CN115804772B - Application of iron death inhibitor in antiviral infection - Google Patents

Abstract

The invention belongs to the technical field of medicines, and discloses a pharmaceutical composition for resisting viral infection and/or reducing the content of viruses, which comprises Liproxstatin-1 or pharmaceutically acceptable salt thereof and pharmaceutical excipients. The pharmaceutical composition has wide application prospect in the field of treating chronic infection.

Description

Application of iron death inhibitor in antiviral infection

Technical Field

The invention relates to the field of biological medicine, and relates to an application of an iron death inhibitor Liproxstatin-1 in antiviral infection.

Background

Chronic infections are metabolically adapted by enhancing lipid uptake or storage, which in turn is related to their dysfunction. Excessive lipid uptake leads to deregulation of lipid metabolism and increased production of Reactive Oxygen Species (ROS) leading to lipid peroxidation. Lipid peroxidation can produce large amounts of deleterious lipid peroxides that disrupt cell membranes and thereby trigger cell-mediated iron death [1], where GPX4 is considered an iron death core regulatory molecule. Cytotoxic cytokine production by cd8+ T cells, which are lipid peroxidized and iron-dead, is reduced, resulting in a greatly impaired anti-tumor capacity [2-4]. However, studies of chronic infections leading to cd8+ cell iron death have not been reported.

Currently known iron death inducers can be broadly divided into four types, depending on the mechanism of action: 1. inhibit type I of the Xc-system (CLASS I FIN); 2. inhibit or degrade GPX4, form II (CLASS II FIN); 3. type III (CLASS III FIN) consuming coenzyme Q10; 4. type IV (CLASS IV FIN) of lipid peroxidation induced by iron or polyunsaturated fatty acid (PUFA) overload. All four types have higher specificity for induction of iron death, namely: during induction of iron death, markers of other types of cell death are not activated substantially. The mechanism of action of different types of iron death inducers is completely different and has no reference significance.

In the prior researches, currently commonly used iron death inhibitors mainly comprise Liproxstatin-1, ferrostatin-1, alpha-Toc, vitE, DFO and the like, wherein Liproxstatin-1 and Ferrostatin-1 are aromatic amine antioxidants, and lipid peroxidation is inhibited by scavenging ROS; alpha-Toc and VitE are lipophilic natural antioxidants, and play an antioxidant role mainly by breaking the chain reaction of autoxidation; DFO iron chelators inhibit the production of hydroxyl radicals by blocking the fenton reaction. Common iron death inducers include targeting System Xc-, erastin affecting GSH synthesis, sulfasalazine, sorafenib, and the like; GPX4 is targeted, RSL3 with reduced antioxidant capacity is adopted; targeting iron ions, dihydroartemisinin which promotes accumulation of cellular ROS, and the like.

It has been found that iron death inhibitors have different biological effects in different cells, tissues or models. For example, iron death inhibitor Ferrostatin-1 can reverse the increase of the lipid peroxidation of CD8+ T cells caused by externally added fatty acid, improve cytokine production [2], and Ferrostatin-1 can also reduce the lipid peroxidation of tumor infiltrating T cells, thereby exerting stronger anti-tumor capability [5]; however, another study found that iron death inhibitor Ferrostatin-1 alone did not improve the killing function of cd8+ T cells [6].

It was found that the biological effects of different inhibitors are different. For example, ferrostatin-1 can inhibit sterol synthesis in macrophages infected with pathogenic yeast histoplasma capsulatum, not only can reduce death of infected macrophages, but also can inhibit growth of pseudomonas capsulata; while another arylamine antioxidant Liproxstatin-1 does not exert the above effects, and is not effective in preventing fungal growth or reducing macrophage death [7]. In addition, liproxstatin-1 was able to inhibit lipid peroxidation, restore GSH and GPX4 expression, and thereby significantly inhibit iron death from occurring in RSL 3-treated oligodendrocytes, whereas DFO effects were not apparent as other iron death inhibitors [8].

