| Background:Erythrocyte membrane-associated protein(ERMAP)has sequence and structural similarities with existing immune checkpoint molecules.ERMAP is considered to be a B7 family related molecule and has sequence homology with the Butyrophilins(BTN)family,including the extracellular immunoglobulin domain IgV,trans-membrane region and intracellular B30.2 domain.ERMAP fusion proteins can improve experimental autoimmune encephalomyelitis(EAE)and type I diabetes mellitus(T1D)by inhibiting autoreactive T cell proliferation and activation,regulating macrophage polarization to M2.Inflammatory bowel disease(IBD)is a group of diseases characterized by chronic inflammation of the gastrointestinal tract.Over-activation of T cells and imbalance of M1/M2 macrophages can exacerbate the development of IBD.M1 macrophages promote the inflammatory response during disease development,and M2 macrophages promote the proliferation of regulatory T cells(Tregs)to produce anti-inflammatory effects and participate in tissue recovery after injury.Whether ERMAP is involved in the occurrence and development of inflammatory bowel disease is unknown,so this project explores the mechanism of ERMAP fusion protein in inflammatory bowel disease.Objective:This project investigated the effect of ERMAP on Dextran Sodium Sulfate(DSS)-induced colitis in mice(C57BL/6)and its effect on macrophage polarization and T cell response.Methods:1.Improvement effect of ERMAP-Ig fusion protein on mouse IBD:30 normal C57BL/6 mice were fed with 3%DSS solution and the mice were divided into 2 groups:control protein(DSS+Control Ig)and ERMAP fusion protein(DSS+ERMAP-Ig)(n=12).(1)Disease activity index(DAI)score and body weight measurement started on day 0 of modeling,Control Ig or ERMAP-Ig was given intraperitoneally on days 3,6,and 9,and the colon was taken for HE staining and immunofluorescence staining on day 12 after modeling.(2)The colonic lamina propria cells were extracted from the colon of the two groups and the macrophage proportion was detected by anti-F4/80/MHC-Ⅱ/CD206 antibody;Tregs proportion was detected by anti-CD4/CD25/Foxp3 staining.(3)The spleen single cell suspension of each group was prepared and the proportion of macrophages was measured by anti-F4/80/MHC-Ⅱ/CD206;cell activation,proliferation and regulatory T cells(Tregs)were detected with anti-CD4/CD8/CD69/Ki67/CD25/Foxp3;after 3 days with anti-CD3 or CD28,PMA,Lonomycin and BFA were added 4-6h earlier on the fourth day,and the expression of cytokinine TNF-α was measured by FACS;The proliferation of T cells was measured by CFSE staining after 5 days of stimulation with anti-CD3 or CD28.(4)Single-cell suspensions of colon and spleen were cultured under PMA/BFA/Lonomycin stimulation for 6h,and the expression of TNF-α,IFN-γ,IL-4,and IL-17A in CD4+cells was detected by flow staining.(5)The serum ELISA of each group was used to detect the content of cytokines TGF-β1 and IL-6.(6)Colons of each group of mice were collected and Western Blot tested for NLRP3,GSDMD,Caspasel and IL-1β,and gene expression of qRT-PCR for Arg-1,CD206,iNOS,NLRP3,GSDMD,Caspasel,IL-1β,TNF-α,IFN-γ,TGF-β1,IL-4,and IL-10.2.In vitro interference with ERMAP expression on macrophage cell line RAW264.7 to explore its effect on macrophage polarization and its effect on T cell activation:(1)Lentivirus interfered with the expression of ERMAP on RAW264.7 and cultured in M1 and M2 differentiation conditions to flow macrophage typing.(2)Lentiviral-scrambled cells were co-cultured with splenocytes of C57BL/6 mice with anti-CD3 coated plates(5 μg/mL),collected 16 hours later to detect cell activation index CD69,and 72 hours later to detect cell proliferation index Ki67.(3)After cell activation,PMA/BFA/Lonomycin was added for 6 h,and the expression of TNF-α,IFN-γ,IL-4 and IL-17A in CD4+cells was measured by flow cytometry.