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Enhanced orbital adipogenesis in a mouse model of T-cell-mediated autoimmunity, zymosan A-treated SKG mice: Implications for Graves' ophthalmopathy.

ABSTRACT: Inflammation and remodelling of orbital tissue associated with enhanced adipogenesis commonly occur in Graves' ophthalmopathy (GO), however, the underlying mechanisms that link immune cells and adipocytes in orbital inflammation are not well-known. The primary aim of this study was to elucidate how a genetically determined shift in the T-cell repertoire toward self-reactive T-cells could drive orbital adipogenesis. To induce the T-cell-mediated autoimmune response, SKG mice were intraperitoneally injected with zymosan A once at 8 weeks of age. After three months, orbital magnetic resonance imaging (MRI), histopathologic studies, and in vitro analyses were performed to evaluate inflammation and adipogenesis. The eyes of the zymosan A-treated SKG mice displayed proptosis and blepharitis. A detailed analysis of orbital adipose tissue showed enhanced orbital adipogenesis and cellular infiltration compared to controls. In addition, increased secretion of adipokines and other cytokines in the periorbital tissue was observed, together with elevated serum concentration of inflammatory cytokines. Orbital adipogenesis was enhanced in zymosan A-treated SKG mice, a novel mouse model for GO-like inflammatory adipose phenotypes most likely induced by T-cell mediated autoimmune responses. This mouse model gives us the opportunity to examine the underlying molecular mechanisms of enhanced adipogenesis in GO, ultimately providing a potential therapeutic target alternative to conventional GO treatment.

SUBMITTER: Park S

PROVIDER: S-EPMC7193596 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Enhanced orbital adipogenesis in a mouse model of T-cell-mediated autoimmunity, zymosan A-treated SKG mice: Implications for Graves' ophthalmopathy.

Park Sera S Park Dae-Young DY Kim Jaeryung J Woo Kyung In KI Kim Yoon-Duck YD Han Jisang J Chung Tae-Young TY Cha Hoon-Suk HS Lim Dong Hui DH

Scientific reports 20200430 1

Inflammation and remodelling of orbital tissue associated with enhanced adipogenesis commonly occur in Graves' ophthalmopathy (GO), however, the underlying mechanisms that link immune cells and adipocytes in orbital inflammation are not well-known. The primary aim of this study was to elucidate how a genetically determined shift in the T-cell repertoire toward self-reactive T-cells could drive orbital adipogenesis. To induce the T-cell-mediated autoimmune response, SKG mice were intraperitoneall ...[more]

PMID: 32355208

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Project description:BackgroundGraves' ophthalmopathy (GO) is a frequent extrathyroidal complication of Graves' hyperthyroidism. Orbital fibroblasts contribute to both orbital tissue inflammation and remodeling in GO, and as such are crucial cellular elements in active GO and inactive GO. However, so far it is largely unknown whether GO disease progression is associated with functional reprogramming of the orbital fibroblast effector function. Therefore, the aim of this study was to compare both the proteome and global DNA methylation patterns between orbital fibroblasts isolated from active GO, inactive GO and healthy controls.MethodsOrbital fibroblasts from inactive GO (n=5), active GO (n=4) and controls (n=5) were cultured and total protein and DNA was isolated. Labelled and fractionated proteins were analyzed with a liquid chromatography tandem-mass spectrometer (LC-MS/MS). Data are available via ProteomeXchange with identifier PXD022257. Furthermore, bisulphite-treated DNA was analyzed for methylation pattern with the Illumina Infinium Human Methylation 450K beadchip. In addition, RNA was isolated from the orbital fibroblasts for real-time quantitative (RQ)-PCR. Network and pathway analyses were performed.ResultsOrbital fibroblasts from active GO displayed overexpression of proteins that are typically involved in inflammation, cellular proliferation, hyaluronan synthesis and adipogenesis, while various proteins associated with extracellular matrix (ECM) biology and fibrotic disease, were typically overexpressed in orbital fibroblasts from inactive GO. Moreover, orbital fibroblasts from active GO displayed hypermethylation of genes that linked to inflammation and hypomethylated genes that linked to adipogenesis and autoimmunity. Further analysis revealed networks that contained molecules to which both hypermethylated and hypomethylated genes were linked, including NF-κB, ERK1/2, Alp, RNA polymerase II, Akt and IFNα. In addition, NF-κB, Akt and IFNα were also identified in networks that were derived from the differentially expressed proteins. Generally, poor correlation between protein expression, DNA methylation and mRNA expression was observed.ConclusionsBoth the proteomics and DNA methylation data support that orbital fibroblasts from active GO are involved in inflammation, adipogenesis, and glycosaminoglycan production, while orbital fibroblasts from inactive disease are more skewed towards an active role in extracellular matrix remodeling. This switch in orbital fibroblast effector function may have therapeutic implications and further studies into the underlying mechanism are thus warranted.