The effect of iron death inhibitors/inducers on different immune cells also varies. For example, in macrophages, iron death inducer RSL3 can induce iron death in M2 type macrophages, but cannot induce iron death in M1 type macrophages [9]. In the tumor microenvironment, iron death inducing agents Erastin and RSL3 at certain concentrations are effective in inducing iron death in tumor cells, but not in CD4+ T cells and CD8+ T cells [10]. In LCMV infected mouse models, feeding vitamin E significantly increases the absolute cell number of cd8+ T cells without affecting the absolute cell number of cd4+ T cells; however, in GPX4 knockout infected mice, vitamin E fed was able to restore the absolute cell number of CD4+ T cells, but not of CD8+ T cells [1].

Hepatitis B Virus (HBV) is a pathogen causing hepatitis B (abbreviated as hepatitis B), belongs to the family of hepadnaviridae, and comprises two genera of orthohepadnaviridae and avian hepadnaviridae, and causes infection of human bodies. The degree of liver cell damage is related to the intensity of immune response of organism, and the mechanism of HBV causing immune pathological damage is that the immune response of organism is low due to virus, drug resistance is generated by virus variation, immune pathological damage is mediated by antibody, immune pathological damage is mediated by cell, and the like. Common drugs include interferon, lamivudine, adefovir dipivoxil, telbivudine, entecavir, tenofovir disoproxil, and the like. Because the current first-line antiviral drugs cannot meet the clinical cure demands of most patients, there is a need to continue developing new antiviral drugs and researching new therapeutic schemes to improve the clinical cure of HB in a limited course of treatment, and the current new antiviral drugs are mainly divided into direct antiviral Drugs (DAAs) targeting the viral life cycle and indirect antiviral drugs participating in immune regulation. DAAs mainly comprise small interfering RNA, antisense oligonucleotides, gene editing silencing, capsid assembly regulator, cytostatic agent, HBsAg release inhibitor; indirect antiviral drugs involved in immunomodulation mainly include TLR7/8 innate immune modulators, therapeutic vaccines, monoclonal antibodies, immune checkpoint inhibitors, and the like.

In HBV chronic infection, there are serious obstacles to the body's innate immune response and acquired immune response. Replication of HBV within hepatocytes is not perceived by the intracellular innate immune system, resulting in impairment of antigen presenting function of DCs [11]. In addition, NK cells and HBV-specific cd4+, cd8+ T cells are severely dysfunctional and depleted, HBV is able to produce and secrete a large amount of viral antigen, gradually changing and depleting the function of HBV-specific T cells, resulting in decreased production of antiviral cytokines such as IFN- γ and TNF- α, ultimately resulting in HBV that is long-lasting in the body and is not readily cleared [12,13]. Current research consensus is that cd4+ and cd8+ T cell mediated immune responses determine HBV treatment outcome. However, even with the current standard therapies, the deficiency of the immune response makes it difficult for patients to restore HBV-specific immune cell function to normal levels, and thus does not effectively control infection.

The mechanism of virus infection is extremely complex, clinical symptoms caused by different virus infections are quite different, the immunological mechanism and principle of different treatment strategies are quite different, whether the inhibitor with a single mechanism can inhibit the virus infection is difficult to expect, and the treatment effects are also quite different. For example, the IAP antagonist APG-1387 for the research of a novel anti-HBV drug can induce the apoptosis of liver cells expressing HBV antigen and can enhance the specific T cell response of HBV [14]; anti-HBV selectively activates IL-2 pathway in cd8+ T cells, reversing the deficiency of cd8+ T cells without significant effect on other immune cells in the IL-2 immunotherapy drug studied AB359 [15]; in anti-HBV treatment, the TLR8 agonist GS-9688 can improve the cytolytic and non-cytolytic effect functions of NK cells, increase the number of HBV specific CD8+ T cells and the production of cytokines of partial patients, increase the number of follicular helper T cells and reduce the number of tregs and monocyte MDSCs [16]; mitochondrial antioxidants MitoQ and MitoTempo were able to significantly increase the effector function of HBV-specific CD8+ T cells, whereas only very weak effects were observed on the total T cell population [17]. In view of this, the mechanism by which different antiviral drugs regulate the antiviral immune response is complex.

