Pertussis Toxin Promotes Experimental Autoimmune Uveitis By Inhibiting Classic WNT Signaling Pathway Of Spleen

ObjectiveAutoimmune uveitis is not only a common eye disease causing blindness,but also a common autoimmune disease,but its pathogenesis has not been fully elucidated.Classical wnt signaling pathway participates in many processes of life activities and plays an important role in a variety of autoimmune diseases.Pertussis toxin is a commonly used adjuvant to establish the Experimental autoimmune uveitis model of experimental autoimmune uveitis in mice.it can bind to the PTX-sensitive G protein on the cell membrane and inhibit the activity of G protein.The activation of G protein promotes the phosphorylation of lipoprotein receptor-related protein 6 and the activation of classical wnt signal pathway.Therefore,this study will study the role of PTX on wnt pathway and T cell function in the pathogenesis of EAU through mouse EAU animal model,so as to provide new ideas for expounding the pathogenesis of EAU,and then lay a theoretical foundation for the prevention and treatment of uveitis.Method1.Establishment of EAU mouse model:photoreceptor vitamin A binding protein651～670 and complete Freund’s adjuvant were injected subcutaneously in combination with PTX intraperitoneal injection to establish C57BL/6J mouse model.2.Clinical observation:the fundus injury was observed and the score was recorded every day from the 10th day after immunization.3.Retinal histopathological score:on the 12th,16th,20th,24th and 28th day after immunization,the mice were killed and their eyeballs were removed for hematoxylin-eosin staining.The changes of retina were observed under microscope and the score was recorded.4.OCT scanning:starting from the 10th day after immunization,the fundus retina images were scanned by OCT every other day to observe the changes of retina.5.real-time fluorescence quantitative PCR:The spleen tissues were taken before immunization and on the 1st,5th,12th,18th and 25th day after immunization,and m RNA was extracted for reverse transcription,and then the expression of APC,SOX9,AXIN2,LEF1 and TCF1 genes was detected.6.Western Blot:the spleen tissues were taken before immunization and on the 1st,2nd,3rd,4th and 5th day after immunization,and the protein was extracted to detect the expression level ofβ-catenin and activeβ-catenin.7.Flow cytometry detection:the splenic single cell suspension was prepared before and 3 days after immunization,and the expression levels of activeβ-catenin in T lymphocytes,B lymphocytes and DC cells in splenic single cell suspension were detected.8.Flow cytometry detection:the single cell suspension of spleen tissue was prepared before immunization and on the third day after immunization,and the proportion of Th1 cells and Th17 cells was detected.9.Using immune drugs alone or in combination to treat mice:according to the characteristics and requirements of the drugs,IRBP,CFA and PTX were divided into six kinds of use:(1)IRBP~-CFA~-PTX~-,(2)IRBP~+CFA~+PTX~+,(3)IRBP~+CFA~+PTX~-,(4)IRBP~-CFA~+PTX~+,(5)IRBP~-CFA~+PTX~-,(6)IRBP~-CFA~-PTX~+.The mode of administration,the dosage and the time of administration shall remain the same,and the drugs shall be given separately according to the above.The mice were killed on the first day and the third day after administration,and the spleen tissue of mice was taken to detect the expression level ofβ-catenin and activeβ-catenin.10.Sorting spleen tissue CD4~+T cells:the spleen tissues of wild-type mice were grinded,split red and passed through the membrane to get single cell suspension.According to the CD4~+T cell sorting kit,CD4~+T cells were obtained by hatching antibodies,incubating magnetic beads and passing through magnetic poles.11.Detection of classical wnt signal expression level of splenic T cells:CD3 antibody was laid in 96-well plate one day in advance,then CD4+T cells of wild-type mice spleen were sorted,CD3 antibody was washed off and coated with CD28 antibody.CD4~+T cells were planted in 96-well plate,and the cells of seed plate were divided into PTX group and control group.The proportion of CD4~+active-β-catenin~+cells was detected on the third day after culture.12.The level of T cell proliferation was detected by carboxyfluorescein diacetate succinimide method:the spleen CD4~+T cells of wild-type mice were selected and stained with CFSE.The labeled CD4~+T cells were seeded in 96-well plate,and the proliferation experiment was carried out under the intervention of PTX.The proliferation level of T cells was detected on the fifth day after proliferation.13.T cell differentiation test:the spleen CD4~+T cells of wild-type mice were sorted and seeded in 96-well plates.Under the intervention of PTX,T cell differentiation experiments were carried out.Differentiation stimulators IL-12 and anti-IL-4 were added to differentiate into Th1 cells,and IL-6,anti-IL-4,TGF-βand IFN-γwere added to differentiate into Th17 cells.The proportion of CD4~+IFN-γ~+cells and CD4~+IL-17~+cells was detected by flow cytometry.Result1.Clinical observation and histopathological observation showed that a stable EAU mouse model was successfully established.2.Western Blot and qPCR experiments showed that the expression level of classical wnt signal pathway in spleen was significantly decreased in the early stage of EAU disease(p<0.05),and began from the third day after immunization.3.Flow cytometry analysis showed that the expression level of classical wnt signal pathway of splenic T and B lymphocytes decreased significantly on the third day after immunization(p<0.05),while the expression level of classical wnt signal pathway of splenic DC cells had no significant change.4.Flow cytometry showed that the proportion of Th1 cells in spleen of early EAU mice was higher than that of Th17 cells.5.Western Blot experiment showed that the expression of classical wnt signal in spleen was down-regulated by intraperitoneal injection of PTX.6.Flow cytometry showed that the expression of classical wnt signal in T cells was down-regulated after PTX treatment.7.T cell proliferation test showed that there was no significant change in splenic T cell proliferation after PTX treatment.8.T cell differentiation assay showed that there was no significant change in the differentiation level of Th17 cells after PTX treatment,but the differentiation level of Th1 cells was significantly up-regulated(p<0.05).Conclusion.1.The classical wnt signal is under-expressed in the spleen of EAU mice.2.In EAU,PTX inhibits the classical wnt pathway by inhibiting the activation level of Gi protein in spleen.3.In EAU disease,PTX can enhance autoimmune response by inhibiting the classical wnt pathway of spleen and promoting the differentiation of Th1 cells,which provides a new idea for the pathogenesis of EAU.