A method for inhibiting proinflammatory pathogenic functions of th17 cells based on low temperature and its application

Abstract

The application belongs to the technical field of biological medicine and immunology, and particularly relates to a method for inhibiting proinflammatory pathogenic functions of Th17 cells based on low temperature and application thereof. + The T cells are cultured at a culture temperature lower than 37 DEG C for Th17 cell differentiation, so that the differentiation and / or proliferation of the Th17 cells are inhibited, and / or the expression of proinflammatory pathogenic genes in the Th17 cells is inhibited, and / or the expression and secretion of the anti-inflammatory cytokine IL-10 in the Th17 cells are promoted. The mechanism not only enhances the understanding of temperature inhibition of immunity, but also provides a low-temperature intervention strategy for autoimmune diseases in which Th17 cells are mainly involved, and has important application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and immunology, specifically relating to a method for inhibiting the pro-inflammatory and pathogenic function of Th17 cells in vitro by lowering the cell culture temperature, Th17 cells prepared using this method, and the application of this method in the preparation of related reagent kits and cell preparations. Background Technology

[0002] Th17 cells are a crucial type of CD4 cell in adaptive immunity. + Helper T cell subsets. Th17 cells are distinguished from other CD4 cells by secreting characteristic inflammatory cytokines such as IL-17A, IL-17F, and IL-22. + Helper T cell subsets such as Th1, Th2, and regulatory T cells (Tregs) are involved. IL-17A and IL-22 can induce the secretion of antimicrobial peptides, induce the expression of pro-inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), participate in the clearance of various pathogens such as Klebsiella pneumoniae, maintain mucosal immunity, promote the secretion of chemokines by astrocytes and microglia to recruit pro-inflammatory cells, damage and disrupt the blood-brain barrier, inhibit the maturation and survival of oligodendrocytes, thereby disrupting the sphingomyelin structure of neurons, and mediate the occurrence and exacerbation of the autoimmune disease multiple sclerosis (MS).

[0003] Th17 cells exhibit high plasticity, and their function is regulated by various external factors, such as cytokines, metabolites, and environmental stress. Studies have shown that Th17 cells induced by IL-6 and TGF-β1 express the anti-inflammatory cytokine IL-10, maintaining tissue and organ homeostasis without inducing inflammation or autoimmune diseases. Furthermore, Th17 cells induced by IL-23 or TGF-β3 co-express the T-box family transcription factor T-bet and secrete interferon-γ (IFN-γ) and GM-CSF, demonstrating high pathogenicity. Therefore, targeting the pathogenic function of Th17 cells has become an important strategy for intervening in inflammatory and autoimmune diseases.

[0004] Body temperature is a crucial biological parameter for maintaining cellular activity and regulating metabolism. Numerous studies have shown that changes in body temperature can participate in various pathological processes, including inflammation, autoimmune diseases, and cancer, by regulating the survival, migration, activation, and cytokine release of immune cells. Previous research has demonstrated that Th17 cells significantly enhance their differentiation capacity at feverish temperatures and promote SUMOylation of SMAD4 protein through TGF-β1 downstream signal transduction, thereby increasing the pro-inflammatory and pathogenic capacity of Th17 cells. However, there are still many gaps in our understanding of the regulatory effects of hypothermia on immune cell function, particularly regarding whether thermosensitive Th17 cells can respond to hypothermic stimuli and alter their characteristics and functions. It is noteworthy that clinical trials have shown that high-intensity hypothermia treatment using liquid cooling suits can lower the body temperature of multiple sclerosis patients and significantly improve visual acuity, motor function, and alleviate fatigue symptoms; however, the underlying mechanisms are unclear, and whether hypothermia can alleviate autoimmunity by regulating immune cell function requires further clarification.

[0005] Currently, interventions targeting the pathogenic function of Th17 cells mainly rely on drug treatments (such as antibody neutralization and small molecule inhibitors), which suffer from high costs and significant side effects. Therefore, there is an urgent need to develop new, simple, and low-cost methods to inhibit the pro-inflammatory and pathogenic function of Th17 cells, providing new intervention strategies for Th17 cell-mediated autoimmune diseases. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a method and its application based on low temperature to inhibit the pro-inflammatory and pathogenic function of Th17 cells. This invention achieves one or more of the following objectives by lowering the cell culture temperature to below normal body temperature (37°C): (1) inhibiting the differentiation and / or proliferation of Th17 cells in vitro; (2) inhibiting the expression of pro-inflammatory and pathogenic genes in Th17 cells; (3) promoting the expression and secretion of the anti-inflammatory cytokine IL-10 in Th17 cells; and (4) reducing the pathogenicity of Th17 cells.

