Systemic Sclerosis Skin Biopsies Research Articles

EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis.

Erythropoietin-producing hepatocellular (Eph)/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models of skin fibrosis. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from patients with SSc and healthy controls, which was followed by in vivo analysis of fibroblast-specific Ephb2-deficient mice. Expression of EphB2 is up-regulated in SSc skin tissue and explanted SSc dermal fibroblasts compared with healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-β (TGF-β) cytokines up-regulate EphB2 in dermal fibroblasts via noncanonical TGF-β/mother against decapentaplegic signaling, and silencing EPHB2 in human dermal fibroblasts is sufficient to dampen TGF-β-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. Our data implicate TGF-β regulation of EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.

Long non-coding RNA H19X as a regulator of mononuclear cell adhesion to the endothelium in systemic sclerosis.

To define the functional relevance of H19 X-linked co-expressed lncRNA (H19X) in endothelial cell (EC) activation as a key process in systemic sclerosis (SSc) vasculopathy. H19X expression in SSc skin biopsies was analyzed from single cell RNA sequencing (scRNA-seq) data. Differential expression and pathway enrichment analysis between cells expressing (H19Xpos) and non expressing H19X (H19Xneg) cells was performed. H19X function was investigated in human dermal microvascular EC (HDMECs) by silencing. H19X and EC adhesion molecules levels were analyzed by RT-qPCR and Western Blot after stimulation with proinflammatory cytokines. Cytoskeletal rearrangements were analyzed by fluorescent staining. Endothelial adhesion was evaluated by co-culture of HDMECs and fluorescent labelled peripheral blood mononuclear cells (PBMCs). Shedding VCAM1 was evaluated by ELISA on HDMEC supernatant. scRNA-seq showed significant upregulation of H19X in SSc compared with healthy EC. In HDMEC, H19X was consistently induced by type I and II interferons. H19X knockdown lead to a significant decrease of the mRNA of several adhesion molecules. Particularly, vascular cell adhesion protein 1 (VCAM1) was significantly reduced at protein and mRNA levels. Co-expression analysis of the scRNA-seq data confirmed a higher expression of VCAM1 in (H19Xpos) EC. EC were also strongly associated with the 'cell adhesion molecule' pathway. Moreover, VCAM1 downstream pathway displayed less activation following H19X knockdown. Contractility of HDMEC, PBMC adhesion to HDMEC and VCAM1 shedding were also reduced following H19X knockdown. lncRNA H19X may contribute to EC activation in SSc vasculopathy, acting as a regulator of expression of adhesion molecules in EC.

Autoantibodies Which Bind to and Activate Keratinocytes in Systemic Sclerosis.

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterised by pathological processes involving autoimmunity, vasculopathy and resultant extensive skin and organ fibrosis. Recent studies have demonstrated activation and aberrant wound healing responses in the epithelial layer of the skin in this disease, implicating the epithelial keratinocytes as a source of pro-fibrotic and inflammatory mediators. In this paper, we investigated the role of Immunoglobulin G (IgG) autoantibodies directed against epithelial cells, as potential initiators and propagators of pathological keratocyte activation and the ensuing SSc fibrotic cascade. A keratinocyte cell-based ELISA is used to evaluate the binding of SSc IgG. SSc skin biopsies were stained by immunofluorescence for the presence of IgG in the keratinocyte layer. Moreover, IgG purified from SSc sera was evaluated for the potential to activate keratinocytes in tissue culture and to induce TLR2 and 3 signalling in reporter cell lines. We demonstrate enhanced binding of SSc IgG to keratinocytes and the activation of these cells leading to the release of IL-1α, representing a potential initiating pathway in this disease.

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.

Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis.

