Systemic Sclerosis Fibroblasts Research Articles

Alternative splicing and intron retention: Their profiles and roles in cutaneous fibrosis of systemic sclerosis

BackgroundAlternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc). MethodsWe integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins. FindingsAS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-β stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features. ConclusionTotally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-β-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc.

Mutual amplification of GLI2/Hedgehog and cJUN/AP1 signaling in fibroblasts in Systemic Sclerosis (SSc) - potential implications for combined therapies.

Deregulation of the cJUN/AP1- and hedgehog/GLI2 signaling pathways have been implicated in fibroblast activation in Systemic Sclerosis (SSc). However, the consequences of their concomitant upregulation are unknown. Here, we tested the hypothesis that mutual amplification of both pathways might drive persistent fibroblast activation. Cultured fibroblasts and skin sections of diffuse SSc-patients and healthy volunteers were analyzed. cJUN/AP1- and hedgehog/GLI2-signaling were inhibited using knockdown and pharmacologic approaches. Hedgehog signaling was activated in mice by fibroblast-specific overexpression of constitutively-active Smoothend. cJUN and GLI2 are concomitantly upregulated and colocalize in fibroblasts of SSc patients compared to healthy controls. Activation of hedgehog/GLI2 signaling induces the expression of cJUN in vitro and in vivo, whereas inactivation of GLI2 inhibits cJUN expression. Likewise, inactivation of cJUN impairs the expression of GLI2. This mutual regulation occurs at the level of transcription with binding of cJUN and GLI2 to specific binding motifs. Interference with this mutual amplification of cJUN- and GLI2-signaling inhibits fibroblast activation and collagen release: Inhibition of cJUN/AP1-signaling ameliorates hedgehog-induced fibroblast activation and skin fibrosis in SmoACT-mice with a reduction of skin thickness of 103 % (p=0.0043) in the treatment group compared to the fibrotic control group. Moreover, combined pharmacological inhibition of cJUN/AP1- and hedgehog/GLI2 exerts additive antifibrotic effects in a model of TGFβ-driven experimental fibrosis (TBRACTmice). The transcription factors cJUN and GLI2 reinforce each other's activity to promote fibroblast activation in SSc. Interruption of this crosstalk by combined inhibition of both pathways exerts additive anti-fibrotic effects at well tolerated doses.

Talabostat, fibroblast activation protein inhibitor, attenuates inflammation and fibrosis in systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by excessive fibrosis, where activated fibroblasts play a pivotal role in disease progression. This study aimed to investigate the potential of Talabostat, a small molecule inhibitor of dipeptidyl peptidases, in alleviating fibrosis and inflammation associated with SSc pathogenesis. Dermal fibroblasts were obtained from skin biopsies of ten diffuse cutaneous SSc patients and healthy controls. These fibroblasts were subjected to treatment with either TGF-β alone or in combination with Talabostat. Immunofluorescence staining was conducted to evaluate FAPα and α-SMA protein levels. The expression of activated fibroblast markers (FAPα and ACAT2), pro-fibrotic (COL1A1 and COL1A2), anti-fibrotic (MMP1, MMP2, and MMP9), and inflammatory (IL-6 and TGFβ1) related genes was measured by quantitative real-time PCR. Talabostat-treated fibroblasts were assessed for their migratory capacity using a scratch assay and for their viability through MTT assay and Annexin V staining. The basal expression of COL1A1 and TGFβ1 was notably higher in healthy subjects, while MMP1 expression showed a significant increase in SSc patients. Furthermore, TGF-β stimulation led to upregulation of activated fibroblast markers, pro-fibrotic, and inflammatory-related genes in SSc-derived fibroblasts, which were attenuated upon Talabostat treatment. Interestingly, Talabostat treatment resulted in an upregulation of MMP9 expression. Moreover, Talabostat exhibited a concentration-dependent inhibition of activated fibroblast viability in both healthy and SSc fibroblasts, and suppressed fibroblast migration specifically in SSc patients. In summary, Talabostat modulates fibrotic genes in SSc, thereby inhibiting myofibroblast differentiation, activation, and migration. These findings suggest promising therapeutic avenues for targeting fibrosis in SSc.