(4)RAW264.7 was sequencing analyzed with ERMAP shRNA RAW264.7 and the expression of related genes was determined by qRT-PCR.3.The effect of ERMAP-/-macrophages on IBD:WT mice(6 per group)were cleared of macrophages by intraperitoneal injection of 100μl of clodronate liposomes on days-1 and day-2.On day 0,WT mice were transplanted with peritoneal macrophages M0(1x106 per mice)from either WT or ERMAP-/-mice.DSS induced colitis 12 hours after macrophage transfer.DAI scoring and body weight measurements were performed daily,and mice were harvested after euthanasia on day 9.(1)Colons were obtained for HE staining.(2)Single-cell suspensions of spleen were prepared and the ratio of cell activation,proliferation and Tregs were measured by anti-CD4/CD8/CD69/Ki67/CD25/Foxp3.(3)Inflammatory cytokines IL-1β,IL-6,IL-23,IFN-γ,TNF-α,and TGF-β were detected by qRT-PCR in the colon.Results:1.Results of a mouse IBD model of ERMAP-Ig fusion protein function:(1)ERMAP-Ig effectively reduced the clinical symptoms and reduced the DAI score in IBD mice.The histopathological staining indicated inflammatory cell infiltration,severe epithelial destruction and crypt loss in Control Ig mice.(2)In the ERMAP-Ig treated group,mice had increased M2 macrophages,decreased M1 cells,increased CD4+CD25+FoxP3+Tregs,decreased CD4+CD69+and CD8+CD69+T cells,decreased CD4+CD67+and Ki67+T cells,and CFSE proliferation showed decreased CD4+T cells.(3)TNF-α+ macrophages were reduced after ERMAP-Ig treatment.ERMAP-Ig has ameliorated DSS-induced colitis symptoms in mice.(4)Peripheral serum from IBD mice showed that the pro-inflammatory cytokines IL-6 and TGF-β 1 were significantly reduced in the ERMAP-Ig group.(5)In the DSS-induced IBD model,the NLRP3,GSDMD,Caspasel,and IL-1β protein content in the colon tissues of ERMAP-Ig group mice were lower compared with Control Ig,and the difference was statistically significant.(6)qRT-PCR detected M2 type macrophage markers Arg-1 and CD206 in the colon of ERMAP-Ig group;the relative expression of upstream and downstream genes,including GSDMD,was higher than ERMAP-Ig group;(7)inflammatory factors IL-6,TNF-α,IFN-γ,IFN-γ and TGF-β 1 were decreased in ERMAP-Ig group,while the expression of IL-4 and IL-10 were increased.2.Interference with ERMAP in RAW264.7 to inhibit M2 type macrophage polarization and promote T cell response:(1)Lentivirus interfered with macrophage cell line RAW264.7,and M1 increased type MHCIIhiCD206loM1 macrophages compared with untreated RAW264.7 and control viruses,and type MHCIIloCD206hi M2 macrophages.(2)Co-culture of ERMAP shRNA RAW264.7 with splenocytes of C57BL/6 mice found that macrophages with ERMAP interference increased activated T cells of CD4+CD69+and CD8+CD69+,and increased proliferation of CD4+Ki67+and CD8+Ki67+T cells.(3)Co-culture with macrophages interfering with ERMAP increased the ratio of Th1(TNF-α+,IFN-γ+)and Th 17(IL-17A+)in CD4+splenocytes.(4)RNA-seq selected NOD-like recepter-related signaling pathways that possibly with ERMAP affected macrophage function,and pyroptosis-related genes associated with ERMAP were verified by qRT-PCR.3.ERMAP-/-macrophages aggravated DSS-induced colitis in WT mice:(1)mice given KOMcp significantly increased DAI scores from days 5 to 9.(2)Histopathological analysis showed that KOMcp significantly increased colon damage in mice with colitis induced by DSS compared with WTMcp.(3)On day 9,the spleens of two groups were taken for flow analysis,and found that the Tregs in KOMcp group were lower than those in WTMcp.(4)Splenic T cell activation and proliferation in WT+KOMcp mice were increased compared with WT+WTMcp mice,indicating that macrophages knocked down by ERMAP promoted the proliferation and activation of T cells.(5)Colcolon of WT+KOMcp mice expressed more inflammatory factors IL-1β,IL-6,IL-23,IFN-γ,TNF-α,and TGF-β.Conclusions:1.ERMAP-Ig improved the colitis symptoms and inflammatory response induced by DSS in mice;2.ERMAP-Ig increased the number of colonic M2-type macrophages;3.ERMAP-Ig inhibited the spleen T cell response and promoted macrophage to M2 polarization in IBD mice;4.Interference with ERMAP in RAW264.7 inhibited M2 type macrophage polarization and promoted T cell responses.5.Adoptive transfer of ERMAP-/-macrophages aggravated DSS-induced colitis in WT mice. |
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