[0007] This invention is specifically achieved through the following technical solutions: The first aspect of the present invention provides a method for inhibiting the pro-inflammatory and pathogenic function of Th17 cells in vitro, comprising: introducing naïve CD4+ into the in vitro cell line... + T cells were cultured at a temperature below 37°C to differentiate into Th17 cells, thereby inhibiting the differentiation and / or proliferation of Th17 cells, and / or inhibiting the expression of pro-inflammatory pathogenic genes in Th17 cells, and / or promoting the expression and secretion of the anti-inflammatory cytokine IL-10 in Th17 cells.

[0008] Furthermore, the culture temperature below 37°C is 34°C-36°C; preferably 35°C.

[0009] Furthermore, the culture medium used for Th17 cell differentiation culture contains the cytokine combination IL-6, TGF-β1, IL-23 and IL-1β; preferably, the concentration of IL-6 is 15 ng / mL, the concentration of TGF-β1 is 2 ng / mL, the concentration of IL-23 is 25 ng / mL and the concentration of IL-1β is 10 ng / mL; the differentiation culture time is 72 hours.

[0010] A second aspect of the invention provides the use of cryogenically treated Th17 cells in the preparation of cell preparations for alleviating Th17 cell-mediated autoimmune diseases, wherein the cryogenic treatment is the treatment of initial CD4 cells at a culture temperature below 37°C. + T cells were used to differentiate into Th17 cells.

[0011] Furthermore, the culture temperature below 37°C mentioned in this application is 34°C-36°C, preferably 35°C; the autoimmune disease mentioned is multiple sclerosis.

[0012] A third aspect of the present invention provides the application of low-temperature conditions in the preparation of a kit for inhibiting the pro-inflammatory and pathogenic function of Th17 cells in vitro, wherein the low-temperature conditions are cell culture temperatures of 34°C-36°C, preferably 35°C.

[0013] Furthermore, the aforementioned cell preparations are pharmaceutical preparations.

[0014] The technical solution provided by this invention has the following beneficial effects: (1) It is the first time that the cell culture temperature can be reduced to significantly inhibit the in vitro differentiation and proliferation of Th17 cells; (2) It is the first time that low temperature conditions can inhibit the expression of pro-inflammatory pathogenic genes (such as Il23r, Csf2, etc.) in Th17 cells, while significantly upregulating the expression and secretion of the anti-inflammatory cytokine IL-10, so that Th17 cells change from a pro-inflammatory pathogenic state to an inhibitory state; (3) RNA-seq analysis reveals that low temperature treatment makes the overall gene expression profile of Th17 cells closer to that of non-pathogenic Th17 cells, while being far away from pathogenic Th17 cells; (4) The in vivo EAE mouse model verifies that Th17 cells treated with low temperature have significantly reduced pathogenicity, and this inhibitory effect depends on the upregulation of IL-10; (5) The temperature regulation method provided by this invention is simpler, safer and lower cost than traditional drug treatment methods, and provides a brand-new technical solution for the preparation of related cell preparations and kits. Attached Figure Description

[0015] Figure 1To investigate the inhibition of Th17 cell differentiation by low-temperature culture. (A) Schematic diagram of Th17 cell differentiation induced by different in vitro temperatures. After isolating wild-type mouse Tn cells, they were activated with CD3 and CD28 antibodies and a combination of cytokines (IL-6 + TGF-β1 + IL-23 + IL-1β) was added to the culture medium. The cells were cultured in cell culture incubators at different temperatures for 3 days to complete differentiation into Th17 cells. Flow cytometry was then used to detect intracellular IL-17A expression. (B) Representative expression map (left) and proportion (right) of IL-17A in Th17 cells at different in vitro culture temperatures (n = 3) detected by flow cytometry. Data are presented as mean ± standard deviation and significance was calculated using one-way ANOVA. *p<0.05; ****p<0.0001; ns, no significance.

[0016] Figure 2 To investigate the inhibition of Th17 cell proliferation in vitro by low-temperature culture, flow cytometry was used to detect representative CellTrace Violet signal maps (left) and corresponding proliferation indices (right) of Th17 cells induced at 37°C and 35°C in vitro (n=4). Results were analyzed using the paired student's t-test, with ***p<0.001.

