Abstract
Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), along with its receptor fibroblast growth factor-inducible (Fn)14, is associated with various biological activities including inflammation. However, its role in the pathogenesis of Graves’ orbitopathy (GO) is unknown. In this study, we investigated the mechanism by which TWEAK regulates inflammatory signaling in orbital fibroblasts from GO patients. We found that TWEAK and tumor necrosis factor-α (TNFA) mRNA levels were upregulated in GO as compared to non-GO tissue samples. TWEAK, TNF receptor (TNFR)1, TNFR2, and TNFR superfamily member 12A mRNA, and TWEAK and Fn14 protein levels were increased by interleukin (IL)-1β and TNF-α treatment. Treatment with exogenous recombinant TWEAK increased the transcript and protein expression of the pro-inflammatory cytokines IL-6, IL-8, and monocyte chemoattractant protein-1 to a greater extent in GO than in non-GO cells, while treatment with the anti-Fn14 antibody ITEM4 suppressed TWEAK-induced pro-inflammatory cytokine release and hyaluronan production. Additionally, the serum level of TWEAK was higher in Graves’ disease patients with (341.86 ± 86.3 pg/ml) as compared to those without (294.09 ± 41.44 pg/ml) GO and healthy subjects (255.33 ± 39.38 pg/ml), and was positively correlated with clinical activity score (r = 0.629, P < 0.001) and thyroid binding immunoglobulin level (r = 0.659, P < 0.001). These results demonstrate that TWEAK/Fn14 signaling contributes to GO pathogenesis. Moreover, serum TWEAK level is a potential diagnostic biomarker for inflammatory GO, and modulating TWEAK activity may be an effective therapeutic strategy for suppressing inflammation and tissue remodeling in GO.
Highlights
Graves’ orbitopathy (GO) is an inflammatory autoimmune disorder of the orbit associated with Graves’ disease (GD) that occurs concurrently with thyroid dysfunction in about 40% of cases[1]
We found that pro-inflammatory cytokine and hyaluronan production by GO fibroblasts in primary cultures was increased in response to exogenous rTWEAK, an effect that was abrogated by anti-Fn14 monoclonal antibody (mAb)
Expression of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor in orbital tissue and fibroblasts To evaluate the expression of TWEAK and its receptor in GO, orbital tissue explants were obtained from GO patients (n = 11) and non-GO healthy controls (n = 7), and relative tumor necrosis factor-α (TNFA), TWEAK, TNFR1, TNFR2, and TNFRSF12A mRNA levels were analyzed by real-time polymerase chain reaction (PCR)
Summary
Recombinant TWEAK, recombinant human IL-1β, and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA). Inhibitors of MAPK kinase 1 (PD98059), p38 MAPK (SB203580), JNK (SP600125), phosphoinositide. 3-kinase (PI3K; LY294002), and NF-κB p65 (SC514) were purchased from Calbiochem (La Jolla, CA, USA). Orbital tissue specimens were collected from patients during orbital decompression surgery for severe GO (n = 11; eight women and three men, aged 39–55 years). Normal orbital tissue was obtained over the course of orbital surgery for other non-inflammatory diseases from ageand sex-matched control subjects with no history of autoimmune thyroid disease or GO (n = 7; four women and three men, aged 34–53 years). The non-GO subjects underwent orbital wall fracture surgery (n = 3), evisceration (n = 2), and orbital benign mass excision (n = 2).
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