Activation of TLR4 by its cognate damage-associated molecular patterns (DAMPs) elicits potent profibrotic effects and myofibroblast activation in systemic sclerosis (SSc), while genetic targeting of TLR4 or its DAMPs in mice accelerates fibrosis resolution. To prevent aberrant DAMP/TLR4 activity, a variety of negative regulators evolved to dampen the magnitude and duration of the signaling. These include radioprotective 105 kDa (RP105), a transmembrane TLR4 homolog that competitively inhibits DAMP recognition of TLR4, blocking TLR4 signaling in immune cells. The role of RP105 in TLR4-dependent fibrotic responses in SSc is unknown. Using unbiased transcriptome analysis of skin biopsies, we found that levels of both TLR4 and its adaptor protein MD2 were elevated in SSc skin and significantly correlated with each other. Expression of RP105 was negatively associated with myofibroblast differentiation in SSc. Importantly, RP105-TLR4 association was reduced, whereas TLR4-TLR4 showed strong association in fibroblasts from patients with SSc, as evidenced by PLA assays. Moreover, RP105 adaptor MD1 expression was significantly reduced in SSc skin biopsies and explanted SSc skin fibroblasts. Exogenous RP105-MD1 abrogated, while loss of RP105 exaggerated, fibrotic cellular responses. Importantly, ablation of RP105 in mice was associated with augmented TLR4 signaling and aggravated skin fibrosis in complementary disease models. Thus, we believe RP105-MD1 to be a novel cell-intrinsic negative regulator of TLR4-MD2-driven sustained fibroblast activation, representing a critical regulatory network governing the fibrotic process. Impaired RP105 function in SSc might contribute to persistence of progression of the disease.

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-β pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.

Association of endothelial to mesenchymal transition and cellular senescence with fibrosis in skin biopsies of systemic sclerosis patients: a cross-sectional study.

Fibrosis is the dominant hallmark of systemic sclerosis (SSc). Several mechanisms have been proposed to drive the disease process, but how these relate to skin fibrosis is poorly understood. We performed a cross-sectional study on archival skin biopsies from 18 SSc patients and four controls. Dermal fibrosis and inflammatory cell infiltration were scored in HE and Masson's Trichrome-stained sections. The presence of senescence was defined by P21 and/or P16 positivity in Ki-67 negative cells. Endothelial to mesenchymal transition (EndMT) was identified by co-localisation of CD31 and α-SMA in immunofluorescent double-stained sections, and by an enclosure of ERG positive endothelial cell nuclei by α-SMA stained cytoplasm in immunohistochemical double staining. The histological dermal fibrosis score of SSc skin biopsies was correlated with the modified Rodnan skin score (rho 0.55, p=0.042). Staining for markers of cellular senescence on fibroblasts was correlated with fibrosis score, inflammatory score, and CCN2 staining on fibroblasts. Moreover, EndMT was more abundant in skin from patients with SSc (p<0.01) but did not differ between groups with different fibrosis severity. The frequency of these EndMT features increased with the abundance of senescence markers and CCN2 on fibroblasts and dermal inflammation. EndMT and fibroblast senescence were more abundant in skin biopsies from SSc patients. This finding indicates that both senescence and EndMT are involved in the pathway leading to skin fibrosis and might be valuable biomarkers and/or possible targets for novel therapeutic interventions.

Gut microbe-derived metabolite trimethylamine N-oxide activates PERK to drive fibrogenic mesenchymal differentiation

SummaryIntestinal dysbiosis is prominent in systemic sclerosis (SSc), but it remains unknown how it contributes to microvascular injury and fibrosis that are hallmarks of this disease. Trimethylamine (TMA) is generated by the gut microbiome and in the host converted by flavin-containing monooxygenase (FMO3) into trimethylamine N-oxide (TMAO), which has been implicated in chronic cardiovascular and metabolic diseases. Using cell culture systems and patient biopsies, we now show that TMAO reprograms skin fibroblasts, vascular endothelial cells, and adipocytic progenitor cells into myofibroblasts via the putative TMAO receptor protein R-like endoplasmic reticulum kinase (PERK). Remarkably, FMO3 was detected in skin fibroblasts and its expression stimulated by TGF-β1. Moreover, FMO3 was elevated in SSc skin biopsies and in SSc fibroblasts. A meta-organismal pathway thus might in SSc link gut microbiome to vascular remodeling and fibrosis via stromal cell reprogramming, implicating the FMO3-TMAO-PERK axis in pathogenesis, and as a promising target for therapy.

Single-cell transcriptomics reveals distinct effector profiles of infiltrating T cells in lupus skin and kidney.

