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Chemokine Expression during Adipogenesis and Inflammation in Orbital Fibroblasts from Patients with Graves' Orbitopathy.

ABSTRACT: PURPOSE:Chemokines are involved in the pathogenesis of various autoimmune diseases, including Graves' orbitopathy (GO), but comprehensive analyses of the dynamics of these cytokines and their receptors in such diseases remain lacking. In this study, we investigated the expressions of chemokines and their receptors during adipogenesis and inflammation in primary cultured orbital fibroblasts from patients with GO. METHODS:The messenger RNA (mRNA) expression levels of chemokines were compared between GO (n = 6) and non-GO (n = 5) orbital tissues by real-time polymerase chain reaction. After adipogenesis was induced in primary cultured orbital fibroblasts from patients with GO (n =5) and following stimulation with interleukin (IL)-1? and tumor necrosis factor (TNF)-?, the mRNA expression levels of chemokines and their receptors were analyzed. RESULTS:Chemokines were significantly downregulated in GO orbital tissues compared to non-GO orbital tissues (p < 0.05). Adipogenesis resulted in a strong increase in mRNA expression levels of chemokines and their receptors at an early stage (day 1); however, expression levels started to decrease thereafter and, eventually, decreased to below basal levels at the end of adipogenesis (day 10). Following stimulation with IL-1? and TNF-?, the mRNA expression levels of chemokines and their receptors increased, showing different responses to various proinflammatory cytokines. CONCLUSIONS:Chemokines were strongly upregulated in the early phase of adipogenesis before decreasing continuously until the end of adipogenesis. Also, overt mature GO tissues showed reduced mRNA expression of chemokines compared to controls, which might indicate the existence of a shorter window for effective medical inflammatory treatment. The heightened levels of chemokines and their receptors observed after stimulation with IL-1? and TNF-? suggest a crucial role of proinflammatory cytokines in the pathogenesis of GO and, further, support the idea that chemokines could be used as biomarkers of GO activity.

SUBMITTER: Lee CE

PROVIDER: S-EPMC7269740 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Chemokine Expression during Adipogenesis and Inflammation in Orbital Fibroblasts from Patients with Graves' Orbitopathy.

Lee Chae Eun CE Choi Soo Hyun SH Yoon Jin Sook JS

Korean journal of ophthalmology : KJO 20200601 3

<h4>Purpose</h4>Chemokines are involved in the pathogenesis of various autoimmune diseases, including Graves' orbitopathy (GO), but comprehensive analyses of the dynamics of these cytokines and their receptors in such diseases remain lacking. In this study, we investigated the expressions of chemokines and their receptors during adipogenesis and inflammation in primary cultured orbital fibroblasts from patients with GO.<h4>Methods</h4>The messenger RNA (mRNA) expression levels of chemokines were ...[more]

PMID: 32495527

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Project description:PurposeTo examine the impact of gypenosides (Gyps) on oxidative stress damage of orbital fibroblasts (OFs) from Graves' ophthalmopathy (GO) patients.MethodsThe relationship between Gyps and GO oxidative stress was understood by bioinformatics analysis. Orbital connective tissues of GO and non-GO patients were obtained for primary OF culture. The proliferation level of OFs was measured by Cell Counting Kit-8 method, and the appropriate intervention concentration of Gyps and H2O2 was obtained. The expression of apoptosis-related protein mRNA was analyzed by RT-qPCR technique. ROS and SOD test suites were employed to detect the oxidative stress level in OFs. Flow cytometry apoptosis detection, TUNEL detection, and lactate dehydrogenase detection were used to analyze the level of apoptosis. Western blotting detection was utilized to examine the regulatory pathway of oxidative stress, apoptosis, and autophagy-related proteins. The changes of cell morphology, autophagosome, and autophagy lysosome were observed by transmission electron microscope.ResultsThe suitable intervention concentration of Gyps is 100 μg/mL, and the suitable intervention concentration of high concentration H2O2 is 350 μM. In comparison with the blank control group, the H2O2 intervention group enhanced the expression of apoptosis-related mRNA, the expression of ROS and SOD, the apoptosis rate, the expression of autophagy activation-related protein and Nrf2/ERK/HO-1 protein, and the number of autophagosomes and autophagy lysosomes. Compared with H2O2 intervention group, the expression of apoptosis-related mRNA decreased, ROS expression decreased, SOD expression increased, apoptosis rate decreased, autophagy activation-related protein expression decreased, Nrf2/ERK/HO-1 protein expression increased, and the quantity of autophagosomes and autophagy lysosomes decreased in H2O2 + Gyps intervention group.ConclusionGyps can decrease the oxidative stress level of OFs generated by H2O2, reduce cell autophagy, and reduce apoptosis. Gyps may regulate the oxidative stress response of OFs in GO patients via the Nrf2/ERK/HO-1 signaling pathway.

Project description:PurposeBruton's tyrosine kinase (BTK) is an essential protein in B-cell antigen receptor (BCR) signaling pathway and is known to be related to pathogenetic effect on B-cell related malignancies and various autoimmune diseases. In this study, we investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK inhibitor in the pathogenesis of Graves' orbitopathy (GO) in in vitro model.MethodsExpression of BTK in orbital tissues from GO and normal control subjects were evaluated by real-time polymerase chain reaction (PCR). Primary cultured orbital fibroblasts from each subject were exposed to ibrutinib and stimulated with interleukin (IL)-1β or insulin like growth factor (IGF)-1. Production of inflammatory cytokines was evaluated by real time PCR and enzyme-linked immunosorbent assays (ELISA). The downstream transcription factors were also determined by western blot assays.ResultsThe expression of BTK in GO tissues were significantly higher than in healthy controls. After stimulation of GO orbital fibroblasts with IL-1β or IGF-1, BTK mRNA and phosphorylated (p)- BTK protein expression was also enhanced. Ibrutinib reduced the expression of BTK mRNA and proteins of p-BTK, and inhibited the IL-1β- and IGF-1-induced production of proinflammatory cytokines including IL-6, IL-8 and COX-2 in both GO and normal cells. Ibrutinib also significantly attenuated phosphorylation of Akt, p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) in IL-1β stimulated GO cells and Akt, JNK, and NF-κB in IL-1ß stimulated normal cells.ConclusionsBTK expression is enhanced in GO tissue and orbital fibroblasts. Ibrutinib, a BTK inhibitor suppresses proinflammatory cytokine production as well as phosphorylation of Akt and NF-κB protein. Our results suggest the potential role of BTK in GO inflammatory pathogenesis and possibility of a novel therapeutic target of GO.

Dataset Information

Chemokine Expression during Adipogenesis and Inflammation in Orbital Fibroblasts from Patients with Graves' Orbitopathy.

Publications

Chemokine Expression during Adipogenesis and Inflammation in Orbital Fibroblasts from Patients with Graves' Orbitopathy.

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