CCR5 hi Application of innate immune memory cells in the preparation of immunotherapeutic drugs for treating sepsis

Abstract

The present invention relates to the field of biomedical technologies, and specifically discloses the application of CCR5<supgt;hi< / supgt> innate immune memory cells in the preparation of immunotherapeutic drugs for the treatment of sepsis. The present invention provides an innate immune memory cell, CD45<supgt;+< / supgt>CD11b<supgt;+< / supgt>Ly6G<supgt;‑< / supgt>Ly6C<supgt;+< / supgt>CCR5<supgt;hi< / supgt> monocytes, which is induced by BCG combined with bacterial lipoprotein to train immunity and has a protective effect on sepsis. Through single-cell sequencing analysis and functional verification, the cell subset characteristics are accurately identified, and its functional mechanism is clarified, thereby filling the key gaps of "unclear effector cell identity and unclear action targets" in the existing technology, providing a theoretical basis and technical solution at the cellular level for the targeted immunotherapy of sepsis, and is expected to achieve better therapeutic effects in clinical practice.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly relates to CCR5 hi The application of innate immune memory cells in the preparation of immunotherapeutic drugs for the treatment of sepsis. Background Art

[0002] Sepsis is a syndrome of multiple organ dysfunction caused by the dysregulation of the host's response to infection, and there is a serious immune imbalance during its pathogenesis. The imbalance between pro-inflammatory and anti-inflammatory mechanisms disrupts the body's homeostasis, leading to disease progression and even threatening life. Currently, the treatment of sepsis mainly relies on anti-infection treatment (such as antibiotics), fluid resuscitation, organ support, etc. However, the overuse of antibiotics has led to the emergence of multi-drug resistant strains, and even given rise to "superbugs" that are completely resistant to all existing antibiotics. This phenomenon poses a severe challenge to traditional anti-infection strategies and highlights the urgent need to develop innovative immunotherapy strategies to improve the prognosis of sepsis.

[0003] Trained immunity (TI) is an emerging immunomodulatory strategy that mainly relies on innate immune cells such as monocytes-macrophages, NK cells, and ILC cells to sense pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), and then induce epigenetic and metabolic reprogramming, enabling them to produce a stronger immune response when encountering the same or different pathogen stimuli again, thereby accelerating pathogen clearance and restoring the body's homeostasis. The immune-enhancing effect of trained immunity shows broad application prospects in anti-viral, anti-bacterial, and anti-fungal infections, especially in the prevention and treatment of sepsis. Research has shown that inducing trained immunity can enhance the pro-inflammatory response of septic mice, improve the ability to clear bacteria, and thus improve survival rate. Therefore, developing effective strategies for inducing trained immunity is expected to break through the limitations of traditional antibiotic treatment and provide new ideas for the immune regulation of sepsis.

[0004] Many biological agents have been proven to be able to induce trained immunity. Among them, the live attenuated vaccine Bacillus Calmette-Guérin (BCG), which is widely used for tuberculosis prevention, is one of the most mature and widely used inducers of trained immunity. As a classic inducer of trained immunity, BCG drives the metabolic reprogramming and epigenetic reprogramming of innate immune cells through PRRs such as Toll-like receptors (TLRs), enhancing the host's non-specific immune defense ability. In recent years, the immunomodulatory potential of BCG has gradually extended to fields such as cancer, viral infections, and autoimmune diseases, and its role in the prevention and treatment of sepsis has also attracted much attention. For example, neonatal BCG vaccination can promote the generation of emergency granulocytes and effectively control early sepsis infections.

[0005] Although BCG can enhance anti-infection ability through trained immunity, its application in the treatment of sepsis still has limitations: ① The inflammatory response induced by BCG may exacerbate the immune imbalance in sepsis patients; ② Its mechanism of action has not been fully elucidated, especially which innate immune cells play a key role in the protection against sepsis induced by BCG remains unclear; ③ Traditional BCG vaccination has certain safety hazards, especially in immunodeficient individuals.

