochondria in the macrophages by using a mito-traker probe, (B) detecting the mitochondrial production by using a flow cytometry method, and (C) observing the mitochondrial condition of the macrophages by using a fluorescence microscope and photographing. (D) Flow cytometry detects macrophage mitochondrial membrane potential. (E) Cells were lysed, the amount of ATP in the cell lysate and total protein were measured, and the amount of macrophage unit ATP production was assessed. (F-G) extracting macrophage mitochondria and lysing, and Western blot detecting expression of (F) mitochondrial carnitine transferase and (G) mitochondrial respiratory chain complexes. * P <0.05, < P <0.01, < P <0.001, scale bar=100 μm.

FIG. 4, spermidine reprogrammed macrophage inactivation sensitivity to LPS

(A-B) after bone marrow-derived macrophages were cultured in a medium containing spermidine (20. Mu.M) for 7 days, the spermidine was washed off with PBS and stimulated with LPS (100 ng/ml) for 24 hours, (A) RNA was extracted and the expression level of the pro-inflammatory macrophage-related gene was detected using Q-PCR. (B) Protein expression levels of iNOS were detected using Western blot detection. And detecting the content of nitric oxide in the culture medium by using the nitric oxide detection kit. * P <0.05, < P <0.01, < P <0.001.

FIG. 5, spermidine modified macrophages effective in alleviating inflammatory bowel disease

(A) Mice were orally administered 4% dss-induced inflammatory bowel disease model daily, and spermidine-pretreated bone marrow-derived macrophages were intraperitoneally injected on days 1,3, and 5 of model induction, mice were sacrificed on day 7, and the colon was isolated for analysis (n=5/group). (B) Mice were monitored daily and recorded for body weight and statistically analyzed. (C-D) isolated colon tissue was taken 2 photographs per group (C), the colon length was measured and statistical analysis was performed (D). (E) The fecal matter in the colon tissue is cleaned and then weighed, and the ratio of the weight of the colon tissue to the length is counted. (F) Treated colon tissue of mice was fixed with 4% pfa, sectioned in paraffin, stained with H & E tissue and photographed. * p <0.05, < p <0.01, < p <0.001, < p <0.0001, < scale bar = 100 μm).

FIG. 6, spermidine treated macrophages with altered expression and secretion profiles for treatment of liver fibrosis

(A-B) after culturing bone marrow-derived macrophages in a medium containing spermidine (20. Mu.M) for 7 days, the expression level of a member of the family of matrix metalloproteinases (A), and the expression level of liver growth factor (B) were detected by Q-PCR after extraction of total cellular RNA. (C) The supernatant of macrophages after spermidine stimulation was collected and the content of HGF in the supernatant was detected by ELISA kit. (D) RIPA lyses cells and protein expression levels of HGF are detected by Western blot detection. * P <0.05, < P <0.01, < P <0.001.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The present inventors have conducted intensive studies to reveal that spermidine can modify macrophages into a stable oxidative phosphorylation-dependent inflammation-inhibiting phenotype by metabolic reprogramming, thereby alleviating the progression of inflammatory diseases. Thus, spermidine can be used as a drug to suppress immune response. In addition, the spermidine can be used as a modifier for in vitro domestication of macrophages, so that immunotherapy of the macrophages is realized. The present invention has been completed on the basis of this finding.

Spermidine and use thereof

Spermidine is an important metabolite of cellular amino acid (arginine) metabolism. Such polyamines play an important role in maintaining homeostasis of the body. The content of spermidine in the human body is mostly reduced with the age, and a large number of aging-related diseases occur. In recent years, a great deal of research shows that spermidine can initiate autophagy and inhibit cell senescence. The inventors have studied intensively and have revealed for the first time: spermidine improves anti-inflammatory ability through metabolic reprogramming, thereby relieving the progress of inflammatory diseases, and simultaneously, the increased inhibition of the expression of metalloproteinase is beneficial to the remodelling of extracellular matrix at lesions.

The inventors found that spermidine enhances the immune modulating ability by enabling reprogramming macrophages to initiate a cell oxidative phosphorylation process that depends on mitochondrial activity, to switch to an anti-inflammatory macrophage phenotype. Firstly, spermidine is able to significantly inhibit the glucose uptake by macrophages, at the same time as mitochondrial biosynthesis increases significantly, while the mitochondrial membrane potential does not rise significantly, but oxidative phosphorylation activity is significantly enhanced and more ATP is produced, whereas active oxidative phosphorylation is necessary for anti-inflammatory macrophages. Furthermore, it has been found that spermidine modified macrophages are relatively stable in maintaining the anti-inflammatory phenotype and inactivate responses to M1 type macrophage stimuli such as LPS, which is more beneficial for the modified macrophages to maintain the anti-inflammatory phenotype in complex microenvironments.