[0017] Figure 3 (A) Schematic diagram of Th17 cell differentiation and sorting induced by different temperatures in vitro. (Isolation) Il17a GFP After fluorescent reporter mouse Tn cells were activated using CD3 and CD28 antibodies, and a combination of cytokines (IL-6 + TGF-β1 + IL-23 + IL-1β) was added to the culture medium, the cells were cultured in cell culture incubators at different temperatures for 3 days to complete differentiation into Th17 cells, and then GFP was sorted by flow cytometry. + (B) qPCR was used to detect the relative levels of mRNA in Th17 cells induced at 37°C and 35°C, in order to... Gapdh(C) Flow cytometry analysis of representative expression of IL-17A and IL-10 in Th17 cells at different culture temperatures (left) and the proportion of IL-10 in Th17 cells (right) (n = 5). (D) ELISA analysis of IL-10 concentration in the supernatant of Th17 cells induced at 37℃ and 35℃ after 2 days of culture (n = 3). (E) Flow cytometry analysis of representative expression of IL-10 in Th17 cells at different culture temperatures (left) and the proportion (right) (n = 3). (F) Volcano plot showing differential gene expression in Th17 cells at 35℃ compared to Th17 cells at 37℃. (G) GO pathway analysis using upregulated genes in Th17 cells at 35℃ to identify significantly enriched immune-related signaling pathways. (H) GO pathway analysis using downregulated genes in Th17 cells at 35℃ to identify significantly enriched immune-related signaling pathways. (I) Enrichment analysis of defined Th17-related gene sets in Th17 cells at 35°C. Left: Gene set of TGF-β1 and IL-6 induced non-pathogenic Th17 cells (GSE39820); Middle: Gene set of TGF-β3 and IL-6 induced pathogenic Th17 cells (GSE39820); Right: Characteristic gene set of Th17 cells infiltrating the central nervous system of EAE mice (GSE74833). NES: Normalized enrichment score. Data are presented as mean ± standard deviation and significance was calculated using student's t-test (B, C, D) and One-way ANOVA (E). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, no significance.

[0018] Figure 4 (A) Schematic diagram of constructing an EAE model using Th17 cell reinfusion to inhibit the pathogenicity of Th17 cells. l17a GFP / - 2d2 TCR Mouse Tn cells were co-cultured with antigen-presenting cells at 37 ℃ or 35 ℃ to differentiate into Th17 cells in vitro and activated after 2 days. GFP-positive Th17 cells were then selected and injected via tail vein into the target cells. Rag2 - / -(A) Th17 cells were injected into mice, and 200 ng of pertussis toxin was injected intraperitoneally on days 0 and 2. (B) Daily EAE disease score statistics of mice infused with hypothermia-induced Th17 or conventional Th17 cells (n = 4, 37℃ Th17 group; n = 5, 35℃ Th17 group). (C) The left image shows a representative image of a cross-section of the spinal cord of mice infused with 35℃ or 37℃ Th17 cells on day 22 (n = 4). Scale bar is 300 μm. (D) Representative expression of IL-10 in different groups of Th17 cells in the EAE model detected by flow cytometry (left) and the proportion of IL-10 (right) (n = 4, 37℃ Th17 group; n = 5, 35℃ Th17 group). (E) Schematic diagram of the use of IL-10 neutralizing antibody or isotype control antibody in the construction of EAE model by Th17 cell infusion. Mice were used to establish the EAE model in (A). Starting on day 4, they were intraperitoneally injected with 150 μg of IL-10 neutralizing antibody or isotype control antibody every other day until sample collection and testing. (F) Daily EAE disease score statistics (n = 5) of mice that received Th17 cells at 35°C or 37°C after administration of IL-10 neutralizing antibody or isotype control. Data are presented as mean ± standard deviation and significance was calculated using student's t-test (C, D) and Two-way ANOVA (B, F). *p<0.05; **p<0.01;***p<0.001;****p<0.0001; ns, no significance. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to these embodiments. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] Example:

[0021] 1. Experimental materials

[0022] 2. Experimental Methods 2.1 In vitro differentiation of Th17 cells Healthy female mice with a C57BL / 6J background, approximately 8 weeks old, were euthanized using CO2 and then immersed in 75% ethanol. Spleen tissue was isolated in a biosafety cabinet. After releasing spleen cells by grinding, the cells were resuspended in 1 mL of PBS containing 3% FBS, centrifuged at 850 g, 4°C, for 7 min, the supernatant was discarded, and the cells were resuspended in erythrocyte lysis buffer (0.15 MNH4Cl, 1 mM NaHCO3, 0.1 mM EDTA), followed by lysis at room temperature in the dark for 10 min. After centrifugation at 850 g, 4°C, for 7 min, the cells were resuspended in PBS containing 3% FBS and analyzed using EasySep™ Mouse Naïve CD4. + Tn cells were isolated using the T Cell Isolation Kit, resuspended in primary T cell 1640 medium (RPMI 1640 + 10% fetal bovine serum + penicillin / streptomycin + 1 mM sodium pyruvate + 0.05 mM β-mercaptoethanol), and counted. Anti-CD3 and Anti-CD28 antibodies were coated with PBS solution to a final concentration of 1 µg / mL in 48-well plates at least 1 h in advance, and the bottom of the wells was washed once with PBS after coating. Differentiation media were prepared by adding different combinations of cytokines, and 0.5 × 10⁶ cells were resuspended in 500 µL of differentiation medium. 6 One Tn cell was added to a 48-well plate and placed in a cell culture incubator for 72 h to complete differentiation. Pathogenic Th17: IL-6 (15 ng / mL), TGF-β1 (2 ng / mL), IL-23 (25 ng / mL), IL-1β (10 ng / mL).