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2Ma X,Xiao L,Liu L,Ye L,Su P,Bi E,et al.CD36-mediated ferroptosis dampens intratumoral CD8+T cell effector function and impairs their antitumor ability.Cell Metab 2021;33:1001-1012.e5.

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Liproxstatin-1 (Lip-1) is a potent inhibitor of iron death that can be used in vivo and in vitro. Lip-1 is easy to penetrate into the lipid bilayer and stays in the lipid bilayer, so that the active site of the Lip-1 is in close directional contact with the lipid peroxidation site, and CH3OO is started to extract hydrogen atoms from the aromatic amine site. After scavenging the lipid peroxide, the Lip-1 free radical formed by Lip-1 can be reduced by other antioxidants in the body (e.g. ubiquinone). Lip-1 inhibits iron death more effectively than other iron death inhibitors, such as deferoxamine, vitamin E, and ferrostatin-1. Lip-1 has been demonstrated to protect mouse myocardium from ischemia/reperfusion injury by reducing VDAC1 levels and restoring GPX4 levels; can inhibit RSL3 induced human renal proximal tubular epithelial cell death and GPX4 deficiency induced acute renal failure.

The mechanism of action of iron death inhibitor Liproxstatin-1 in antiviral infections has not been known so far, and no report has been made of Liproxstatin-1 in antiviral infections.

Disclosure of Invention

In order to overcome the technical problems in the prior art, the invention provides a new application of an iron death inhibitor Liproxstatin-1, in particular to an application in improving the effector function of CD8+T cells.

The invention is realized by the following technical scheme.

Use of an iron death inhibitor Liproxstatin-1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing cd8+ T cell effector function or for combating viral infection.

Preferably, the method comprises the steps of,

The CD8+ T cells are CD8+ T cells and antigen specific CD8+ T cells in a chronic hepatitis B model.

As a preferred technical scheme of the invention: the use is to reduce the lipid peroxidation level of cd8+ T cells and antigen specific cd8+ T cells.

As a preferred technical scheme of the invention: the use is to increase the production of cd8+ T cells and antigen specific cd8+ T cell cytotoxic cytokines.

As a preferred technical scheme of the invention: the application is to inhibit iron death of CD8+ T cells and antigen-specific CD8+ T cells.

As a preferred technical scheme of the invention: the application is to improve the antiviral capacity of CD8+ T cells.

Preferably, the virus is HBV virus.

The invention also provides a medicine for resisting viral infection, the active ingredient of which is iron death inhibitor Liproxstatin-1 or pharmaceutically acceptable salt thereof. The virus is HBV.

Drawings

Fig. 1A: effect on cd8+ T cell lipid peroxidation in CHB patients after co-incubation with iron death inhibitor Liproxstatin-1.

Fig. 1B: antigen specific cd8+ T cell loop gate strategy was labeled using MHC tetramers.

Fig. 1C: effect of antigen-specific cd8+ T lipid peroxidation following co-incubation with iron death inhibitor Liproxstatin-1.

Fig. 1D: effect on cd8+ T cells IFN- γ and TNF- α production following co-incubation with iron death inhibitor Liproxstatin-1.

Fig. 1E: effects of antigen-specific CD8+ T cells IFN-gamma and TNF-alpha following co-incubation with iron death inhibitor Liproxstatin-1.

Fig. 1F: effects on cd8+ T cell GPX 4-related expression following co-incubation with iron death inhibitor Liproxstatin-1.

Fig. 2A: treatment protocol diagram of mice.

Fig. 2B: effect on lipid peroxidation of cd8+ T cells in the liver following treatment with iron death inhibitor Liproxstatin-1.

Fig. 2C: effects on intra-hepatic cd8+ T cell IFN- γ and TNF- α production following treatment with iron death inhibitor Liproxstatin-1.

Fig. 2D: effect on HBsAg in mice during treatment with iron death inhibitor Liproxstatin-1. Fig. 2E: effect on mouse HBVDNA following treatment with iron death inhibitor Liproxstatin-1;

FIG. 2F is the effect on mouse body weight following treatment with iron death inhibitor Liproxstatin-1.

Fig. 2G: effects on liver pathology in mice following treatment with iron death inhibitor Liproxstatin-1.

