The Long Non-coding RNA NRIR Drives IFN-Response in Monocytes: Implication for Systemic Sclerosis

TLR4 activation initiates a signaling cascade leading to the production of type I IFNs and of the downstream IFN-stimulated genes (ISGs). Recently, a number of IFN-induced long non-coding RNAs (lncRNAs) that feed-back regulate the IFN response have been identified. Dysregulation of this process, collectively known as the “Interferon (IFN) Response,” represents a common molecular basis in the development of autoimmune and autoinflammatory disorders. Concurrently, alteration of lncRNA profile has been described in several type I IFN-driven autoimmune diseases. In particular, both TLR activation and the upregulation of ISGs in peripheral blood mononuclear cells have been identified as possible contributors to the pathogenesis of systemic sclerosis (SSc), a connective tissue disease characterized by vascular abnormalities, immune activation, and fibrosis. However, hitherto, a potential link between specific lncRNA and the presence of a type I IFN signature remains unclear in SSc. In this study, we identified, by RNA sequencing, a group of lncRNAs related to the IFN and anti-viral response consistently modulated in a type I IFN-dependent manner in human monocytes in response to TLR4 activation by LPS. Remarkably, these lncRNAs were concurrently upregulated in a total of 46 SSc patients in different stages of their disease as compared to 18 healthy controls enrolled in this study. Among these lncRNAs, Negative Regulator of the IFN Response (NRIR) was found significantly upregulated in vivo in SSc monocytes, strongly correlating with the IFN score of SSc patients. Weighted Gene Co-expression Network Analysis showed that NRIR-specific modules, identified in the two datasets, were enriched in “type I IFN” and “viral response” biological processes. Protein coding genes common to the two distinct NRIR modules were selected as putative NRIR target genes. Fifteen in silico-predicted NRIR target genes were experimentally validated in NRIR-silenced monocytes. Remarkably, induction of CXCL10 and CXCL11, two IFN-related chemokines associated with SSc pathogenesis, was reduced in NRIR-knockdown monocytes, while their plasmatic level was increased in SSc patients. Collectively, our data show that NRIR affects the expression of ISGs and that dysregulation of NRIR in SSc monocytes may account, at least in part, for the type I IFN signature present in SSc patients

CXCL4 triggers monocytes and macrophages to produce PDGF-BB, culminating in fibroblast activation: Implications for systemic sclerosis

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Serum microRNA screening and functional studies reveal miR-483-5p as a potential driver of fibrosis in systemic sclerosis

Abstract Objective MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. Methods The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjogren's syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. Results 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. Conclusions Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

Background and objective S ystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. Methods C hromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFNresponsive gene expression. Results 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFN\u3b1 induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. Conclusion SS c monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc

Association of MicroRNA-618 Expression With Altered Frequency and Activation of Plasmacytoid Dendritic Cells in Patients With Systemic Sclerosis

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

Exploring the role of monocyte dysregulation in the pathogenesis of Systemic Sclerosis