P27 Chloride intracellular channel 4 is a global regulator of type 1 interferon signalling in systemic sclerosis skin

Abstract Introduction and aims Systemic sclerosis (SSc) is an autoimmune disease in which an immune-related injury induces fibrosis of the skin and can progress to affect the internal organs in the most serve cases. Aberrant type 1 interferon (IFN) signalling in the keratinocytes plays a major role in SSc disease progression. We have previously shown the chloride intracellular channel 4 (CLIC)4 is upregulated in SSc skin fibroblasts and plays an important role in SSc fibrosis by enhancing transforming growth factor-β signalling. In this study, we investigated the role of CLIC4 in SSc keratinocyte biology. Methods Skin biopsies from patients with SSc (N = 6) and healthy controls (HCs) (N = 6) were analysed by immunohistochemistry for the expression of CLIC4 and phosphorylated signal transducer and activator of transcription (STAT)1. The skin keratinocyte cell line HaCaTs was stimulated with a range of type 1 IFN signalling agonists including POLY I:C, POLY dA:dT, ODN 2216 and IFN-α. CLIC4 was inhibited with the chloride channel inhibitors NPPB and IAA-94 or small interfering RNA specific to CLIC4. Conditioned media from HC or SSc dermal fibroblasts was employed for indirect coculture of HaCaTs. Results Skin biopsies from SSc showed high levels of CLIC4 in SSc skin fibroblasts, keratinocytes and endothelial cells compared with HC. Indirect coculture of HaCaTs with SSc fibroblast media induced CLIC4 expression in the HaCaTs. The increased CLIC4 expression played an important role in type 1 IFN signalling in keratinocytes as inhibition of CLIC4 blocked Toll-like receptor (TLR)3, TLR9 and cGAS-mediated activation of the type 1 IFN signalling pathway transcription factor STAT1. Additionally, inhibition of CLIC4 prevented SSc fibroblast media from inducing a type 1 IFN response in keratinocytes. Conclusions The data presented in this study suggests that CLIC4 is a global regulator of the type 1 IFN signalling cascade in SSc epithelial cells.

Upregulation of GPX4 drives ferroptosis resistance in scleroderma skin fibroblasts

The pathogenesis of systemic sclerosis (SSC) fibrosis involves the rapid proliferation of skin fibroblasts, and current anti-fibrotic treatments are limited. This study investigated the relationship between ferroptosis and SSC skin fibroblasts. We observed that erastin-induced ferroptosis was suppressed in SSC fibroblasts. RSL3, a direct inhibitor of Glutathione Peroxidase 4 (GPX4), significantly reduced the viability of the fibroblasts, and upregulation of GPX4 in the SSC fibroblasts contributed to ferroptosis resistance. Furthermore, we demonstrated that transferrin receptor 1 (TfR1) was a crucial transporter for iron deposition in the fibroblasts. Collectively, our results highlight that GPX4 inhibition could enhance the sensitivity to ferroptosis by SSC fibroblasts, which showed distinct characteristics of iron metabolism that were not observed in normal fibroblasts in this study. Taken together, these results suggest that targeting ferroptosis could be a therapeutic strategy for the treatment of SSC.

Interferon-γ Induces Interleukin-6 Production and Alpha-smooth Muscle Actin Expression in Systemic Sclerosis Fibroblasts.

