Method for regulating MO-MDSCs differentiation by PAD2 and application thereof

Abstract

The invention relates to a method for regulating and controlling MO-MDSCs differentiation through PAD2 and application of the method, and belongs to the technical field of biomedicine. Experiments prove that the immunosuppression capability of the MO-MDSCs can be enhanced by inhibiting the expression of PAD2 in the MO-MDSCs. In-vitro induction experiments show that high expression of PAD2 in the MO-MDSCs can promote the MO-MDSCs to be differentiated into M phi or DCs; the invention also finds that after the expression of PAD2 in the MO-MDSCs is inhibited, the MO-MDSCs are adoptively transferred, so that the morbidity of CIA mice can be obviously inhibited. The PAD2 is used for regulating and controlling the immunosuppression function of the MO-MDSCs, so that the plasticity regulation of the MO-MDSCs is realized, and a new thought is provided for treating rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to a method and application of PAD2 regulating MO-MDSCs differentiation, and belongs to the technical field of biomedicine. Background Art

[0002] Rheumatoid arthritis (RA) is caused by multiple factors, including environment, lifestyle, and genetics, which ultimately lead to the destruction of immune tolerance, which is manifested by the production of low-titer autoantibodies, mainly anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF), accompanied by increased cytokines and metabolic disorders, which usually occur before the development of obvious clinical joint diseases. Some triggering factors such as infection can further promote the production of ACPA, which is closely related to HLA-II genes and can cause T cell autoimmunity.

[0003] Myeloid cells are a highly diverse population. The mononuclear cell line in the bone marrow can terminally differentiate into macrophages (Mφ) and dendritic cells (DCs). Monocytes differentiate into Mφ and DCs in tissues under inflammatory conditions. Activation of these cells begins in response to strong signals, including pathogen-associated molecμlar patterns (PAMPs) and danger-associated molecμlar patterns (DAMPs). This activation is relatively short-lived, manifested by vigorous phagocytosis, respiratory bursts, and the release of a large number of inflammatory factors, and ends when the stimulus stops. In contrast, stimuli related to chronic infection, inflammation or cancer are relatively weak and persistent signals. The signals in this state can also induce the production of bone marrow cells. However, the myeloid-derived suppressor cells (MDSCs) generated in this state, although similar in morphology and phenotype to neutrophils and monocytes, have obviously different genomes, and their biological characteristics and functions have also changed greatly. The main feature of these cells is that they have the function of inhibiting various immune responses. The uncontrolled immune response caused by this extensive tissue damage and the continuous inflammatory stimulation prompt the body to produce a protective effect, which ultimately forms a mechanism for the accumulation of MDSCs. However, the role of MDSCs in different pathological conditions is not the same. For example, in the tumor environment, the powerful immunosuppressive function of MDSCs is conducive to the immune escape of tumors, while the MDSCs produced in the state of autoimmune diseases want to use their immunosuppressive ability to control the hyperinflammatory state of autoimmune diseases.

[0004] MDSCs include two subpopulations. The first is granulocyte-like or polymorphonuclear MDSCs (PMN-MDSCs), which are similar to neutrophils in phenotype and morphology; the second is monocytic MDSCs (MO-MDSCs). Immunosuppression is the main function of MDSCs, and the main target of MDSCs to exert their inhibitory function is T cells. MO-MDSCs can express nitric oxide (NO) and related cytokines to inhibit T cell responses in an antigen-specific and non-specific manner.

[0005] At present, studies on the role of MDSCs in RA have shown that, on the one hand, adoptive transfer of MDSCs in the spleen or bone marrow of CIA mice promotes disease progression and the differentiation of Th17 cells; on the other hand, MDSCs have the effect of alleviating disease progression in experimental models of autoimmune arthritis. Although the role of MDSCs in RA is not fixed, the number of MDSCs is undoubtedly increased in rheumatoid arthritis. Whether it has the effect of inhibiting or promoting inflammation, it is possible to provide a new method for the treatment of RA through plastic regulation of MDSCs. Therefore, whether in humans or in animal models, it is necessary to further study the effects of various factors on the immunosuppressive function of MDSCs and find suitable methods for MDSCs cell immunotherapy of RA. Summary of the invention

[0006] In order to overcome the defects in the prior art, the present invention provides a method and application of PAD2 regulating the differentiation of MO-MDSCs. The purpose of the present invention is to innovatively clarify the expression of PAD and citrullinated proteins in MDSCs and their subpopulations, that is, MO-MDSCs undergo citrullinated modification, thereby providing a new treatment idea for rheumatoid arthritis (RA).

[0007] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0008] The present invention first provides an application of PAD2 in regulating MO-MDSCs cell differentiation, wherein the application is achieved by regulating the expression level of PAD2.

[0009] Among them, the expression level of PAD2 is positively correlated with the degree of MO-MDSCs cell differentiation.