Cutaneous lupus is commonly present in patients with systemic lupus erythematosus (SLE). T cells have been strongly suspected to contribute to the pathology of cutaneous lupus; however, our understanding of the relevant T cell phenotypes and functions remains incomplete. Here, we present a detailed single-cell RNA-Seq profile of T and NK cell populations present within lesional and nonlesional skin biopsies of patients with cutaneous lupus. T cells across clusters from lesional and nonlesional skin biopsies expressed elevated levels of IFN-simulated genes (ISGs). Compared with T cells from control skin, however, T cells from cutaneous lupus lesions did not show elevated expression profiles of activation, cytotoxicity, or exhaustion. Integrated analyses indicated that skin lymphocytes appeared less activated and lacked the expanded cytotoxic populations prominent in lupus nephritis kidney T/NK cells. Comparison of skin T cells from lupus and systemic sclerosis skin biopsies further revealed an elevated ISG signature specific to cells from lupus biopsies. Overall, these data represent the first detailed transcriptomic analysis to our knowledge of the T and NK cells in cutaneous lupus at the single-cell level and have enabled a cross-tissue comparison that highlights stark differences in composition and activation of T/NK cells in distinct tissues in lupus.

Acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, demonstrates efficacy in systemic sclerosis preclinical mouse models

BackgroundUncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterized by generalized fibrosis affecting particularly the lungs and skin. Costimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension.MethodsExpression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, acazicolcept was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 μg of acazicolcept or a molar-matched dose of an Fc control protein twice a week for 6 weeks. After 6 weeks, skin and lung were evaluated.ResultsICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc control and acazicolcept groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of acazicolcept treatment. In mice challenged with HOCL, acazicolcept induced a significant decrease in dermal thickness, collagen content, myofibroblast number, and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, acazicolcept treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of acazicolcept-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, acazicolcept reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by acazicolcept was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice.ConclusionsOur results confirm the importance of costimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by acazicolcept decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

Single-cell transcriptome analysis identifies skin-specific T-cell responses in systemic sclerosis

ObjectivesAlthough T cells have been implicated in the pathogenesis of systemic sclerosis (SSc), a comprehensive study of T-cell-mediated immune responses in the affected skin of patients with progressive SSc is...

AB013. CXCL4-DNA immune complexes drive inflammation in systemic sclerosis by amplifying TLR9-mediated interferon-α production

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation-driven tissue fibrosis and vasculopathy. CXCL4 is a chemokine overexpressed in SSc and represents an early serum biomarker of severe disease. CXCL4 likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we combine X-ray scattering, confocal microscopy, immune cell experiments, and analysis of SSc skin biopsies to elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc. We find that CXCL4 organizes “self” and microbial DNA into liquid crystalline immune complexes that drastically amplify Toll-like receptor 9 (TLR9)-mediated plasmacytoid dendritic cell (pDC) hyperactivation and interferon-α production. Surprisingly, this immunomodulatory activity is independent of CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) levels in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Taken together, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and identify a potential therapeutic opportunity to disrupt inflammation in SSc by inhibiting the self-assembly of CXCL4-DNA complexes. Our findings are consistent with an emerging general paradigm in which nucleic acid-scaffolding proteins like antimicrobial peptides, bacterial amyloids, and chemokines can drive autoimmunity by modulating innate immune receptors without being direct agonists.

Gut Microbe-Derived Metabolite Trimethylamine N-Oxide Activates PERK to Drive Mesenchymal Differentiation and Fibrosis

Intestinal dysbiosis is prominent in systemic sclerosis (SSc), but it remains unknown if and how it contributes to synchronous microvascular injury and fibrosis that characterize this disease. Trimethylamine (TMA) is generated in the gut and converted by flavin-containing monooxygenase (FMO3) to trimethylamine N-oxide (TMAO), which has been implicated in chronic cardiovascular diseases. We now show that TMAO reprograms skin fibroblasts, vascular endothelial cells and adipocytic progenitor cells to myofibroblasts in a process mediated via R-like endoplasmic reticulum kinase (PERK), a TMAO-binding protein. Remarkably, FMO3, principally expressed in the liver, was found also to be present in both isolated skin fibroblasts and in skin explants, and its expression was elevated in pericytes and fibroblasts in SSc skin biopsies and explanted dermal fibroblasts. A TMAO meta-organismal pathway in SSc thus potentially links the gut microbiome to vasculopathy and fibrosis via stromal cell reprogramming, implicating this pathway in pathogenesis, and as a target for therapy.