[0023] 2.2 Cell Trace Violet for tracing cell proliferation Cell Trace Violet reagent is dissolved in DMSO to a storage concentration of 5 mM and can be stored at -20°C. After washing the cells to be labeled with PBS, the cell pellet is obtained by centrifugation. The pellet is then resuspended in an appropriate volume of preheated PBS (37°C) to achieve a final concentration of 1 × 10⁻⁶. 6Cells were cultured at a concentration of 5 µM (5 cells / mL) and 1000× staining stock solution was added to achieve a working concentration. After mixing, the cells were incubated in a 37°C cell culture incubator in the dark for 20 min. Then, 5 volumes of pre-warmed 37°C complete culture medium were added and mixed well. The cells were incubated in a 37°C cell culture incubator for another 5 min to remove unbound dye. The supernatant was removed by centrifugation, and the cell pellet was resuspended in fresh pre-warmed 37°C culture medium and incubated for 10 min. Flow cytometry was then performed, or the cells were seeded into wells and cultured in a cell culture incubator.

[0024] 2.3 Extraction of total RNA from cells and real-time quantitative PCR Total RNA extraction from cells must be performed in a clean, RNase-free environment. After centrifugation, the cells are collected in a 1.5 mL EP tube, the culture medium is discarded, and the cells are rinsed once with pre-chilled PBS solution. The cells are then centrifuged again to obtain a cell pellet. 1 mL of TRIzol solution is added, and the cell pellet is resuspended by pipetting to ensure complete lysis. The pellet can be temporarily stored at -20 °C. 200 µL of chloroform solution is added, and the mixture is vigorously vortexed for 15 seconds, then incubated at room temperature for 3 minutes. Finally, it is centrifuged at 12000 rpm at 4 °C for 15 minutes. After centrifugation, the solution separates into three layers: a pink organic phase at the bottom, a white intermediate layer, and a colorless aqueous phase at the top. RNA is mainly present in the top aqueous phase. The aqueous phase (approximately 500 µL) is transferred to a new 1.5 mL EP centrifuge tube, avoiding the aspirate of the protein and organic phase layers. Add Glycogen to a final concentration of 50 µg / mL, mix well, and then add an equal volume of isopropanol solution to the water. Mix thoroughly by inverting the container and incubate at -20 °C for 15 min. Then centrifuge at 12000 rpm, 4 °C for 15 min to obtain the RNA precipitate. Wash the precipitate twice with 70% ethanol solution prepared with DEPC water. Carefully discard the ethanol solution and heat in a 40 °C metal bath to evaporate any remaining water and ethanol. Dissolve the dried RNA precipitate in an appropriate amount of DEPC water and determine the concentration using Nanodrop. It can be stored at -80 °C.

[0025] Prepare a 20 µL reverse transcription system: 1 µg RNA + 4 µL 5× PrimeScript RT Master Mix + DEPC water. The reverse transcription program is 37 ℃ for 15 min, 85 ℃ for 1 min, to obtain cDNA.

[0026] The cDNA solution was diluted 5-fold with DEPC water to obtain template DNA for real-time fluorescent quantitative PCR (QPCR). The QPCR reaction mixture was prepared using the QuantiNova SYBR Green PCR Kit: 5 µL 2× SYBR Green Mix + 0.05 µL ROX Dye + 1.4 µL 10 µM primers (0.7 µL each for forward and reverse) + 1 µL template DNA + 2.55 µL DEPC water. The reaction program was: 95 °C for 2 min, 95 °C for 5 s + 60 °C for 30 s (40 cycles). -ΔΔCt The relative quantification of genes among groups was calculated.

[0027] 2.4 Enzyme-linked immunosorbent assay (ELISA) The collected cell culture supernatant and other reagents were used to determine the IL-10 content using the Thermo Fisher-Invitrogen IL-10 Mouse Uncoated ELISA Kit. The procedure was performed according to the manufacturer's instructions, including: preparing the Capture Antibody with Coating Buffer and adding it to a 96-well plate overnight; washing the plate three times for 2 min each time; sealing the plate with ELISA / ELISPOT at room temperature for 1 h; washing the plate once; diluting the standard powder with ELISA / ELISPOT at 2× dilution to a total of 8 concentrations, adding it to the plate, and adding the test sample to selected wells, using ELISA / ELISPOT as a blank control; sealing the plate and incubating at room temperature for 2 h or overnight at 4 °C; washing the plate three to five times for 2 min each time; adding the Detection Antibody and incubating at room temperature for 1 h; washing three to five times for 2 min each time; adding Streptavidin-HRP and incubating at room temperature for 30 min; washing the plate five to seven times for 2 min each time; adding 1×TMB substrate and incubating at room temperature for 5-15 min; adding 1 M... The color development was terminated with sulfuric acid solution; the final plate reading was obtained by subtracting the absorbance at 570 nm from the absorbance at 450 nm. A standard curve was plotted based on the concentration and absorbance of the standard. The final concentration was calculated by subtracting the blank control group reading from the sample reading and substituting the result into the standard curve.

