Application of CCR5hi innate immune memory cell in preparation of immunotherapy drug for treating sepsis

Abstract

The invention relates to the technical field of biological medicines, particularly discloses an application of a CCR5hi innate immune memory cell in preparation of an immunotherapy drug for treating sepsis, and provides an innate immune memory cell CD45 + CD11b + Ly6G-Ly6C + CCR5hi mononuclear cell which is generated by induction training immunity of bacillus calmette guerin vaccine and bacterial lipoprotein and has a protection effect on sepsis. Through single cell sequencing analysis and functional verification, the cell subset characteristics of the sepsis are accurately identified, and the functional mechanism of the sepsis is clarified, so that the key blank of unclear effector cell identity and unclear action target in the prior art is filled, and a cellular theoretical basis and a technical scheme are provided for targeted immunotherapy of the sepsis; and a better treatment effect is expected to be achieved in clinical practice.

Description

Technical Field

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

[0002] Sepsis is a multi-organ dysfunction syndrome caused by a dysregulated host response to infection, and its pathogenesis is accompanied by severe immune imbalance. The dysregulation of pro-inflammatory and anti-inflammatory mechanisms destroys the body's homeostasis, leading to disease progression and even life-threatening. At present, the treatment of sepsis mainly relies on anti-infection therapy (such as antibiotics), fluid resuscitation, organ support, etc. However, the overuse of antibiotics has led to the emergence of multidrug-resistant strains and even spawned "super bacteria" that are completely resistant to all existing antibiotics. This phenomenon has posed severe challenges to traditional anti-infection strategies and highlighted 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 perceive pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), thereby inducing epigenetic and metabolic reprogramming, so that they produce stronger immune responses when they encounter the same or different pathogenic stimuli again, thereby accelerating pathogen clearance and restoring body homeostasis. The immune-enhancing effect of trained immunity has given it broad application prospects in antiviral, antibacterial, and antifungal infections, especially in the prevention and treatment of sepsis. Studies have shown that inducing trained immunity can enhance the pro-inflammatory response of septic mice, improve bacterial clearance, and thus improve survival. Therefore, the development of an effective trained immunity induction strategy 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 shown to induce trained immunity. Among them, Bacillus Calmette-Guérin (BCG), a live attenuated vaccine widely used in tuberculosis prevention, is one of the most mature and widely used trained immunity inducers. As a classic trained immunity inducer, BCG drives metabolic reprogramming and epigenetic reprogramming of innate immune cells through PRRs such as Toll-like receptors (TLRs), thereby enhancing the host's nonspecific immune defense capabilities. In recent years, the immunomodulatory potential of BCG has gradually expanded to fields such as cancer, viral infection, and autoimmune diseases, and its role in the prevention and treatment of sepsis has also attracted much attention. For example, BCG vaccination of newborns can promote emergency granulocyte production and achieve effective control of early sepsis infection.

[0005] Although BCG can enhance anti-infection ability by training immunity, its application in the treatment of sepsis still has limitations: ① BCG-induced inflammatory response may aggravate the immune imbalance of patients with sepsis; ② Its mechanism of action has not been fully clarified, especially which innate immune cells play a key role in the protection process of BCG-induced sepsis is still unclear; ③ Traditional BCG vaccination has certain safety risks, especially in immunocompromised individuals.

[0006] Therefore, developing safer and more efficient trained immune induction strategies and clarifying their core effector cell types and mechanisms of action are crucial to optimizing the immune regulation of sepsis. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a CCR5 hi Application of 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, which is produced by BCG combined with bacterial lipoprotein-induced immune training and has a protective effect on sepsis. hi Monocytes, through single-cell sequencing analysis and functional verification, can accurately identify their cell subpopulation characteristics and clarify their functional mechanisms, thus filling the key gaps in the existing technology of "unclear identity of effector cells and unknown targets of action", and providing a cellular-level theoretical basis and technical solutions for targeted immunotherapy of sepsis.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme: The present invention provides a CCR5 hi Application of innate immune memory cells in the preparation of immunotherapeutic drugs for the treatment of sepsis.

[0009] Specifically, the CCR5 hi Innate immune memory cells are a type of immune cells produced by BCG combined with bacterial lipoprotein-induced training immunity and express CD45 + CD11b + 6G - Ly6C + CCR5 hi cell subpopulations.

