A method for regulating immune regulation of sepsis based on PIAS3-STAT3 SUMOylation regulation of regulatory T cell differentiation and application thereof

Abstract

This invention proposes a sepsis immunomodulation method based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation and its application, belonging to the fields of biomedicine and immunomodulation technology. Through immunofluorescence co-localization, immunoprecipitation, and molecular docking, it is clearly demonstrated that exosome-derived PD-L1 can enter CD4+. + Intracellular interaction with PIAS3 protein within T cells and influencing PIAS3 protein expression fills a gap in current research on the intracellular regulatory mechanism of exosomal PD-L1, and breaks through the traditional understanding that PD-L1 only functions through membrane surface receptors; by constructing a signaling axis involving PIAS3-STAT3 SUMOylation in the regulation of T cell immune differentiation, the balance of RORγt / Foxp3 expression and Foxp3 expression can be regulated. + Treg differentiation status. This method utilizes in vitro human CD4... + Both T cells and animal models of sepsis have shown clear immunomodulatory effects, providing new technical means for elucidating the immunosuppressive mechanism of sepsis and developing related immunotherapy strategies.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and immune regulation technology, specifically relating to a method for sepsis immune regulation based on PIAS3-STAT3SUMOylation to regulate the differentiation of regulatory T cells and its application. Background Technology

[0002] Sepsis is a severe systemic inflammatory response syndrome caused by infection and is one of the leading causes of death in critical care medicine. Recent studies have shown that the pathological process of sepsis includes not only an early inflammatory response but also a significant later state of immunosuppression. The main characteristics of the immunosuppressive phase include decreased immune cell function, immune dysregulation, and the depletion of regulatory T cells (Foxp3). + An abnormal increase in regulatory T cells (Tregs) leads to a decrease in the body's ability to clear pathogens and increases the risk of secondary infections.

[0003] In recent years, the role of the immune checkpoint molecule PD-L1 (Programmed Death Ligand 1) in immunosuppression has received widespread attention. PD-L1 is not only present on the cell membrane surface but also exists in body fluids in the form of exosomes. Exosomes are small extracellular vesicles with a diameter of approximately 30-200 nm, capable of carrying proteins and nucleic acid molecules to participate in intercellular signal transduction and playing an important role in immune regulation. Current research indicates that the occurrence of sepsis-related immunosuppression is closely related to abnormal activation of the PD-1 / PD-L1 immune checkpoint signaling pathway. The expression levels of PD-1 and PD-L1 are significantly elevated in sepsis patients and are closely associated with decreased immune cell function and poor patient prognosis. Current research generally believes that PD-L1 mainly inhibits T cell activation and cytokine release by binding to the PD-1 receptor on the surface of T cells, thereby leading to impaired immune function. For exosome-derived PD-L1, current research also mainly focuses on its binding mechanism with receptors on the surface of target cells. Whether it can enter the target cell and further participate in intracellular signal regulation is still lacking in systematic research.

[0004] Signal transducer and activator of transcription 3 (STAT3) is an important signal transduction molecule that plays a central role in T cell differentiation, regulation of inflammatory responses, and the formation of immune tolerance. STAT3 activity can be regulated through various post-translational modifications. Most existing research focuses on the regulation of STAT3 function by phosphorylation, while studies on other post-translational modifications are relatively limited. Recent studies have found that SUMOylation (small ubiquitin-like modification) can alter STAT3 stability and transcriptional activity, representing a key modification for regulating STAT3 function; however, its specific role and regulatory mechanism in immune function regulation remain unclear.

[0005] PIAS3 (Protein inhibitor of activated STAT3) belongs to the E3SUMO ligase family and is a specific E3SUMO ligase for STAT3. It catalyzes the SUMOylation modification of STAT3, thereby regulating the STAT3 signaling pathway. Current research reports that PIAS3 participates in the regulation of the STAT3 signaling pathway, but its specific mechanism of action in the immune regulation of sepsis remains fully elucidated.

[0006] In addition, Foxp3 + Treg cells play an important role in maintaining immune homeostasis and regulating immunosuppression. Foxp3 is present in sepsis patients. + A significantly elevated Treg ratio is one of the important factors leading to a decline in the body's immune function. However, current research on the regulatory mechanisms of Treg differentiation mainly focuses on transcriptional regulation, and there is still a significant gap in research on its upstream signaling regulation mechanisms.

[0007] In summary, while existing technologies have reported on the PD-L1 signaling pathway, exosome transport mechanisms, STAT3 signaling regulation, and PIAS3-mediated SUMOylation modification, they still suffer from the following core deficiencies: 1) They primarily focus on the mechanism of PD-L1's action on the cell membrane surface, lacking systematic research on the intracellular action of exosome-derived PD-L1; 2) They mainly focus on STAT3 phosphorylation modification, lacking systematic research on the role of STAT3 SUMOylation modification in immune regulation; 3) The key mechanism of PIAS3 in sepsis immune regulation has not been clearly elucidated; 4) There is a lack of systematic technical solutions based on stabilizing PIAS3 protein expression and regulating STAT3 SUMOylation modification to restore immune function, and precise methods for regulating sepsis immunity at the intracellular signaling modification level are still relatively scarce. These technological gaps severely limit the development of new strategies for precise regulation of sepsis immunosuppression. Summary of the Invention

[0008] This application addresses the problems in the prior art by providing a method for immunomodulation of sepsis based on PIAS3-STAT3SUMOylation to regulate the differentiation of regulatory T cells, and its application.