[0028] 2.5 Intracellular staining flow cytometry For flow cytometry detection of cytokines, cells need to be stimulated at 37 °C for 4 h beforehand. The stimulation medium consists of complete medium supplemented with a final concentration of 50 ng / mL PMA and 500 ng / mL Ionomycin, with 2 µM GolgiStop added during the last 2 h of stimulation. After stimulation, cells are washed once with PBS, centrifuged at 850 g, 4 °C, and 7 min, and then stained with cell surface antibodies and Fixed viability dye eFluor506 at 4 °C for 30 min. After washing cells with PBS, cells are fixed with 200 µL of Fixation / Permeabilization Solution Kit at 4 °C for 30 min. Antibodies against intracellular factors are prepared using permeabilization buffer and stained at 4 °C for 60 min. After washing cells with permeabilization buffer at 2300 g, 4 °C, and 7 min, the cell pellet is resuspended in an appropriate amount of PBS and filtered through a 70 µm filter into flow cytometry tubes for final analysis using a flow cytometer. If flow cytometry staining of nuclear transcription factors is involved, replace the fixation and permeabilization kit with the FOXP3 / Transcription Factor Staining Buffer Set.

[0029] 2.6 Isolation of immune cells from the mouse central nervous system After CO2 euthanasia of mice, the whole brain and spine were removed. The spinal cord was flushed out of the spine using HBSS solution. The brain and spinal cord were thoroughly minced and ground in 2 mL tubes. The tissue fragments were resuspended in 2 mL HBSS solution, and Collagenase D and DNase I were added to a final concentration of 2 mg / mL. Digestion was carried out at 37 °C for 20 min, with shaking to mix during digestion. After digestion, the sample was placed on ice to stop digestion. The tissue sample was thoroughly ground, filtered through a 70 µm filter membrane, and collected in a 15 mL tube. Centrifugation was carried out at 500 g, 4 °C for 7 min. The supernatant was discarded, and the pellet was resuspended in 10 mL of 37% Percoll (prepared with HBSS). Centrifugation was carried out at 500 g with the acceleration / deceleration set to 0, at room temperature for 20 min. The supernatant was aspirated. The cell pellet was resuspended in erythrocyte lysis buffer and lysed at room temperature in the dark for 10 min. The cell pellet was then obtained by incubation at 850 g, 4 °C for 7 min for subsequent analysis.

[0030] 2.7 Paraffin section preparation and Luxol Fast Blue (LFB) staining A 5 mm sample was taken from the lumbar region of the mouse spinal cord and fixed in 4% paraformaldehyde solution at room temperature for at least 30 min. Dehydration was then performed using ethanol-water solutions of progressively increasing concentrations (50%, 60%, 70%, 80%, 90%, 95%, 100%), for 10 min at each concentration. The sample was then pretreated in a 1:1 xylene and ethanol solution at room temperature for 5 min, followed by clearing in xylene for 5–10 min. The sample was then encapsulated in an embedding cassette and immersed in a 1:1 xylene and paraffin solution at 65 °C for 30 min. The sample was then transferred to a 65 °C paraffin solution for treatment three times: the first two times for 1 h, and the last time overnight. After paraffin immersion, the sample was encapsulated in a sectioning and embedding cassette and treated at -20 °C for 12 h before storage. Paraffin sections were prepared with a thickness of 5 µm and dried at 42 °C overnight.

[0031] Before LFB staining, sections were dewaxed twice in xylene for 5 min each time, followed by rehydration in 100% ethanol twice and 95% ethanol twice for 5 min each time. Staining was performed using the Luxol Fast Blue Myelin Stain Kit, with the following main steps: sections were stained with Solid Blue solution overnight at room temperature; excess staining solution was removed by rinsing in 95% ethanol for 30 s, followed by rinsing in Milli-Q for 5 min; sections were differentiated in lithium carbonate solution for 20 s, followed by further differentiation in 70% ethanol for 5 s; sections were quickly rinsed in Milli-Q for 5 min, and microscopic examination revealed clear differentiation between gray and white matter; sections were stained with tar violet for 30 s to 1 min; sections were quickly rinsed in Milli-Q for 5 min, and microscopic examination showed the gray matter stained purple; sections were dehydrated sequentially in 95% ethanol and 100% ethanol, and cleared twice in xylene; sections were mounted with neutral resin and dried in a fume hood. Finally, the results were observed and photographed under a microscope.

