Periprosthetic infection treatment target and application thereof

Abstract

The invention belongs to the technical field of biomedicine, particularly discloses a periprosthetic infection treatment target and application thereof, and particularly discloses application of an antagonist or an expression inhibitor aiming at Treg cell immune checkpoint molecules CXCR6 in any one of the following aspects: preparing a product for preventing, treating, relieving or improving periprosthetic infection; preparing a product for inhibiting occurrence and development of periprosthetic infection; preparing a product for inhibiting proliferation and / or growth of periprosthetic infected tissue cells; preparing a product for promoting or inducing apoptosis of periprosthetic infected tissue cells; preparing a product for inhibiting inflammation, necrosis or fibrosis of periprosthetic infected tissues; according to the invention, a target CXCR6 knockout experiment is carried out on Treg, and a CXCR6-targeted product is found to have a potential therapeutic effect on reduction of PJI-related bacterial colonization and biofilm formation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a target for the treatment of periprosthetic infection and its application. This case is a divisional application of application number CN202410226724.1, and the invention name is "target for the treatment of periprosthetic infection and its application". Background Art

[0002] Periprosthetic Joint Infection (PJI) is a serious complication after joint replacement. Although the incidence of complications after artificial joint replacement has been significantly reduced in recent years with the continuous improvement of prosthesis design and the advancement of medical level, PJI still has an incidence of about 1-2%. With the popularization of joint replacement, the number of patients undergoing total knee arthroplasty (TKA) and total hip arthroplasty (THA) due to various orthopedic diseases has increased year by year, and the number of revisions has also increased year by year, which not only brings great pain to patients but also adds burden to the medical system. Patients with PJI usually require surgical treatment, and the current gold standard for the treatment of PJI is still a two-stage revision. However, the difficulty of PJI revision is different from that of revision caused by aseptic loosening around the prosthesis. PJI is difficult to debride. In order to completely remove the infected microorganisms, a large amount of infected tissue must be removed and flushed multiple times. The functional recovery of the joint after surgery is often poor, and there is a high recurrence rate. Finding more treatments for PJI is of great significance for early treatment of patients and restoration of joint function.

[0003] There are obvious differences in gene expression between the PJI synovium and periprosthetic aseptic loosening, which indicates that the molecular pathophysiology in PJI has changed. Long-term chronic infection leads to increased expression of immunosuppressive genes, which may be an important reason why PJI is difficult to treat. There is currently no relevant research that has conducted in-depth research on the periprosthetic microenvironment of PJI. The composition of PJI synovial cells is diverse. The changes in various cells such as fibroblasts, endothelial cells, T cells, and macrophages in PJI are very critical for subsequent treatment research. This study used single-cell sequencing combined with conventional transcriptome sequencing to explore the changes in the PJI microenvironment and explore the mechanism of occurrence, in order to have a more comprehensive understanding of PJI and provide a scientific basis for finding possible drug targets. Summary of the invention

[0004] The main purpose of the present invention is to provide a periprosthetic infection treatment target and its application, providing a new direction for PJI treatment.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: The present invention has found that the antagonist or expression inhibitor of Treg cell immune checkpoint molecules can be used in any of the following a1)-a5): a1) Preparation of products for preventing, treating, alleviating or ameliorating periprosthetic infection; a2) Preparation of products that inhibit the occurrence and development of periprosthetic infection; a3) Preparation of products that inhibit proliferation and / or growth of cells in periprosthetic infected tissues; a4) Preparation of products that promote or induce apoptosis of cells in periprosthetic infected tissues; a5) Preparation of products that inhibit inflammation, necrosis or fibrosis of infected tissues around prostheses; The immune checkpoint molecules are CTLA4 and / or CXCR6.

[0006] In this article, "Treg" refers to regulatory T cells (Treg for short), which are a type of T cell subpopulation that controls the body's autoimmune reactivity.

[0007] For "immune checkpoint molecules", as understood by those skilled in the art, they generally exist and function in the form of ligand-receptor pairs. In this article, immune checkpoint protein receptors and their ligands are collectively referred to as immune checkpoint molecules. For example, for Treg cell immune checkpoint molecules, the receptors in the ligand-receptor pair are generally expressed in NK cells, while the corresponding ligands are expressed in other types of cells. Through the interaction between receptor-ligand, the activation and immune effector functions of Treg cells are suppressed. Therefore, when referring to "immune checkpoint molecules", both ligands and receptors are covered. For example, when referring to CTLA4 and / or CXCR6, CTLA4 and / or CXCR6 and their ligands HLA-E (human) and Qa-1 (mouse) are covered.