[0009] To achieve the above objectives, the technical solution adopted in this application is as follows: Firstly, a sepsis immunomodulation method based on PIAS3-STAT3SUMOylation to regulate regulatory T cell differentiation includes the following steps: Step 1: Isolate and obtain PD-L1 highly expressed exosomes; Step 2: Combine the PD-L1 highly expressed exosomes with CD4 + T cell co-culture allows exosomes to be enzymatically converted to CD4+. + T cell uptake, exosome-derived PD-L1 in CD4 + Intracellular interaction with PIAS3 protein in T cells stabilizes PIAS3 protein expression, promotes PIAS3-mediated SUMOylation of STAT3, and constructs the PIAS3-STAT3 SUMOylation signaling axis. Step 3: Adjust CD4 via the PIAS3-STAT3SUMOylation signal axis. + The expression balance between RORγt and Foxp3 in T cells promotes Foxp3. + Regulatory T cell differentiation enables immune regulation of sepsis.

[0010] Preferably, in step one, the isolation and acquisition of PD-L1 highly expressed exosomes specifically includes: Cell culture supernatant or animal / human serum samples were collected. Cells and cell debris were removed by centrifugation at 2000×g for 10-20 min, followed by centrifugation at 10000×g for 20-40 min to remove larger particles. The supernatant was then ultracentrifuged at 100000×g for 60-90 min to obtain exosome precipitate. The precipitate was resuspended in PBS buffer to obtain PD-L1 highly expressed exosomes. The morphology of the obtained PD-L1 highly expressed exosomes was observed using transmission electron microscopy, particle size distribution was detected by nanoparticle tracking analysis, and the expression of exosome marker proteins (CD9, TSG101, HSP70) and PD-L1 protein was detected by Western blot, thus completing the identification of PD-L1 highly expressed exosomes. The PD-L1 highly expressed exosomes are derived from lung epithelial cells or cells treated with inflammatory stimulation; the cells are cultured in a serum-free culture medium containing exosomes to reduce interference from exogenous vesicles and improve exosome purity.

[0011] Preferably, in step two, the amount of PD-L1 highly expressed exosomes added to the co-culture system is 10-100 μg / mL, preferably 20-50 μg / mL; and the co-culture time is 24 h.

[0012] Preferably, in step two, the CD4 + T cells were isolated using a magnetic bead sorting method, specifically: peripheral blood samples were collected, and peripheral blood mononuclear cells were separated by density gradient centrifugation, using CD4+. + Positive sorting was performed using a T-cell magnetic bead sorting kit to obtain CD4+ cells with a purity ≥90%. + T cells; in animal model validation, CD4 can also be obtained by isolating mouse spleen samples. + T cells. The CD4 + T cells were cultured in RPMI-1640 medium at 37°C and 5% CO2.

[0013] Preferably, in step two, the interaction and binding sites between PD-L1 and PIAS3 proteins are detected by one or more of the following methods: immunoprecipitation, immunofluorescence co-localization, and molecular docking analysis.

[0014] As a preferred embodiment, in step three, flow cytometry is used to detect the proportion of CD25 and Foxp3 double-positive cells, and to evaluate Foxp3... + The degree of differentiation of regulatory T cells was determined; the expression balance levels of Foxp3, RORγt and STAT3-related proteins were detected by Western blot.

[0015] As a preferred option, the method also includes an optional step four: Step four: By regulating the expression level of miR-497-5p, the expression level of PD-L1 in exosomes is adjusted, thereby regulating the activation level of the PIAS3-STAT3SUMOylation signaling pathway. Specifically, miR-497-5p expression level in cells or animals is regulated by transfecting miR-497-5p mimics or inhibitors, preferably by transfecting miR-497-5p inhibitors or administering antagomiR-497 in vivo. The upstream regulatory effect of miR-497-5p on PD-L1 and exosomal PD-L1 loading levels is evaluated by detecting PD-L1 expression levels in cells and PD-L1 expression levels in exosomes derived from serum or cell culture supernatant.

[0016] Secondly, the application of a drug based on PIAS3-STAT3SUMOylation-mediated regulation of regulatory T cell differentiation in sepsis immunomodulation.