[0032] 2.8 Th17 reinfusion experimental autoimmune encephalomyelitis (EAE) disease model Around 8 weeks old, C57BL / 6J background simultaneously carries genotype l17a GFP / - and 2D2 + / -Female mice were used to isolate spleens, and Tn cells were isolated using a kit. Wild-type mice (WT) with a C57BL / 6J background were also used to isolate spleens, and T cell-free spleen cells were obtained through negative selection using the EasySep™ Mouse CD90.2 Positive Selection Kit II. These cells were used as antigen-presenting cells (APCs). Tn and APCs were mixed at a ratio of 3:1 and resuspended in 2 mL of differentiation medium. The mixtures were then evenly seeded into 12-well plates and placed in a cell culture incubator. The differentiation medium consisted of RPMI 1640 + 10% FBS + P / S + 1 mM sodium pyruvate + 0.05 mM β-mercaptoethanol + 15 ng / mL IL-6 + 2 ng / mL TGF-β1 + 25 ng / mL IL-23 + 10 ng / mL IL-1β + 10 µg / mL Anti-IL-4 + 10 µg / mL Anti-IFN-γ + 20 ng / mL MOG 35-55 + 2 µg / mL Anti-CD28. Five days after differentiation, cells were collected in 15 mL tubes, centrifuged at 850 g for 7 min at 4 ℃, and the supernatant was discarded. The cell pellet was resuspended in 2 mL of primary T cell culture medium containing 10 µg / mL Anti-IL-4 and 10 µg / mL Anti-IFN-γ, and evenly spread in six-well plates pre-coated with 1 µg / mL Anti-CD3 and 1 µg / mL Anti-CD28. The plates were then incubated for 2 days for activation. Subsequently, CD4-positive and GFP-positive viable cell populations were sorted using a BD sorting instrument after cell surface staining. The cells were then centrifuged at 850 g for 7 min at 4 ℃, the supernatant was discarded, and the cells were resuspended in an appropriate volume of PBS and counted. 4 × 10⁴ cells were then reinfused into approximately 8-week-old female mice lacking the Recombination Activating Gene 2 (Rag2) via tail vein injection. 6 Select cells. Simultaneously, administer 200 ng of pertussis toxin intraperitoneally, marking the day as day 0. On day 2, administer another 200 ng of pertussis toxin intraperitoneally. Model establishment is complete. Subsequently, observe and record the mice's behavior daily, scoring them according to the following criteria: 0 points, normal behavior; 1.0 point, strong tail but ataxia; 2.0 point, ataxia and frequent head turning; 3.0 point, unable to walk in a straight line; 4.0 point, lying on its side, unable to move normally; 4.5 point, continuous involuntary rolling; 5.0 point, near death or death. Samples are collected and relevant indicators are tested when any mouse reaches a score of 4.0.

[0033] 2.9 RNA Sequencing Analysis Total RNA was extracted and sent to Beijing Novogene Technology Co., Ltd. for paired-end 150 bp conventional 6G eukaryotic transcriptome sequencing using the Illumina NovaSeq 6000 platform. The returned raw data underwent FastQc data quality monitoring and trimmomatic software to remove adapters and low-quality bases. Paired FastQ files were aligned to the mouse Ensemble genome GRCm39 using STAR software, and featureCounts were used for quantitative counting. The raw counts were then normalized and differentially expressed using the R package DESeq2. Downstream pathway analysis was performed using the R package ClusterProfiler. Gene set enrichment analysis was performed using GSEA software, and plotting was done using the ggplot2 package.

[0034] 3. Experimental Results 3.1 Low temperature inhibits the in vitro differentiation of Th17 cells Changes in body temperature are closely related to the function of the body's immune system. Th17 cells, in particular, are highly plastic and can change their function in response to various external stimuli, such as high temperatures. However, whether low temperature, another common extracellular environmental stress, also has a regulatory effect on Th17 cells remains to be thoroughly investigated.

[0035] To avoid interference from complex in vivo environments and reactions that could affect the regulatory effect of low temperature on Th17 cells, the inventors first established an in vitro primary Th17 cell induction system. After isolating mouse Tn cells, cytokines (IL-6 + TGF-β1 + IL-23 + IL-1β) were added to the culture medium, and the cells were cultured in a cell culture incubator for 3 days to complete Th17 cell differentiation. By changing the cell culture incubator temperature, a low-temperature parameter was introduced, and the regulatory effect of low temperature on Th17 cells was investigated with temperature as the sole variable. The inventors first placed primary Tn cells under different low-temperature culture conditions (34℃, 35℃, 36℃) for 3 days to complete the differentiation into Th17 cells. By comparing Th17 cells cultured at normal temperature (37℃), the regulatory effect of low temperature on Th17 cell differentiation was explored. Figure 1 (A). By detecting the expression of the characteristic cytokine IL-17A in cells using flow cytometry, the inventors found that the expression ratio of intracellular IL-17A gradually decreased with decreasing culture temperature. A decreasing trend was observed at 36℃, but not significantly; the expression ratio of IL-17A decreased significantly at 35℃; and decreased drastically at 34℃. Figure 1 The results (B) indicate that low temperature can significantly inhibit the in vitro differentiation of Th17 cells.