Application of spermidine in preparing macrophage preparation

The inventors next studied whether spermidine modified macrophages could treat inflammatory bowel disease, notably that spermidine modified macrophages still effectively reduce the progression of inflammatory bowel disease. Based on the above new findings of the present inventors, the present invention provides that said spermidine can metabolize the reprogrammed macrophage phenotype under in vivo or ex vivo conditions and enhance immunotherapy of inflammatory diseases in modulating the immune microenvironment. One way is, for example, to administer an effective dose of spermidine directly to a subject in need thereof, thereby causing the overall modification of the immune microenvironment, increasing the reserves of external Zhou Kangyan macrophages, and reaching a specific site by recruitment migration. Another way is to isolate macrophages or mononuclear cells (PBMCs) from peripheral blood in a patient in need of treatment, treat and culture with spermidine ex vivo, modify and stabilize the reprogramming Cheng Jikang inflammatory phenotype, and reinfusion, for example. It is to be understood that other modes of administration that are contemplated as being feasible by those skilled in the art based upon the novel findings of this invention are also encompassed by this invention.

Based on the results of the inventors' studies, the spermidine also has: improving the expression of liver growth factors in macrophages; enhancing the expression of macrophage matrix metalloproteinase family members MMP8, MMP9, MMP12, and MMP 13. Thus, the present invention also provides the use of spermidine for the preparation of macrophage preparations for the immunotherapy of liver fibrosis. The extracellular matrix-reconstituted phenotype macrophage preparation can be a cell fluid preparation of spermidine modified macrophages. The spermidine treatment may be an in-vivo treatment or an ex-vivo treatment.

As used herein, the term "comprising" means that the various ingredients may be applied together in a mixture or composition of the invention.

As used herein, the term "effective dose" refers to an amount that is functional or active in and acceptable to a patient with an inflammatory disease.

The pharmaceutical compositions may contain liquids such as water, saline, glycerol and ethanol in combination with the pharmaceutically acceptable carrier. In addition, auxiliary substances, such as lubricants, retention aids, wetting or emulsifying agents, pH buffering substances, and the like, may also be present in these carriers.

The compositions of the invention contain a safe and effective amount of the cells and a carrier compatible with the cells. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical formulations should be compatible with the mode of administration and the compositions of the invention may be formulated as injectables, e.g., as prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount.

In using the compositions of the invention, a safe and effective amount of the cells of the invention is administered to a mammal. Of course, the particular dosage and number of administrations should also take into account factors such as the weight, age, health, etc. of the patient to be administered, and are within the skill of those skilled in the art.

The composition of the invention can be directly used for increasing the proportion and the number of anti-inflammatory macrophages and regulating the inflammatory microenvironment. In addition, it may be used in combination with other therapeutic agents or adjuvants.

The invention will be further elucidated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to manufacturer's recommendations.

Example 1: spermidine is effective in relieving inflammatory bowel disease

C57BL/6 mice with inflammatory bowel disease were intraperitoneally injected with 50mg/kg of spermidine daily as shown in FIG. 1A. As a result, spermidine was found to significantly alleviate DSS-induced loss of body weight, shortening of the colon, and thickening of the colon wall in mice, as shown in fig. 1B-D. The pathological analysis result also shows that the colon tissue structure of the mice in the spermidine treatment group is basically complete, and inflammatory cell infiltration and colon goblet cell and crypt loss caused by DSS are also obviously improved. In summary, the administration of spermidine can significantly inhibit inflammation due to colon tissue damage.

Example 2: spermidine is capable of reprogramming macrophages to a stable anti-inflammatory phenotype both in vitro and in vivo

Macrophages of a pro-inflammatory phenotype play an important role in inflammatory bowel disease and are an important participant in the colonic inflammation microenvironment. Therefore, in vitro using bone marrow-derived macrophages as a study object, it was investigated whether spermidine has reprogramming effect on tumor-promoting macrophages, so that it can inhibit inflammation and promote repair, and we first add spermidine during the macrophage culturing process, and as a result, found that spermidine can promote polarization of macrophages to anti-inflammatory phenotype, as shown in fig. 2A-D. Furthermore, this anti-inflammatory phenotype may be stable for at least 3 days, as shown in fig. 2E. To determine the effect of spermidine on macrophages in vivo, mice were injected daily with 50mg/kg of spermidine during the induction of mouse abdominal megaphagy, with the result that almost all of the macrophages removed had the phenotype of anti-inflammatory macrophages, as shown in figures 2F and 2G. In summary, spermidine is capable of acclimating macrophages to relatively stable anti-inflammatory phenotyped (M2) macrophages in vitro as well as in vivo.