[0006] Therefore, developing a safer and more efficient strategy for inducing trained immunity and clarifying its core effector cell types and mechanism of action are crucial for optimizing the immune regulation of sepsis. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an application of CCR5 hi innate immune memory cells in the preparation of immunotherapeutic drugs for the treatment of sepsis. The present invention provides an innate immune memory cell - CCR5 hi monocytes, which are induced by BCG combined with bacterial lipoproteins to generate trained immunity, have a protective effect on sepsis. Through single-cell sequencing analysis and functional verification, the characteristics of its cell subsets are accurately identified, and its functional mechanism is elucidated, thus filling the key gap of "unclear effector cell identity and unclear action target" in the prior art, and providing a theoretical basis and technical solution at the cellular level for the targeted immunotherapy of sepsis.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides an application of CCR5 hi innate immune memory cells in the preparation of immunotherapeutic drugs for the treatment of sepsis.

[0010] Specifically, the CCR5 hi innate immune memory cells are a cell subset that is induced by BCG combined with bacterial lipoproteins to generate trained immunity and simultaneously expresses CD45 + CD11b + Ly6G - Ly6C + CCR5 hi .

[0011] Specifically, the dosage form of the drug is an injection.

[0012] Specifically, the administration routes of the drug include intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection.

[0013] Specifically, the drug further includes a pharmaceutically acceptable carrier.

[0014] Specifically, the carrier includes one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant, and a preservative.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The present invention provides a cell population - CCR5 hi monocytes with innate immune memory characteristics induced by the combination of Bacillus Calmette-Guérin (BCG) and bacterial lipoprotein (BLP). This cell subset has antibacterial activity and enhanced ability to respond to inflammatory responses. It rapidly responds during the occurrence of sepsis, enhances the body's immune defense ability, and can significantly improve the prognosis of sepsis. The present invention is expected to break through the limitations of existing antibiotic treatments, provide a theoretical basis for the precise regulation of trained immunity, and open up a new direction for the immunotherapy of sepsis.

[0017] (2) To overcome the limitations of BCG-induced trained immunity in the treatment of sepsis, the present invention proposes a strategy of optimizing trained immunity by combining BCG and bacterial lipoprotein (BLP). As a TLR2-specific agonist, BLP is different from the strong pro-inflammatory response mediated by LPS-TLR4. It can promote the maturation of macrophage phagosomes by upregulating Rab10 and activate the CXCL2-CXCR2 pathway to recruit neutrophils, thereby bypassing the TLR4-dependent inflammatory pathway and directly enhancing the pathogen clearance ability, forming an "anti-inflammatory - high bactericidal" immune protection mode. To deeply analyze the mechanism of trained immunity induced by BCG+BLP, the present invention combines 10X Genomics single-cell sequencing and identifies a CCR5 hi monocyte with innate immune memory characteristics. This subset rapidly responds during the occurrence of sepsis, enhances the host's immune defense ability, and significantly improves the prognosis of sepsis; CCR5 hi monocytes can pre-activate innate immunity before infection, enabling the host to rapidly release an appropriate amount of inflammatory factors after contact with pathogenic microorganisms, accelerating the recruitment of phagocytes and pathogen recognition, thereby effectively clearing pathogens before the infection gets out of control and blocking the vicious cycle of "inflammatory imbalance - tissue damage - secondary inflammation exacerbation", ultimately improving the survival rate of neonatal sepsis; different from the immune regulation mode of traditional anti-inflammatory drugs such as glucocorticoids, this strategy not only reduces inflammation-related side effects but also enhances the early immune response and improves the pathogen clearance ability.

[0018] (3) The present invention provides new ideas and methods for the treatment of sepsis, and is expected to achieve better treatment effects in clinical practice. By precisely regulating CCR5 hi monocytes, it is possible to more effectively control the development of sepsis, reduce the mortality rate of sepsis, improve the prognosis of patients, and has important clinical application value and broad market prospects.

