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Item does not contain fulltext, OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology. METHODS: In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, Itgax-Cre:Runx3 (f/f) mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model. RESULTS: Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the RUNX3 gene, which is associated with polymorphism rs6672420, correlates with lower RUNX3 expression and SSc susceptibility. Hypoxia is another factor that decreases RUNX3 level in pDC. Mouse pDCs deficient of Runx3 show enhanced maturation markers on CpG stimulation. In vivo, deletion of Runx3 in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis. CONCLUSIONS: We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of Runx3 in SSc model further underscoring the role of pDCs in this disease.

Contains fulltext : 177298.pdf (publisher's version ) (Closed access), OBJECTIVE: Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNalpha than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. METHODS: We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. RESULTS: Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFNalpha, mimicking the PDC phenotype observed in SSc patients. CONCLUSION: Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFNalpha, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

Objective. Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFN alpha than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. Methods. We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. Results. Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFN alpha, mimicking the PDC phenotype observed in SSc patients. Conclusion. Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFN alpha, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

Contains fulltext : 136617.pdf (publisher's version ) (Open Access), BACKGROUND: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. METHODS: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. RESULTS: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (+/-SD) level of CXCL4 in patients with systemic sclerosis was 25,624+/-2652 pg per milliliter, which was significantly higher than the level in controls (92.5+/-77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346+/-1011 pg per milliliter), ankylosing spondylitis (1368+/-1162 pg per milliliter), or liver fibrosis (1668+/-1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. CONCLUSIONS: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypert

Contains fulltext : 97587.pdf (publisher's version ) (Closed access), OBJECTIVE: Suppressor of cytokine signalling (SOCS) proteins constitute a class of intracellular proteins that are key physiological regulators of immune cell function. It has previously been shown that antigen-presenting cells (APCs) overexpressing SOCS3 steer T helper immune responses and protect against experimental arthritis. A study was undertaken to investigate the contribution of SOCS3 in regulating invariant natural killer T (iNKT) cell responses during collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunised with type II collagen and adenoviruses encoding SOCS3 were administered intravenously before the clinical onset of arthritis. Murine APCs overexpressing SOCS3 were co-cultured with an iNKT cell hybridoma and interleukin 2 (IL-2) release was measured by Luminex multi-analyte technology. The frequency and activation of primary iNKT cells was assessed by flow cytometry. Murine APCs were analysed for cytokine and CD1d expression following viral SOCS3 gene transfer. Results : Viral overexpression of SOCS3 in APCs resulted in reduced activation of the iNKT cell hybridoma. Importantly, during initiation of CIA, adenovirus-mediated overexpression of SOCS3 in hepatic and splenic APCs inhibited iNKT cell expansion in both organs. The iNKT cell population from SOCS3-treated mice showed low expression of the early activation marker CD69 and primary liver iNKT cells produced less interferon gamma and IL-4 upon alpha-galactosylceramide stimulation. No differences in CD1d surface expression were observed, but SOCS3-transduced APCs produced decreased levels of proinflammatory cytokines and increased levels of IL-10. CONCLUSION: These results demonstrate a critical role for SOCS3 in controlling the immunostimulatory capacities of APCs, which has direct implications for the effector function of iNKT cells during arthritis.