Systemic sclerosis (SSc) is an autoimmune systemic disease that is characterized by immune dysregulation, inflammation, vasculopathy, and fibrosis. Tissue fibrosis plays an important role in SSc and can affect several organs such as the dermis, lungs, and heart. Dysregulation of interferon (IFN) signaling contributes to the SSc pathogenesis and interferon regulatory factor 1 (IRF1) has been indicated as the main regulator of type I IFN. This study aimed to clarify the effect of IFN-gamma (-γ) and dexamethasone (DEX) on the IRF1, extracellular signal-regulated kinase 1/2 (ERK1/2), and the expression of alpha-smooth muscle actin (α-SMA) in myofibroblasts and genes involved in the inflammation and fibrosis processes in early diffuse cutaneous systemic sclerosis (dcSSc). A total of 10 early dcSSc patients (diffuse cutaneous form) and 10 unaffected control dermis biopsies were obtained to determine IFNγ and DEX effects on inflammation and fibrosis. Fibroblasts were treated with IFNγ and DEX at optimum time and dose. The expression level of genes and proteins involved in the fibrosis and inflammation processes have been quantified by quantitative real-time PCR (RT-qPCR) and western blot, respectively. IFNγ could up-regulate some of the inflammation-related genes (Interleukin-6; IL6) and down-regulate some of the fibrosis-related genes (COL1A1) in cultured fibroblasts of patients with early dcSSc compared to the untreated group. Besides, it has been revealed that IFNγ can induce fibroblast differentiation to the myofibroblast that expresses α-SMA. Concerning the inhibitory effect of IFNγ on some fibrotic genes and its positive effect on the inflammatory genes and myofibroblast differentiation, it seems that IFNγ may play a dual role in SSc.

The β-Secretase BACE1 Drives Fibroblast Activation in Systemic Sclerosis through the APP/β-Catenin/Notch Signaling Axis

The beta-amyloid precursor protein cleaving enzyme 1 (BACE1) is well known for its role in the development of Alzheimer’s disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in SSc patient skin. Inhibition of BACE1 with small molecule inhibitors or siRNA blocked SSc and fibrotic stimuli mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on β-catenin and Notch signalling. The Neurotropic factor BDNF negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from SSc patients show higher Aβ and lower BDNF levels compared to healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimer’s disease and could be repurposed to ameliorate fibrosis progression.

The Crosstalk between N-Formyl Peptide Receptors and uPAR in Systemic Sclerosis: Molecular Mechanisms, Pathogenetic Role and Therapeutic Opportunities.

Systemic Sclerosis (SSc) is a heterogeneous autoimmune disease characterized by widespread vasculopathy, the presence of autoantibodies and the progressive fibrosis of skin and visceral organs. There are still many questions about its pathogenesis, particularly related to the complex regulation of the fibrotic process, and to the factors that trigger its onset. Our recent studies supported a key role of N-formyl peptide receptors (FPRs) and their crosstalk with uPAR in the fibrotic phase of the disease. Here, we found that dermal fibroblasts acquire a proliferative phenotype after the activation of FPRs and their interaction with uPAR, leading to both Rac1 and ERK activation, c-Myc phosphorylation and Cyclin D1 upregulation which drive cell cycle progression. The comparison between normal and SSc fibroblasts reveals that SSc fibroblasts exhibit a higher proliferative rate than healthy control, suggesting that an altered fibroblast proliferation could contribute to the initiation and progression of the fibrotic process. Finally, a synthetic compound targeting the FPRs/uPAR interaction significantly inhibits SSc fibroblast proliferation, paving the way for the development of new targeted therapies in fibrotic diseases.

SLC39A7 upregulation links to skin fibrosis in systemic sclerosis via TGF-β/SMAD pathway.

Systemic sclerosis (SSc) is a multisystem connective tissue disease. Skin fibrosis, the hallmark of this disease, is defined as the excess deposition and accumulation of extracellular matrix, mainly type 1 collagen, in the dermis. SLC39A7 is an intracellular zinc transporter that plays a unique role in connective tissue formation. Therefore, we investigated the expression and role of SLC39A7 in SSc. Using immunohistochemical staining we demonstrated the overexpression of SLC39A7 in the skin of SSc patients. Quantitative real-time PCR and western blot data analysis showed that both SLC39A7 mRNA and protein levels were significantly upregulated in dermal fibroblasts from SSc patients compared to healthy controls. We used the shRNA lentiviral particle transduction system to stably knockdown the expression of SLC39A7 in SSc fibroblasts. The results showed that knockdown of SLC39A7 suppressed the production of type 1 collagen. These findings provide evidence that SLC39A7 is involved in the pathogenesis of SSc and that SLC39A7 plays a positive role in its progression.

Characteristics and impact of infiltration of B-cells from systemic sclerosis patients in a 3D healthy skin model.