Project description:Graves' ophthalmopathy (GO) is a common orbital disease that threatens visual function and appearance. Orbital fibroblasts (OFs) are considered key target and effector cells in GO. In addition, hyaluronan (HA) production, inflammation, and orbital fibrosis are intimately linked to the pathogenesis of GO. In this study, we explored the therapeutic effects of dihydroartemisinin (DHA), an antimalarial drug, on GO-derived, primary OFs. CCK8 and EdU assays were applied to evaluate the antiproliferative effect of DHA on OFs. Wound healing assays were conducted to assess OF migration capacity, while qRT-PCR, western blotting, ELISA, and immunofluorescence were used to determine the expression of fibrosis-related and pro-inflammatory markers in these cells. Moreover, RNA sequencing was conducted to identify differentially expressed genes (DEGs) in DHA-treated OFs, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs was performed to explore potential mechanisms mediating the antifibrotic effect of DHA on GO-derived OFs. Results showed that DHA dose-dependently inhibited OF proliferation and downregulated, at the mRNA and protein levels, TGF-β1-induced expression of fibrosis markers, including alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). Furthermore, DHA inhibited TGF-β1 induced phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3), which suggested that DHA exerted antifibrotic effects via suppression of the ERK and STAT3 signaling pathways. In addition, DHA suppressed the expression of pro-inflammatory cytokines and chemokines, including IL-6, IL-8, CXCL-1, MCP-1, and ICAM-1, and attenuated HA production induced by IL-1β in GO-derived OFs. In conclusion, our study provides first-time evidence that DHA may significantly alleviate pathogenic manifestations of GO by inhibiting proliferation, fibrosis- and inflammation-related gene expression, and HA production in OFs. These data suggest that DHA may be a promising candidate drug for treatment of GO.

Project description:PurposeChemokines 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.MethodsThe 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)-1β and tumor necrosis factor (TNF)-α, the mRNA expression levels of chemokines and their receptors were analyzed.ResultsChemokines 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-1β and TNF-α, the mRNA expression levels of chemokines and their receptors increased, showing different responses to various proinflammatory cytokines.ConclusionsChemokines 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-1β and TNF-α 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.

Project description:BackgroundGraves' ophthalmopathy (GO) is a disorder, in which orbital connective tissues get in inflammation and increase in volume. Stimulants such as thyroid-stimulating hormone (TSH), insulin-like growth factor 1(IGF-1), IL-1, interferon γ, and platelet-derived growth factor cause differentiation into adipocytes of orbital fibroblasts (OFs) in the orbital fat and extraocular muscles. Human placental mesenchymal stem cells (hPMSCs) are known to have immune modulation effects on disease pathogenesis. Some reports suggest that hPMSCs can elicit therapeutic effects, but to date, research on this has been insufficient. In this study, we constructed PRL-1 overexpressed hPMSCs (hPMSCsPRL-1) in an attempt to enhance the suppressive function of adipogenesis in GO animal models.MethodsIn order to investigate the anti-adipogenic effects, primary OFs were incubated with differentiation medium for 10 days. After co-culturing with hPMSCsPRL-1, the characteristics of the OFs were analyzed using Nile red stain and quantitative real-time polymerase chain reaction. We then examined the in vivo regulatory effectiveness of hPMSCsPRL-1 in a GO mouse model that immunized by leg muscle electroporation of pTriEx1.1Neo-hTSHR A-subunit plasmid. Human PMSCsPRL-1 injection was performed in left orbit. We also analyzed the anti-adipogenic effects of hPMSCsPRL-1 in the GO model.ResultsWe found that hPMSCsPRL-1 inhibited adipogenic activation factors, specifically PPARγ, C/EBPα, FABP4, SREBP2, and HMGCR, by 75.1%, 50%, 79.6%, 81.8%, and 87%, respectively, compared with naïve hPMSCs in adipogenesis-induced primary OFs from GO. Moreover, hPMSCsPRL-1 more effectively inhibited adipogenic factors ADIPONECTIN and HMGCR by 53.2% and 31.7%, respectively, than hPMSCs, compared with 15.8% and 29.8% using steroids in the orbital fat of the GO animal model.ConclusionOur findings suggest that hPMSCsPRL-1 would restore inflammation and adipogenesis of GO model and demonstrate that they could be applied as a novel treatment for GO patients.

Dataset Information

Enhanced orbital adipogenesis in a mouse model of T-cell-mediated autoimmunity, zymosan A-treated SKG mice: Implications for Graves' ophthalmopathy.

Publications

Enhanced orbital adipogenesis in a mouse model of T-cell-mediated autoimmunity, zymosan A-treated SKG mice: Implications for Graves' ophthalmopathy.

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