Detailed Description

The invention is further described below, the embodiments presented in this description are only exemplary and do not limit the scope of the invention. It will be understood by those skilled in the art that the details and forms of the invention may be modified or substituted without departing from the spirit and scope of the invention.

Definition of terms

As used herein, the term "cd8+ T cell" refers to a cytotoxic T lymphocyte, a killer T cell, that can participate in immunization by secretion of various cytokines.

As used herein, the term "iron death" is a novel mode of cell death first proposed by the Brent R Stockwell doctor's laboratory in 2012. Iron death is different from pyro-death, apoptosis, necrosis and autophagic cell death. This process of iron death is manifested by the accumulation of lethal ROS and lipid peroxidation products (LPOs) during iron metabolism.

As used herein, the term "Lip-1", i.e., iron death inhibitor Liproxstatin-1, is a potent iron death inhibitor. By inhibiting the generation of lipid free radicals, lipid peroxidation and iron death are inhibited.

As used herein, the term "Med" is a cell culture medium, i.e., a primary cell culture medium.

As used herein, the term "Pep" is an HBV antigenic peptide, the sequence of which is HBV-core (aa 18-27: FLPSDFFPSV).

As used herein, the term "MHC tetramer" is a combination of soluble MHC proteins and polypeptides, and is a simple and effective method for identifying, isolating and studying antigen-specific T cells from a variety of biological samples.

As used herein, the term "IFN- γ", i.e. interferon γ, is an important cytokine capable of performing an immunomodulatory function. It was found due to its antiviral activity. IFN-gamma plays a key role in host defense through antiviral, antiproliferative and immunomodulatory functions. Can reflect immune cell effector functions.

As used herein, the term "TNF- α," tumor necrosis factor α, is involved in normal inflammatory and immune responses, and may synergistically regulate the production of other cytokines, cell survival and death to coordinate tissue homeostasis. Cytotoxicity to various tumor cells is an important factor in mediating the immune response to viral infection. Can reflect immune cell effector functions.

As used herein, the term "GPX4", glutathione peroxidase-4, plays a key role in degrading deleterious lipid peroxides to maintain homeostasis as an iron death core regulatory molecule.

This patent is described in further detail below in conjunction with the drawings and specific experiments. Unless otherwise indicated, all reagents, instruments, devices and methods used in this patent are those commonly available in the art.

Example 1

In vitro experiments.

Isolation of PBMCs: PBMCs were isolated from fresh heparinized blood using Ficoll-Hypaque density gradient.

Specific T cell treatment: isolated PBMCs from CHB patients were cultured in RPMI1640 containing 10% fetal bovine serum. HBV-specific CD8+ T cells were further analyzed by stimulating PBMCs with HBV peptide (1 μM) for 10 days in the presence or absence of iron death inhibitor Liproxstatin-1 (50 nM). Sorting and activation of cd8+ T cells: CD8+ T cells were isolated from PBMC using a negative selection kit (Biolegend) and stimulated with the addition of human T activating antibodies CD3/CD28 (10. Mu.g/ml, biolegend) and IL-2 (10 ng/ml). Cd8+ T cells were stimulated with HBV peptide (1 μm) for 10 days in the presence or absence of iron death inhibitor Liproxstatin-1.

Lipid peroxidation measurement: the experiments were performed according to the manufacturer's protocol. Briefly, cells were incubated with lipid peroxidation sensor C11 BODIPY 581/591 (Invitrogen) in cell culture medium for 30 minutes in a 37℃incubator containing 5% carbon dioxide. After incubation, cells were washed and flow cytometry detection was performed with FACSCELESTA (BD) within 2 hours after staining.

MHC tetramer staining: to 25. Mu.L of peripheral blood mononuclear cell suspension (2X 10 ⁷/mL) was added 1. Mu. LMHC tetrameric dye (HelixGen) and incubated for 60 minutes in ice protected from light. After incubation, FACSBuffer (pbs+2% calf serum+0.1% sodium azide) was added for washing and then resuspended in 200 μl of fixative (pbs+1% paraformaldehyde) for fixation.