[0019] (4)The present invention clarifies CCR5 hi As an effector cell population, monocytes can perform more precise therapeutic interventions, reducing the impact on non-related cells and tissues, lowering the side effects and risks during treatment, improving the safety of treatment, and overcoming the problem of insufficient understanding of the safety and effectiveness of existing technologies in treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is an expression diagram of CCR5 in the cluster5 monocyte subset. Among them, A is a KEGG enrichment analysis diagram of 715 up-regulated genes in the Cluster5 monocyte subset; B is a t-SNE diagram of the expression level of the Ccr5 gene in monocytes, and each point represents a cell, and the point color represents the average expression level of the Ccr5 gene; C is a violin diagram of the expression of Ccr5 in each monocyte subset.

[0021] Figure 2 for CCR5 hi Flow sorting protocol diagram of monocytes.

[0022] Figure 3 It is a diagram of the bacteriophage ability of monocytes in different groups. Among them, A is a colony diagram of Escherichia coli phagocytosed by monocytes in different groups on an LB culture dish; B is a colony statistical chart of Escherichia coli phagocytosed by monocytes in different groups (n = 4), CCR5 lo represents CD45 + CD11b + Ly6G - Ly6C + CCR5 lo cells; CCR5 hi represents CD45 + CD11b + Ly6G - Ly6C + CCR5 hi cells.

[0023] Figure 4 It is a diagram of the expression level of inflammatory factors in the cell supernatant of different groups. Among them, A is the expression level of IL-6 in the cell supernatant after 12 h of LPS stimulation (n = 4); B is the expression level of TNF-α in the cell supernatant after 12 h of LPS stimulation (n = 4), CCR5 lo represents CD45 + CD11b + Ly6G - Ly6C + CCR5 lo cells; CCR5 hi represents CD45 +CD11b + Ly6G - Ly6C + CCR5 hi Cells, * indicates P < 0.05, ** indicates P < 0.01.

[0024] Figure 5 It is a comparison graph of the improvement effects of different groups on septic mice. Among them, A is the survival curve graph of mice in different groups (n = 10 or 12); B is the HE staining result graph of mouse lung tissue, scale bar = 100 μm; C is the statistical graph of pathological damage scores of HE staining of lung tissue (n = 4), * indicates P < 0.05, *** indicates P < 0.001. Specific implementation manners

[0025] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0026] It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are all purchased through commercial channels.

[0027] The BCG used in the embodiments of the present invention was donated by the Suzhou Center for Disease Control and Prevention, and the type of Escherichia coli is ATCC8099.

[0028] Example 1

[0029] CCR5 hi Sorting of cell subsets

[0030] 1. Establishment of a trained immune model

[0031] Newborn C57BL / 6 mice were randomly divided into four groups, with 10 mice in each group. Bacillus Calmette-Guérin (BCG) and bacterial lipoprotein (BLP) were selected as the trained immune inducers. First, weigh the body weights of 7-day-old mice, and intraperitoneally inject BCG (250 μg / g mouse body weight) and BLP (5 μg / g mouse body weight) to induce trained immunity. After injection, place the mice in a suitable breeding environment and continue to be fed by the mother mouse for 3 days.

[0032] 2. Preparation of cecal slurry (CS)

[0033] Five male and female C57BL / 6 mice aged 8 weeks were selected, anesthetized with pentobarbital (3%, 1 μL / g mouse body weight) and then immersed in 75% alcohol. The following operations were performed in a biosafety cabinet: the cecal tissue of the mouse was obtained with the help of sterile scissors and tweezers and placed in a sterile culture dish, and a 50 mL centrifuge tube was weighed in advance, the weight of which was M1; a small hole was cut at one end of the cecum with scissors, and then the cecal contents were collected into the above centrifuge tube using sterile tweezers, and weighed again as M2, and M2-M1 was the weight of the cecal contents; the corresponding volume of sterile 0.5% glucose saline was added to the centrifuge tube containing the cecal contents, so that the final concentration of the cecal slurry was 100 mg / mL, and after thorough vortex mixing, the undissolved residue was removed by 100 μm and 70 μm filters in turn, and then the cecal slurry was divided while vortexing to ensure that the concentration of each tube was consistent, and finally stored in a -80°C refrigerator for use within 3 months.

