M1-Like Macrophages Modulate Fibrosis and Inflammation of Orbital Fibroblasts in Graves' Orbitopathy: Potential Relevance to Soluble Interleukin-6 Receptor

Background: Graves' orbitopathy (GO) is a disfiguring and sight-threatening autoimmune disease. Previous studies have shown the infiltration of macrophages in GO orbital connective tissues. However, the immunophenotypes of macrophages and their modulatory effects on orbital fibroblasts (OFs) have not been examined so far. In this study, we sought to determine the pathophysiology of macrophages in GO. Methods: In this case-control study, orbital connective tissues collected from 40 GO patients and 20 healthy controls were immunohistochemically stained for cytokines and macrophage cell surface antigens. The polarization of orbital-infiltrating macrophages was investigated by flow cytometry and immunofluorescence. Effects of interleukin (IL)-6 combined with soluble IL-6 receptor (sIL-6R) on the proliferation, differentiation, and inflammation of different OF subsets were examined by CCK-8, Western blotting, and Luminex assays, respectively. The antigen-presenting abilities of different OF subsets under IL-6/sIL-6R signaling were studied by proteomics. Finally, the differentiation of CD8+ IL-17A-producing T cells by sIL-6R was tested. Results: GO orbital connective tissues displayed increased IL-6, sIL-6R, STAT3, and IL-17A levels. CD86+ M1-like macrophages were predominant in active GO patients, while stable GO patients tended to have more CD163+ M2-like macrophages. The expression of IL-6 was higher in M1-like macrophages, and the expression of transforming growth factor-β was higher in M2-like macrophages both in GO orbital connective tissues in situ in vivo and in cell culture system in vitro. The IL-6/sIL-6R stimulation promoted the fibrosis of both CD34+ and CD34- OFs. Monocyte chemoattractant protein-1 expression was also induced by IL-6/sIL-6R stimulation in both OF subsets. IL-6/sIL-6R stimulation enhanced the antigen processing of CD34+ OFs through upregulating the intact major histocompatibility complex I and antigen transporters. However, the protein expressions of the thyrotropin receptor and insulin-like growth factor 1 receptor could not be directly increased by IL-6/sIL-6R stimulation in CD34+ OFs. Furthermore, sIL-6R was conducive to the differentiation of CD8+ IL-17A-producing T cells. Conclusions: Our study demonstrated the immunophenotypes of orbital-infiltrating macrophages that may activate OFs depending on the IL-6/sIL-6R signaling in GO. Our preclinical findings implicate, at least in part, the molecular rationale for blocking sIL-6R as a promising therapeutic agent for GO.