Background Systemic sclerosis (SSc) is a rare autoimmune disease in which the connective tissue in the skin and internal organs of patients thickens and hardens due to the excessive production of extracellular matrix (ECM) proteins. This process is also referred to as the development of fibrosis. Fibrosis affecting the internal organs such as the lungs or the cardiovascular system is a common cause of death for these patients. SSc is a complex disease in which a high degree of heterogeneity is observed in the patient population. What causes the onset of fibrosis is not exactly known, but clinical observations and the results of previous studies indicate the presence of vascular abnormalities and a persistent inflammation before the onset of fibrosis. Although various genetic changes have been identified as risk factors, the heredity of SSc is very limited. In addition to the genetic factors, epigenetic changes probably play an important role in SSc development. These epigenetic changes regulate the expression of genes without changing the genetic code. Examples of such regulatory mechanisms are the expression of non-coding RNAs such as microRNAs and long non-coding RNAs, DNA methylation and histone modifications. The theory behind the development of this deforming disease is therefore that environmental factors cause epigenetic changes in people genetically predisposed to SSc, leading to immune activation and subsequent stimulation of ECM overproduction by fibroblasts. In recent years, extensive research efforts have been spent to obtaining a better understanding of the pathogenesis of SSc. One of the recurring observations in previous studies is the presence of large amounts of monocytes in the affected skin of patients, which are very likely to contribute to the activation of fibroblasts and thereby stimulate the overproduction of ECM. Additionally, it is known that the concentration of several signaling proteins, including platelet factor 4 (CXCL4) and angiopoietin 2 (ANG2), is increased in the blood of SSc patients. However, the effects of these factors on the different immune cells are not well understood. Finally, the activation of both the innate and adaptive immune systems and the increased expression of type 1 interferon responsive genes, also known as the IFN signature, have been associated with the pathogenesis of SSc. Aim of this thesis The aim of this thesis was to study monocyte dysregulation in patients with SSc and to evaluate the effects of this dysregulation on the pathogenesis of SSc. To realize this, we first looked at changes in the frequency of monocytes in the circulation, changes at the level of gene expression in these cells and the histone modifications that may underlie this aberrant gene expression. In the second part of the thesis we studied the response of monocytes to CXCL4 and ANG2, two signaling factors associated with SSc, and looked at how this response can contribute to the inflammation and fibrosis observed in SSc patients. Summary of the findings In chapter 2, I describe the results of a study looking at the frequency of 44 different types of circulating immune cells in two cohorts of healthy people, patients with SSc and patients with other autoimmune conditions. The results indicated that an increased frequency of monocytes was present in the circulation of SSc patients compared with healthy people. This increase was not present in patients with other autoimmune diseases. In SSc patients, the frequency of circulating monocytes was directly correlated with the disease severity. For example, the degree of skin fibrosis was found to be related to the monocyte frequency and we found that SSc patients with lung involvement had more circulating monocytes compared with patients without lung involvement. In addition, the increase in monocytes was found to coincide with an increase in two signaling proteins, CXCL10 and CXCL11, which were previously shown to be related to SSc disease progression. The increase in monocytes was also present in so-called "early" SSc patients, indicating that the increased number of monocytes in circulation may be linked to disease progression. In chapter 3 we took a closer look at the dysregulation of monocytes in SSc patients. Using a global gene expression analysis, we identified genes with an abnormal expression pattern in patient monocytes compared with those of healthy people. The genes with increased expression were found to mainly play a role in the regulation of cell processes that are part of the immune response system, the production of cytokines and the IFN response. Concomitantly, we looked at epigenetic modifications of the histones that form the packaging material of the DNA and regulate its accessibility. We found that many alterations of these modifications are present in patients and show that these changes are linked to the sustained dysregulation of gene expression. We identified several histone-modifying enzymes whose aberrant gene expression may underlie the observed histone modifications. In a proof-of-concept experiment, we demonstrated that the effects of the altered histone modifications can be reversed with an inhibitor, JQ1, implying that epigenetic changes may be a pharmacological target for restoring homeostasis in the monocytes of SSc patients. Using a similar method as the one described in chapter 3, in chapter 4 we studied the changes of epigenetic histone modifications in monocytes from patients with SSc, systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) compared with healthy people. In this study, we looked at two different modifications, one of which is associated with the activation of gene expression while the other modification inhibits gene expression. In the monocytes of SSc and SLE patients, we found both unique and overlapping differences in these modifications compared with healthy monocytes. The genes associated with an increased amount of the activating modification had, on average, a higher expression level and were found to play a role in cellular processes associated with the pathogenesis of the various autoimmune disorders. Although a decrease in the inhibitory modification does not directly result in increased gene expression, it is known that it allows the expression of nearby genes to be quickly induced upon activation of the cell by exogenous or endogenous factors or during differentiation. In monocytes from SSc patients, genes with the inhibitory modification removed were found to encode pro-fibrotic factors or ECM molecules, such as THBS1, FAM20A and collagens, as well as growth factors such as PDGFB which we investigated further as described in chapter 6. In the second part of this thesis, we looked at the effects of SSc related signaling molecules on monocytes and evaluated how this can contribute to the inflammation and fibrosis which is observed in SSc patients. In chapter 5 we investigated the effect of the imbalance between angiopoietin (ANG) 1 and 2 observed in SSc patients on monocytes. Although ANG1 and ANG2 are primarily known as regulators of vascularization by binding to the TIE-2 receptor on endothelial cells, it has recently been shown that monocytes also express these receptors. Our results show that in SSc monocytes, the TIE-2 signaling induced by ANG2 causes the production of the pro-inflammatory cytokines IL-6 and IL-8. We found the same result after the stimulation of healthy monocytes with serum from SSc patients. In addition, our experiments indicated that the neutralization of ANG2 in the serum of patients or the inhibition of the TIE2 receptor could nullify this effect. This shows that ANG2 neutralization could be a promising therapeutic target in inhibiting inflammation to treat SSc. Finally, chapter 6 of this thesis describes how CXCL4 leads to activation of monocytes and macrophages and reveals how this activation contributes to inflammation and fibrosis in SSc patients. CXCL4 is a signaling protein of which the concentration is greatly increased in the circulation and affected tissue of SSc patients. The stimulation of monocytes and macrophages with CXCL4 increases the secretion of the growth factor PDGF-BB. This effect appears to be specific for CXCL4 as it was not observed after stimulation of monocytes with interferon, transforming growth factor (TGF) beta or ligands for the Toll-like receptors. In skin fibroblasts, PDGF-BB contributes to the induction of the expression of pro-inflammatory cytokines and proteins that play a role in the chemotaxis of immune cells. In addition, PDGF-BB has a pro-fibrotic function as it stimulates the production of ECM molecules such as collagen and fibronectin in fibroblasts. That the CXCL4 activation of macrophages can directly contribute to fibrosis was shown in experiments where the culture medium of CXCL4- stimulated macrophages was found to induce inflammation and ECM production in fibroblasts. Blocking the PDGF receptor with Crenolanib, a specific inhibitor molecule, prevented this response which implies that intervention with the CXCL4/PDGF-bb signaling axis in monocytes may hold therapeutic value. In summary, we can state that there is a clear dysregulation of monocytes in patients with SSc. This dysregulation is strongly related to the disease severity and is therefore very likely directly involved in the pathogenesis of SSc. Future research will hopefully show how the prevention of monocyte dysregulation can contribute to curing or preventing SSc