[0010] The present invention also provides a method for regulating the differentiation of MO-MDSCs cells, wherein the method comprises regulating the differentiation of MO-MDSCs cells by regulating the expression level of PAD2 in MO-MDSCs cells.

[0011] When the expression level of PAD2 is upregulated, the differentiation of MO-MDSCs cells is promoted; when the expression level of PAD2 is downregulated, the differentiation of MO-MDSCs cells is inhibited.

[0012] The present invention also provides a product obtained by the method for regulating MO-MDSCs cell differentiation, wherein the product includes MO-MDSCs cells with downregulated PAD2 expression obtained by inhibiting MO-MDSCs cell differentiation.

[0013] Furthermore, the product also includes Mφ cells or DCs cells obtained by promoting the differentiation of MO-MDSCs cells.

[0014] The present invention also provides the use of the product obtained by the method for regulating MO-MDSCs cell differentiation in the preparation of a drug for treating rheumatoid arthritis.

[0015] The present invention also provides a medicine for treating rheumatoid arthritis, wherein the medicine comprises a product obtained by the method for regulating MO-MDSCs cell differentiation.

[0016] Compared with the existing achievements, the beneficial effects of the present invention are as follows:

[0017] Citrullination (Cit) was first discovered by Rogers and Simmonds in 1958. It is a catalytic reaction process in which arginine attached to a peptide is converted to citrulline, and is a type of post-translational modification. The catalytic enzyme involved in citrullination is peptidyl arginine deiminase (PAD). Five PADs have been found in the human body, including PAD1, PAD2, PAD3, PAD4, and PAD6. PAD and its mediated citrullination have always been a hot topic in RA research. Citrullinated fibrinogen in RA joints was the first protein discovered to be a substrate for ACPA, and related studies have shown that citrullination in RA can occur extracellularly. Therefore, studying whether this modification exists in MDSCs and the regulatory effect of this modification on the function of MDSCs is expected to bring new ideas to the cell immunotherapy of MDSCs.

[0018] (1) The present invention provides for the first time the application of PAD2 in regulating the differentiation of MO-MDSCs cells. The present invention first detects the mRNA expression levels of five isozymes (PAD1, PAD2, PAD3, PAD4 and PAD6) in the spleen of CIA mice and MO-MDSCs by qRT-PCR, Western Blot, confocal microscopy, etc., and finds that PAD2 and PAD4 are expressed in MO-MDSCs cells. The present invention further knocks down the expression of PAD2 or PAD4 in MO-MDSCs cells and finds that knocking down PAD2 can inhibit the process of MO-MDSCs cells differentiating into Mφ and DCs, indicating that PAD2 can serve as a switch for MO-MDSCs cell differentiation.

[0019] (2) The present invention also provides a cell preparation for treating rheumatoid arthritis, the cell preparation comprising MO-MDSCs cells with downregulated PAD2 expression. The present invention found that downregulated PAD2 expression in MO-MDSCs cells can enhance the immunosuppressive function of MO-MDSCs cells. Furthermore, the present invention adoptively transferred MO-MDSCs cells with downregulated PAD2 expression into CIA mice and found that MO-MDSCs cells with downregulated PAD2 expression can effectively alleviate the onset of CIA mice, which provides a new idea for the treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a purity identification diagram of MO-MDSCs. Figure A is the result of magnetic bead sorting, and Figure B is the Wright-Giemsa staining diagram of MO-MDSCs cells.

[0021] Figure 2 This is the result of qRT-PCR detection of PAD mRNA expression in MDSCs and its subpopulations.

[0022] Figure 3 This is a graph showing the expression results of PAD2 and PAD4 in MO-MDSC detected by Western Blot.

[0023] Figure 4 This is the result of immunofluorescence detection of the expression of PAD2 and PAD4 in MO-MDSC.

[0024] Figure 5 This is a graph showing the expression of citrullinated proteins in MDSCs and their subsets.

[0025] Figure 6 PAD2 and PAD4 inhibit CD4 in MO-MDSCs +The results of the effect of T cell proliferation are shown in Figure A, which shows the results of the negative control and positive control groups, and Figure B shows the results of the CTRL group, si-NC group, si-PAD2 group and si-PAD4 group. Figure C shows the results of the maternal CD4 + T cells, 1st to 3rd generation CD4 + T cells and CD4 + Result graph of the proportion of T cells.

[0026] Figure 7 This is a graph showing the effect of PAD2 on the expression of NO and Arginase in MO-MDSCs.

[0027] Figure 8 The results show the effect of PAD2 or PAD4 on the differentiation of MO-MDSCs into Mφ and their maturity during the differentiation process.

[0028] Fig. 9 The results show the effect of PAD2 or PAD4 on the differentiation of MO-MDSCs into DCs and their maturity during the differentiation process.

[0029] Fig.10 This figure shows the effect of adoptive transfer of MO-MDSCs on the incidence and clinical scores of CIA mice after downregulating PAD2. Figure 1 shows the comparison of incidence and clinical scores between Si-NC-MO-MDSCs and Si-PAD2-MO-MDSCs groups, and Figure 2 shows the comparison of incidence and clinical scores between MO-MDSCs and AFM30a-MO-MDSCs groups.