[0010] Specifically, the dosage form of the drug is an injection.

[0011] Specifically, the administration routes of the drug include intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection.

[0012] Specifically, the drug also includes a pharmaceutically acceptable carrier.

[0013] 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.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a cell population with inherent immune memory characteristics, namely CCR5, which is generated by Bacillus Calmette-Guérin (BCG) combined with bacterial lipoprotein (BLP) induced immune training hi Monocytes, this cell subpopulation has the characteristics of antibacterial activity and enhanced ability to respond to inflammation. They respond quickly when sepsis occurs, enhance the body's immune defense capabilities, 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 immune training, and open up new directions for the immunotherapy of sepsis.

[0015] (2) In order 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 with 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 ability to clear pathogens, forming a "low inflammation-high bactericidal" immune protection model. In order to deeply analyze the mechanism of trained immunity induced by BCG+BLP, the present invention combined with 10X Genomics single-cell sequencing to identify a CCR5 with inherent immune memory characteristics. hi Monocytes, this subset responds rapidly when sepsis occurs, enhances the host's immune defense, and significantly improves the prognosis of sepsis; CCR5 hi Monocytes can pre-activate innate immunity before infection, allowing the host to quickly release appropriate amounts of inflammatory factors after contact with pathogenic microorganisms, accelerating the recruitment of phagocytes and pathogen recognition, thereby effectively eliminating pathogens before the infection gets out of control, blocking the vicious cycle of "inflammatory imbalance-tissue damage-secondary inflammation intensification", and ultimately improving the survival rate of sepsis in newborn mice; 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 early immune responses and improves pathogen clearance capabilities.

[0016] (3) This invention provides new ideas and methods for the treatment of sepsis, and is expected to achieve better therapeutic effects in clinical practice by precisely regulating CCR5 hi Monocytes can more effectively control the progression of sepsis, reduce the mortality rate of sepsis, and improve the prognosis of patients. They have important clinical application value and broad market prospects.

[0017] (4) The present invention clarifies that CCR5 hi As an effector cell population, monocytes can perform therapeutic intervention more accurately, reduce the impact on non-related cells and tissues, reduce side effects and risks during treatment, improve the safety of treatment, and overcome the problem of insufficient understanding of treatment safety and effectiveness in existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The expression diagram of CCR5 in cluster5 monocyte subsets, where A is the KEGG enrichment analysis diagram of 715 upregulated genes in Cluster5 monocyte subsets; B is the t-SNE diagram of the expression level of Ccr5 gene in monocytes, each point represents a cell, and the point color represents the average expression level of Ccr5 gene; C is the violin plot of Ccr5 expression in each monocyte subset.

[0019] Figure 2 CCR5 hi Diagram of the flow cytometry scheme for monocyte isolation.

[0020] Figure 3 Figure 2 is the phagocytic ability of monocytes in different groups, where A is the LB culture plate colony diagram of monocytes in different groups phagocytosing E. coli; B is the colony statistics of monocytes in different groups phagocytosing E. coli (n=4), CCR5 lo CD45 + CD11b + 6G - Ly6C + CCR5 lo Cell; CCR5 hi CD45 + CD11b + 6G - Ly6C + CCR5 hi cell.

[0021] Figure 4 Figure 2 shows the expression levels of inflammatory factors in the cell supernatants of different groups, where A is the expression level of IL-6 in the cell supernatant after 12 hours of LPS stimulation (n=4); B is the expression level of TNF-α in the cell supernatant after 12 hours of LPS stimulation (n=4), CCR5 lo CD45 + CD11b + 6G - Ly6C + CCR5 lo Cell; CCR5 hi CD45 + CD11b+ 6G - Ly6C + CCR5 hi Cells, * indicates P < 0.05, ** indicates P < 0.01.

[0022] Figure 5 The figures are comparative diagrams of the improvement effects of different groups on septic mice, where A is the survival curve of mice in different groups (n=10 or 12); B is the HE staining result of mouse lung tissue, scale=100μm; C is the pathological injury score statistical diagram of HE staining of lung tissue (n=4), * indicates P<0.05, *** indicates P<0.001. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0024] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0025] The BCG used in the embodiments of the present invention was donated by Suzhou Center for Disease Control and Prevention, and the type of Escherichia coli was ATCC8099.