In systemic sclerosis (SSc), B-cells are activated and present in the skin and lung of patients where they can interact with fibroblasts. The precise impact and mechanisms of the interaction of B-cells and fibroblasts at the tissular level are poorly studied. We investigated the impact and mechanisms of B-cell/fibroblast interactions in cocultures between B-cells from patients with SSc and 3-dimensional reconstituted healthy skin model including fibroblasts, keratinocytes and extracellular matrix. The quantification and description of the B-cell infiltration in 3D cocultures were performed using cells imagery strategy and cytometry. The effect of coculture on the transcriptome of B-cells and fibroblasts was studied with bulk and single-cell RNA sequencing approaches. The mechanisms of this interaction were studied by blocking key cytokines like IL-6 and TNF. We showed a significant infiltration of B-cells in the 3D healthy skin model. The amount but not the depth of infiltration was higher with B-cells from SSc patients and with activated B-cells. B-cell infiltrates were mainly composed of naïve and memory cells, whose frequencies differed depending on B-cells origin and activation state: infiltrated B-cells from patients with SSc showed an activated profile and an overexpression of immunoglobulin genes compared to circulating B-cells before infiltration. Our study has shown for the first time that activated B-cells modified the transcriptomic profile of both healthy and SSc fibroblasts, toward a pro-inflammatory (TNF and IL-17 signaling) and interferon profile, with a key role of the TNF pathway. B-cells and 3D skin cocultures allowed the modelization of B-cells infiltration in tissues observed in SSc, uncovering an influence of the underlying disease and the activation state of B-cells. We showed a pro-inflammatory effect on skin fibroblasts and pro-activation effect on infiltrating B-cells during coculture. This reinforces the role of B-cells in SSc and provide potential targets for future therapeutic approach in this disease.

Expression of collagen-related piRNA is dysregulated in cultured dermal fibroblasts derived from patients with scleroderma.

PIWI-interacting RNA (piRNA) is a class of recently discovered small non-coding RNAs. piRNAs derive from an initial transcript encompassing a piRNA cluster via a unique biosynthesis process, interact with PIWI proteins, bind to specific targets, and recruit chromatin modifiers to enable transcriptional repression. Abnormal expression of PIWI proteins and piRNAs has been reported in some human cancers, with participation of some PIWI/piRNAs complexes in tumorigenesis and association with cancer prognosis. Their expression in patients with systemic sclerosis (SSc) has not been widely elucidated. PIWI/piRNAs and their role in the pathogenesis of collagen accumulation in SSc was therefore investigated; no difference was found in the PIWIL1-4 levels between normal and cultured SSc dermal fibroblasts. Among piRNAs predicted to target SSc-related molecules, we first found significant piR-32364 up-regulation in SSc dermal fibroblasts, likely due to intrinsic TGF-β signaling. Forced piR-32364 overexpression in normal fibroblasts significantly reduced COL1A1 expression both at mRNA and protein levels, but not COL1A2. Thus, piR-32364 overexpression in SSc fibroblasts may be the negative feedback against collagen up-regulation, which could suggest the potential of piRNAs as a therapeutic target.

TLR8 aggravates skin inflammation and fibrosis by activating skin fibroblasts in systemic sclerosis.

Innate immunity significantly contributes to SSc pathogenesis. TLR8 is an important innate immune mediator that is implicated in autoimmunity and fibrosis. However, the expression, mechanism of action, and pathogenic role of TLR8 in SSc remain unclear. The aim of this study was to explore the roles and underlying mechanisms of TLR8 in SSc. The expression of TLR8 was analysed, based on a public dataset, and then verified in skin tissues and skin fibroblasts of SSc patients. The role of TLR8 in inflammation and fibrosis was investigated using a TLR8-overexpression vector, activator (VTX-2337), inhibitor (cu-cpt-8m), and TLR8 siRNA in skin fibroblasts. The pathogenic role of TLR8 in skin inflammation and fibrosis was further validated in a bleomycin (BLM)-induced mouse skin inflammation and fibrosis model. TLR8 levels were significantly elevated in SSc skin tissues and myofibroblasts, along with significant activation of the TLR8 pathway. In vitro studies showed that overexpression or activation of TLR8 by a recombinant plasmid or VTX-2337 upregulated IL-6, IL-1β, COL I, COL III and α-SMA in skin fibroblasts. Consistently, both TLR8-siRNA and cu-cpt-8m reversed the phenotypes observed in TLR8-activating fibroblasts. Mechanistically, TLR8 induces skin fibrosis and inflammation in a manner dependent on the MAPK, NF-κB and SMAD2/3 pathways. Subcutaneous injection of cu-cpt-8m significantly alleviated BLM-induced skin inflammation and fibrosis in vivo. TLR8 might be a promising therapeutic target for improving the treatment strategy for skin inflammation and fibrosis in SSc.