[0034] 3. Establishment of sepsis model

[0035] The present invention uses the method of intraperitoneal injection of CS to construct a multi-strain sepsis model in neonatal mice. The specific operation is as follows: take out CS (100 mg / mL) from the -80°C refrigerator, thaw and thoroughly vortex mix. Subsequently, weigh the body weight of 10-day-old mice, and use an insulin needle to inject CS at a dose of 1.0 mg / g mouse body weight. After 1h, 12h, and 24h after sepsis modeling, use pentobarbital (3%, 1μL / g mouse body weight) to anesthetize and retain specimens for subsequent experiments, and monitor the survival, health status and weight changes of mice in each group for 8 consecutive days.

[0036] 4. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

[0037] After the modeling, the mice were anesthetized, and the heart was blooded with an insulin needle. The whole blood was collected in an anticoagulant tube containing EDTA, and the whole blood was diluted with an equal volume of PBS. 3 mL of separation solution was added to a 15 mL centrifuge tube, and the diluted whole blood was spread on the surface of the separation solution with a Pasteur pipette to form a layer; then centrifuged at room temperature for 500 g for 30 min, and the speed of the centrifuge was adjusted to 1. After the centrifugation, obvious stratification appeared. The white film layer cells were carefully aspirated into a new 15 mL centrifuge tube with a pipette, which was PBMCs.

[0038] 5. Single-cell sequencing to identify CCR5 hi Monocyte subsets

[0039] Single-cell sequencing was performed on the PBMCs isolated in the previous step, and cell subsets were annotated. A total of eight cell populations were identified: monocytes (CD14, Csf1r, Lyz2), B lymphocytes (CD79a, Ms4a1, CD19), T lymphocytes (Cd3d, Cd3e, CD3g), neutrophils (Ngp, S100a8), dendritic cells (Cd74, Itgax), megakaryocytes (Ppbp), red blood cells (Hba-a1, Hba-a2), and natural killer cells (Nkg7, Klrb1c). Monocytes were re-annotated and classified, and cluster5 showed specific expansion in the trained immunity induced by BCG+BLP.

[0040] As can be seen from Figure 1 Figure A, KEGG enrichment analysis showed that 715 differentially expressed genes upregulated in cluster5 were biologically related to endocytosis. Phagocytosis is a special type of endocytosis. As can be seen from Figure 1 Figure B, compared with other monocytes, the phagocytosis-related gene CCR5 was significantly upregulated in the cluster5 monocyte subset. As can be seen from Figure 1 Figure C, the violin plot further showed that CCR5 was expressed in all monocyte populations, and CCR5 was observed to be particularly abundant in the cluster5 monocyte subset, demonstrating that CCR5 hi Monocytes are an innate immune memory subset mediated by BCG+BLP.

[0041] 6. Flow cytometry detection of CCR5 hi Monocytes

[0042] ① Take a part of the mouse PBMCs suspension prepared by density gradient centrifugation and adjust the cell concentration to about 1×10 6 cells / mL.

[0043] ② Take 50 μL of the cell suspension and add 50 μL of the flow antibody solution premixed with fluorescence-activated cell sorting buffer (FACS), including CD11b, LY-6G, Ly-6C, CCR5, ensuring that the volume ratio of the final cell suspension to the antibody stock solution is 100:1, and incubate in the dark on ice for 30 min.

[0044] ③ After antibody incubation, add 300 μL of FACS buffer to each tube and centrifuge at 500 g, 4 °C for 5 min.