[0030] Fig.11 This is a graph showing the effects of adoptive transfer of MO-MDSCs on toe swelling and joint structure in CIA mice after downregulating PAD2. Figure A is a real photo of the mouse, and Figure B is a joint section of the CIA mouse.

[0031] Fig.12 Figure 2 shows the effect of adoptive transfer of MO-MDSCs on the levels of total IgG, anti-CⅡ antibodies, IFN-γ, and IL-17 in the serum of CIA mice after downregulation of PAD2. A is the level of total IgG, B is the level of anti-CⅡ antibodies, and C is

[0032] IFN-γ level, and D is IL-17 level. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0034] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0035] The CIA mice described in the examples are commonly used experimental animal models. The model construction method is a conventional method. The DBA1 / J mice used to construct the model were obtained from the Animal Experiment Center of Jiangsu University.

[0036] Example 1: Phenotypic and morphological identification of MDSCs

[0037] (1) Obtaining spleen cells from CIA mice:

[0038] Blood was collected from the eyeballs of CIA mice, and the CIA mice were killed by CO2 asphyxiation. The killed CIA mice were soaked in 75% alcohol, and then placed in a clean bench glass dish. The spleen of the CIA mouse was taken out, placed on a sieve, and the spleen was ground with a grinding rod. PBS (phosphate buffered saline) and RPMI-1640 culture medium (Gibco) were repeatedly washed to obtain a spleen single cell suspension. The spleen single cell suspension was centrifuged at 500g for 5 minutes, and the supernatant was discarded. ACK (Red Blood Cell Lysis Buffer) in a 37°C water bath was added to mix the spleen cells. After lysis for 5 minutes, 5 mL of RMPI1640 culture medium containing 10% calf serum was added to mix, and lysis was terminated; centrifuged again at 500g for 5 minutes, the supernatant was discarded, and 10 mL of PBE (phosphate buffered EDTA) solution at 4°C was added to mix, and washed to obtain CIA mouse spleen cells.

[0039] (2) Magnetic bead separation of PMN-MDSCs and MO-MDSCs:

[0040] According to every 10 8 The spleen cells of CIA mice were resuspended in 350 μL PBE (Phosphate Buffered Saline-EDTA) and mixed to obtain a spleen single cell suspension, each containing 10 8 Add 50 μL of FcR Blocking Reagent to a 15 mL centrifuge tube containing 10 spleen cells, mix well, and place on ice for 10 min. 8 Add 40 μL of Anti-Ly-6G-Biotin standard reagent to each spleen cell, flick it evenly and incubate it on ice for 30 minutes. During this period, mix it evenly every 10 minutes. Then add 10 mL of PBE and mix evenly. Centrifuge it at 500 g for 5 minutes at 4°C and discard the supernatant. 850 μL of Anti-Biotin-MicroBeads standard reagent was added to each spleen cell, and the mixture was incubated on ice for 30 min after flicking. The mixture was mixed every 10 min. Then 10 mL of PBE was added for washing, and the supernatant was discarded after centrifugation at 500 g for 5 min at 4 °C. 500 μL of PBE was added for mixing and then passed through the column to obtain the magnetic column filtrate of PMN-MDSCs. The cells coated with magnetic beads were adsorbed on the magnetic column (the magnetic column was LS Separationcolumns, purchased from Miltenyi Biotec).

[0041] Add 5 mL of PBE to the magnetic column, push the liquid in the magnetic column with the piston, add 5 mL of PBE again, push the piston once to flush out the cells adsorbed on the magnetic column (purchased from Miltenyi Biotec, model LS Separation columns), and obtain 10 mL of PMN-MDSCs cell suspension. Collect the remaining magnetic column filtrate from the PMN-MDSCs separation and sorting, add 10 mL of PBE to resuspend and mix, centrifuge at 500g for 5 minutes at 4°C, and discard the supernatant. 8 Add 40 μL of Anti-Gr-1-Biotin standard reagent to each cell, flick it to mix well, and incubate on ice for 30 minutes. During this period, flick it every 10 minutes. Then add 10 mL of PBE to mix and wash, centrifuge at 4°C and 500g for 5 minutes, and discard the supernatant. 8 Add 50 μL of Anti-Biotin-MicroBeads standard reagent to each cell, flick it evenly and incubate it on ice for 30 minutes, mixing it every 10 minutes. Then add 10 mL of PBE to wash. Add 500 μL of PBE and mix it well before passing it through the column. Rinse the magnetic column and wash off the cells adsorbed on the magnetic column to obtain 10 mL of MO-MDSCs cell suspension. Take a small amount of cells for counting and temporarily place the cells on ice for later use.