Caspase 1 Enhances Transport and Golgi Organization Protein 1 Expression to Promote Procollagen Export From the Endoplasmic Reticulum in Systemic Sclerosis Contributing to Fibrosis.

Transport and Golgi Organization protein 1 (TANGO1) is a protein that regulates the export of procollagen from the endoplasmic reticulum and has a role in the organization of exit sites for general protein export. What regulates the expression of TANGO1 and the role of TANGO1 in fibrosis is poorly understood and has never been studied in the setting of systemic sclerosis (SSc). We undertook this study to determine the role of TANGO1 in SSc fibrosis. SSc (n=15) and healthy (n=12) primary fibroblast lung cell lines were investigated for the expression of TANGO1. Histologic analyses for TANGO1 were performed on lung biopsy samples (n=12 SSc patient samples and n=8 healthy control samples). SSc fibroblasts showed increased expression of TANGO1 protein in cultured fibroblasts. TANGO1 colocalizes with α-smooth muscle actin (α-SMA)-positive cells in SSc lung tissue and is highly up-regulated in the neointima of SSc vessels. TANGO1 expression was dependent on the inflammasome activation of caspase 1. It was also dependent on signaling from the interleukin-1 (IL-1) and transforming growth factor β (TGFβ) receptors. The decrease in TANGO1 down-regulated export of larger cargos including collagen and laminin. Reduced TANGO1 protein had no effect on smaller molecular weight cargoes; however, the secretion of elastin was significantly reduced. TANGO1 is markedly increased in SSc fibroblasts and was found to be elevated in lung tissue in association with α-SMA-positive cells. TANGO1 expression is driven by inflammasome-dependent caspase 1 activation and is mediated by IL-1 and TGFβ downstream signaling. These observations suggest that during fibrosis, caspase 1 promotes the up-regulation of TANGO1 and the organization of endoplasmic reticulum exits sites, ultimately contributing to procollagen export and fibrosis.

Amelioration of Fibrotic Remodeling of Human 3-Dimensional Full-Thickness Skin by Transglutamase 2 Inhibition.

Fibrotic tissues are characterized by excessive crosslinking between extracellular matrix (ECM) proteins, rendering them more resistant to degradation. Although increased crosslinking of ECM is thought to play an important role for progression of tissue fibrosis, enhanced ECM crosslinking has not yet been targeted therapeutically in systemic sclerosis (SSc). Here, we investigated the role of transglutaminase 2 (TG2), a central crosslinking enzyme, in the activation of SSc fibroblasts. We assessed TG2 expression and activity using TG2 staining, Western blotting, and TG2 activity assays. We inhibited TG2 in fibroblasts cultured under standard 2-dimensional conditions and in a 3-dimensional full-thickness equivalent skin model using monoclonal inhibitory anti-TG2 antibodies. TG2 expression was increased in the skin of patients with SSc compared with healthy controls, with levels particularly high in patients with SSc-associated interstitial lung disease. TG2 expression and TG2 activity were also increased in SSc dermal fibroblasts. Moreover, the levels of circulating TG2 in the plasma samples from SSc patients were increased versus samples from healthy controls. Anti-TG2 antibodies did not show consistent antifibrotic effects across different fibroblast cell lines under 2-dimensional culture conditions; however, anti-TG2 antibodies effectively reduced transforming growth factor β-induced dermal thickening, myofibroblast differentiation, and collagen accumulation in the 3-dimensional full-thickness model of human skin. We provide the first evidence, to our knowledge, that inhibition of TG2 might be a potential antifibrotic approach in SSc. Our findings have translational potential as anti-TG2 antibodies are currently evaluated in a phase II clinical trial in chronic allograft injury and would thus be available for clinical studies in SSc (ClinicalTrials.gov identifier: NCT04335578).

