So Young Han1,2, Soo Hyun Choi2, Jeon-Soo Shin3, Eun Jig Lee4, Sueng-Han Han2, Jin Sook Yoon2. 1. 1 Department of Ophthalmology, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. 2. 2 Institute of Vision Research, Department of Ophthalmology, Severance Hospital; Severance Hospital, Institute of Endocrine Research; Yonsei University College of Medicine, Seoul, Republic of Korea. 3. 3 Department of Microbiology, BK21 PLUS Project for Medical Science, Institute for Immunology and Immunological Diseases and Severance Biomedical Science Institute; Severance Hospital, Institute of Endocrine Research; Yonsei University College of Medicine, Seoul, Republic of Korea. 4. 4 Department of Endocrinology, Severance Hospital, Institute of Endocrine Research; Yonsei University College of Medicine, Seoul, Republic of Korea.
Abstract
Background: High-mobility group box 1 (HMGB1) has been implicated in the pathogenesis of inflammatory autoimmune diseases. This study investigated the influence and mechanisms of HMGB1 in Graves' orbitopathy (GO). Methods: HMGB1 and its receptors (receptor for advanced glycation end products [RAGE], Toll-like receptor [TLR] 2, and TLR4) mRNA levels were evaluated by real-time polymerase chain reaction (RT-PCR) in GO and non-GO orbital tissues. The mRNA expressions of HMGB1 and its receptors were evaluated in primary cultured orbital fibroblasts from six GO patients and five healthy control subjects under interleukin (IL)-1β or tumor necrosis factor (TNF)-α stimulation using RT-PCR. HMGB1 secretions under IL-1β or TNF-α stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA). The effects of an anti-HMGB1 antibody, RAGE antagonist (FPS-ZM1), and anti-TLR2 antibody on the expressions of IL-1β or TNF-α induced pro-inflammatory cytokines and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells were evaluated using ELISA and Western blot analysis, respectively. The plasma levels of HMGB1 were compared among patients with active GO (n = 51), inactive GO (n = 48), Graves' disease without GO (n = 30), and healthy control subjects (n = 46) by ELISA. Results: The genes encoding HMGB1 and its receptors, as well as HMGB1 protein expression, were increased in GO orbital tissues compared to non-GO tissues. IL-1β and TNF-α stimulation increased the mRNA levels of HMGB1, RAGE, and TLR2 and the secretion of HMGB1 protein further in GO cells. Anti-HMGB1 antibody, FPS-ZM1, and anti-TLR2 antibody reduced IL-1β- or TNF-α-induced production of pro-inflammatory cytokines and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells. The plasma levels of HMGB1 were highly increased in patients with active GO, and were significantly correlated with the clinical activity score (r = 0.566, p = 0.002) and levels of thyrotropin binding inhibitory immunoglobulin (r = 0.506, p < 0.001). Conclusions: This study demonstrates an association of HMGB1 and its receptors in the inflammatory mechanisms of GO. HMGB1, RAGE, and TLR2 blockers reduced the production of pro-inflammatory molecules, providing a rationale for blocking the HMGB1 pathway to treat patients with GO. HMGB1 proteins were secreted further in the plasma of patients with active GO, suggesting that HMGB1 can be used as a biomarker of GO activity.
Background: High-mobility group box 1 (HMGB1) has been implicated in the pathogenesis of inflammatory autoimmune diseases. This study investigated the influence and mechanisms of HMGB1 in Graves' orbitopathy (GO). Methods:HMGB1 and its receptors (receptor for advanced glycation end products [RAGE], Toll-like receptor [TLR] 2, and TLR4) mRNA levels were evaluated by real-time polymerase chain reaction (RT-PCR) in GO and non-GO orbital tissues. The mRNA expressions of HMGB1 and its receptors were evaluated in primary cultured orbital fibroblasts from six GO patients and five healthy control subjects under interleukin (IL)-1β or tumor necrosis factor (TNF)-α stimulation using RT-PCR. HMGB1 secretions under IL-1β or TNF-α stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA). The effects of an anti-HMGB1 antibody, RAGE antagonist (FPS-ZM1), and anti-TLR2 antibody on the expressions of IL-1β or TNF-α induced pro-inflammatory cytokines and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells were evaluated using ELISA and Western blot analysis, respectively. The plasma levels of HMGB1 were compared among patients with active GO (n = 51), inactive GO (n = 48), Graves' disease without GO (n = 30), and healthy control subjects (n = 46) by ELISA. Results: The genes encoding HMGB1 and its receptors, as well as HMGB1 protein expression, were increased in GO orbital tissues compared to non-GO tissues. IL-1β and TNF-α stimulation increased the mRNA levels of HMGB1, RAGE, and TLR2 and the secretion of HMGB1 protein further in GO cells. Anti-HMGB1 antibody, FPS-ZM1, and anti-TLR2 antibody reduced IL-1β- or TNF-α-induced production of pro-inflammatory cytokines and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells. The plasma levels of HMGB1 were highly increased in patients with active GO, and were significantly correlated with the clinical activity score (r = 0.566, p = 0.002) and levels of thyrotropin binding inhibitory immunoglobulin (r = 0.506, p < 0.001). Conclusions: This study demonstrates an association of HMGB1 and its receptors in the inflammatory mechanisms of GO. HMGB1, RAGE, and TLR2 blockers reduced the production of pro-inflammatory molecules, providing a rationale for blocking the HMGB1 pathway to treat patients with GO. HMGB1 proteins were secreted further in the plasma of patients with active GO, suggesting that HMGB1 can be used as a biomarker of GO activity.
Entities:
Keywords:
Graves' orbitopathy; Toll-like receptor 2; high mobility group box 1; inflammation; receptor for advanced glycation end products
Authors: Yeon Jeong Choi; Charm Kim; Eun Woo Choi; Seung Hun Lee; Min Kyung Chae; Hyung Oh Jun; Bo-Yeon Kim; Jin Sook Yoon; Sun Young Jang Journal: PLoS One Date: 2022-08-18 Impact factor: 3.752