[0042] (3) Swiss Giemsa staining to observe mouse MO-MDSCs:

[0043] The MO-MDSCs cell suspension separated by magnetic beads in step (2) was diluted to 5×10 3Resuspend the cells at a concentration of 100 cells, then apply them on a glass slide, wait for natural drying, place on a staining rack, add 3-5 drops of Swiss Giemsa staining kit (purchased from Sigma-Aldrich) I solution to the cell smear area, and stain for 1 minute. Add 3-5 drops of II solution to the cell smear area, blow evenly with an ear wash bulb, and stain for 4-6 minutes (20-25°C). Under running water, gently rinse one end of the glass slide, and rinse off the staining solution. Be careful not to rinse repeatedly. Use absorbent paper to drain the remaining water on the glass slide. After the glass slide is naturally air-dried, observe under a microscope.

[0044] The MO-MDSCs separated by magnetic beads were tested by flow cytometry to detect cell phenotype. The test results showed that the purity of MO-MDSCs was over 90% ( Figure 1 A). Wright-Giemsa staining of MO-MDSCs showed that the nuclei of MO-MDSCs were large and round or quasi-round ( Figure 1 B).

[0045] Example 2: PAD expression and citrullinated protein expression of MO-MDSCs in the spleen of CIA mice

[0046] (1) Construction of CIA mouse model:

[0047] The DBA1 / J mice were first immunized with type II bovine collagen (purchased from chondrex) and Freund's complete adjuvant (purchased from Sigma) on day 0, and secondly immunized with type II bovine collagen and Freund's incomplete adjuvant (purchased from Sigma) on day 21. The CIA mouse model was established by this method. The expression of PAD and its citrullinated protein in the spleen MDSCs (abbreviated as d28-MDSCs, d35-MDSCs, and d42-MDSCs) of CIA mice were analyzed on days 28, 35, and 42 after the first immunization.

[0048] (2) qRT-PCR detection of PAD mRNA expression:

[0049] The MO-MDSCs cell suspension obtained in Example 1 was taken and at least 2×10 6cells, then washed with PBS, added with 1mL of Trizol, allowed to stand at room temperature for 5min, and centrifuged at 12,000g for 15min at 4℃. Add 200μL of chloroform, mix thoroughly by inversion, let stand for 10min, and after separation, centrifuge at 12,000g for 15min at 4℃. At this time, the reagent in the EP tube presents an upper aqueous phase containing RNA; a middle phase containing DNA; and a lower chloroform phase containing protein. Transfer the RNA aqueous phase to a new EP tube, add 500μL of isopropanol, shake and mix, let stand at room temperature for 10min, centrifuge at 12,000g for 15min at 4℃, and carefully discard the supernatant. Add 1mL of 75% ethanol to the EP tube, mix by inversion, centrifuge at 7500g for 5min at 4℃, and carefully discard the supernatant. Use a fan in the clean bench for 5-15min to dry the RNA precipitate, dissolve it in DEPC water, and store it in a -80℃ low-temperature refrigerator for later use. Measure RNA concentration, the sample loading volume is 1μL, adjust to zero with DEPC water, A260 / A280 is preferably 1.8-2.0, and record the test results. For reverse transcription, prepare imported large, medium and small gun tips, disposable plastic gloves, markers, reverse transcription kits, and thaw in a 4℃ ice box. Prepare the reverse transcription system (see Table 1 for details). After the sample is added, centrifuge it instantly, use the PCR instrument to amplify, and start the machine for preheating in advance. Create a temperature setting, select a cycle gradient, heat at 37℃ for 30min, heat at 85℃ for 5s, and then cycle at 4℃. After the reverse transcription sample is taken out, cover the lid after the machine cools down to obtain the reverse transcription sample, that is, cDNA, which can be stored at 4℃ for a short time, or -20℃ for a long time. The material preparation is the same as the reverse transcription step, add 8-well tubes or 96-well plates, design and calculate the number of sample wells and qRT-PCR system (see Table 2 for details). qRT-PCR requires an internal reference gene as a control. This experiment uses actin. Each sample is repeated 3 to 5 times to eliminate sample interference. After adding the sample and reagent, centrifuge it instantly, cover it to keep it clean, and store it at 4°C if it cannot be detected immediately. For machine operation, use the CFX Manager quantitative detector, set the scheduled denaturation for 10 minutes, adjust the denaturation temperature to 95°C for 1s, adjust the annealing temperature to 60°C for 60s, adjust the extension temperature to 72°C for 60s, and perform 42 cycles of denaturation, annealing and extension. Observe the melting curve, obtain the Ct value, and calculate the relative expression of PAD.