[0026] Example 1 CCR5 hi Isolation of cell subsets 1. Establishment of training immune model C57BL / 6 newborn mice were randomly divided into four groups, 10 in each group, and Bacillus Calmette-Guérin (BCG) and bacterial lipoprotein (BLP) were selected as training immune inducers. First, the 7-day-old mice were weighed, and BCG (250µg / g mouse body weight) and BLP (5µg / g mouse body weight) were intraperitoneally injected to induce training immunity. After injection, the mice were placed in a suitable feeding environment and continued to be fed by the mother mice for 3 days.

[0027] 2. Preparation of cecal slurry (CS) 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.

[0028] 3. Establishment of sepsis model 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.

[0029] 4. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) 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.

[0030] 5. Single-cell sequencing to identify CCR5 hi Monocyte subsets Single-cell sequencing was performed on the PBMCs isolated in the previous step, and cell subsets were annotated. A total of eight groups of cells 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), erythrocytes (Hba-a1, Hba-a2) and natural killer cells (Nkg7, Klrb1c). Monocytes were annotated and classified again, and cluster5 showed specific expansion in BCG+BLP-induced training immunity.

[0031] from Figure 1 As can be seen in Figure A, KEGG enrichment analysis showed that 715 differentially expressed genes upregulated in cluster 5 were biologically related to endocytosis. Phagocytosis is a special type of endocytosis. Figure 1 As can be seen in Figure B, the phagocytosis-related gene CCR5 was significantly upregulated in the cluster 5 monocyte subset compared with other monocytes. Figure 1 As can be seen in Figure C, the violin plot further shows that CCR5 is expressed in all monocyte populations, and CCR5 is particularly abundant in the cluster5 monocyte subset, proving that CCR5 hi Monocytes are a subset of innate immune memory mediated by BCG+BLP.

[0032] 6. Flow cytometry detection of CCR5 hi Monocytes ① Take part of the mouse PBMCs suspension prepared by density gradient centrifugation and adjust the cell concentration to about 1×10 6 Pieces / mL.

[0033] ②Take 50 μL of cell suspension and add 50 μL of flow cytometry antibody solution premixed with fluorescence activated cell sorting buffer (FACS), including CD11b, LY-6G, Ly-6C, and CCR5, ensuring that the final volume ratio of cell suspension to antibody stock solution is 100:1. Incubate on ice in the dark for 30 minutes.

[0034] ③After antibody incubation, add 300 μL FACS buffer to each tube and centrifuge at 500 g, 4°C, for 5 min.

[0035] ④Discard the supernatant, add 300 μL FACS buffer to each tube to resuspend the cell pellet, mix thoroughly and transfer to the flow tube, use flow cytometer for detection, and use Flow jo software to analyze the results. The flow detection scheme is as follows: Figure 2 As shown, the relevant steps are as follows: (1) Set up a live cell gate based on the forward scatter area (FSC-A) and side scatter area (SSC-A) parameters to exclude debris and dead cells; (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; (3) Use anti-CD45 antibody staining to screen CD45 + Leukocyte population; (4) In CD45 + In the population, myeloid cell subsets were screened by positive expression of CD11b; (5) Double staining using anti-LY6G antibody and anti-LY6C antibody - / LY6C + Characteristic surface marker combinations specifically sort monocyte subsets from myeloid cells; (6) In the monocyte population, CCR5 was finally identified based on the CCR5 expression intensity. hi Monocyte subsets.

[0036] Example 2 CCR5 hi Antimicrobial activity of monocytes and their ability to respond to inflammation This experiment designed two groups of experiments: a control group (PBS) and a training immune group (BB). The control group (PBS) was injected with 50 µL PBS into the peritoneal cavity of mice, and the training immune group (BB) was injected with BCG (250 µg / g mouse body weight) and BLP (5 µg / g mouse body weight) into the peritoneal cavity of mice. Flow cytometry was used to sort CD45 from the bone marrow of the two groups of mice. + CD11b + 6G - Ly6C + CCR5 lo Cells and CD45 + CD11b + 6G - Ly6C + CCR5 hi Cells were divided into four groups, denoted as PBS-CCR5 lo , PBS-CCR5 hi ,BB-CCR5 lo and BB-CCR5 hi These cells were then inoculated in cell culture plates at a density of 1×10 5 coli infection or LPS stimulation, and then the cells and supernatant were collected for functional experiments to verify their phagocytic and inflammatory response capabilities.