[0045] ④ Discard the supernatant, add 300 μL of FACS buffer to each tube to resuspend the cell pellet, mix well and transfer to a flow tube, and use a flow cytometer for detection. Analyze the results using Flow jo software. The flow detection protocol is as shown in Figure 2As shown below, the relevant steps are as follows:

[0046] (1) Set up a live cell gate based on the parameters of the forward scatter area (FSC-A) and the side scatter area (SSC-A) to exclude debris and dead cells;

[0047] (2) Exclude adherent cells based on FSC-A vs FSC-H and SSC-A vs SSC-W to ensure that the object is a single cell;

[0048] (3) Stain with anti-CD45 antibody to screen for CD45 + leukocyte population;

[0049] (4) In the CD45 + population, screen for the myeloid cell subset through positive expression of CD11b;

[0050] (5) Double stain with anti-LY6G antibody and anti-LY6C antibody, and specifically sort the monocyte subset from myeloid cells through the characteristic surface marker combination of LY6G - / LY6C + ;

[0051] (6) In the monocyte population, finally delineate the CCR5 hi monocyte subset according to the CCR5 expression intensity.

[0052] Example 2

[0053] The antibacterial activity of CCR5 hi monocytes and their response ability to inflammation

[0054] In this experiment, two groups of experiments were designed: a control group (PBS) and a trained immune group (BB). In the control group (PBS), 50 μL of PBS was injected into the abdominal cavity of mice, and in the trained immune group (BB), BCG (250 μg / g mouse body weight) and BLP (5 μg / g mouse body weight) were injected into the abdominal cavity of mice. Using flow cytometry sorting technology, CD45 + CD11b + Ly6G - Ly6C + CCR5 lo cells and CD45 + CD11b + Ly6G - Ly6C + CCR5 hi cells were sorted from the bone marrow of mice in both groups and divided into four groups, denoted as PBS-CCR5 lo and PBS-CCR5 hi and BB-CCR5 lo and BB-CCR5hi , Subsequently, these cells were respectively inoculated into cell culture plates at an inoculation density of 1×10 5 cells / well, and were respectively given E.coli infection or LPS stimulation. Subsequently, the cells and supernatants were collected for functional experiments to verify their phagocytic ability and inflammatory response ability.

[0055] The four groups of sorted monocytes were co-incubated with Escherichia coli in a bacterial incubator for 30 min. The number of monocytes was 1×10 5 cells, and Escherichia coli was 8×10 6 CFU. The dilution coating method was used to quantify the phagocytic intensity of CCR5 lo / CCR hi monocytes. The results are as Figure 3 shown. As can be seen from Figure 3 , compared with the control group of CCR5 lo and CCR5 hi monocytes, the number of Escherichia coli phagocytosed by BB-CCR5 hi monocytes increased significantly; compared with BB-CCR5 lo monocytes, the phagocytosis level of BB-CCR5 hi monocytes increased significantly; while compared with BB-CCR5 lo monocytes and PBS-CCR5 hi , PBS-CCR5 lo monocytes, there was no significant difference in phagocytosis.

[0056] The four groups of sorted monocytes were stimulated with LPS (100 ng / mL) for 12 h, and the cell supernatants of each group were collected. ELISA was used to detect the expression levels of inflammatory factors in the cell supernatants of PBS-CCR5 lo , PBS-CCR5 hi , BB-CCR5 lo and BB-CCR5 hi . The results are as Figure 4 shown. As can be seen from A in Figure 4 , compared with PBS-CCR5 hi , the level of IL-6 secreted by BB-CCR5 hi monocytes increased significantly; compared with BB-CCR5 lo , the level of IL-6 secreted by BB-CCR5 hi monocytes also increased significantly. In addition, as can be seen from B in Figure 4 , compared with BB-CCR5 lo , the level of TNF-α secreted by BB-CCR5 hi monocytes increased significantly; while BB-CCR5 hiThe level of TNF-α released by the BB-CCR5 group was slightly higher than that of the PBS-CCR5 group, but the difference did not reach statistical significance. hi The above results indicate that the antibacterial activity of monocytes and the ability to respond to inflammation in the BB-CCR5 group were enhanced.

[0057] Example 3 hi Transfer of adoptive CCR5 monocytes was used to verify their effect on neonatal mouse sepsis.