[0050] Table 1. Reverse transcription system

[0051]

[0052] Table 2. Real-time fluorescence quantitative PCR system

[0053]

[0054] (3) Western Blot detection of PAD and citrullinated protein expression in MO-MDSCs:

[0055] Prepare 5% concentrated gel and 12% separation gel, add 50μg protein sample to each sample well, add pre-stained Marker 26616 as a marker to monitor electrophoresis and estimate mobility, and add loading buffer to both ends to flatten. Perform electrophoresis at 80V voltage. When the sample is electrophoresed to the separation gel part, adjust the voltage to 100V and continue electrophoresis until the Marker reaches the bottom of the gel and stops electrophoresis. Take out the gel immersed in the transfer buffer and place it on a white plate. According to the molecular weight of the pre-stained Marker, cut the gel at the corresponding molecular weight position of PAD and citrullinated protein. Cut the PVDF membrane slightly smaller than the gel according to the size of the gel, soak the PVDF membrane in anhydrous methanol for 30s to prevent protein contamination and activate the PVDF membrane. Soak the PVDF membrane treated with anhydrous methanol in the transfer buffer for 5min, and the filter paper placed on both sides of the splint must also be completely soaked in the transfer buffer. After soaking, carefully spread the electrophoresis gel on the filter paper, and then cover it with the marked PVDF membrane. Expel bubbles during the operation and clamp the splint. Load the splint into the transfer tank as required, connect the positive and negative power supplies, and place the transfer device in ice. Adjust the power supply parameters and transfer the membrane at 0.35A for 90 minutes. Take out the PVDF membrane after the transfer, wash it once with 1× TBS-T, and then soak the PVDF membrane in 5% skim milk solution, and slowly shake it on a shaker for 1 hour to complete the blocking.

[0056] Incubate with mouse anti-PAD antibody or rabbit anti-citrullinated protein antibody (purchased from AMC-Merck Millipore), dilute the antibody with TBS-T solution at 1:5000, and then add it to the blocked PVDF membrane. Incubate overnight at 4°C. Wash the antibody-incubated PVDF membrane with TBS-T on the second day, and wash it three times on a shaker. Incubate with goat anti-mouse and goat anti-rabbit HRP-IgG antibodies, dilute goat anti-mouse HRP-IgG antibodies with TBS-T at 1:10000, incubate at room temperature for 2h, and wash the PVDF membrane with TBS-T, and wash it three times on a shaker. Expose, add ECL detection reagent to the PVDF membrane for color development, and obtain the bands on the PVDF membrane.

[0057] (4) Detection of PAD expression in MO-MDSCs using fluorescent antibody technology:

[0058] First, take a 75% alcohol-sterilized thin glass slide and dry it on an alcohol lamp. After cooling, plant it in a 24-well plate. Pipette the magnetic bead-sorted MO-MDSCs cell suspension and distribute 1×10 6cells, then resuspended to 1 mL with PBS and evenly seeded in the wells. After standing for 10 minutes, place in a horizontal centrifuge and centrifuge at 500g for 5 minutes at room temperature. After centrifugation, discard the supernatant and fix with 4% paraformaldehyde for 10 minutes at room temperature. Then wash twice with PBS, 5 minutes each time, and finally discard the supernatant. Add 0.5% Triton (purchased from Sinopharm Chemical Reagent Co., Ltd.) to the wells, cover the bottom of the wells, and stand for 30 minutes (Triton is dissolved in 1% bovine serum albumin (BSA) and placed in a wet box). Then wash twice with 500μL PBS, 5 minutes each time. Block with 5% BSA (prepared with PBS) for 1 hour, then wash 3 times with 500μL PBS, 5 minutes each time. Incubate with 1:200 diluted PE-labeled CD11b, or 1:1000 diluted anti-mouse PAD2 or PAD4 antibodies in a wet box at 4°C overnight. The next day, wash 3 times with 500 μL of PBS, 5 minutes each time. Add mouse secondary antibody that shows green fluorescence and incubate at room temperature for 2 hours. Then wash 3 times with PBS, 5 minutes each time. Add nuclear dye for 10 minutes to show the nuclear position and distinguish non-specific signals. Then wash 3 times with 500 μL of PBS. Seal the slides and observe using a confocal microscope. Find the field of view with nuclear staining, and observe the expression of CD11b, PAD2 and PAD4 in the red and green bands.

[0059] qRT-PCR was used to detect the mRNA expression levels of the five isozymes in the spleen and MO-MDSCs of CIA mice. The results showed that MO-MDSCs expressed PAD2 and PAD4, but not PAD1, PAD3, and PAD6. Figure 2 ). Western Blot was used to detect the expression of PAD in d28-MDSCs, d35-MDSCs and d42-MDSCs. The results showed that PAD2 and PAD4 were expressed in MO-MDSCs ( Figure 3 ). PAD2 was observed to be expressed in the nucleus of MO-MDSCs cells under confocal microscopy ( Figure 4 ). Western Blot was used to detect the expression of citrullinated proteins in MDSCs and their subpopulations. The results showed that MO-MDSCs expressed high levels of citrullinated proteins ( Figure 5 ).