[0017] Preferably, a sepsis immunomodulation method based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation can be used to prepare a signaling pathway for regulating the PIAS3-STAT3 SUMOylation signaling axis and modulating Foxp3. + Drugs or reagents that regulate T cell differentiation, remodel the immune differentiation state of sepsis-related T cells, or intervene in at least one function of sepsis-related immunosuppression signaling pathways.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are as follows: This application presents a sepsis immunomodulation method based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation and its application. Through immunofluorescence co-localization, immunoprecipitation, and molecular docking, it is clearly demonstrated that exosome-derived PD-L1 can enter CD4+. + Inside T cells, it forms a protein complex with PIAS3 protein and stabilizes PIAS3 protein expression, filling the gap in the existing technology for studying the intracellular regulatory mechanism of exosomal PD-L1 and breaking through the traditional understanding that PD-L1 only functions through membrane surface receptors. This application is the first to systematically demonstrate that PIAS3, as a STAT3-specific E3SUMO ligase, can regulate the functional state of STAT3 through mediating STAT3SUMOylation modification, thereby affecting the expression balance between RORγt and Foxp3, and ultimately promoting Foxp3 expression. +Regulatory T cell differentiation was studied, and a complete signaling regulatory chain of PIAS3-STAT3SUMOylation-RORγt-Foxp3 was constructed, clearly elucidating the core role of STAT3SUMOylation modification in immune regulation. This application verifies that miR-497-5p can directly target and regulate the expression level of PD-L1 in cells and exosomes, thereby regulating the activity of the PD-L1-mediated PIAS3-STAT3 SUMOylation signaling pathway in exosomes. A multi-level regulatory mechanism of miR-497-5p—PD-L1—PIAS3—STAT3 has been established, providing a new intervention target and strategy for regulating sepsis immunosuppression from the source. By modulating the exosomal PD-L1 and PIAS3-STAT3 SUMOylation signaling axes, Foxp3 can be significantly modulated. + Tregs have the ability to differentiate and regulate sepsis-related immune function. Their immunomodulatory effects have been clearly verified in both in vivo and in vitro models. This provides a new technical means for the recovery of sepsis-related immune function and immunomodulatory therapy for related diseases, and has good prospects for clinical translation and application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Figure 1 shows the identification results of PD-L1 highly expressed exosomes in Example 1 of this application; Figure A (1-A) is a transmission electron microscopy morphology image of the exosomes; Figure B (1-B) is a particle size distribution map of the exosomes; Figure C (1-C) shows the exosome marker proteins (CD9, TSG101, HSP70); Figure D (1-D) is a Western blot result of PD-L1 expression level detection in serum-derived exosomes from sepsis model mice. Figure 2 This image shows the results of CD4+ T cells taking up PD-L1 highly expressed exosomes observed by laser confocal microscopy in Example 1 of this application; the green fluorescence represents the PKH67-labeled exosome signal, and the blue fluorescence represents the DAPI-stained cell nuclear signal. Figure 3Figure 1 shows the detection results of the PIAS3-STAT3 SUMOylation signaling axis regulatory mechanism mediated by PD-L1 exosomes in Example 1 of this application. Figure A (3-A) is a volcano plot of differentially expressed genes in CD4+ T cells after treatment with PD-L1 exosomes; Figure B (3-B) is the result of Western blot detection of the protein expression levels of P-STAT3, STAT3, PIAS3, RORγt, and Foxp3 in CD4+ T cells after treatment with PD-L1 exosomes; Figure C (3-C) is a simulated result of the docking of the intracellular tail of PD-L1 with PIAS3 protein molecules; Figure D (3-D) is the result of co-immunoprecipitation (Co-IP) detection of the interaction between PD-L1 and PIAS3 proteins; Figure E (3-E) is the result of co-immunoprecipitation detection of the interaction between PIAS3 and STAT3; and Figure F (3-F) is the result of SUMO-IP detection of STAT3 SUMOylation levels. Figure 4 In Example 1 of this application, exosomal PD-L1 regulates Foxp3 + Figure 1 shows the results of flow cytometry analysis of regulatory T cell differentiation; Figure A (4-A) shows human CD4 cells after exosome PD-L1 treatment. + CD4 in T cells + CD25 + FoxP3 + The results of regulatory T cell proportion detection are shown in Figure B (4-B), which shows the proportion of Foxp3+ Tregs in wild-type and PD-L1 deficiency-related sepsis model mice; Figure C (4-C) shows the proportion of Foxp3+ Tregs after intervention with different doses of PD-L1 antibody. + The results of the detection of changes in Treg ratios; Figure D (4-D) shows the Foxp3 levels after GW4869 inhibited exosome release. + Graph showing the detection results of Treg ratio changes; Figure 5Figure 1 shows the results of miR-497-5p regulation of PD-L1 and exosomal PD-L1 expression detection in Example 1 of this application. Figure A (5-A) shows the results of immunofluorescence detection of PD-L1 expression levels in lung tissue of septic model mice injected with miR-497-5p inhibitors; Figure B (5-B) shows the results of Western blot detection of PD-L1 expression levels in serum-derived exosomals of septic mice injected with miR-497-5p inhibitors; Figure C (5-C) shows the results of dual-luciferase reporter gene assay detecting the target binding relationship between miR-497-5p and PD-L1; Figure D (5-D) shows the results of flow cytometry detection of changes in PD-L1 expression levels in BEAS-2B cells transfected with miR-497-5p inhibitors; Figure E (5-E) shows the results of Western blot detection of PD-L1 expression levels in BEAS-2B cells transfected with miR-497-5p inhibitors. Figure 5 shows the results of blot detection of changes in PD-L1 protein expression in BEAS-2B cells after miR-497-5p inhibition; Figure F (5-F) shows the results of Western blot detection of PD-L1 expression level in exosomes derived from cell culture supernatant regulated by miR-497-5p. Figure 6 For example, in Embodiment 1 of this application, exosomal PD-L1 enters CD4 + Immunofluorescence detection results of T cells co-localizing with PIAS3 intracellularly; where green fluorescence is PKH67-labeled exosome signal, purple fluorescence is PIAS3 protein signal, red fluorescence is PD-L1 protein signal, and blue fluorescence is DAPI-stained cell nuclear signal. Figure 7 Figure 1 shows the detection results of PIAS3-STAT3 SUMOylation signaling axis related proteins in CD4+ T cells derived from the spleen of septic mice in Example 1 of this application. Figure A (7-A) shows the results of detecting SUMO1 levels after immunoprecipitation (IP) with STAT3, which is used to evaluate the changes in STAT3 SUMOylation modification after treatment with exosomes that highly express PD-L1. Figure B (7-B) shows the results of Western blot detection of the protein expression levels of PIAS3, P-STAT3, STAT3, RORγt and Foxp3. Detailed Implementation