[0058] CD45, CD11b, Ly6G, Ly6C, and CCR5 cells sorted from the bone marrow of mice in the control group (PBS) and the trained immune group (BB) were injected via the tail vein into recipient mice and divided into three groups: the group injected with PBS (NC) via the vein, the group injected with control group CCR5 monocytes (Ctrl-CCR5), and the group injected with trained immune group CCR5 monocytes (BB-CCR5). The injection volume was 100 μL and the number of cells was 1×10. Subsequently, a polymicrobial sepsis model was established, samples were collected to detect corresponding indicators, and the survival of the above three groups of mice was observed. The results are shown as follows.

[0059] Transfer of adoptive CCR5 hi monocytes was used to verify their effect on neonatal mouse sepsis.

[0060] CD45, CD11b, Ly6G, Ly6C, and CCR5 cells sorted from the bone marrow of mice in the control group (PBS) and the trained immune group (BB) were injected via the tail vein into recipient mice and divided into three groups: the group injected with PBS (NC) via the vein, the group injected with control group CCR5 + CD11b + Ly6G - Ly6C + CCR5 hi cells (Ctrl-CCR5 group), and the group injected with trained immune group CCR5 hi monocytes (BB-CCR5 hi group). The injection volume was 100 μL and the number of cells was 1×10 hi . Subsequently, a polymicrobial sepsis model was established, samples were collected to detect corresponding indicators, and the survival of the above three groups of mice was observed. The results are shown as follows. hi As can be seen from A, 8 mice (8 / 10) in the NC group died within 36 h after sepsis modeling, and the survival rate at 72 h was 20% (2 / 10); 8 mice (8 / 12) in the Ctrl-CCR5 group died within 36 h after sepsis modeling, and the survival rate at 72 h was 33.3% (4 / 12); 4 mice (4 / 12) in the BB-CCR5 group died within 48 h after sepsis modeling, and the survival rate at 72 h was 66.7% (8 / 12). At the same time, compared with the Ctrl-CCR5 group, the survival rate of the BB-CCR5 group was significantly increased (P = 0.048). 6 For the mice, samples were collected to detect corresponding indicators, and the survival of the above three groups of mice was observed. The results are shown as follows. Figure 5 shown.

[0061] From Figure 5 A, it can be seen that 8 mice (8 / 10) in the NC group died within 36 h after sepsis modeling, and the survival rate at 72 h was 20% (2 / 10); 8 mice (8 / 12) in the Ctrl-CCR5 hi group died within 36 h after sepsis modeling, and the survival rate at 72 h was 33.3% (4 / 12); 4 mice (4 / 12) in the BB-CCR5 hi group died within 48 h after sepsis modeling, and the survival rate at 72 h was 66.7% (8 / 12). At the same time, compared with the Ctrl-CCR5 hi group, the survival rate of the BB-CCR5 hi group was significantly increased (P = 0.048).

[0062] HE staining was performed on the lung tissue of mice 24 h after sepsis modeling. The results are shown in B and C. Compared with the lung tissue of mice in the NC group, the Ctrl-CCR5 Figure 5 grouphi and BB-CCR5 hi In the group of mice, lung tissue inflammation and alveolar damage were significantly improved; compared with the Ctrl-CCR5 hi group of mice, in the BB-CCR5 hi group of mice, the infiltration of inflammatory cells and the degree of damage in the lung tissue were significantly reduced.

[0063] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, based on the present invention, some modifications and improvements can be made. Therefore, any modification or improvement made without departing from the spirit of the present invention falls within the scope of protection required by the present invention.

Claims

1. CCR5 hi Use of innate immune memory cells in the preparation of an immunotherapeutic drug for treating sepsis, characterized in that, The CCR5 hi The innate immune memory cells are a cell subset produced by training immunized mice with BCG combined with bacterial lipoprotein and simultaneously express CD45 + CD11b + Ly6G - Ly6C + CCR5 hi The innate immune memory cells are a cell subset that expresses CCR5, and the innate immune memory cells expressing CCR5 hi are used to prepare an immunotherapeutic drug for treating septic mice.

2. The application according to claim 1, characterized in that, The dosage form of the drug is an injection.

3. The application according to claim 1, characterized in that, The administration routes of the drug include intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection.

4. The application according to claim 1, wherein The drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The carrier includes one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant and a preservative.

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