P161 Systemic Sclerosis Dermal Fibroblast-derived Exosomes trigger a Type I Interferon Response in Keratinocytes, through Tank Binding Kinase 1

Abstract Background/Aims Activation of Type I IFN response has been shown to be present in blood and tissue in systemic sclerosis (SSc) and correlates with disease pathogenesis and therapy response. It is currently unknown whether the tissue-specific Type I IFN activation is a consequence of the response observed in blood or rather its source. SSc fibroblasts share many cellular and molecular features with cancer-associated fibroblasts (CAF), and there is a wealth of evidence supporting the role of CAF in inducing Type I IFN activation in epithelial cancer cells. Here, we aimed to determine whether keratinocytes in SSc show evidence of Type I IFN activation and the potential role of SSc in the process. Methods Skin biopsies were obtained from healthy and SSc patients’ forearms and processed for immunohistochemistry and subculture of primary fibroblasts and keratinocytes. The human skin keratinocyte cell line (HaCats) were stimulated with conditioned media from healthy (n = 3) and SSc (n = 3) fibroblasts. Exosomes were isolated from healthy and SSc patient fibroblast cultured supernatant by a combination of ultracentrifugation and polymer-based precipitation and used to stimulate HaCats. Cellular exosomes were investigated by immunoblotting and electron microscopy. TANK-binding kinase (TBK) was inhibited using a small molecule inhibitor (GSK8612). RT-PCR based IFN array and western blot for pSTAT1 (Tyr701) were employed to assess Type I IFN activation in these cells. Results Consistent with previous studies we found high levels of pSTAT1 in keratinocytes from SSc patient biopsies. Contrary to observations in dermal fibroblasts, primary keratinocytes isolated from SSc skin biopsies lost IFN activation once cultured in-vitro. Conversely, conditioned media from SSc patient fibroblasts triggered ISG induction; MX1 (2.7-fold, p < 0.05), CXCL10 (2.8-fold, p < 0.01) and CXCL11 (1.9-fold, p < 0.05). Media fractionation by ultracentrifugation indicated that the effect on keratinocytes was driven mainly by pelleted micro-vesicles, suggesting a potential role for cellular exosomes. Fibroblasts from SSc patients shed cellular exosomes of similar quantities and biophysical qualities, compared to healthy. SSc exosomes induced a 2.2-fold increase in phosphorylation of STAT1 (p < 0.05), as well as an induction in ISGs; MX1 (1.45-fold, p < 0.05), CXCL10 (1.84-fold, p < 0.01), CXCL11 (2.11-fold, p < 0.01) and OAS (1.24-fold, p < 0.05). Suppression of TBK activity by 10μM of GSK8612 suppressed the induction of pSTAT1. Consistent with these findings IFN array of HaCats stimulated with SSc exosomes showed significant (p < 0.05) upregulation of 16 ISGs by at least 1.2-fold compared to the effect of healthy control exosomes. Conclusion We have shown for the first time that scleroderma keratinocytes ex-vivo IFN activation is lost in-vivo and can be regained following exposure to fibroblast-derived exosomes though a TBK-dependent mechanism. Our data indicates that exosomes from SSc fibroblasts may carry the “signal zero” of local Type I IFN activation, potentially though activation of toll-like receptors and/or pattern recognition receptors. Disclosure J.A. Bryon: None. C. Wasson: None. R.L. Ross: None. E. Zeqiraj: None. F. Del Galdo: Consultancies; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.. Grants/research support; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.