[0008] Further, as some specific embodiments, the expression inhibitor includes any one of the following b1)-b5): b1) Products that inhibit the expression or activity of CTLA4 and / or CXCR6 genes; b2) inhibiting the transcription of CTLA4 and / or CXCR6 genes into mRNA products; b3) inhibiting the translation of CTLA4 and / or CXCR6 genes into protein products; b4) Products that inhibit the activity or function of CTLA4 and / or CXCR6 proteins; b5) Products that reduce the levels of CTLA4 and / or CXCR6 protein.

[0009] In specific embodiments, it is understood that "inhibition", "antagonism" and "blocking" and other ways of affecting the functions of CTLA4 and / or CXCR6 genes or proteins should have equivalent meanings, and the product form of CTLA4 and / or CXCR6 inhibitors can be one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses, and the product form of antagonists can be antibodies or antigen-binding fragments thereof against CTLA4 and / or CXCR6 or their ligand HLA-E, such as anti-CTLA-4, an antagonist of CTLA4.

[0010] The method of inhibiting the expression or activity of CTLA4 and / or CXCR6 genes can be achieved by gene mutation, gene silencing, gene knockout or gene knockdown techniques known to those skilled in the art. Gene knockdown techniques include RNA interference, Morpholino interference, antisense nucleic acid, ribozyme or dominant negative inhibitory mutation, etc., but are not limited thereto. Furthermore, it is well known to those skilled in the art to silence genes by inhibiting gene expression using shRNA or siRNA expressed by viruses (such as lentiviruses, adeno-associated viruses).

[0011] Furthermore, as some more specific embodiments, the product that inhibits the expression or activity of CTLA4 and / or CXCR6 genes is a substance that silences, knocks down or knocks out CTLA4 and / or CXCR6 genes.

[0012] Furthermore, the substance for silencing, knocking down or knocking out the CTLA4 and / or CXCR6 gene is miRNA, siRNA, dsRNA or shRNA.

[0013] The substance for knocking out the CTLA4 and / or CXCR6 gene can be any substance that can achieve the body's failure to produce the functional protein product of the CTLA4 and / or CXCR6 gene, and the specific method can be to remove all or part of the CTLA4 and / or CXCR6 gene sequence, introduce deletion mutations and / or insertion mutations and / or base substitutions in the CTLA4 and / or CXCR6 gene, etc. Usually, the knockout is performed at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0014] Furthermore, the substance for knocking out CTLA4 and / or CXCR6 genes is a gene editing tool for knocking out CTLA4 and / or CXCR6 genes, and the gene editing tool includes: zinc finger protein ZFN gene editing system or TALENs gene editing system or CRISPR / Cas9 gene editing system.

[0015] The present invention also provides a pharmaceutical composition comprising an antagonist or expression inhibitor for a Treg cell immune checkpoint molecule, wherein the immune checkpoint molecule is CTLA4 and / or CXCR6, and the pharmaceutical composition has the application in any one of the following a1)-a5), a1) Preparation of products for preventing, treating, alleviating or ameliorating periprosthetic infection; a2) Preparation of products that inhibit the occurrence and development of periprosthetic infection; a3) Preparation of products that inhibit proliferation and / or growth of cells in periprosthetic infected tissues; a4) Preparation of products that promote or induce apoptosis of cells in periprosthetic infected tissues; a5) Preparation of products for inhibiting inflammation, necrosis or fibrosis of infected tissues around prostheses.

[0016] The "pharmaceutical composition" of the present invention comprises an inhibitor or antagonist for Treg cell CTLA4 and / or CXCR6 targets, and optionally one or more pharmaceutically acceptable carriers, excipients and / or diluents, and the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications within the scope of reasonable medical judgment, and are commensurate with a reasonable benefit / risk ratio. As used herein, the phrase "pharmaceutically acceptable carrier, excipient and / or diluent" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid or solvent encapsulating material, which maintains the stability, solubility or activity of the inhibitor of the present disclosure.

[0017] The "pharmaceutical composition" disclosed in the present invention can be administered by various routes and formulated according to different routes of administration. Including but not limited to parenteral administration, subcutaneous injection, intravenous injection (including push injection), intramuscular injection and intra-arterial injection. Since the administration of parenteral dosage forms usually bypasses the patient's natural defense against pollutants, parenteral dosage forms are preferably sterile or can be sterilized before being administered to the patient. Examples of parenteral dosage forms include but are not limited to solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable injection vehicle, suspensions ready for injection, controlled release parenteral dosage forms, and emulsions.

[0018] The present invention also provides a use of a substance in preparing a product for detecting or evaluating periprosthetic infection, wherein the substance is any one of the following c1) to c3), c1) substances that detect at least one gene or protein among CTLA4, CXCR6, and CXCL16; c2) a substance for detecting Treg cells, wherein the Treg cells express FOXP3 and CTLA4; c3) Detecting a substance of MDSCs cells, wherein the MDSCs cells are CD11b+CD33+HLA-DR- cells.