[0021] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0022] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0023] Example 1, the reagents and materials used in this example are as follows: CD4 + T-cell magnetic bead sorting kit was purchased from Miltenyi Biotec; RPMI-1640 medium, fetal bovine serum, and penicillin / streptomycin antibiotics were purchased from Gibco; PKH67 fluorescent dye was purchased from Sigma; Western blot-related antibodies (CD9, TSG101, HSP70, PD-L1, PIAS3, STAT3, SUMO1, Foxp3, RORγt, β-actin) were purchased from Cell Signaling Technology; immunoprecipitation kit and SUMO-IP kit were purchased from Thermo Fisher Scientific; miR-497-5p mimic, inhibitor, and negative control were purchased from Ribobio; C57BL / 6 mice used in the experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were SPF-grade. like Figures 1-7 As shown, a sepsis immunomodulation method based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation is described below: Step 1: Isolation, purification, and systematic identification of PD-L1 highly expressed exosomes, specifically: A1; Sample Collection: Human lung epithelial cell lines were cultured for 48 h in RPMI-1640 medium containing 10% exosome-free fetal bovine serum. The cell culture supernatant was collected for the isolation of cell-derived exosomes. Separately, serum samples were collected from sepsis model mice for in vivo validation of exosome PD-L1 expression levels. In in vitro functional experiments, human peripheral blood-derived CD4+ T cells were used as the main recipient cells.

[0024] A2: Exosome isolation: Exosomes were isolated using a combination of differential centrifugation and ultracentrifugation. The specific steps were as follows: The collected samples were centrifuged at 2000×g for 15 min at 4℃ to remove cells and cell debris; the supernatant was collected and centrifuged at 10000×g for 30 min to remove larger particulate impurities; the supernatant was transferred to an ultracentrifuge tube and ultracentrifuged at 100000×g for 70 min, the supernatant was discarded, and the exosome precipitate was obtained; the precipitate was resuspended in pre-cooled PBS buffer and ultracentrifuged again at 100000×g for 70 min, the supernatant was discarded, and the resulting precipitate was the purified exosomes, which were resuspended in 100 μL of pre-cooled PBS and stored at -80℃ for later use.

[0025] A3: Exosome identification: (1) Transmission electron microscopy observation: 10 μL of exosome resuspension was dropped onto a copper grid, allowed to stand at room temperature for 2 min, excess liquid was absorbed with filter paper, 2% phosphotungstic acid solution was added for negative staining for 2 min, and after drying at room temperature, the morphology of exosomes was observed and photographed using a transmission electron microscope. The results showed that the obtained particles had a typical cup-shaped vesicle structure, which was consistent with the morphological characteristics of exosomes. The results are attached. Figure 1 -A is shown; (2) Particle size distribution detection: The particle size distribution of the exosome resuspension was detected by nanoparticle tracking analysis (NTA). The results showed that the particle size was concentrated in the range of 30-200 nm, which is consistent with the particle size characteristics of exosomes. The results are attached. Figure 1 -B is shown.