[0036] 3.2 Low temperature inhibits the proliferation of Th17 cells In addition to its effect on cell differentiation, the inventors further explored the regulatory role of low temperature on Th17 cell proliferation. They used CellTrace Violet labeling to differentiate Tn cells isolated from wild-type mice into Th17 cells at 37°C and 35°C. Three days later, flow cytometry was used to detect CellTrace Violet signaling in Th17 cells. The flow cytometry results showed ( Figure 2 As the cell proliferation level increased, the CellTrace Violet signal decreased progressively (the "0" peak, which had never divided, generated subsequent "1", "2", "3", and "4" peaks through successive cell divisions). Compared to Th17 cells cultured at 37°C, the proliferation peaks induced by Th17 cells cultured at 35°C were reduced, and the signal was concentrated in the "1" peak after one division. The corresponding proliferation parameters also showed lower proliferation parameters for Th17 cells at 35°C. These results indicate that the low temperature of 35°C can significantly inhibit the proliferation of Th17 cells.

[0037] 3.3 Low temperature suppresses the pro-inflammatory phenotype of Th17 cells Previous results indicated that low-temperature culture (35°C) not only weakened the proliferation of Th17 cells in vitro but also inhibited their differentiation, further reducing Th17 cell production. Since Th17 cells exert their pro-inflammatory and pathogenic effects, they also depend on the secretion of pro-inflammatory cytokines. Therefore, the inventors further investigated the effect of low-temperature culture on the pro-inflammatory capacity of Th17 cells. Given that the proportion of Th17 cells induced in vitro was not 100%, to accurately investigate the IL-17A-positive Th17 cell population, the inventors first introduced IL-17A reporter mice tagged with green fluorescent protein (GFP). After isolating Tn cells from these mice, they differentiated them into Th17 cells at 37°C and 35°C. Subsequently, GFP cells were sorted using flow cytometry. + Further research will be conducted on the cell population. Figure 3 (A). The inventors first used qPCR technology to detect characteristic cytokine genes in Th17 cells differentiated at different temperatures. Il17a , Il17f The expression of the gene was found to be not significantly different, but the GM-CSF gene, which plays a pathogenic role in MS, was different. Csf2 Reduced expression of the IL-23 receptor gene, which mediates pro-inflammatory signaling in Th17 cells. Il23r The levels also decreased. In addition, the inventors detected a significant and dramatic increase in anti-inflammatory cytokines in Th17 cells differentiated at 35°C. Il10 Gene expression ( Figure 3Flow cytometry also detected higher expression of IL-10 protein in Th17 cells at 35°C. Figure 3 The C), and ELISA results also showed that Th17 cells at 35℃ could secrete more IL-10 protein (C), and the ELISA results also showed that Th17 cells at 35℃ could secrete more IL-10 protein (C). Figure 3 (D). Furthermore, the inventors compared the effects of different low-temperature conditions on promoting IL-10 expression in Th17 cells. Flow cytometry results showed that compared to 37℃, IL-10 expression in Th17 cells at 36℃ only showed an increasing trend, while the increase was most significant at 35℃. Further lowering the culture temperature to 34℃ did not significantly cause a further increase in IL-10. Figure 3 E).

[0038] To further analyze the regulation of pro-inflammatory capacity of Th17 cells by low temperature, the inventors used RNA-seq to deeply mine genes showing significant changes in Th17 cells at 35°C compared to those at normal temperature (37°C). The results showed that, in addition to... Il10 Low temperature conditions of 35°C significantly increased the levels of Treg-related genes. Tgfbr1 and FoxP3 The expression of [certain substances] was inhibited, while key pro-inflammatory factors and regulatory genes in Th17 cells, such as [other substances], were suppressed. Stat4 , Tgfb3 , Ccl20 , Ccr6 , Itga3 The expression ( Figure 3 The F). Further using gene ontology (GO) pathway enrichment analysis, the inventors found that genes significantly promoted at 35°C in Th17 cells were enriched in the negative immune regulatory pathway (F). Figure 3 The genes that are significantly suppressed at 35°C are mostly enriched in immune-related pathways, such as T cell activation and immune cell proliferation. Figure 3 Furthermore, the inventors used gene set enrichment analysis (GSEA) to compare the enrichment levels of representative gene sets of defined Th17 cells in different functional states at 35°C and 37°C. The results showed that Th17 cells induced at 35°C were closer to non-pathogenic Th17 cells, and further away from in vitro induced and in vivo EAE-induced pathogenic Th17 cells. Figure 3 (I).

[0039] These data indicate that, in addition to inhibiting the differentiation and proliferation of Th17 cells, low temperature can also suppress the pro-inflammatory phenotype of Th17 cells.