[0019] The study in this invention shows that myeloid-derived suppressor cells (MDSC) recruit Treg cells expressing FOXP3 and CTLA4 through CXCL16-CXCR6, leading to the formation of an immunosuppressive microenvironment in PJI, making PJI difficult to cure.

[0020] As some specific embodiments, quantitative detection of at least one gene of CTLA4, CXCR6, and CXCL16 can be used to evaluate peri-prosthetic infection, and detection of Treg cells and / or MDSCs cells can also be used to evaluate peri-prosthetic infection.

[0021] In specific embodiments, it can be understood that "determining" and "detecting" have equivalent meanings, and "detecting" can be carried out via a method for detecting amplification products (for example, but not limited to PCR, RT-PCR, q-PCR, etc.), or other suitable methods for detecting nucleic acids / genes / proteins / markers known in the art can be used.

[0022] As other specific embodiments, CTLA4 and / or CXCR6 are used as targets in any of the following d1)-d5), a1) Preparation of products for preventing, treating, alleviating or ameliorating periprosthetic infection; a2) Preparation of products that inhibit the occurrence and development of periprosthetic infection; a3) Preparation of products that inhibit proliferation and / or growth of cells in periprosthetic infected tissues; a4) Preparation of products that promote or induce apoptosis of cells in periprosthetic infected tissues; a5) Preparation of products for inhibiting inflammation, necrosis or fibrosis of infected tissues around prostheses.

[0023] The products described in the present invention include but are not limited to kits, biochips or medicines.

[0024] In a specific embodiment, the kit has a physical form, for example, the kit can be a container having one or more spaces, wherein the one or more spaces are used to accommodate materials or devices such as controls, standards, and detection reagents.

[0025] The present invention also provides a method for constructing an animal model of periprosthetic infection, which increases or improves the content and / or activity of CTLA4 and / or CXCR6 proteins in the animal, thereby obtaining an animal model of periprosthetic infection with severe lesions.

[0026] The technical effects achieved by the present invention are: The present invention identified Treg cells by the expression of FOXP3 and CTLA4, and GSVA enrichment analysis found that Treg in the PJI group had stronger immunosuppressive function. Through flow cytometry verification, we found that Treg in the PJI synovium was significantly higher than that in the AF and OA groups; Then the present invention performed flow cytometry to verify the MDSCs subpopulation, and used CD11b+CD33+HLA-DR- to define the MDSCs subpopulation. The results showed that compared with the OA group and AF group, the proportion of MDSCs subpopulation in human synovial tissue PJI was significantly higher than that in the OA and AF groups, which was statistically significant (P < 0.01) ( Figure 4 ).

[0027] The present invention uses Cellchat to perform cell interaction analysis on MDSCs and Tregs, and finds that there is a rich interaction relationship between the two cells. Myeloid-derived suppressor cells (MDSCs) recruit Tregs through CXCL16-CXCR6, leading to the formation of an immunosuppressive microenvironment for PJI. PJI is difficult to cure, and CXCR6 can be used as a therapeutic target for PJI to improve the cure rate of PJI. Experimental verification shows that knocking out the target CXCR6 of Tregs and using anti-CTLA4 have potential therapeutic effects on reducing bacterial colonization and biofilm formation associated with PJI. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1: Overall landscape of single cell subsets in the synovium of the knee joint. A: UMAP of synovial cell subsets in PJI, osteoarthritis, and prosthesis loosening. B: Harmony merged UMAP map after removing batch effects. C: UMAP map of markers of various synovial cell subsets. D: Bar graph of cell subset ratios. E: Bar graph of cell subset number. F: Violin plot of markers of various synovial cell subsets.

[0029] Figure 2: Treg cells increased significantly in PJI and highly expressed CXCR4. A: UMAP of T cell subsets. B: UMAP of T cell subset markers. C: Treg cell ratio bar graph, Treg cell subsets increased significantly in PJI. D: T cell subset ratio bar graph. E: T cell subset number bar graph. F: GSVA enrichment analysis of Treg cell subsets, Treg in PJI was more enriched in immunosuppression-related pathways. GH: The proportion of Treg cells in the PJI group was significantly higher than that in the AF and OA groups (p < 0.05).

[0030] Figure 3:Treg cells increased significantly in PJI and highly expressed CXCR4. A: UMAP of T cell subsets. B: UMAP of T cell subset markers. C: Treg cell ratio bar graph, Treg cell subsets increased significantly in PJI. D: T cell subset ratio bar graph. E: T cell subset number bar graph. F: GSVA enrichment analysis of Treg cell subsets, Treg in PJI is more enriched in immunosuppression-related pathways.

[0031] Figure 4 : Validation results by flow cytometry; We found that the number of Treg in the synovium of the PJI group was significantly higher than that of the AF and OA groups (p < 0.05).