[0026] (3) Protein expression detection: Total protein was extracted from exosomes and quantified using the BCA method. The expression of exosome marker proteins CD9, TSG101, and HSP70 was detected by Western blot, and the expression level of PD-L1 protein was also detected. The results showed that the obtained exosomes highly expressed CD9, TSG101, and HSP70, as shown in the figure below. Figure 1 As shown in -C; PD-L1 expression was significantly increased in serum-derived exosomes from the selected sepsis model mice, as shown in the attached figure. Figure 1 -D is shown; Step 2: Exosomes enter CD4 + The verification of T cells and their interaction with PIAS3 is as follows: B1: CD4 + T cell isolation and culture: Human peripheral blood samples were collected, and peripheral blood mononuclear cells were separated by density gradient centrifugation and cultured according to CD4+. + The T-cell magnetic bead sorting kit instructions were followed to perform positive sorting and obtain human CD4. + T cells. Cell purity was assessed using flow cytometry, and the obtained CD4+ cells were... + T cell purity ≥90%. The sorted CD4 cells... + T cells were seeded in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37°C in a 5% CO2 incubator. Subsequent assays included exosome uptake, immunofluorescence co-localization, immunoprecipitation, STAT3 SUMOylation, and Foxp3 assays. + Treg differentiation assays were all performed using human CD4. + T cells were the primary experimental subject.

[0027] B2: Exosome fluorescent labeling and uptake verification: PD-L1 highly expressed exosomes obtained in Example 1 were labeled with PKH67 fluorescent dye according to the dye instructions. After labeling, unbound dye was removed by ultracentrifugation with PBS. The labeled exosomes were then combined with CD4+. + T cells were co-cultured with exosomes at a final concentration of 30 μg / mL for 24 h. After culture, cells were fixed with 4% paraformaldehyde, and nuclear staining was performed with DAPI. Exosomes were observed on CD4+ using a laser confocal microscope. + Distribution of CD4 within T cells. Results showed... + A distinct green fluorescence signal of PKH67 was observed intracellularly in T cells, demonstrating that PD-L1-highly expressed exosomes can be converted by CD4+. + Effective uptake by T cells, as shown in the attached document Figure 2 As shown, the results indicate that PD-L1-overexpressing exosomes can be effectively taken up by CD4+ T cells and enter the cell.

[0028] B3: Validation of the interaction between PD-L1 and PIAS3: (1) Immunofluorescence co-localization: PD-L1 highly expressed exosomes were combined with CD4 + After T cell co-culture for 24 h, the cells were fixed, permeabilized, and incubated overnight at 4°C with PD-L1 and PIAS3 antibodies. Then, fluorescent secondary antibody was added and incubated at room temperature for 1 h. Nuclei were stained with DAPI, and protein co-localization was observed using a laser confocal microscope. Results showed that PD-L1 and PIAS3 co-localized on CD4+. + There is a clear colocalization phenomenon within T cells, as shown in the appendix. Figure 6 As shown in the merged images, PD-L1 highly expressed exosomes can be converted by CD4. + T cells effectively took up PD-L1, and exosome-derived PD-L1 and PIAS3 showed significant co-localization within the cell; Pearson's correlation coefficient analysis on the right side further indicated a high spatial correlation between the two, suggesting that exosome-derived PD-L1 enters CD4+. + T cells can then interact intracellularly with PIAS3.

[0029] (2) Co-immunoprecipitation (Co-IP): Collect CD4 after co-culture + T cells were used to extract total protein, and PIAS3 antibody was added. The mixture was incubated overnight at 4°C, followed by the addition of Protein A / G agarose beads and incubation at 4°C for 4 hours. After washing, the protein was eluted, and Western blot analysis was performed to detect PD-L1 binding. The results showed that PD-L1 protein expression was detected in the PIAS3 antibody-precipitated complex, demonstrating that exosome-derived PD-L1 can bind to CD4+. +T cells form a protein complex with PIAS3 protein, and the results are shown in the attached figure. Figure 3 -D is shown; simultaneously, an interaction between PIAS3 and STAT3 proteins can also be detected, as shown in the attached figure. Figure 3 As shown in -E, this further supports the view that exosomal PD-L1 can promote PIAS3-mediated STAT3 SUMOylation modification.

[0030] Step 3: PIAS3 mediates STAT3 SUMOylation and Foxp3 + Treg differentiation regulation, specifically: C1: The PD-L1 highly expressed exosomes obtained in Example 1 were combined with CD4 + T cells were co-cultured, with a control group (with equal volume of PBS), an exosome treatment group (final exosome concentration 30 μg / mL), and an exosome + PD-L1 blocking antibody group, co-cultured for 24 h; after treatment with exosomes expressing high PD-L1, CD4... + Volcano plot analysis of differentially expressed genes on T cells showed upregulation of PIAS3 and Foxp3 expression, as shown in the attached figure. Figure 3 As shown in Figure A; docking simulations of the PD-L1 intracellular tail with the PIAS3 protein molecule show a stable binding conformation, as shown in the attached figure. Figure 3 -C is shown.

