Gene Expression Profiling of IL-17A-treated Synovial Fibroblasts from the Human Temporomandibular Joint
ABSTRACT: Synovial fibroblasts contribute to the inflammatory temporomandibular joint under pathogenic stimuli. Synovial fibroblasts and T cells participate in the perpetuation of joint inflammation in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. IL-17 is an inflammatory cytokine produced primarily by Th17 cells that plays critical roles in the pathogenesis of numerous autoimmune and inflammatory diseases. Here, we investigated the roles of IL-17A in temporomandibular joint disorders (TMD) by using genome-wide analysis of synovial fibroblasts isolated from patients with TMD. We analyzed the gene expression profiles of synovial fibroblasts that were treated with or without IL-17A. IL-17 induced gene expression in synovial fibroblasts from human temporomandibular joint was measured at 4 hours after treated with IL-17A (10 ng/ml) and untreated control samples. This experiment used one donor sample.
Project description:In psoriasis lesions, a diverse mixture of cytokines is upregulated which influence each other generating a complex inflammatory situation. Although this is the case, the inhibition of Interleukin-17A (IL-17A) alone showed unprecedented clinical results in patients, indicating that IL-17A is a critical inducer of psoriasis pathogenesis. To elucidate IL-17A-driven keratinocyte-intrinsic signaling pathways, we treated monolayers of normal human epidermal keratinocytes in vitro with a mixture of 6 cytokines (IL-17A, TNF-a, IL-17C, IL-22, IL-36g and IFN-g) involved in psoriasis, to mimic the inflammatory milieu in psoriasis lesions. Microarray and gene set enrichment analysis revealed that this cytokine mixture induced similar gene expression changes with the previous transcriptome studies using psoriasis lesions. Importantly, we identified a set of IL-17A-regulated genes in keratinocytes, which recapitulate typical psoriasis genes exemplified by DEFB4A, S100A7, IL19 and CSF3, based on differences in the expression profiles of cells stimulated with 6 cytokines versus cells stimulated with only 5 cytokines lacking IL-17A. Furthermore a specific IL-17A-induced gene, NFKBIZ, which encodes IkappaB-zeta, a transcriptional regulator for NF-kappaB, was demonstrated to have a significant role for IL-17A-induced gene expression. Thus, we present novel in vitro data from normal human keratinocytes that would help elucidating the IL-17A-driven keratinocyte activation in psoriasis. Cytokine mixture-induced gene expression in primary normal human epidermal keratinocytes (NHEKs) was measured at 24 hours after exposure. NHEKs were exposed to the combination of selected six cytokines (IL-17A: 100 ng/ml, TNF-a: 10 ng/ml, IFN-g: 10 ng/ml, IL-17C: 100 ng/ml, IL-22: 100 ng/ml, IL-36g: 500 ng/ml) , or to the different combinations of five of the six cytokines (in total, 7 different treatments and one untreated control). No replicate experiments were conducted.
Project description:The etiology of post-inflammatory gastrointestinal (GI) motility dysfunction, after resolution of acute symptoms of inflammatory bowel diseases (IBD) and intestinal infection, is largely unknown. Here, using an established mouse model of experimental enteritis, we show that enhancement of smooth muscle cell (SMC) contraction by interleukin (IL)-17A may be involved in postinflammatory GI hypermotility. To examine the effect of IL-17 in the small intestinal smooth muscle, we used whole genome microarray expression profiling to find out the genes which respond to IL-17 stimulus. The smooth muscle strips were peeled off from mouse small intestine and incubated 24h with or without IL-17. And we also examined the effect of 6-shogaol, which is one of the ingredients of Japanese traditional medicine for intestinal mortor disorder, Daikenchuto, in IL-17 stimulated small intestinal smooth muscle strip. The smooth muscle strips were peeled off from mice small intestine and incubated in the culture media for 24h with or without IL-17. A part of IL-17 stimulated strips were co-incubated with 6-shogaol. Six independent experiments were performed.