[0032] Figure 5 : Diagram of the interaction between MDSCs and Treg cells. AB: Interaction intensity and number between MDSCs cell subsets and T cell subsets; CD: Bar chart comparing interaction pathways between OA, AF, and PJI groups, the CXCL interaction pathway was significantly enhanced in PJI; EF: The CXCL16-CXCR6 interaction between MDSCs and Treg cells was most obvious in the PJI group.

[0033] Figure 6 : PCR identification results; the genotypes determined by F1 / R1 primers include (flox / +) heterozygote, (flox / flox) homozygote, and (+ / +) wild type. F2 / R1 primers are used to breed tool mice and confirm that the identified mice carry the corresponding specific Cre gene to determine whether the target tissue has been specifically knocked out. The results indicate that the conditional knockout mouse (Cxcr6[fox / flox], Foxp3YFP-Cre[KI / +]) was successfully constructed.

[0034] Figure 7 :The successful construction of the mouse PII model. (A) Photos of the surgical area of ​​the control group and PJI group mouse models; (B) X-rays of the knee joints of mice loaded with prostheses in extension and flexion positions showed that the prosthesis was in a good position after implantation.

[0035] Figure 8 : The results of single-cell sequencing analysis of knee joint tissue 35 days after surgery in the mouse PII model verified the successful modeling and the successful construction of CXCR6 conditional knockout mice (Cxcr6[flox / flox,Foxp3YFP-Cre[KI / +]) (CKO-CXCR6). Detection chart: The bar graph shows the proportion of each T cell subset in the PJI, AF and OA groups (top); the dot plot shows the expression of marker genes in each T cell subset (bottom).

[0036] Fig. 9:Bone mass and signs of bone infection, including periosteal reaction and osteolysis, were detected in each group by X-ray and micro-CT (left). Milder signs of bone infection were observed in the CKO-CXCR6 group and the anti-CTLA4 group. Using micro-CT analysis to evaluate changes in knee microstructure and bone remodeling, we found that targeted CXCR6 knockout of Tregs promoted defense against infection and resulted in higher BV / TV and Tb.N values ​​than those observed in the PJI group (statistical charts) (comparative analysis, * P < 0.05. ** P < 0.01, *** P < 0.001).

[0037] Fig.10 : AB: Representative images of live bacteria around implants counted by diffusion plate and statistical data of diffusion plate assay. Data are shown as mean ± SD, n >= 5 (comparative analysis, * P < 0.05. ** P < 0.01, *** P < 0.001). C: Representative images of SEM microscopy of Staphylococcus aureus and related biofilm formation on the implant surface of each group. Scale bar, 10 μm.

[0038] Fig.11 :GJ:representative histological images of sagittal sections of knee joints of mice in each group 35 days after surgery and statistical analysis of histopathological scores of sections; K is the evaluation of weight-bearing activity of mice, representative images of gait ink of mice in each group: RH, right posterior (red); LH, left posterior (red); RF, right anterior (dark blue); LF, left anterior (dark blue). L is the statistical analysis of weight-bearing activity scores between groups, data are shown as mean ± SD, n >= 5 (comparative analysis, * P < 0.05. ** P < 0.01, *** P < 0.001). DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

[0040] For commodities purchased in the test method, if the specific conditions are not specified, the test shall be carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, conventional products can be purchased from the market.

[0041] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice and testing of the present disclosure.

[0042] As used herein, the following words and terms shall have the meanings indicated: The GenBank number of the "CXCR6 gene" in the present invention is: 10663 / 80901 The "CXCR6 protein" in the present invention refers to the functional protein obtained by transcription and translation of the CXCR6 gene.

[0043] The GenBank number of the "CTLA4 gene" in the present invention is: 1493 / 12477 The "CTLA4 protein" in the present invention refers to the functional protein obtained by transcription and translation of the CTLA4 gene.

[0044] Example 1 Preliminary exploration of the immune microenvironment of prosthetic loosening (AF), periprosthetic joint infection (PJI), and osteoarthritis (OA) using single-cell transcriptome sequencing Peri-prosthetic Joint Infection (PJI) is a common catastrophic complication after total joint replacement. Our team found that in the PJI microenvironment, Treg constitutes the key cells of immunosuppression and is the key factor that makes PJI difficult to cure. So far, no studies have performed single-cell sequencing on PJI to explore the periprosthetic immune microenvironment. Single-cell transcriptome sequencing is currently a particularly suitable technical means for studying disease microenvironment. Our team previously performed single-cell sequencing analysis on the knee synovium of patients with osteoarthritis (OA), aseptic prosthetic replacement failure (AF, APL) and PJI, dividing the synovial tissue into multiple cell subpopulations in order to analyze the role of each subpopulation in the disease separately. The R language was used for data analysis to divide the tissue into fibroblasts, endothelial cells, macrophages, T cells, mast cells, proliferative cells, and parietal cell subpopulations ( Figure 1 ). The samples were harmnoyed to remove the batch effect, so the fusion was good. The differences in the proportion of subpopulations between groups were found. The relative proportion of OA and PJI macrophages in the inflammatory environment was higher, but there was also heterogeneity between different samples.