[0031] C2: STAT3 SUMOylation Level Detection: Cells from each group were collected, and proteins were extracted using the SUMO-IP kit and immunoprecipitated. Following the kit instructions, the STAT3 SUMOylation level was detected by Western blot, along with the expression levels of PIAS3 and total STAT3 proteins. Results showed that compared to the control group, the exosome-treated group exhibited significantly increased PIAS3 protein expression and STAT3 SUMOylation levels. The addition of PD-L1 blocking antibody significantly inhibited these changes, demonstrating that exosomal PD-L1 can promote PIAS3-mediated STAT3 SUMOylation modification by binding to PIAS3, successfully constructing the PIAS3-STAT3 SUMOylation signaling axis. Results are attached. Figure 3 -F is shown; C3: Total protein was extracted from cells in each group, and the expression levels of Foxp3 and RORγt proteins were detected by Western blot. The results showed that compared with the control group, Foxp3 protein expression was significantly increased and RORγt protein expression was significantly decreased in the exosome-treated group. This regulatory effect was significantly reversed after the addition of PD-L1 blocking antibody, demonstrating that the PIAS3-STAT3 SUMOylation signaling axis can regulate the balance of RORγt and Foxp3 expression, as shown in the appendix. Figure 3-B is shown; C4: CD4 was detected by flow cytometry. + CD25 + FoxP3 + The proportion of regulatory T cells. Results showed that, compared to the control group, the exosome-treated group had a higher proportion of Foxp3. + The proportion of Treg cells increased significantly; PD-L1 blocking antibody and Foxp3 were added. + The increase in the Treg ratio was significantly reduced, proving that the PIAS3-STAT3 SUMOylation signal axis in Foxp3... + It plays a key regulatory role in the differentiation of regulatory T cells; among them, exosomes with high expression of PD-L1 treat CD4 + T cells followed by Foxp3 + The proportion of Tregs increased significantly, and the results are shown in the attached figure. Figure 4 As shown in -A; anti-PD-L1 antibodies can dose-dependently inhibit exosomal PD-L1-induced Foxp3. + Treg differentiation, as shown in the appendix Figure 4 -C is shown; furthermore, in animal model validation, there was a difference in the proportion of Tregs between wild-type and PD-L1 deficiency-related sepsis model mice, suggesting that PD-L1 plays an important role in the regulation of Treg differentiation in vivo, as shown in the appendix. Figure 4 -B shows that after inhibiting exosome release with GW4869, Foxp3 + The decreased proportion of Tregs further suggests that the exosome pathway is involved in PD-L1-mediated regulation of Treg differentiation, as shown in the attached figure. Figure 4 As shown in Figure -D, this further demonstrates that exosomal PD-L1 regulates Foxp3. + They play a crucial role in the differentiation of regulatory T cells.

[0032] Step 4: upstream regulation of PD-L1 and downstream signaling pathways by miR-497-5p, specifically as follows: D1: Cell transfection: BEAS-2B lung epithelial cells were seeded in culture plates. When the cell confluence reached 70%, miR-497-5p inhibitor and corresponding negative control were transfected at a concentration of 50 nM. The transfection procedure was performed according to the Lipofectamine 3000 reagent instructions.

[0033] D2: In vivo miR-497-5p inhibition experiment in animals: After establishing a septic mouse model, antagomiR-497 was injected via the tail vein to inhibit miR-497-5p expression in vivo, while the control group received the corresponding negative control. Lung tissue and serum samples were collected after administration. Immunofluorescence was used to detect PD-L1 expression levels in lung tissue, and serum-derived exosomes were separated using differential centrifugation combined with ultracentrifugation. Total protein was extracted from the exosomes, and PD-L1 expression levels were detected by Western blot. Results showed that compared with the negative control group, antagomiR-497 treatment enhanced PD-L1 expression in the lung tissue of septic mice and increased PD-L1 expression in serum-derived exosomes, suggesting that inhibiting miR-497-5p can promote PD-L1 and exosomal PD-L1 expression in vivo. (See attached image) Figure 5 As shown in -A and 5-B; D3: In vitro validation of miR-497-5p's targeted regulation of PD-L1: Dual-luciferase reporter gene assay results showed that miR-497-5p can bind to the PD-L1 3′UTR region and inhibit reporter gene activity, suggesting that PD-L1 is a potential target gene of miR-497-5p. (See attached image) Figure 5 -C is shown. Further analysis in BEAS-2B cells revealed that inhibition of miR-497-5p increased the expression level of PD-L1 on the cell surface, enhanced PD-L1 expression in total cellular protein, and synchronously increased PD-L1 expression in exosomes derived from cell culture supernatant, as shown in the attached figure. Figure 5 As shown in -D, 5-E, and 5-F.

