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Shannon J. C. Shan, Tong Wu, Tunde Mester, Raymond Douglas; TSH/M22 induces TNFα production in human fibrocytes: mechanistic aspects. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2603.
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© ARVO (1962-2015); The Authors (2016-present)
Fibrocytes (FC) are bone marrow-derived progenitor cells implicated in various conditions of inflammation and fibrosis. In Graves disease (GD) and Graves orbitopathy (GO), circulating FCs become more abundant and can infiltrate the thyroid and orbit. FCs express high levels of thyrotropin receptor (TSHR) and insulin growth factor-1 receptor (IGF-1R), two putative pathogenic autoantigens in GO. The role of the FCs and these receptors in the pathogenesis of GO remains to be fully delineated. Treatment of FCs with thyroid stimulating hormone (TSH) or M22 (activating TSHR antibody) induces the production of a multitude of cytokines, including the key pro-inflammatory cytokine, tumor necrosis factor α (TNFα). This study aims to characterize the molecular mechanisms underlying TSH/M22-induced TNFα production in FCs.
Circulating and cultured FCs from healthy and GD patients were treated with various combinations of TSH, M22, MG132 and AKTi (inhibitors of NF-kB and Akt, respectively), and teprotumumab (TMB, a human monoclonal IGF-1R blocking antibody). Extracellular and intracellular TNFα protein production was measured by Luminex and flow cytometry, respectively. Messenger RNA expression was quantified by real time PCR.
Treatment with TSH or M22 induced significant TNFα protein and mRNA production by FCs. TSH-induced TNFα protein production in cultured FCs was reduced by 53% and 81% when pretreated with MG132 and AKTi, respectively (p<0.0001). Similarly, TSH-induced TNFα mRNA expression was reduced from 93-fold to 16-fold expression with MG132 (p<0.001), and from 23-fold to 1.3-fold expression with AKTi (p<0.0001). TMB decreased TSH-induced TNFα protein production in circulating FCs from mean fluorescent index (MFI) value of 2.92 to 1.91, and mRNA expression in cultured FCs from 141- to 52-fold expression (p<0.0001). TMB also decreased M22-induced TNFα protein production from MFI of 1.67 to 1.12, and mRNA expression from 6- to 3-fold expression (p<0.0001).
TSH/M22 stimulates FCs to produce TNFα. Both steady state mRNA and protein levels are increased. This process involves the transcription factor NF-kB and its regulator Akt. TMB attenuates this activating effect of TSH/M22, suggesting that TSHR downstream signaling may be partially dependent on IGF-1R signaling. By modulating the proinflammatory properties of FC such as TNFα production, TMB may be a promising therapeutic agent for GO.
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