[0045] We selected fibroblasts, macrophage subsets, and T cells for subsequent analysis. By comparing the cell proportions, we found that fibroblasts account for the largest proportion of cell subsets in the synovium; there is a higher proportion of immune cells in the PJI synovium, especially macrophages and T cells, which play an important role in PJI.

[0046] Synovial T cells can be divided into many different subpopulations, thus exerting different functions. Among them, Treg cells are most related to immunosuppression. A higher proportion of Treg cells can inhibit immune responses, promote the proliferation and spread of pathogens, lead to the colonization of Staphylococcus aureus, and are difficult to be cleared by the body itself. There are highly expressed chemokine receptors on Treg, which will be affected by the chemokine ligands corresponding to other cell subpopulations, and be recruited to the corresponding parts through ligand receptor interactions to play a role. CCR is a chemokine receptor that is selectively expressed in T cell subpopulations, especially Treg cells. In tumor cells that highly express their corresponding ligand CCL, it can be recruited to the tumor area through CCL-CCR interaction, leading to consequences such as tumor resistance. Therefore, it can be used as a therapeutic target to develop related drugs for treatment in clinical practice. In the preliminary experiment, we identified Treg cells by the expression of FOXP3 and CTLA4. GSVA enrichment analysis found that Treg in the PJI group had stronger immunosuppressive function. Through flow cytometry verification, we found that Treg in the PJI synovium was significantly higher than that in the AF and OA groups (Figure 2).

[0047] Example 2 Myeloid-derived suppressor cells (MDSC) recruit Tregs through CXCL16-CXCR6, leading to the formation of an immunosuppressive microenvironment in PJI. PJI is difficult to cure. CXCR6 can be used as a therapeutic target for PJI to improve the cure rate of PJI.

[0048] The reason why prosthetic loosening (AF), inflammatory osteolysis and periprosthetic joint infection (PJI) are difficult to treat may be related to the immunosuppressive microenvironment of chronic inflammation. Studies have shown that myeloid-derived suppressor cells (MDSCs) play an important role in the persistence of periprosthetic inflammation and PJI infection. MDSCs are a subset of innate immune cells that are known to reduce the host's immunoregulatory capacity to prevent collateral damage caused by excessive host immune response. MDSCs are divided into two subtypes, including polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Myeloid precursor cells abnormally differentiate in the bone marrow to produce a large number of MDSCs, which are further recruited to the site of infection to form an immunosuppressive microenvironment. The recruitment effect produced by chronic inflammatory stimulation is the key to ensuring that MDSCs function in the immune microenvironment, and blocking this recruitment mechanism is expected to improve the immunosuppression of the microenvironment.

[0049] The macrophage subsets were extracted separately for subsequent analysis and divided into 5 subsets, among which MDSCs were a less abundant subset, but it can be seen that the content in PJI was significantly higher than that in the OA group and AF group. We defined the function of each macrophage subset through GSVA enrichment analysis. MDSCs were enriched in chemokines, bacterial attraction, and T cell function inhibition-related pathways, indicating that there was a significant immunosuppressive function. In addition, we also verified the results through MDSCs gene scoring and found that the subgroup we defined as MDSCs had a higher score, and the MDSCs score of PJI was significantly higher among the three groups ( Figure 3 ).

[0050] Next, we performed flow cytometry to verify the MDSCs subpopulations, and used CD11b+CD33+HLA-DR- to define the MDSCs subpopulations. The results showed that compared with the OA group and the AF group, the proportion of MDSCs subpopulations in the human synovial tissue PJI was significantly higher than that in the OA and AF groups, which was statistically significant (P < 0.01) ( Figure 4 ).

[0051] We used Cellchat to analyze the cell interactions between MDSCs and Tregs and found that there were rich interactions between the two cells. Among them, the CXCL interaction pathway in the PJI group was significantly richer than that in the AF and OA groups. After comparing the interaction intensity and number between different cell subsets, it was found that the MDSCs cell subset had the strongest interaction with Treg. Further comparing the types of CXCL-related cell interactions, we found that the intensity of CXCL16-CXCR6 in PJI was significantly higher than that in the AF and OA groups. The above analysis shows that MDSCs are likely to recruit Tregs through CXCL16-CXCR6 ( Figure 5 ).