Production assay of T cell cytokines: cell activation medium (100 IU/mL IL-2+1. Mu.g/mL ionomycin+50 ng/mL PMA+10. Mu.g/mL brefeldin A) was prepared, 1mL activation medium was added to 1X 10 ⁶ PBMC cells, and incubated in 24-well plates for 4 hours. After labeling CD3-Percp-Cy5.5 (Biolegend), CD8-BV421 (Biolegend) and MHC tetramer-APC, the samples were subjected to fixation-permeabilization blocking, labeling with IFN-. Gamma. -PE (Biolegend) and TNF-. Alpha. -PE-CF594 (Biolegend), and washing. GPX4 markers: the permeabilized blocked cells were blocked by adding 1/400 dilution of recombinant Anti-Glutathione Peroxidase antibody (Abcam) for 2h at 4deg.C, and goat serum was added to block, goat Anti-rabbit IgG-FITC (Abclonal) was used as secondary antibody at a dilution of 1/2000.

As shown in FIG. 1A, lip-1 significantly reduced the lipid peroxidation level of CD8+ T cells in PBMC of CHB patients.

As shown in fig. 1B, the main cell population was circled in the sorted cd8+ T cells, after diagonal deblocking, the single cell population was circled, and finally the tetramer positive cell population, i.e., HBV-specific cd8+ T cells, was circled.

As shown in FIG. 1C, lip-1 significantly reduced the lipid peroxidation level of HBV-specific CD8+ T cells in PBMC of CHB patients.

As shown in FIG. 1D, lip-1 significantly increased cytokine production (IFN-. Gamma.and TNF-. Alpha.) by CD8+ T cells in PBMC of CHB patients.

As shown in FIG. 1E, lip-1 was able to increase the production of cytokines (IFN-. Gamma.and TNF-. Alpha.) by HBV-specific CD8+ T cells in PBMC of CHB patients.

As shown in FIG. 1F, lip-1 significantly increased the level of GPX4 in CD8+ T cells and HBV-specific CD8+ T cells in PBMC of CHB patients.

(Wherein "ns" represents no significant difference, "" represents p <0.05, "" represents p < 0.01)

Example 2

In vivo experiments.

Construction of HBV-carrier mouse model: taking C57BL/6J mice of 5-6 weeks old, injecting 8 mu gpAAV/HBV1.2 plasmid into tail vein under high pressure, taking peripheral blood after 5-6 weeks to separate serum, and detecting the HBsAg level in the serum, wherein the serum HBsAg concentration is higher than 500ng/mL, which is HBV-carrier mice successfully molded.

Treatment strategy: mice with successful molding were intraperitoneally injected with 30mg/kg of iron death inhibitor Liproxstatin-1 daily, and body weight changes were detected every two days for 15 days of treatment.

Mice were bled to isolate serum: fixing the mice in the fixer under the aseptic state, and exposing tails of the mice; gently wiping the tail of the mice with 70% alcohol for disinfection; the tail end of the mouse was cut at about 2mm, and the tail was massaged from the tail root to the tail tip, and the blood flowing out of the tail tip was collected. Standing at room temperature for 30min, centrifuging at 3000rpm for 15min, collecting supernatant, and storing at-80deg.C.

Obtaining liver mononuclear cells: dissecting and separating the liver of the mouse, grinding and then collecting the liver into a 15mL centrifuge tube; 100g, centrifuging for 1 min, and collecting a supernatant; centrifuging 400g for 5 min, and discarding supernatant to obtain precipitate; 4ml of 40% Percoll solution (cytiva) was added to resuspend the cells; centrifuging at 800g for 25 min, discarding supernatant to retain precipitate; adding erythrocyte lysate to resuspend cells, standing at 4 ℃ for 10 minutes, stopping erythrocyte lysis, and filtering to separate tubes.