[0060] Example 3: Down-regulating the expression of PAD2 in MO-MDSCs enhances the immunosuppressive ability of MO-MDSCs

[0061] (1) siRNA knockdown:

[0062] The lyophilized powders of siRNA-PAD2, siRNA-PAD4 and siRNA Negative Control (siRNA-NC) (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.) were centrifuged and dissolved in sterile DEPC water, mixed by pipetting to form a storage solution with a concentration of 20 μM, and stored at -80°C. 1×10 6 cells, and the system was supplemented to 450 μL with RPMI-1640 culture medium; take a clean EP tube, add 1.25 μL of siRNA-PAD2, siRNA-PAD4 and siRNA-NC storage solution, and then use RPMI-1640 culture medium to make up to 25 μL, and finally prepare a 50 nM concentration of siRNA dilution; take a clean EP tube, add EntranceTM-R (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.) transfection reagent, take 50 nM working concentration as an example, add 1 μL of transfection reagent, and finally use RPMI-1640 culture medium to make up to 25 μL, and let it stand at room temperature for 5 minutes; add the dilution of transfection reagent to the corresponding siRNA-PAD2, siRNA-PAD4 and siRNA-NC dilutions, and then immediately mix thoroughly, and let it stand at room temperature for 30 minutes; add 50 μL of the final system transfection complex dropwise to MO-MDSCs and mix gently; complete the transfection step 6 hours after transfection and observe the cell status. The Western Blot method described in Example 2 was used to detect the inhibitory effect on MO-MDSCs after transfection.

[0063] (2)MO-MDSCs and CD4 + T cell co-culture and CFSE staining to detect the suppressive function of MO-MDSC:

[0064] 50 μL of 2 μg / mL anti-mouse CD3 antibody coating solution (purchased from eBioscience) was used to coat a 96-well concave bottom culture plate and incubated at 4°C overnight. Negative control wells did not require CD3 coating. Magnetic beads were used to separate CD4 + T cells, per 10 8 Resuspend spleen cells in 350 μL PBE and mix the spleen single cell suspension. Add 50 μL FcR Blocking Reagent to each 15 mL centrifuge tube, mix well and incubate on ice for 10 min. 8Add 40 μL of anti-CD4-Biotin-Microbeads standard reagent (purchased from eBioscience) to each spleen cell, mix well and incubate on ice for 30 minutes, mixing every 10 minutes. Rinse the magnetic column with PBE once, add the spleen cells incubated with the antibody, filter the cell suspension until the last drop flows out, and then add 500 μL of PBE to filter until the last drop flows out. Then add 3 mL of PBE until the last drop flows out, repeat 3 times until the last drop flows out, carefully remove the magnetic column, insert it into a 15 mL glass centrifuge tube, add 5 mL of PBE, use the piston to push the liquid out, flush out the cells adsorbed on the magnetic column, add 5 mL of PBE again to rinse once, and get 10 mL of CD4 + T cell suspension, aspirate a small amount of cells for counting, and place the cells on ice for later use. Resuspend and mix the CD4 T cells sorted by magnetic beads in preheated PBS containing 0.1% BSA. + The final concentration of T cell suspension was adjusted to 10 6 ~10 7 cells / mL. Add 2.5-5μmol / L CFSE dye and incubate in a 37℃ water bath for 10 minutes, gently shaking and mixing to ensure the staining effect. After the incubation, stop the staining with 5 times the volume of pre-cooled 10% fetal bovine serum RPMI-1640 culture medium. After mixing, let it stand on ice for 5 minutes, then centrifuge and precipitate the cells, discard the supernatant, and wash it twice with 10% fetal bovine serum RPMI-1640 culture medium, discard the supernatant and resuspend the cells, adjust the cell concentration, and prepare the seed plate.

[0065] The number of cells seeded in each well of a 96-well plate was 5 × 10 5 cells, set up the following groups:

[0066] Negative control group (Unstimulated): containing 5×10 5 CD4 + T cells, no treatment

[0067] Positive control group (Stimulated): containing 5×10 5 CD4 + T cells, CD3 coated in advance and CD28 added (CD3 and CD28 are both known and public materials);

[0068] MO-MDSCs and CD4 + T cell co-culture group (also called experimental group): MO-MDSCs cells and CD4 + T cells were added in a ratio of 1:2, with 2.5×10 5 and 5×10 5cells, pre-coated with CD3 and added with CD28;

[0069] MO-MDSCs and CD4 + The T cell co-culture group was divided into the following 4 groups according to the MO-MDSCs treatment method:

[0070] MO-MDSCs were not treated (CTRL group);

[0071] MO-MDSCs were transfected with siRNA-NC (Si-NC group);

[0072] MO-MDSCs were transfected with siRNA-PAD2 (Si-PAD2 group);

[0073] MO-MDSCs were transfected with siRNA-PAD4 (Si-PAD4 group).

[0074] Then, anti-mouse CD28 monoclonal antibody was added to different treatment groups to a final concentration of 2 μg / mL, but not added to the negative control group. The final volume of each well was maintained at 200 μL. If the volume of the system was insufficient, it was supplemented with RPMI-1640 culture medium containing 10% fetal bovine serum. The cells were cultured at 37°C and 5% CO2 for 72 hours.

