Mechanism Of T Helper 17 In The Pathogenesis Of Graves Orbitopathy

ObjectivesGraves orbitopathy(GO)is an autoimmune disease.Studies have shown that T helper 17(Th17)might be involved in the development of GO.In this study,we studied the regulation of Th17 cells on orbital fibroblasts(OFs)which are target cells of GO,and studied the crosstalk between Th17 cells and CD34+fibrocytes which are precursor cells of GO and its mechanism.The GO immune microenvironment was also identified.Materials and Methods1.Blood samples and orbital tissues from GO patients and healthy controls were collected.2.Western Blot,immunofluorescence staining was used to detect the regulation of Th17 cells on OFs,and the mechanism was confirmed by PathScan intracellular signal detection and protein mass spectrometry.The relationship between Th17 cells and fibroblasts was investigated by co-culture,quantitative RT-PCR,Luminex and transwell experiments.3.We identified the orbital immune microenvironment by single cell sequencing.The phenotypic of different cell subpopulations was examined by multicolor flow cytometry.Co-culture experiments and Western blot analysis were used to determine the mechanism of the Th17 cell pathway in GO orbits.Results1.For CD90+OFs,IL-17A promoted TGF-?-induced fibrosis,but for CD90-OFs,IL-17 inhibited 15d-PGJ2-induced adipogenesis.Th17 cells promoted the secretion of proinflammatory cytokines by CD90+and CD90-OFs.At the same time,CD90+and CD90OFs could promote the differentiation of Th17 cells by producing PGE2.In addition,Th17 cells could up-regulate the expression levels of costimulatory molecules on OFs.2.GO patients had increased CD34+fibroblasts,and IL-17A stimulated the production of cytokines in fibroblasts.Compared with normal fibroblasts,GO fibroblasts were more responsive to IL-17A.Autologous Th17 cells promoted inflammation and antigen presentation in GO fibrocytes.At the same time,fibrocytes also enhanced Th17 cell function and recruited more Th17 cells through macrophage inflammatory protein 3(MIP3)/CC chemokine type 6(CCR6)pathway.3.The GO orbital microenvironment consisted of natural killer cells,dendritic cells,macrophages,T cells,plasma cells and CD34+OFs.Th2 and Th17 type immune responses existed locally in the orbits;and Th17 cells exhibited a Th1 cell-like phenotype.CD34+OFs promoted the expression of IL-23R and IL-1R on T cells and promoted their phenotypic transformation to Th17 cells via the prostaglandin E2(PGE2)-EP2/EP4-cAMP signaling pathway.Conculsions1.Th17 cells is pathogenic in the development of GO.2.The crosstalk between CCR6+Th17 cells and CD34+fibroblasts promote the development of GO.3.The GO orbital immune microenvironment is heterogeneous,and the phenotype of local immune elements in GO orbits is identified here.