Sijie Fang1,2,3,4, Yazhuo Huang1,2,3,4, Ningjian Wang5, Shuo Zhang1,2, Sisi Zhong1,2, Yinwei Li1,2, Jing Sun1,2, Xingtong Liu1,2, Yang Wang1,2, Ping Gu1,2, Bin Li2,3,4, Huifang Zhou1,2, Xianqun Fan1,2. 1. Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China. 2. Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, China. 3. Shanghai Institute of Immunology, Shanghai JiaoTong University School of Medicine, Shanghai, China. 4. Department of Immunology and Microbiology, Shanghai JiaoTong University School of Medicine, Shanghai, China. 5. Department of Endocrinology and Metabolism, Shanghai Ninth People's Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China.
Abstract
CONTEXT: Unique features of local immunity in thyroid-associated ophthalmopathy (TAO) may affect disease progression. OBJECTIVE: To investigate the association between the orbital immune microenvironment and TAO development. DESIGN/SETTING/PARTICIPANTS: TAO and control orbital connective tissues were collected. MAIN OUTCOME MEASURES: Single-cell sequencing examined orbital lymphocytic infiltrates. Multicolor flow cytometry explored the phenotypes of different cell subsets and in vitro models for cell functional studies. Coculture experiment and western blotting assay were used to determine underlying mechanism of the enhanced T helper 17 (Th17) cell pathway. RESULTS: The TAO orbital microenvironment was composed of natural killer cells, dendritic cells, macrophages, T cells, plasma cells, and CD34+ orbital fibroblasts, but few B cells. Increases in CD3+CD8- IL-17A-producing and RAR-related orphan receptor (ROR)γt-expressing T cells and in CD3+CD8- IL-13-producing and GATA3-expressing T cells suggested Th17 and Th2 cell responses in TAO orbits. Increased interferon-γ (IFN-γ)-producing and RORγt+Tbet+ T cells indicated a Th1-like phenotype of orbital-infiltrating Th17 cells. Higher IL-23R and IL-1R expression and lower IL-21R expression were also observed on Th17 cells in TAO orbits. Multivariate analyses revealed that the Th17 pathway [IL-17A (P = 0.001), IFN-γ (P = 0.009), RORγt (P = 0.003), IL-23R (P = 0.033), IL-21R (P = 0.019)], and Th2 pathway [IL-13 (P = 0.015), GATA3 (P = 0.012)] were associated with TAO. IL-17A, IL-23R, and IL-1R correlated with clinical activity score and visual acuity. CD34+ orbital fibroblasts exhibited distinct cell surface marker expression and promoted IL-23R and IL-1R expression on T cells to facilitate the Th17-cell phenotype through prostaglandin E2-EP2/EP4-cAMP signaling. CONCLUSION: Our study addresses the importance of retroorbital immunity and suggests possible means of disrupting TAO pathogenesis.
CONTEXT: Unique features of local immunity in thyroid-associated ophthalmopathy (TAO) may affect disease progression. OBJECTIVE: To investigate the association between the orbital immune microenvironment and TAO development. DESIGN/SETTING/PARTICIPANTS: TAO and control orbital connective tissues were collected. MAIN OUTCOME MEASURES: Single-cell sequencing examined orbital lymphocytic infiltrates. Multicolor flow cytometry explored the phenotypes of different cell subsets and in vitro models for cell functional studies. Coculture experiment and western blotting assay were used to determine underlying mechanism of the enhanced T helper 17 (Th17) cell pathway. RESULTS: The TAO orbital microenvironment was composed of natural killer cells, dendritic cells, macrophages, T cells, plasma cells, and CD34+ orbital fibroblasts, but few B cells. Increases in CD3+CD8- IL-17A-producing and RAR-related orphan receptor (ROR)γt-expressing T cells and in CD3+CD8- IL-13-producing and GATA3-expressing T cells suggested Th17 and Th2 cell responses in TAO orbits. Increased interferon-γ (IFN-γ)-producing and RORγt+Tbet+ T cells indicated a Th1-like phenotype of orbital-infiltrating Th17 cells. Higher IL-23R and IL-1R expression and lower IL-21R expression were also observed on Th17 cells in TAO orbits. Multivariate analyses revealed that the Th17 pathway [IL-17A (P = 0.001), IFN-γ (P = 0.009), RORγt (P = 0.003), IL-23R (P = 0.033), IL-21R (P = 0.019)], and Th2 pathway [IL-13 (P = 0.015), GATA3 (P = 0.012)] were associated with TAO. IL-17A, IL-23R, and IL-1R correlated with clinical activity score and visual acuity. CD34+ orbital fibroblasts exhibited distinct cell surface marker expression and promoted IL-23R and IL-1R expression on T cells to facilitate the Th17-cell phenotype through prostaglandin E2-EP2/EP4-cAMP signaling. CONCLUSION: Our study addresses the importance of retroorbital immunity and suggests possible means of disrupting TAO pathogenesis.