Project description:Psoriasis is a chronic inflammatory disease of the skin for which no cure has emerged. Its complex etiology requires the development of an in vitro model that appropriately recapitulates the physiopathology of this disease. In this study, we exploited the self-assembly method in order to develop a new tissue-engineered model of psoriatic skin substitutes. To circumvent the addition of immune cells, we supplemented the reconstructed psoriatic substitutes with a cocktail of four cytokines, TNF-α, IL-1α, IL-6 and IL-17, and monitored their impact on global gene expression by DNA microarray. The cytokines-supplemented substitutes have a more irregular epidermis, with protuberances and much thinner areas. Most interestingly, gene profiling on microarrays identified several genes reported as being deregulated psoriasis skin in vivo. Indeed, expression of the S100A12, IL8, DEFB4A and KYNU genes increased dramatically compared to their level in normal skin substitutes (P <0.005 to <0.05). In addition, the ACSBG1 gene, reported to be repressed in psoriasis, was also repressed in the cytokines-supplemented psoriatic substitutes compared to the controls (P <0.005). The product encoded by the genes deregulated in the cytokines-supplemented substitutes belong to biological pathways, such as the inflammatory and the immune responses, that are similarly altered in psoriasis in vivo. In conclusion, addition of cytokines to involved psoriatic substitutes alters the transcriptome of these cells in a manner similar to that observed with psoriasis in vivo. The addition of this pro-inflammatory cocktail, comparable cytokine in vivo psoriasis, prepares us for the next step: the characterization of the model once added immune cells. Tissue-engineered psoriatic human skin (TEPHS) cultivated with (number of replicates: 3) or without (number of replicates: 3) Cytokines (IL-17a, IL-6, IL1a, TNF-a).
Project description:Atherosclerosis is a chronic inflammatory disease. Lesion progression is primarily mediated by cells of the monocyte/macrophage lineage. Interleukin-17A is a pro-inflammatory cytokine, which modulates immune cell trafficking and is involved inflammation in (auto)immune and infectious diseases. But the role of IL-17A still remains controversial. In the current study we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. 26-weeks old apolipoprotein E-deficient (Apoe-/-) mice were fed a standard chow diet and treated either with IL-17A mAb (n=15) or irrelevant immunoglobulin (n=10) for 16 weeks. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied in vitro. Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (P=0.001) by reducing inflammatory burden and cellular infiltration (P=0.01) and improved lesion stability (P=0.01). In vitro experiments showed that IL-17A plays a role in chemoattractance, monocyte adhesion, sensitization of antigen-presenting cells toward pathogen-derived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a pro-inflammatory milieu and up-regulation of molecules expressed by the IL-17A-induced macrophage subtype. We here show for the first time that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabilization in advanced lesions in Apoe-/- mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte / macrophage lineage. In addition, translational experiments underline the relevance for the human system. Effects of IL-17A on human monocyte-derived macrophages were assessed (n=2 per group).
Project description:Rheumatoid arthritis (RA) and spondyloarthropathy (SpA) are chronic inflammatory disorders primarily targeting joints. Although the type of inflammatory cells present in affected joints implicated in the disease pathogenesis is the same, the diseases differ in terms of disease activity and manifestation. So to comprehend the two diseases in better terms, we carried out a comparative gene expression profiling using the inflammatory mononuclear cells from knee aspirated synovial fluid samples as this joint fluid bathes the affected region thereby giving us a closer view of the pathological site. We identified 1469 differentially regulated genes. Out of 1469 differentially regulated genes, 1366 genes were upregulated in SpA and 103 genes were downregulated in SpA versus RA. The proposed study carried out mRNA expression profiling of mononuclear cells from synovial fluid samples of seven RA and five SpA cases. We have used 4X44K and 1X44K arrays from Agilent Technologies platform. Based on the RNA integrity, selected molecules were processed further using single-color microarray based gene expression. The raw data files obtained from this study were eventually analyzed with R package of the BioConductor suite.
Project description:NLRP3 inflammasome assembles in response to stress or danger signals and leads to unconventional secretion of proinflammatory IL-1. FADD is an NLRP3 inflammasome component. Here we found that classical NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion, which required potassium efflux, functional NLRP3 sensor, ASC adaptor and caspase-1 scaffold molecule. FADD is a leaderless protein unconventionally secreted through plasma membrane-derived microvesicles. Blood-derived monocytes from rheumatoid arthritis (RA) patients secreted more FADD following NLRP3 inflammasome activation than those from healthy donors, and we found increased levels of FADD in the sera (ESPOIR cohort) and synovial fluids from RA patients. Levels of synovial FADD correlated with the inflammatory status of the joint. These data reveal that FADD secretion occurs during inflammatory disease in vivo.