New insights into the genetics and epigenetics of systemic sclerosis

Systemic sclerosis (SSc) is a severe autoimmune disease that is characterized by vascular abnormalities, immunological alterations and fibrosis of the skin and internal organs. The results of genetic studies in patients with SSc have revealed statistically significant genetic associations with disease manifestations and progression. Nevertheless, genetic susceptibility to SSc is moderate, and the functional consequences of genetic associations remain only partially characterized. A current hypothesis is that, in genetically susceptible individuals, epigenetic modifications constitute the driving force for disease initiation. As epigenetic alterations can occur years before fibrosis appears, these changes could represent a potential link between inflammation and tissue fibrosis. Epigenetics is a fast-growing discipline, and a considerable number of important epigenetic studies in SSc have been published in the past few years that span histone post-translational modifications, DNA methylation, microRNAs and long non-coding RNAs. This Review describes the latest insights into genetic and epigenetic contributions to the pathogenesis of SSc and aims to provide an improved understanding of the molecular pathways that link inflammation and fibrosis. This knowledge will be of paramount importance for the development of medicines that are effective in treating or even reversing tissue fibrosis

Characterization of Long Non-Coding RNAs in Systemic Sclerosis Monocytes: A Potential Role for PSMB8-AS1 in Altered Cytokine Secretion

Systemic sclerosis (SSc) is a chronic autoimmune disease mainly affecting the connective tissue. In SSc patients, monocytes are increased in circulation, infiltrate affected tissues, and show a pro-inflammatory activation status, including the so-called interferon (IFN) signature. We previously demonstrated that the dysregulation of the IFN response in SSc monocytes is sustained by altered epigenetic factors as well as by upregulation of the long non-coding RNA (lncRNA) NRIR. Considering the enormously diverse molecular functions of lncRNAs in immune regulation, the present study investigated the genome-wide profile of lncRNAs in SSc monocytes, with the aim to further unravel their possible role in monocyte dysregulation and disease pathogenesis. Transcriptomic data from two independent cohorts of SSc patients identified 886 lncRNAs with an altered expression in SSc monocytes. Differentially expressed lncRNAs were correlated with neighboring protein coding genes implicated in the regulation of IFN responses and apoptotic signaling in SSc monocytes. In parallel, gene co-expression network analysis identified the lncRNA PSMB8-AS1 as a top-ranking hub gene in co-expression modules implicated in cell activation and response to viral and external stimuli. Functional characterization of PSMB8-AS1 in monocytes demonstrated that this lncRNA is involved in the secretion of IL-6 and TNF\u3b1, two pivotal pro-inflammatory cytokines altered in the circulation of SSc patients and associated with fibrosis and disease severity. Collectively, our data showed that lncRNAs are linked to monocyte dysregulation in SSc, and highlight their potential contribution to disease pathogenesis