Serum HBsAg levels in mice peripheral blood were measured by CLIA method: diluting the serum sample according to experimental requirements; add 50. Mu.L of sample or standard (0, 0.05, 0.8, 10, 85, 250 ng/mL) to the corresponding coated wells, respectively; adding 50 mu L of enzyme conjugate into each hole, gently shaking and mixing, pasting a sealing plate film, and incubating for 1h at 37 ℃; after the incubation is finished, the sealing plate film is torn off, liquid in the holes is forcibly thrown off, the coated plate is washed by HBsAg washing liquid, the washing is repeated for 5 times, and finally, the coated plate is patted to be dry as much as possible; mixing the luminous substrate A and the luminous substrate B according to the proportion of 1:1, adding 50 mu L of the mixed luminous substrate into the coating hole, gently shaking, and standing for 10min at room temperature in a dark place. And detecting the luminous intensity by using a Synergy 2 multifunctional enzyme-labeled instrument, making a standard curve, and calculating the concentration of HBsAg in the sample. Serum HBV DNA detection: to 200. Mu.L of the sample, 450. Mu.L of DNA extract I and 4. Mu.L of internal standard solution were added, and the mixture was shaken and mixed for 15 seconds, followed by instantaneous centrifugation to remove water droplets on the lid and the tube wall. Treating at 100deg.C for 10+ -1 min, and centrifuging at 12000rpm for 5min. The corresponding amount of PCR reaction liquid and Taq enzyme (HBV PCR reaction liquid 27 mu L/tube+Taq enzyme 3 mu L/tube) are taken according to the proportion, and are fully mixed and packaged into PCR reaction empty tubes suitable for each instrument according to 30 mu L/tube for standby. And respectively adding the extracted sample nucleic acid to be detected, HBV negative quality control product, HBV strong positive quality control product, HBV critical positive quality control product and positive quantitative reference product into the HBV reaction tube by using a suction nozzle with a filter element, centrifuging for a plurality of seconds at 8000rpm, and transferring to an amplification detection area. The PCR reaction conditions were as follows (DA 7600):

TABLE 1 PCR reaction conditions for detecting peripheral blood serum DNA

As shown in FIG. 2A, HBV-carrier mice after 5 weeks of successful molding were intraperitoneally injected with 30mg/kg of iron death inhibitor Liproxstatin-1 per day, and body weights were measured every two days for 15 days of treatment.

As shown in fig. 2B, the lipid peroxidation level of cd8+ T cells in the liver of HBV-carrier mice was significantly higher than that of wild-type mice, and Lip-1 treatment significantly reduced the lipid peroxidation level of HBV-carrier mice.

As shown in fig. 2C, lip-1 treatment significantly improved cytokine production by cd8+ T cells (IFN- γ and TNF- α) in the liver of HBV-carrier mice.

As shown in fig. 2D, lip-1 treatment was able to reduce HBsAg levels in HBV-carrier mouse serum.

As shown in fig. 2E, lip-1 treatment was able to reduce the level of HBVDNA in HBV-carrier mouse serum.

As shown in fig. 2F, lip-1 treatment reduced HBV-carrier mice weight at day 5 and gradually recovered to normal at day 10.

As shown in fig. 2G, lip-1 treatment did not show damage to the liver of mice.

(Wherein "ns" represents no significant difference, "" represents p <0.05, "" represents p <0.01, "" represents p < 0.0001)

The present study also explored the effect of other iron death inhibitors on antigen-specific cd8+ T cells of HBV infection, found that edaravone, ferrostatin-1, vitamins E, UAMC-3203 and defetoxamine, etc. all had no significant activating/stimulating effect, nor observed therapeutic effect that could significantly benefit in vivo in anti-HBV infection studies.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention.

Claims (2)

1. A method of making antigen-specific cd8+ T cells, comprising the steps of:

Step 1) isolated PBMCs from patients with chronic hepatitis B are incubated in RPMI 1640 containing 10% fetal bovine serum, and the PBMCs are stimulated with HBV peptide in the presence of iron death inhibitor Liproxstatin-1 for 10 days;

step 2) CD8+ T cells are isolated from PBMC and stimulated with the addition of human T activating antibodies CD3/CD28 and IL-2; stimulating cd8+ T cells with HBV peptide in the presence of iron death inhibitor Liproxstatin-1 for 10 days;

The HBV peptide is FLPSDFFPSV.

2. The method according to claim 1, wherein the concentration of Liproxstatin-1 used in step 1) and step 2) is 50nM.