[0075] MO-MDSCs and CD4 + In the T cell co-culture group, MO-MDSCs and CD4 + The control groups of the other groups in the T cell co-culture group. + T cell proliferation Figure 6 As shown, the negative control group CD4 + T cells did not proliferate, and CD4 + T cell proliferation was obvious ( Figure 6 A), compared with the experimental group with MO-MDSCs added ( Figure 6 B) Compared with CD4 + The proliferation of T cells was inhibited; the expression of PAD4 and CD4 + MO-MDSCs were added to the T cell proliferation system, and CD4 + There was no significant change in the proportion of T cells ( Figure 6 C); inhibit the expression of PAD2 and CD4 + MO-MDSCs were added to the T cell proliferation system, and mother CD4 + The proportion of T cells increased from 51.2%±4.1% to 61.3%±3.3% (P<0.05). +The proportion of T cells decreased from 41.3%±1.2% to 34.2%±2.3% (P<0.05). + The proportion of T cells decreased from 11.2% ± 1.1% to 5.0% ± 0.9% (P < 0.05) ( Figure 6 C).

[0076] The NO detection kit (purchased from Biotech) and ECL kit (purchased from Milipore) were used to detect the expression of NO and Arg-1 in different treatment groups. Inhibition of PAD2 expression in MO-MDSCs increased the NO expression level in MO-MDSCs from 6.53±1.02μM to 14.21±1.12μM (P<0.01), and the Arg-1 expression level in MO-MDSCs increased from 3.13±0.22U / L to 5.13±0.21U / L (P<0.05) ( Figure 7 ). These results indicate that downregulating the expression of PAD2 can enhance the immunosuppressive function of MO-MDSCs.

[0077] Example 4: Down-regulation of PAD2 expression in MO-MDSCs inhibits differentiation of MO-MDSCs

[0078] (1) Inducing MO-MDSCs to differentiate into Mφ in vitro: Cut the femur of healthy mice, cut both ends and rinse to obtain bone marrow cells (BMCs), resuspend the BMCs single cell suspension in 1 mL of RPMI-1640 medium containing 10% fetal calf serum, and induce in the system with 10 ng of macrophage colony stimulating factor (M-CSF) and 10 ng of interleukin-6 (IL-6). After the induction differentiation into MDSCs, discard the culture supernatant and use a large, medium and small gun tips to aspirate to complete the operation of discarding all supernatants. Prepare an M-CSF solution with a final concentration of 10 nmol / mL in RPMI-1640 with 10% fetal bovine serum, and gently add 1 mL along the wall of the well. Carefully operate the whole process to ensure that the cells are evenly distributed at the bottom during the culture process. Culture at 37°C and 5% CO2 for 72 hours. After the culture is finished, the cells are collected by repeatedly blowing each well, centrifuged at 500g for 5min, the supernatant is discarded, and the cells are resuspended and mixed with 100μL of PBS and collected in EP tubes. 0.5μL of PE-Cy5-stained anti-CD11b antibody and 0.5μL of PE-stained anti-F4 / 80 antibody are added to the EP tube and incubated at 4℃ for 30min. After the incubation, the cells are resuspended to 1mL with PBS, and then centrifuged at 500g for 5min at 4℃, the supernatant is discarded, and 200μL of PBS is added to resuspend and mix. The proportion of Mφ in cells at the end of induced differentiation is detected by FCM.

[0079] (2) Inducing MO-MDSCs to differentiate into DCs in vitro: After the induction of BMCs to differentiate into MDSCs, discard the culture supernatant and use a large, medium and small pipette tip to remove all the supernatant. Prepare 10 ng / mL granulocyte-macrophage colony stimulating factor (GM-CSF) (STEMCELL Technologies Inc.) and 2.5 ng / mL interleukin-4 (IL-4) solution (STEMCELL Technologies Inc.) with 10% fetal bovine serum RPMI-1640, and gently add 1 mL along the wall of the well. Be careful throughout the process to ensure that the cells are evenly distributed at the bottom of the culture. Culture at 37°C and 5% CO2 for 72 h. After the culture is over, the cells are repeatedly blown and hit in each well to collect the cells, centrifuged at 500g for 5min, the supernatant is discarded, and the cells are resuspended and mixed with 100μL of PBS. 0.5μL of PE-Cy5-stained anti-MHC-II antibody (purchased from eBioscience) and 0.5μL of PE-stained anti-CD11c antibody (purchased from eBioscience) are added to the EP tube and incubated at 4°C for 30min. After the incubation, the cells are resuspended to 1mL with PBS, and then centrifuged at 500g for 5min at 4°C, the supernatant is discarded, and 200μL of PBS is added to resuspend and mix. The proportion of DCs in the cells after induced differentiation is detected by FCM.