[0034] D4: Based on the above in vitro and in vivo results, miR-497-5p can negatively regulate PD-L1 expression in cells and exosomal PD-L1. Combined with the aforementioned ability of PD-L1-overexpressing exosomal tracts to enter human CD4+, this demonstrates the potential for PD-L1 overexpression. + The results, which showed that T cells were affected by the PIAS3-STAT3 SUMOylation signaling axis, indicate that miR-497-5p can act as an upstream regulator of exosomal PD-L1 levels, providing a basis for further regulation of the PD-L1 / PIAS3 / STAT3 SUMOylation axis and T cell immune differentiation status. Example 1 demonstrates the immunomodulatory effect of a sepsis immunomodulatory method based on PIAS3-STAT3 SUMOylation regulating regulatory T cell differentiation in a mouse model of sepsis. Specifically: Experimental animal grouping: Male C57BL / 6 mice aged 6-8 weeks, weighing 20-25 g, were selected and housed in an SPF-grade environment. After one week of acclimatization feeding, they were randomly divided into a sham-operated group, a CLP model group, and a PD-L1 high-expression exosome intervention group, with 6 mice in each group. (See attached...) Figure 4The experimental objectives shown in -B and 4-D are to set up a PD-L1 deficiency-related validation group or a GW4869 intervention group to evaluate the role of PD-L1 and exosome release in the regulation of Treg differentiation in vivo.

[0035] Sepsis Model Establishment: A mouse sepsis model was established using the CLP method. Mice were anesthetized with sodium pentobarbital via intraperitoneal injection. The abdomen was prepared and disinfected, and a midline abdominal incision was made to expose the cecum. The cecum was ligated at the distal half, and two penetrating punctures were performed using a 21G puncture needle. A small amount of intestinal contents was gently squeezed out, and the cecum was then repositioned. The abdominal wall incision was sutured layer by layer. In the sham surgery group, only the cecum was exposed through abdominal incision; no ligation or puncture was performed, and the remaining procedures were the same as in the model group. Postoperatively, all mice received a subcutaneous injection of 1 mL of sterile saline for fluid replacement.

[0036] Drug intervention: Two hours after CLP modeling, mice in the exosome intervention group were injected via tail vein with PD-L1-overexpressing exosomes obtained in Example 1 at a dose of 1×10⁻⁶. 9 Each mouse was given a particle, dissolved in 100 μL PBS; mice in the sham-operated group and CLP model group were injected with an equal volume of sterile PBS via the tail vein; the GW4869 intervention group was given GW4869 to inhibit exosome release; the anti-PD-L1 monoclonal antibody intervention group was given anti-PD-L1 monoclonal antibody in addition to treatment with high-expression PD-L1 exosomes to evaluate the effect of PD-L1 blockade on the PIAS3-STAT3 SUMOylation signaling axis and related protein expression; the PD-L1 deficiency-related validation group was used to evaluate the effect of PD-L1 deficiency on Foxp3. + The impact of Treg differentiation.

[0037] Sample collection and testing: 24 hours after modeling, mice in each group were sacrificed by cervical dislocation, and spleen tissue and peripheral blood samples were collected aseptically.

[0038] (1) Flow cytometry detection: A single-cell suspension of spleen was prepared, and CD4 in the spleen was detected by flow cytometry. + CD25 + FoxP3 + The proportion of regulatory T cells. Results showed that, compared to the sham-operated group, the CLP model group mice had significantly higher levels of Foxp3 in their spleens. + The proportion of Treg cells was significantly increased; compared with the CLP model group, the Foxp3 concentration in the spleen of mice in the high PD-L1 exosome intervention group was significantly increased. + The proportion of Tregs further increased significantly, demonstrating that the PD-L1-overexpressing exosomes of the present invention can significantly promote Foxp3 in vivo. + Regulatory T cell differentiation, and Foxp3 in PD-L1 loss-related models. +The decrease in Treg proportions can be partially reversed by exogenous supplementation of PD-L1-overexpressing exosomes, as shown in the attached figure. Figure 4 -B shows; GW4869 inhibits exosome release after Foxp3 + The proportion of Treg decreased, as shown in the attached figure. Figure 4 As shown in Figure -D, both PD-L1 and exosome release processes are involved in the regulation of Treg differentiation in vivo.

[0039] (2) Protein levels and STAT3 SUMOylation detection: Proteins were isolated and extracted from mouse spleen-derived CD4+ T cells, and the expression levels of PIAS3, Foxp3, RORγt, STAT3, and P-STAT3 proteins were detected by Western blot. Simultaneously, the STAT3 SUMOylation modification level was detected using immunoprecipitation combined with Western blot. Specifically, after immunoprecipitation with STAT3 antibody, the levels of P-STAT3 and SUMO1 were detected to evaluate the changes in STAT3 SUMOylation modification after treatment with high-expression PD-L1 exosomes. The results showed that under high-expression PD-L1 exosome treatment, significant STAT3 SUMOylation signals were detected in mouse spleen-derived CD4+ T cells. Western blot results showed that compared with the high-expression PD-L1 exosome treatment group, the expression of PIAS3 and Foxp3 proteins decreased, the expression of RORγt protein increased, and the phosphorylation level of STAT3 changed accordingly after anti-PD-L1 monoclonal antibody intervention, while the total STAT3 protein expression showed no significant change. The above results suggest that exosome-derived PD-L1 can participate in the regulation of PIAS3 expression and STAT3 SUMOylation modification in vivo, and further affect the RORγt / Foxp3 expression balance. The results are attached. Figure 7 As shown.