P158 Defining distinct fibroblast populations in systemic sclerosis skin using functional characterization and single-cell RNA sequencing

Abstract Background/Aims Fibroblasts isolated by traditional explant culture from skin biopsies in systemic sclerosis (SSc) have a profibrotic and “migratory” phenotype. Single cell RNAseq (scRNAseq) studies suggest other fibroblast subpopulations may be important in SSc pathogenesis. We have isolated a second fibroblast population termed “resident” from whole skin in SSc. We used bulk and scRNAseq to further characterise these populations. Methods Forearm skin from dcSSc (n = 3) and HC (n = 3) was biopsied. “Migratory” fibroblasts were isolated by standard explant culture. The remaining biopsy fragments underwent collagenase digestion to yield a second “resident” fibroblast population. Functional characterization included 3-D collagen gel contraction, migratory scratch-wound assays and western blot comparing expression of pro-collagen I, CTGF and αSMA. Significant difference in gene expression was defined by fold change≥1.5, and adjusted p-value<0.05. scRNAseq was performed on 12 SSc whole skin biopsies, and 3 HC, using the 10X Genomics platform. Results Both SSc fibroblast populations showed a fibrotic phenotype with overexpression of COL1, CTGF and αSMA compared to HC, but with reduced expression αSMA in resident compared to migratory SSc fibroblasts (p < 0.05). RNAseq confirmed 1483 genes being significantly differentially expressed between the SSc fibroblast populations (Table 1). All four populations clustered separately on hierarchical clustering. 10 fibroblast clusters were identified using scRNAseq of whole skin. Of these, cluster 0 and 4 showed greater expression of COL10A1, and STC2 in keeping with migratory fibroblasts. Increased expression of CCL2, and EGR1 were seen in clusters 3 and 6, representing the resident fibroblast population. Gene set enrichment analysis (GSEA) confirmed concordance between KEGG pathway upregulation of the migratory and resident fibroblast clusters and bulk RNAseq analysis. Resident fibroblasts showed upregulation of complement and coagulation genes, and JAK/STAT signalling pathway. There was upregulation of glycolysis gluconeogenesis, and PPAR signalling in migratory fibroblasts. There was agreement in GSEA findings between relevant scRNAseq clusters and bulkRNAseq for resident and migratory subpopulations. Conclusion We have isolated for the first time two distinct profibrotic fibroblast populations from SSc skin with altered expression of key genes associated with SSc pathogenesis. Future work will define their role in SSc pathogenesis, potentially facilitating a more targeted treatment approach. Disclosure K.E. Clark: None. S. Xu: None. V.H. Ong: None. C.D. Buckley: None. C. Denton: None.

OA07 Clinical and pathogenic significance of S100A4 overexpression in systemic sclerosis

Abstract Background/Aims Preclinical studies suggest that the S100A4, a Damage Associated Molecular Pattern (DAMP) protein upregulated upon stress or injury, may be a target for antifibrotic therapy. We have studied expression and function of S100A4 in systemic sclerosis (SSc) to further explore its relevance as a driver of fibroblast activation. Methods S100A4 protein concentration was measured by ELISA (Cusabio, USA) in serum of SSc (n = 94) and healthy controls (n = 15). Association of serum level with major organ complications and skin score (mRSS) was determined. S100A4 protein expression in supernatant and cell layer of skin fibroblast cultures from diffuse cutaneous SSc (SScF, n = 6) and healthy controls (NF, n = 6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were used to investigate activation of profibrotic molecular and functional phenotype in SSc and control fibroblasts. Results Median [range] S100A4 (ng/ml) was higher in serum of SSc (89.9 [15.0-240.0]) than healthy controls (71.4 [7.9-131.8]; p = 0.027). There was significant association with SSc-ILD (p = 0.025, n = 55), scleroderma renal crisis (p = 0.026, n = 4) and trend of increase in pulmonary hypertension (p = 0.075, n = 7). There was a trend for S100A4 to fall in longer disease duration (r=-0.18, p = 0.09) and a weak (r = 0.28, p = 0.07) correlation with current mRSS but not peak mRSS in dcSSc. Median [range] S100A4 (ng/ml) was higher in culture supernatants of SScF (4.19 [0.52-8.42]) than NF controls (0.28 [0.02-3.29]; p < 0.0001) and SSc cell layer had increased levels by Western blot analysis (mean [sd] RDU NF = 0.50 [0.91]; SScF 8.16 [2.49]; p < 0.0001). S100A4 induced a fibrotic phenotype in NF with concentration dependent increased in Col1, SMA and CTGF expression, and promotion of migration (scratch wound assay) and 3D type I collagen gel contraction that was attenuated by AX-202. AX-202 also reduced the constitutive profibrotic gene and protein expression phenotype of SScF. The effect of S100A4 was significantly attenuated by SB431524, a specific inhibitor of ALK5 (p < 0.0001 ANOVA) using qPCR assays across treatment groups for Col1, SMA and CTGF. Gene expression data (qPCR) confirmed protein expression. Genome-wide RNAseq analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (FDR<0.001 and FC > 1.5) induced in NF by S100A4 were also constitutively overexpressed in SScF, and significantly downregulated by AX-202. Pathway mapping of these S100A4 dependent transcripts in SSc showed the most significant enriched Kegg® pathways (FDR<0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold). Conclusion Our findings provide compelling evidence supporting a profibrotic role for S100A4 in fibroblast activation in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. Studies to elucidate the therapeutic potential of targeting S100A4 in SSc are warranted. Disclosure C.P. Denton: None. S. Xu: None. R.H. Maclean: None. K.E. Clark: None. S. Borchert: Corporate appointments; Employee of ARXX Therapeutics. R. Hussain: Corporate appointments; Employee of ARXX Therapeutics. J. Klingelhöfer: Corporate appointments; Employee of ARXX Therapeutics. J. Hallén: Corporate appointments; Employee of ARXX Therapeutics. V.H. Ong: None.