[0052] The following is the specific test effect verification process: Example 3 Construction of mouse Cxcr6 gene conditional knockout model (C57BL / 6J-Cxcr6em1Cflox / CyaCKOCMP-80901-Cxcr6-B6J-VA) 1. Methods (1) Construction of CRISPR-AI conditional knockout mice To further verify the effect of CXCR6+Treg as an intervention target on PJI, we first commissioned Saiye (Suzhou) Biotechnology Co., Ltd. to construct and obtain CRISPR-AI conditional knockout Cxcr6[flox / flox] mice, and then bred and cultivated the mice.

[0053] (2) Breeding of Cxcr6 transgenic mice The obtained CRISPR-AI conditional knockout mice were subsequently bred into homozygous form and mated with Foxp3YFP-Cre mice (Product No.: C001047) to obtain (Cxcr6[flox / flox, Foxp3YFP-Cre]) mice of any gender, 6-8 weeks of age, for subsequent experiments.

[0054] (3) Transgenic mouse verification We verified the transgenic mice by homology arm PCR and extracted mouse tail DNA. The primer information is as follows: Primer set 1 F1: 5'-AGTCTCTCAGTTCATTTGAGGAGG-3' R1: 5'-GAAGATCTTTCACCCACAAAAGTG-3' (flox / flox) homozygous: 368 bp (flox / +) heterozygotes: 368 bp and 299 bp (WT) Wild type: 299 bp Primer set 2: Foxp3YFP-Cre primers Cre-F: 5'-CAGAAGAACGGCATCAAGGTGAAC-3' Cre-R: 5'-TTGTTCAGCTTCACCAGGCA-3' Cre positive: ~452 bp After breeding the tool mice and confirming that the identified mice carry the corresponding specific Cre gene, add primer set 3 to determine whether specific knockout of the target tissue occurs: Primer set 3 F2: 5'-CATAGATGCCGGGAACTGTTAGAG-3' R1: 5'-GAAGATCTTTCACCCACAAAAGTG-3' Target tissue deletion: 479 bp 2. Results PCR verification results Figure 6 As shown in the figure, the genotypes determined by the F1 / R1 primers include (flox / +) heterozygotes, (flox / flox) homozygotes, and (+ / +) wild types. The F2 / R1 primers are used to breed tool mice and confirm that the identified mice carry the corresponding specific Cre gene, and then determine whether the target tissue has been specifically knocked out. The results indicate that the transgenic mice have been successfully constructed.

[0055] Example 4 Construction of PJI model combined with 3D printed tibial prosthesis implantation surgery in mice 1. Methods All animals used in this study and the related protocols have been approved by the Clinical Research and Experimental Animal Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. Female C57BL / 6 mice aged 9 to 16 weeks were used in all experiments and were housed under standard SPF conditions in the animal facility. A total of 24 mice received unilateral proximal tibial implant implantation (right tibia). Specific surgical method: An incision was made on the right knee side, the quadriceps fibers were separated medially, and then the anterior cruciate ligament and meniscus were removed, and the patella was dislocated to expose the tibial plateau. The tibial articular cartilage and proximal epiphysis were smoothed. Using a high-speed drill, a hole with a diameter of 0.9 mm was drilled in the tibial medullary canal, and the 3D-printed titanium implant was pressed and inserted into the hole, after which the extensor muscle was closed with resorbable sutures and the skin was closed with non-absorbable sutures. After suturing the joint incision site, half of the animals (n=12) were injected intraarticularly with approximately 10^4 Staphylococcus aureus colony units (CFU). The accuracy of the bacterial dose at the time of inoculation was confirmed by plating an equal amount of the syringe injection onto agar plates containing kanamycin and counting colonies after overnight culture. After surgery, mice were subcutaneously injected with buprenorphine (0.05 mg / kg) every 12 hours for 48 hours and closely monitored for their return to normal ambulation. Mice were injected intraperitoneally with 250 μg of anti-CTLA4 or isotype control antibody starting two days before surgery and infection and every two weeks thereafter until euthanasia on day 35.

[0056] 2. Results The results are as follows Figure 7 As shown, the constructed PJI mouse model and the sham operation group are compared as follows ( Figure 7 Left), obvious pus infiltration can be seen in the joint cavity of the PJI group. X-rays of the knee joints of mice loaded with prostheses were taken in extension and flexion positions, showing that the prosthesis was well in place ( Figure 7 right).

[0057] Example 5 Single-cell transcriptome sequencing detection and comparative analysis of immune microenvironment changes in Cxcr6 conditional knockout mice under PJI modeling 1. Methods Flow cytometry analysis was performed using a CYTEK Aurora flow cytometer. Cell suspensions were prepared and stained with FixableViability Stain 700 (BD Biosciences, 1:1000, 564997) at 4°C in the dark for 20 min. Subsequently, cells were incubated with Human Fc block (BD Pharmingen, 564219) at 4°C in the dark for 15 min. Fluorescent dye-labeled antibodies (CD45, CD3e) were added to the cells and incubated at 37°C for 20 min. Data analysis was performed using FlowJo software (Treestar, v10.8.1). Flow sorting was performed using a BD FACSAria III cell sorter equipped with a nozzle with a diameter of 70 mm. T cells (CD45+CD3e+) and suspensions were collected for single-cell sequencing after detection on a BD FACSAria III instrument. Sequencing data were further analyzed using bioinformatics technology.