[0037] The four groups of sorted monocytes were incubated with Escherichia coli in a bacterial incubator for 30 min, and the number of monocytes was 1×10 5 Escherichia coli is 8×10 6 CFU, quantification of CCR5 using serial dilution plating lo / CCR hi The phagocytic activity of monocytes is as follows: Figure 3 As shown, from Figure 3 It can be seen that compared with the control group CCR5 lo and CCR5 hi Compared with monocytes, BB-CCR5 hi The number of Escherichia coli phagocytosed by monocytes increased significantly; lo Compared with monocytes, BB-CCR5 hi The phagocytic level of monocytes was significantly increased; lo Monocytes and PBS-CCR5 hi , PBS-CCR5 lo There was no statistical difference in phagocytosis between monocytes and cerebrospinal fluid.

[0038] The four groups of monocytes were stimulated with LPS (100 ng / mL) for 12 h, and the cell supernatants of each group were collected. PBS-CCR5 was detected by ELISA. lo , PBS-CCR5 hi ,BB-CCR5 lo and BB-CCR5 hi The expression levels of inflammatory factors in the cell supernatant were as follows Figure 4 As shown, from Figure 4 As can be seen in A, with PBS-CCR5 hi In comparison, BB-CCR5 hi The level of IL-6 secreted by monocytes increased significantly; lo In comparison, BB-CCR5 hi The level of IL-6 secreted by monocytes also increased significantly. Figure 4 As can be seen in B, with BB-CCR5 lo In comparison, BB-CCR5 hi The level of TNF-α secreted by monocytes increased significantly; however, BB-CCR5 hi The level of TNF-α released by the group was slightly higher than that of PBS-CCR5 hi groups, but the difference did not reach statistical significance.

[0039] The above results indicate that BB-CCR5 hiThe antimicrobial activity of monocytes and their ability to respond to inflammation are enhanced.

[0040] Example 3 Adoptive transfer of CCR5 hi Verification of the role of monocytes in sepsis in neonatal mice CD45 isolated from the bone marrow of mice in the control group (PBS) and the trained immune group (BB) + CD11b + 6G - Ly6C + CCR5 hi The cells were injected into the tail vein and then returned to the recipient mice, which were divided into three groups: intravenous injection of PBS (NC) group, intravenous injection of control group CCR5 hi Monocytes (Ctrl-CCR5 hi ) group, intravenous injection training immunization group CCR5 hi Monocytes (BB-CCR5 hi ) group, in which the injection volume was 100 μL and the number of cells was 1×10 6 Then, a multi-strain sepsis model was established, samples were collected to detect relevant indicators, and the survival of the above three groups of mice was observed. The results are as follows Figure 5 shown.

[0041] from Figure 5 As can be seen in A, 8 mice in the NC group died within 36 hours after sepsis modeling (8 / 10), and the survival rate at 72 hours was 20% (2 / 10); hi Eight mice in the BB-CCR5 group died within 36 hours after sepsis modeling (8 / 12), and the survival rate at 72 hours was 33.3% (4 / 12). hi Four mice in the group died within 48 hours after sepsis modeling (4 / 12), and the survival rate at 72 hours was 66.7% (8 / 12). hi Compared with the mice in the BB-CCR5 hi The survival rate of mice in the control group was significantly improved (P=0.048).

[0042] HE staining was performed on the lung tissues of mice 24 hours after sepsis modeling. Figure 5 B and C show that compared with the lung tissue of NC mice, Ctrl-CCR5 hi and BB-CCR5 hi The lung tissue inflammation and alveolar damage of mice in the Ctrl-CCR5 group were significantly improved. hi Compared with the lung tissues of mice in the BB-CCR5 hi The inflammatory cell infiltration and damage degree of lung tissue in group A mice were significantly reduced.

[0043] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. CCR5 hi The use of innate immune memory cells in the preparation of immunotherapeutic drugs for treating sepsis is characterized in that: CCR5 hi Innate immune memory cells are a type of immune cells produced by BCG combined with bacterial lipoprotein-induced training immunity and express CD45 + CD11b + 6G - Ly6C + CCR5 hi cell subpopulations.

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

3. The use 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 use according to claim 1, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

5. The use 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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