Example 3: transition of spermidine metabolism reprogrammed macrophages from glycolysis to oxidative phosphorylation

As previously mentioned, spermidine is an important metabolite of spermidine metabolism, maintaining cellular function mainly by inducing autophagy. The spermidine was found to be able to reprogram the glycometabolism of macrophages. In vitro experiments showed a significant decrease in glucose uptake by bone marrow-derived macrophages of mice treated with spermidine and freshly isolated macrophages from the abdominal cavity of mice injected daily with spermidine, as shown in fig. 3A. At the same time, the number of mitochondria in these macrophages was significantly increased, as shown in fig. 3B and 3C. Although there was no significant change in mitochondrial membrane potential, as in fig. 3D, there was a significant increase in mitochondrial activity, manifested by more ATP production, more carnitine transferase expression, and an increase in oxidative phosphorylation complex, as in fig. 3E-G. This result suggests that spermidine can reduce the utilization of glucose by macrophages, which in turn translates into a mitochondrial dependent oxidative phosphorylation process.

Example 4: spermidine reprogrammed macrophages inactivate sensitivity to LPS

Because of the reduced barrier function of the damaged colon tissue, high levels of inflammatory factors are present in the microenvironment, as well as LPS, so that the acclimatized anti-inflammatory macrophages are able to maintain an anti-inflammatory phenotype without being reprogrammed to pro-inflammatory macrophages exacerbating the inflammatory level. Thus, the addition of spermidine modified macrophages to LPS-containing medium for a further 24 hours showed significant inhibition of polarization of pro-inflammatory phenotype macrophages without spermidine compared to untreated macrophages, as shown in fig. 4A-B. This result suggests that spermidine confers an immunological memory to macrophages, blunting the response to LPS, making it more difficult to reprogram macrophages to a pro-inflammatory phenotype.

Example 5: spermidine modified macrophages are effective in alleviating inflammatory bowel disease

To verify the feasibility of spermidine modified macrophage immunotherapy, mice bone marrow-derived macrophages were treated with spermidine in vitro, and returned 5×10 by intraperitoneal infusion on days 1,3,5 established in the inflammatory bowel disease model ⁶ Cells, as shown in fig. 5A, were used as controls in mice injected with normal macrophages. It was found that spermidine modified macrophages significantly reduced weight loss and knots caused by inflammatory bowel diseaseThe intestines shorten and thicken as shown in fig. 5B-E. Histopathological analysis also showed that spermidine modified macrophages are effective in alleviating inflammatory cell infiltration, disorder of tissue structure, destruction of goblet cells and loss of crypts caused by colon tissue damage, as shown in figure 5F. In conclusion, spermidine in vitro modified macrophages can exert anti-inflammatory effects in complex inflammatory microenvironments. The inventors believe that this finding can provide a new approach for the clinical use of spermidine and macrophage immunotherapy.

Example 6: altered secretion profiles of spermidine-treated macrophages are useful in the treatment of liver fibrosis

Spermidine modified macrophages have great potential not only in inhibiting inflammatory regulatory immune responses, but also in reconstructing extracellular matrix. There was a significant increase in matrix metalloproteinase expression in spermidine treated macrophages as shown in figure 6A. In addition, the expression of liver growth factor (HGF) was significantly elevated after spermidine treatment, as shown in FIGS. 6B-D. Taken together, spermidine directs macrophages to regulate the immune microenvironment, suggesting a potential role for spermidine and related macrophage preparations in liver fibrosis.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (4)

1. Use of spermidine treated macrophages in the preparation of a medicament for the treatment of inflammatory bowel disease.

2. The use of claim 1, wherein said therapeutic agent further comprises a carrier compatible with said macrophages.

3. The use of claim 2, wherein the carrier comprises one or more of saline, buffer, glucose, water, glycerol, ethanol.

4. The use according to claim 3, wherein the carrier further comprises an adjuvant, the adjuvant being one or more of a lubricant, a retention aid, a wetting agent, an emulsifier, a pH buffer.