[0040] The above in vivo experimental results indicate that, in a sepsis animal model, the technical solution of this invention can regulate the stability of PIAS3, promote STAT3 SUMOylation modification, and thus regulate the expression balance between RORγt and Foxp3, thereby promoting Foxp3 expression. + Regulatory T cell differentiation has a clear immunomodulatory effect on sepsis in vivo, which verifies the effectiveness and feasibility of the technical solution of the present invention.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for immunomodulation of sepsis based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation, characterized in that, Includes the following steps: Step 1: Isolate and obtain PD-L1 highly expressed exosomes; Step 2: Combine the PD-L1 highly expressed exosomes with CD4 + T cell co-culture allows exosomes to be enzymatically converted to CD4+. + T cell uptake, exosome-derived PD-L1 in CD4 + The PIAS3 protein binds directly to T cells, stabilizing PIAS3 protein expression, promoting PIAS3-mediated SUMOylation of STAT3, and constructing the PIAS3-STAT3 SUMOylation signaling axis. Step 3: Adjust CD4 via the PIAS3-STAT3SUMOylation signal axis. + The expression balance between RORγt and Foxp3 in T cells promotes Foxp3. + Regulatory T cell differentiation enables immune regulation of sepsis.

2. The method for sepsis immunomodulation based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation according to claim 1, characterized in that, In step one, the isolation and acquisition of PD-L1 highly expressed exosomes specifically involves: Collect cell culture supernatant or animal / human serum samples. First, centrifuge at 2000×g for 10-20 min to remove cells and cell debris, then centrifuge at 10000×g for 20-40 min to remove larger particles. Then, centrifuge the supernatant at 100000×g for 60-90 min to obtain exosome precipitate. Resuspend the precipitate in PBS buffer to obtain PD-L1 highly expressed exosomes. The PD-L1-overexpressing exosomes are derived from lung epithelial cells or cells treated with inflammatory stimulation; the cells are cultured in a serum-free medium containing exosomes.

3. A method for sepsis immunomodulation based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation, as described in claim 1, characterized in that... In step two, the amount of PD-L1 highly expressed exosomes added to the co-culture system is 10-100 μg / mL, preferably 20-50 μg / mL; the co-culture time is 24 h.

4. A method for sepsis immunomodulation based on PIAS3-STAT3SUMOylation to regulate regulatory T cell differentiation, as described in claim 1, characterized in that... In step two, the CD4 + T cells were isolated using a magnetic bead sorting method, specifically: peripheral blood samples were collected, and peripheral blood mononuclear cells were separated by density gradient centrifugation, using CD4+. + Positive sorting was performed using a T-cell magnetic bead sorting kit to obtain CD4+ cells with a purity ≥90%. + T cells; in animal model validation, CD4 cells can also be obtained by collecting mouse spleens and isolating them. + T cells. The CD4 + T cells were cultured in RPMI-1640 medium at 37°C and 5% CO2.

5. The method for sepsis immunomodulation based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation according to claim 1, characterized in that, In step two, one or more of the following methods are used to detect the interaction and binding sites between PD-L1 and PIAS3 proteins: immunoprecipitation, immunofluorescence co-localization, and molecular docking analysis.

6. The method for sepsis immunomodulation based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation according to claim 1, characterized in that, In step three, flow cytometry was used to detect the proportion of CD25 and Foxp3 double-positive cells and to evaluate Foxp3 levels. + The degree of differentiation of regulatory T cells was determined; the expression balance levels of Foxp3, RORγt and STAT3-related proteins were detected by Western blot.

7. The method for sepsis immunomodulation based on PIAS3-STAT3 SUMOylation to regulate regulatory T cell differentiation according to claim 1, characterized in that, It also includes an optional step four: Step four: By regulating the expression level of miR-497-5p, the expression level of PD-L1 in exosomes is regulated, thereby regulating the activation level of the PIAS3-STAT3SUMOylation signaling pathway. Specifically, by transfecting miR-497-5p mimics or inhibitors, preferably by transfecting miR-497-5p inhibitors or by administering antagomiR-497 in vivo, the expression level of miR-497-5p in cells or animals is regulated, and the upstream regulatory effect of miR-497-5p on PD-L1 and exosomal PD-L1 loading level is evaluated by detecting the PD-L1 expression level in cells and the PD-L1 expression level in exosomes derived from serum or cell culture supernatant.

8. The application of the septic immune regulation based on PIAS3-STAT3SUMOylation to regulate the differentiation of regulatory T cells in a drug, as described in any one of claims 1-7.

9. The application of the septic immune regulation based on PIAS3-STAT3SUMOylation regulating regulatory T cell differentiation in a drug according to claim 8, characterized in that, It can be used to prepare signals for regulating the PIAS3-STAT3 SUMOylation signal axis and adjusting Foxp3. + Drugs or reagents that regulate T cell differentiation, remodel the immune differentiation state of sepsis-related T cells, or intervene in at least one function of sepsis-related immunosuppression signaling pathways.

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