[0080] GM-CSF and IL-6 were used to induce BMCs to differentiate into MDSCs, and siRNA was used to knock down the expression of PAD2 or PAD4 in MO-MDSCs. The results showed that inhibiting the expression of PAD2 in MO-MDSCs reduced the proportion of MO-MDSCs differentiated into Mφ from 32.8%±3.7% to 14.9%±2.3% (P<0.01), and the maturity of total cells in the differentiation system decreased ( Figure 8 ); the proportion of DCs differentiated into DCs decreased from 15.4%±2.7% to 8.3%±1.0% (P<0.01), and the maturity of total cells in the differentiation system decreased ( Fig. 9 ), while inhibition of PAD4 expression did not result in significant changes. Compared with untreated MO-MDSCs, the proportion of MO-MDSCs differentiated into Mφ decreased from 32.8%±3.7% to 12.2%±1.4% (P<0.01) ( Figure 8 ), the proportion of DCs differentiated into DCs decreased from 15.4%±2.7% to 9.6%±1.0% (P<0.01) ( Fig. 9 ). These results indicate that PAD2 can promote the differentiation of MO-MDSCs into Mφ or DCs in vitro.

[0081] Example 5: After downregulating the expression of PAD2, adoptive transfer of MO-MDSCs can significantly inhibit the onset of CIA in mice

[0082] The expression of PAD2 in MO-MDSCs was downregulated by siRNA or AFM-30a (purchased from MCE), a specific inhibitor of PAD2. The effect of adoptive transfer of MO-MDSCs on the pathogenesis of CIA mice was then observed. The time of adoptive transfer of MO-MDSCs was 14 days after the first immunization, and the number of adoptive transfer of MO-MDSCs per mouse was 2×10 6 The CIA mice group adoptively transferred with an equal amount of PBS was used as the blank control group, recorded as CIA+PBS; the CIA mice group adoptively transferred with MO-MDSCs cells without any treatment was used as the negative control group, recorded as MO-MDSCs; the CIA mice group adoptively transferred with MO-MDSCs cells treated with Si-NC, Si-PAD2 and AFM30a were used as experimental groups, recorded as Si-NC-MO-MDSCs, Si-PAD2-MO-MDSCs and AFM30a-MO-MDSCs, respectively.

[0083] The toes of CIA mice in each group were photographed and analyzed, the joints were sliced ​​and analyzed, and serum IgG, anti-CⅡIgG, IFN-γ and IL-17 were detected using ECL kits (purchased from). The effect of SiRNA interference with PAD2 can be observed by comparing the Si-PAD2-MO-MDSCs and Si-NC-MO-MDSCs groups, while the effect of inhibitor downregulation of PAD2 can be observed by comparing the AFM-30a-MDSCs and MO-MDSCs groups.

[0084] The experimental results showed that adoptive transfer of MO-MDSCs could reduce the arthritis score of mice after downregulating the expression of PAD2 in MO-MDSCs, regardless of whether Si-RNA or inhibitor AFM-30a was used (P<0.05 or P<0.01) ( Fig.10 ), the redness and swelling of the toes are reduced ( Fig.11 A), the degree of joint damage is reduced ( Fig.11 B), serum IgG ( Fig.12 A) Anti-CⅡIgG( Fig.12 B) IFN-γ ( Fig.12 C) and IL-17 ( Fig.12 D)(P<0.05 or P<0.01). These results indicate that adoptive transfer of MO-MDSCs can more effectively alleviate the pathogenesis of CIA mice after downregulating the expression of PAD2 in MO-MDSCs, which provides a new idea for cellular immunotherapy of RA.

[0085] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail in this specification with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Application of PAD2 in regulating MO-MDSCs cell differentiation.

2. The use of PAD2 in regulating MO-MDSCs cell differentiation according to claim 1, characterized in that: The application is achieved by regulating the expression level of PAD2.

3. The use of PAD2 in regulating MO-MDSCs cell differentiation according to claim 2, characterized in that: The expression level of PAD2 is positively correlated with the differentiation degree of MO-MDSCs cells.

4. A method for regulating MO-MDSCs cell differentiation, characterized in that: The method comprises regulating the differentiation of MO-MDSCs cells by regulating the expression amount of PAD2 in MO-MDSCs cells.

5. The method for regulating MO-MDSCs differentiation according to claim 4, characterized in that: When the expression level of PAD2 is upregulated, the differentiation of MO-MDSCs cells is promoted; when the expression level of PAD2 is downregulated, the differentiation of MO-MDSCs cells is inhibited.

6. The product obtained by the method for regulating MO-MDSCs differentiation according to claim 4 or 5, characterized in that: The product includes MO-MDSCs cells with downregulated PAD2 expression obtained by inhibiting MO-MDSCs cell differentiation.

7. The product according to claim 6, characterized in that The products also include Mφ cells or DCs cells obtained by promoting the differentiation of MO-MDSCs cells.

8. Use of the product according to claim 6 in the preparation of a drug for treating rheumatoid arthritis.

9. A drug for treating rheumatoid arthritis, characterized in that: The medicament comprises the product of claim 6.

Images