Project description:IL-17A is a pro-inflammatory cytokine that promotes host defense against infections and contributes to the pathogenesis of chronic inflammatory diseases. Dendritic cells (DC) are antigen-presenting cells responsible for adaptive immune responses. Here, we report that IL-17A induces intense remodeling of lipid metabolism in human monocyte-derived DC, as revealed by microarrays analysis. In particular NR1H3/LXR-a and its target genes were significantly upregulated in response to IL-17A. IL-17A induced accumulation of Oil Red O-positive lipid droplets in DC leading to the generation of lipid-laden DC. A lipidomic study established that all the analyzed lipid species, i.e phospholipids, cholesterol, triglycerides, cholesteryl esters were elevated in IL-17A-treated DC. The increased expression of membrane lipid transporters in IL-17A-treated DC as well as their enhanced ability to uptake the fatty acid Bodipy-FL-C16 suggested that lipid uptake was the main mechanism responsible for lipid accumulation in response to IL-17A. IL-17A-induced lipid laden DC were able to stimulate allogeneic T cell proliferation in vitro as efficiently as untreated DC, indicating that IL-17A-treated DC are potently immunogenic. This study, encompassed in the field of immunometabolism, points out for the first time IL-17A as a modulator of lipid metabolism in DC and provides a rationale to delineate the importance of lipid-laden DC in IL-17A-related inflammatory diseases. We used microarrays analysis to understand the impact of IL-17A on human monocyte-derived human dendritic cells. We found overexpression of many genes involved in lipid metabolism in IL-17A-treated dendritic cells compared to untreated dendritic cells. In particular NR1H3/LXR-a and its target genes were significantly upregulated in response to IL-17A. IL-17A induced accumulation of Oil Red O-positive lipid droplets in DC leading to the generation of lipid-laden DC. A lipidomic study established that all the analyzed lipid species, i.e phospholipids, cholesterol, triglycerides, cholesteryl esters were elevated in IL-17A-treated DC. The increased expression of membrane lipid transporters in IL-17A-treated DC as well as their enhanced ability to uptake the fatty acid Bodipy-FL-C16 suggested that lipid uptake was the main mechanism responsible for lipid accumulation in response to IL-17A. IL-17A-induced lipid laden DC were able to stimulate allogeneic T cell proliferation in vitro as efficiently as untreated DC, indicating that IL-17A-treated DC are potently immunogenic. This study, encompassed in the field of immunometabolism, points out for the first time IL-17A as a modulator of lipid metabolism in DC and provides a rationale to delineate the importance of lipid-laden DC in IL-17A-related inflammatory diseases. RNA was extracted from untreated in vitro-generated DC at day 0 (DC, 4 biological replicates ) or DC cultured for 12 days with IL-17A, in the absence or presence of IFN-g (DC-17 and DC-G17, 5 biological replicates)
Project description:Inflammatory tissues are characterized by low oxigen concentrations (hypoxia). These conditions are very different from that usually present in tissue cultures where transcriptomic profiles of human fibroblasts from inflammatory tissues have been previously analysed. The aim of this study was to characterize the changes on gene expression induced by hypoxia in human synovial fibroblasts. We used microarray expression profiling in paired normoxic and hypoxic cultures of healthy and rheumatoid arthritis (RA) synovial fibroblasts (HSF and RASF). Hypoxia induces significant changes on the expression of large groups of genes in both HSF and RASF. The hypoxic and normoxic profiles are also different between both groups. These data demonstrate that hypoxia induces significant changes on gene expression in HSF and RASF and identify differences between RASF and HSF. Synovial fibroblasts obtained from 6 patients with rheumatoid arthritis (RASF) and 6 sex and age matched adult healthy donors (HSF) were used. SF cultures were incubated for 22 hours under normoxic or hypoxic (0.5% O2) conditions. Nine experiments per group were performed, single experiments with three SF lines, and duplicated in other three lines per group. All 18 normoxia-hypoxia experiments (36 microarray data) were used for paired analysis of the changes induced by hypoxia in HSF or RASF.
Project description:We examined the effect of IL-17 signaling pathway on extracellular matrix (ECM) expression and the involvement of IL-17 signaling pathway in pathogenesis of SSc. To identify differences in the expression pattern of ECM genes in IL-17A- or IL-17F-treated cells, we performed PCR array analysis, consisting of 84 ECM-related genes. Normal human dermal fibroblasts were cultured until they were confluent, and then stimulated with IL-17A or IL-17F for 12 hours, and total RNA was extracted. A mixture of equal amounts of mRNAs from three normal fibroblasts was prepared in the presence or absence of IL-17A or -17F, and mRNA expression profile was evaluated using PCR Array. Normal fibroblasts were obtained by skin biopsies from 3 healthy donors. Fibroblasts from donors were used and treated separately as indicated in the summary. Equal amount total RNA from each donor was pooled prior to gene expression analysis.