[0040] 3.4 Low temperature inhibits the pathogenicity of Th17 cells To more intuitively evaluate the inhibitory effect of low temperature on the pro-inflammatory function of Th17 cells, the inventors introduced a Th17 reinfusion-mediated EAE mouse model, and isolated... Il17a GFP 2d2 TCR Mouse Tn cells differentiated into Th17 cells at 37°C and 35°C, and were subsequently sorted for GFP. + Th17 cells were then reinfused into immunodeficient mice. Rag2 - / - In vivo, constructing an EAE disease model ( Figure 4 (A). The inventors, through continuous observation of the disease scores of mice that had received different Th17 cells, found that the disease scores of mice that received Th17 cells at 35°C were significantly lower than those of mice that received Th17 cells at 37°C. Figure 4 (B), and LFB staining of pathological sections also showed that the degree of demyelination in the spinal cord of mice that had been reinfused with Th17 cells at 35°C was lower (B). Figure 3 The C value indicates a milder disease severity. These results suggest that hypothermia significantly inhibits the pathogenicity of Th17 cells. Furthermore, previous results indicated that hypothermia significantly promoted the expression of the anti-inflammatory cytokine IL-10 in Th17 cells. To clarify whether hypothermia inhibits the pathogenicity of Th17 cells by inducing more IL-10, the inventors first monitored the difference in IL-10 expression in the central nervous system of mice using flow cytometry. The results showed that, consistent with pre-infusion levels, Th17 cells at 35°C still exhibited higher IL-10 levels after infusion into mice compared to Th17 cells at 37°C. Figure 4 (D). Subsequently, during the construction of the Th17 reinfusion EAE model, the inventors injected each group of mice with 150 μg of IL-10 neutralizing antibody or isotype control antibody every other day, starting from day 4. Figure 4 The results showed that the use of IL-10 neutralizing antibodies in mice that had been re-infused with 35℃ Th17 cells caused their disease scores to rise again. Figure 4 These data suggest that low temperature inhibits the pathogenicity of Th17 cells by promoting high expression of IL-10.

[0041] In summary, this invention investigated the functional regulation of Th17 cells by using an in vitro experimental system with temperature as the sole variable. It found that lowering the cell culture temperature (e.g., to 35°C) significantly reduced Th17 cell proliferation while inhibiting Th17 cell differentiation. Furthermore, low temperature significantly suppressed the expression of pro-inflammatory genes in Th17 cells while enhancing the secretion of the anti-inflammatory factor IL-10, resulting in a non-pathogenic state for Th17 cells overall. The establishment of a mouse EAE model by reinfusing Th17 cells further clarified that low temperature can inhibit the pathogenicity of Th17 cells by upregulating IL-10. In conclusion, this invention identifies the role of low temperature in inhibiting the pro-inflammatory and pathogenic capabilities of Th17 cells, expanding our understanding of how low temperature regulates immune system function. It also provides a novel and simple method to inhibit the pro-inflammatory and pathogenic functions of Th17 cells in vitro (compared to more common drug treatments), and offers a low-temperature intervention strategy for autoimmune diseases primarily involving Th17 cells, demonstrating significant application potential.

Claims

1. A method for inhibiting the pro-inflammatory and pathogenic function of Th17 cells in vitro, characterized in that, include: Initial CD4 + T cells were cultured at a temperature below 37°C to differentiate into Th17 cells, thereby inhibiting the differentiation and / or proliferation of Th17 cells, and / or inhibiting the expression of pro-inflammatory pathogenic genes in Th17 cells, and / or promoting the expression and secretion of the anti-inflammatory cytokine IL-10 in Th17 cells.

2. The method according to claim 1, characterized in that, The culture temperature below 37°C is 34°C-36°C; preferably 35°C.

3. The method according to claim 1 or 2, characterized in that, The culture medium used for Th17 cell differentiation culture contains the cytokine combination IL-6, TGF-β1, IL-23 and IL-1β; preferably, the concentration of IL-6 is 15 ng / mL, the concentration of TGF-β1 is 2 ng / mL, the concentration of IL-23 is 25 ng / mL and the concentration of IL-1β is 10 ng / mL; the differentiation culture time is 72 hours.

4. The application of cryogenically treated Th17 cells in the preparation of cell preparations for alleviating Th17 cell-mediated autoimmune diseases, characterized in that, The low-temperature treatment refers to the incubation of initial CD4 cells at a temperature below 37°C. + T cells were used to differentiate into Th17 cells.

5. The application according to claim 4, characterized in that, The culture temperature below 37°C is 34°C-36°C, preferably 35°C; the autoimmune disease is multiple sclerosis.

6. The application of low-temperature conditions in the preparation of a reagent kit for inhibiting the pro-inflammatory and pathogenic function of Th17 cells in vitro, characterized in that, The low-temperature condition is a cell culture temperature of 34℃-36℃, preferably 35℃.

7. The application according to claim 4 or 5, characterized in that, The cell preparation is a pharmaceutical preparation.

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