The Role Of PGRN In The Pathogenesis Of Experimental Autoimmune Uveitis

Objective:Progranulin（PGRN）exerts an important role in inflammation and immune regulation.However,the role of PGRN in autoimmune uveitis is still unknown.We previously found that individuals with active Behcet’s disease（BD）and Vogt-Koyanagi-Harada syndrome（VKH）have lower PGRN expression in their serum and peripheral blood mononuclear cells（PBMC）than healthy people.As a result,we investigated the role of PGRN in experimental autoimmune uveitis（EAU）and the potential underlying mechanism.Methods:To investigate the effect of exogenous PGRN on EAU,we subcutaneously injected human retinoid-binding protein(IRBP_161-180)to establish an EAU model in B10.RIII mice.Mice with intraperitoneal injection of exogenous recombinant PGRN were served as the PGRN-treated EAU group,while EAU mice in the control group were intraperitoneally injected with the same amount of PBS.To investigate the effect of PGRN gene knockout on EAU,we subcutaneously injected IRBP_651–670to establish an EAU model in C57BL/6 mice.After modeling,both strains of EAU mice were clinically scored by slit lamp microscopy.On the 13th day after modeling,the eyeballs were collected for histopathological evaluation.RT-PCR was used to detect the m RNA expression of proinflammatory factors in the retina,and flow cytometry was used to detect the proportion of Th1,Th17 and Treg cells in the spleen.The retina and spleen tissues of WT and PGRN-/-EAU mice were collected for m RNA sequencing（RNA-seq）.The differentially expressed genes were screened by the DESeq2 package in R.GO function annotation and KEGG pathway enrichment analysis were performed.The STRING database and Cytoscape software were used to construct protein-protein interaction network（PPI）and to identify the hub genes.Finally,GSEA enrichment analysis were performed.In vitro,the isolated splenocytes from WT EAU mice were stimulated with IRBP_651-670peptides in the presence or absence of PGRN,and the proportions of Th1,Th17 and Treg cells were detected by flow cytometry.The na?ve CD4⁺T cells were separated from the spleen of normal C57BL/6 mice by magnetic beads,and stimulated with Th1,Th17,and Treg cell polarization conditions in the presence or absence PGRN.The proportion of Th1,Th17 and Treg cells was detected by flow cytometry.Results:The m RNA expression of PGRN in the retina of EAU mice was significantly increased on day 14 after immunization and decreased rapidly on day 21.Exogenous PGRN can attenuate ocular inflammation in EAU mice,as demonstrated by reduced clinical and histopathological scores and decreased m RNA expression of proinflammatory factors in retina,such as IFN-γ,IL-17,IL-1β,MCP-1,IL-6 and TNF-α.PGRN gene knockout can aggravate ocular inflammation in EAU mice,as demonstrated by increased clinical and histopathological scores and increased m RNA expression of proinflammatory factors in retina,such as IFN-γ,IL-17,IL-1β,MCP-1,IL-6 and TNF-α.PGRN gene knockout significantly altered the m RNA expression profiles in the retina and spleen of EAU mice.Compared with WT EAU,a total of 160 differentially expressed genes（96 up-regulated and 64 down-regulated）were screened in the retina of PGRN^-/-EAU mice,and 226 differentially expressed genes（126 up-regulated and 100 down-regulated）were screened in the spleen.Twenty-three hub genes were identified in the retina,and 24 key genes were identified in the spleen.Five hub genes were shared in the retina and spleen,which were Tnf,Il6,Il1b,Cxcl2 and Ccl2.Both KEGG pathway enrichment analysis and GSEA analysis showed that MAPK signaling pathway and cytokine-cytokine receptor interaction pathway were the most significantly enriched pathways in spleen and retina,respectively,while the common pathway significantly enriched in both spleen and retina was TNF signal pathway.In vivo,exogenous PGRN could down-regulate the proportion of Th1 and Th17 cells in the spleen of EAU mice,but had no influence on the proportion of Treg cells.PGRN knockout can up-regulate the proportion of Th1 and Th17 cells in the spleen of EAU mice,and down-regulate the proportion of Treg cells.In vitro,PGRN inhibits the proportion of antigen-specific Th1 and Th17 cells,while up-regulating the proportion of antigen-specific Treg cells.PGRN had no effect on the differentiation of na?ve CD4⁺T cells into Th1 and Th17 cells,but promoted the differentiation of Treg cells.Conclusion:PGRN is involved in the pathogenesis of experimental autoimmune uveitis,and exerts an anti-inflammatory protective effect on experimental autoimmune uveitis by down-regulating the proinflammatory effector T cells（Th1,Th17）and up-regulating the regulatory T cells（Treg）,in which TNF signaling pathway may play an important role.