1455 Identification and characterization of specific subsets in systemic sclerosis fibroblast cultures

1455 Identification and characterization of specific subsets in systemic sclerosis fibroblast cultures

Enhancement of Zyxin Promotes Skin Fibrosis by Regulating FAK/PI3K/AKT and TGF-β Signaling Pathways via Integrins.

Skin fibrosis is a common pathological manifestation in systemic sclerosis (SSc), keloid, and localized scleroderma (LS) characterized by fibroblast activation and excessive extracellular matrix (ECM) deposition. However, few effective drugs are available to treat skin fibrosis due to its unclear mechanisms. In our study, we reanalyzed skin RNA-sequencing data of Caucasian, African, and Hispanic SSc patients from the Gene Expression Omnibus (GEO) database. We found that the focal adhesion pathway was up-regulated and Zyxin appeared to be the primary focal adhesion protein involved in skin fibrosis, and we further verified its expression in Chinese skin tissues of several fibrotic diseases, including SSc, keloid, and LS. Moreover, we found Zyxin inhibition could significantly alleviate skin fibrosis using Zyxin knock-down and knock-out mice, nude mouse model and skin explants of human keloid. Double immunofluorescence staining showed that Zyxin was highly expressed in fibroblasts. Further analysis revealed pro-fibrotic gene expression and collagen production increased in Zyxin over-expressed fibroblasts, and decreased in Zyxin interfered SSc fibroblasts. In addition, transcriptome and cell culture analyses revealed Zyxin inhibition could effectively attenuate skin fibrosis by regulating the FAK/PI3K/AKT and TGF-β signaling pathways via integrins. These results suggest Zyxin appears a potential new therapeutic target for skin fibrosis.

Centromere defects, chromosome instability, and cGAS-STING activation in systemic sclerosis

Centromere defects in Systemic Sclerosis (SSc) have remained unexplored despite the fact that many centromere proteins were discovered in patients with SSc. Here we report that lesion skin fibroblasts from SSc patients show marked alterations in centromeric DNA. SSc fibroblasts also show DNA damage, abnormal chromosome segregation, aneuploidy (only in diffuse cutaneous (dcSSc)) and micronuclei (in all types of SSc), some of which lose centromere identity while retaining centromere DNA sequences. Strikingly, we find cytoplasmic “leaking” of centromere proteins in limited cutaneous SSc (lcSSc) fibroblasts. Cytoplasmic centromere proteins co-localize with antigen presenting MHC Class II molecules, which correlate precisely with the presence of anti-centromere antibodies. CENPA expression and micronuclei formation correlate highly with activation of the cGAS-STING/IFN-β pathway as well as markers of reactive oxygen species (ROS) and fibrosis, ultimately suggesting a link between centromere alterations, chromosome instability, SSc autoimmunity, and fibrosis.