The Long Non-coding RNA NRIR Drives IFN-Response in Monocytes : Implication for Systemic Sclerosis

TLR4 activation initiates a signaling cascade leading to the production of type I IFNs and of the downstream IFN-stimulated genes (ISGs). Recently, a number of IFN-induced long non-coding RNAs (lncRNAs) that feed-back regulate the IFN response have been identified. Dysregulation of this process, collectively known as the "Interferon (IFN) Response," represents a common molecular basis in the development of autoimmune and autoinflammatory disorders. Concurrently, alteration of lncRNA profile has been described in several type I IFN-driven autoimmune diseases. In particular, both TLR activation and the upregulation of ISGs in peripheral blood mononuclear cells have been identified as possible contributors to the pathogenesis of systemic sclerosis (SSc), a connective tissue disease characterized by vascular abnormalities, immune activation, and fibrosis. However, hitherto, a potential link between specific lncRNA and the presence of a type I IFN signature remains unclear in SSc. In this study, we identified, by RNA sequencing, a group of lncRNAs related to the IFN and anti-viral response consistently modulated in a type I IFN-dependent manner in human monocytes in response to TLR4 activation by LPS. Remarkably, these lncRNAs were concurrently upregulated in a total of 46 SSc patients in different stages of their disease as compared to 18 healthy controls enrolled in this study. Among these lncRNAs, Negative Regulator of the IFN Response (NRIR) was found significantly upregulated in vivo in SSc monocytes, strongly correlating with the IFN score of SSc patients. Weighted Gene Co-expression Network Analysis showed that NRIR-specific modules, identified in the two datasets, were enriched in "type I IFN" and "viral response" biological processes. Protein coding genes common to the two distinct NRIR modules were selected as putative NRIR target genes. Fifteen in silico-predicted NRIR target genes were experimentally validated in NRIR-silenced monocytes. Remarkably, induction of CXCL10 and CXCL11, two IFN-related chemokines associated with SSc pathogenesis, was reduced in NRIR-knockdown monocytes, while their plasmatic level was increased in SSc patients. Collectively, our data show that NRIR affects the expression of ISGs and that dysregulation of NRIR in SSc monocytes may account, at least in part, for the type I IFN signature present in SSc patients

The Long Non-coding RNA NRIR Drives IFN-Response in Monocytes : Implication for Systemic Sclerosis

TLR4 activation initiates a signaling cascade leading to the production of type I IFNs and of the downstream IFN-stimulated genes (ISGs). Recently, a number of IFN-induced long non-coding RNAs (lncRNAs) that feed-back regulate the IFN response have been identified. Dysregulation of this process, collectively known as the "Interferon (IFN) Response," represents a common molecular basis in the development of autoimmune and autoinflammatory disorders. Concurrently, alteration of lncRNA profile has been described in several type I IFN-driven autoimmune diseases. In particular, both TLR activation and the upregulation of ISGs in peripheral blood mononuclear cells have been identified as possible contributors to the pathogenesis of systemic sclerosis (SSc), a connective tissue disease characterized by vascular abnormalities, immune activation, and fibrosis. However, hitherto, a potential link between specific lncRNA and the presence of a type I IFN signature remains unclear in SSc. In this study, we identified, by RNA sequencing, a group of lncRNAs related to the IFN and anti-viral response consistently modulated in a type I IFN-dependent manner in human monocytes in response to TLR4 activation by LPS. Remarkably, these lncRNAs were concurrently upregulated in a total of 46 SSc patients in different stages of their disease as compared to 18 healthy controls enrolled in this study. Among these lncRNAs, Negative Regulator of the IFN Response (NRIR) was found significantly upregulated in vivo in SSc monocytes, strongly correlating with the IFN score of SSc patients. Weighted Gene Co-expression Network Analysis showed that NRIR-specific modules, identified in the two datasets, were enriched in "type I IFN" and "viral response" biological processes. Protein coding genes common to the two distinct NRIR modules were selected as putative NRIR target genes. Fifteen in silico-predicted NRIR target genes were experimentally validated in NRIR-silenced monocytes. Remarkably, induction of CXCL10 and CXCL11, two IFN-related chemokines associated with SSc pathogenesis, was reduced in NRIR-knockdown monocytes, while their plasmatic level was increased in SSc patients. Collectively, our data show that NRIR affects the expression of ISGs and that dysregulation of NRIR in SSc monocytes may account, at least in part, for the type I IFN signature present in SSc patients