[0058] 2. Results The results are as follows Figure 8 As shown in the figure, the proportion of T cell groups in NC, PJI and KO groups of mice sequenced by single cells was significantly lower than that in the control group. Tregs of mice are also characterized by high expression of Foxp3 and Ctla4, which are unique immunosuppressive cells. By comparing the proportion of subgroups, it can be seen that the proportion of Tregs in the KO group is significantly lower than that in the PJI group. Tregs of mice are also characterized by high expression of Foxp3 and Ctla4, which are unique immunosuppressive cells.

[0059] Example 6 Comparative analysis of the therapeutic effects of Cxcr6 conditional knockout on PJI by X-ray and microCT in mice 1. Methods The knee joints were fixed in 4% paraformaldehyde for 24 hours. The mouse knee joints were scanned using high-resolution micro-CT (BRUKER, USA) and all images were acquired under uniform parameters. 3D reconstruction of the bones of the knee joint was performed by drawing the outlines of transverse / longitudinal image slices in uniformly set regions of interest (ROI). These image data of the knee joints were used to reconstruct and quantify the cortical bone destruction and related bone mass changes around the implantation site of the 3D printed prosthesis. Trabecular bone measurements were performed in the implantation area of ​​the distal tibia to evaluate the trabecular bone microstructural parameters, including BV fraction (BV / TV) and trabecular bone number (Tb. N).

[0060] 2. Results Obvious signs of bone infection, including periosteal reaction and osteolysis, were detected on knee X-rays in the PJI and IgG groups. Mild signs of bone infection were observed in the CKO-CXCR6 and anti-CTLA4 groups ( Fig. 9Using micro-CT analysis to assess changes in knee microstructure and bone remodeling, we found that targeted CXCR6 knockout of Tregs promoted defense against infection and resulted in higher BV / TV and Tb.N values ​​than those observed in the PJI group ( Fig. 9 ).

[0061] Example 7 The CKO-CXCR6 group had milder inflammatory response and higher antibacterial ability 1. Methods (1) Bacterial plate count On day 35 post-infection, mice were euthanized to harvest implants and hindlimbs. To detect bacterial load around the prosthetic joint, 200ul of exudate was excised from the hindlimb infected with S. aureus and then homogenized in 800ul PBS. The homogenate was vortexed and serially diluted in PBS (10x, 100x). Each sample was incubated on TSB agar plates at 37°C for 12h, and colonies were counted.

[0062] (2) Electron microscopy detection of bacterial biofilm The prosthesis was observed using an electron microscope, and the morphological characteristics of the bacterial biofilm were: elliptical or spherical structure, and the size of the bacteria was between 0.05μm and 5μm.

[0063] 2. Results Compared with the PJI group, the CKO-CXCR6 group had fewer Staphylococcus aureus and related biofilms. The number of viable bacteria in the secretions around the knee implant was counted by plate method. Representative images of the four groups of plates 12 hours after inoculation are shown in Figure 10 A. The analysis showed that the number of viable bacteria (CFU) in the PJI and IgG groups was significantly higher than that in the CKO-CXCR6 and anti-CTLA4 groups (P < 0.01) ( Fig.10 B). According to the SEM (scanning electron microscopy) results, Staphylococcus aureus (S. aureus) and biofilm aggregates were observed on the implant surfaces of both the periprosthetic joint infection (PJI) group and the homologous control group. In contrast, fewer bacteria and related biofilms were found on the implant surfaces of the CXCR6- and anti-CTLA4-groups, and the inhibitory effect of CKO-CXCR6 was more significant than that of anti-CTLA4 ( Fig.10 C).

[0064] Example 8 CKO-CXCR6 can effectively inhibit the progression of PJI and improve the functional recovery of PJI 1. Methods (1) Preparation and sectioning of paraffin sections of mouse knee joints and hematoxylin-eosin staining (HE staining) After obtaining the joint tissues of each group of mice, mechanical damage was avoided as much as possible, and they were fixed with 4% paraformaldehyde for 24-48 hours, embedded in paraffin with EDTA, and sliced ​​and spread with a thickness of 5um. They were further stained with HE and imaged for analysis.

[0065] (2) Evaluation of weight-bearing activity in mice The weight-bearing activity of rats was assessed using inkblot analysis, and the weight-bearing activity of each rat was divided into full weight-bearing (3 points), partial weight-bearing (2 points), toe touching (1 point), or non-weight-bearing (0 points). The front paws of the mice were stained with dark blue ink, and the hind paws were stained with red ink, and the scoring results were statistically analyzed.

[0066] 2. Results To further investigate the sustained nature of the inflammatory and immune responses, we paraffin-embedded sections of the knee joints of each group of mice on day 35 and performed histological analysis by HE staining. The results are shown in Figure 11. The amount of tissue inflammation, necrosis, fibrotic tissue formation, and implant bone integration were scored using the HE staining scoring system for knee joint sections. H&E-stained histological sections of the knee joints of uninfected mice (NC) showed a mild inflammatory response. In contrast, more diffuse abscesses formed in the tissues of infected mice (PJI, IgG groups), with extensive tissue destruction in and around the joints and necrotic granuloma reactions surrounded by fibrotic sheets, but the above manifestations were significantly improved in CKO-CXCR6 ( Fig.11 GJ). We analyzed the joint function recovery and local inflammatory response of each group of mice through weight-bearing activity evaluation. The results showed that the mice in the CXCR6 group had higher scores, and the difference was statistically significant (P<0.05), indicating that the CXCR6 group had better joint function recovery and local inflammatory response ( Fig.11 These results suggest that CKO-CXCR6 can effectively inhibit the progression of PJI in the face of persistent and extensive tissue inflammation and ongoing infection.

Claims

1. Use of a CXCR6 antagonist or expression inhibitor in any of the following a1)-a5): a1) Preparation of products for preventing, treating, alleviating or ameliorating periprosthetic infection; a2) Preparation of products that inhibit the occurrence and development of periprosthetic infection; a3) Preparation of products that inhibit proliferation and / or growth of cells in periprosthetic infected tissues; a4) Preparation of products that promote or induce apoptosis of cells in periprosthetic infected tissues; a5) Preparation of products for inhibiting inflammation, necrosis or fibrosis of infected tissues around prostheses.

2. The application according to claim 1, characterized in that: The expression inhibitor includes any one of the following b1) to b5): b1) Products that inhibit the expression or activity of the CXCR6 gene; b2) inhibiting the transcription of CXCR6 gene into mRNA products; b3) inhibiting the translation of CXCR6 gene into protein products; b4) Products that inhibit the activity or function of CXCR6 protein; b5) Products that reduce the CXCR6 protein content.

3. The use according to claim 2, characterized in that: The product that inhibits the expression or activity of the CXCR6 gene is a substance that silences, knocks down or knocks out the CXCR6 gene.

4. The use according to claim 3, characterized in that: The substance for silencing, knocking down or knocking out the CXCR6 gene is miRNA, siRNA, dsRNA or shRNA.

5. The use according to claim 3, characterized in that: The substance for knocking out the CXCR6 gene is a gene editing tool for knocking out the CXCR6 gene, and the gene editing tool includes: a zinc finger protein ZFN gene editing system or a TALENs gene editing system or a CRISPR / Cas9 gene editing system.

6. Use of a pharmaceutical composition containing a CXCR6 antagonist or expression inhibitor in any of the following a1) to a5): a1) Preparation of products for preventing, treating, alleviating or ameliorating periprosthetic infection; a2) Preparation of products that inhibit the occurrence and development of periprosthetic infection; a3) Preparation of products that inhibit proliferation and / or growth of cells in periprosthetic infected tissues; a4) Preparation of products that promote or induce apoptosis of cells in periprosthetic infected tissues; a5) Preparation of products for inhibiting inflammation, necrosis or fibrosis of infected tissues around prostheses.

7. Use of a substance in the preparation of a product for detecting or evaluating periprosthetic infection, characterized in that: The substance is any one of the following c1)-c3), c1) substances that detect at least one gene or protein among CTLA4, CXCR6, and CXCL16; c2) detecting a substance of Treg cells, wherein the Treg cells express FOXP3 and CTLA4; c3) Detecting a substance of MDSCs cells, wherein the MDSCs cells are CD11b+CD33+HLA-DR- cells.

8. Use of CXCR6 as a target in any of the following d1)-d5), a1) Preparation of products for preventing, treating, alleviating or ameliorating periprosthetic infection; a2) Preparation of products that inhibit the occurrence and development of periprosthetic infection; a3) Preparation of products that inhibit proliferation and / or growth of cells in periprosthetic infected tissues; a4) Preparation of products that promote or induce apoptosis of cells in periprosthetic infected tissues; a5) Preparation of products that inhibit inflammation, necrosis or fibrosis of infected tissues around prostheses; None of the applications described include disease diagnosis or treatment.

9. A method for constructing an animal model of periprosthetic infection, characterized in that: The CXCR6 protein content and / or activity in animals is increased or enhanced, and combined with mouse 3D printed tibial prosthesis implantation surgery, to obtain an animal model of severe periprosthetic infection.

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