Targeting CD38-Dependent NAD+ Metabolism to Mitigate Multiple Organ Fibrosis

Synchronous fibrosis in multiple organs is the defining hallmark of systemic sclerosis (SSc), but underlying mechanisms remain poorly understood. Organismal decline in nicotinamide adenine dinucleotide (NAD+), the sole substrate for enzymes critical for cellular metabolism, is implicated in age-related pathologies, and is due to NAD+ consumption catalyzed by CD38. We now show that CD38 is upregulated in SSc skin biopsies, and its levels associate with molecular fibrosis signatures as well as clinical skin fibrosis scores, while key NAD+ -synthesizing enzyme levels are unaltered. Boosting NAD+ via CD38 targeting or by NAD+ precursor supplementation protected older mice from skin and lung fibrosis. Mechanistically, CD38 reduced NAD+ levels and sirtuin activity to augment fibrotic responses, while inhibition of CD38 had the opposite effect. Thus we identify upregulation of CD38 and resulting disrupted NAD+ homeostasis as a fundamental pathogenic mechanism driving fibrosis in SSc, suggesting that CD38 might represent a target for therapy.

Regulation of Fibroblast Apoptosis and Proliferation by MicroRNA-125b in Systemic Sclerosis.

To analyze the expression, regulation, and role of microRNA-125b (miR-125b) in systemic sclerosis (SSc). MiR-125b expression was assessed by quantitative polymerase chain reaction (qPCR) of RNA from dermal fibroblasts and whole skin biopsy specimens from healthy controls and SSc patients. To identify downstream effectors, RNA from healthy control fibroblasts was sequenced after miR-125b knockdown and further validated using qPCR and Western blotting. Fibrosis, apoptosis, and proliferation were assessed by Caspase-Glo 3/7 assay, Western blotting, immunofluorescence staining for cleaved caspase 3, and annexin V real-time assay in dermal fibroblasts. Expression of miR-125b was significantly down-regulated in SSc skin biopsy specimens by 53% (median fold change 0.47 [interquartile range 0.35-0.69]; P < 0.001) and in SSc dermal fibroblasts by 47% (median fold change 0.53 [interquartile range 0.36-0.58]; P < 0.001) compared to healthy control skin biopsy specimens and fibroblasts, respectively (n = 10 samples per group). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin A significantly decreased the expression of miR-125b in dermal fibroblasts. MiR-125b knockdown significantly reduced cell proliferation and α-smooth muscle actin (α-SMA) expression at the messenger RNA (mRNA) and protein levels. RNA-Seq identified BAK1, BMF, and BBC3 as potential targets of miR-125b. Quantitative PCR confirmed that knockdown of miR-125b up-regulated these genes (P < 0.01; n = 12). Bcl-2 homologous antagonist killer 1 showed the strongest induction confirmed at the protein level (P < 0.01; n = 10). Consequently, miR-125b knockdown increased apoptosis compared to scrambled control. Accordingly, miR-125b overexpression decreased apoptosis. Our findings indicate that miR-125b is down-regulated in SSc skin and primary dermal fibroblasts. MiR-125b down-regulation increases apoptosis and decreases proliferation and α-SMA expression in dermal fibroblasts, indicating that its compensatory, antifibrotic mechanism may be a potential novel therapeutic option.

OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.

Slit2/Robo4 axis may contribute to endothelial cell dysfunction and angiogenesis disturbance in systemic sclerosis

ObjectiveIn systemic sclerosis (SSc), early microvascular injury is followed by impaired angiogenesis and peripheral capillary loss. Here, we investigated the possible contribution of the neurovascular guidance molecule Slit2 and its...

TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung.

Persistent fibrosis in multiple organs is the hallmark of systemic sclerosis (SSc). Recent genetic and genomic studies implicate TLRs and their damage-associated molecular pattern (DAMP) endogenous ligands in fibrosis. To test the hypothesis that TLR4 and its coreceptor myeloid differentiation 2 (MD2) drive fibrosis persistence, we measured MD2/TLR4 signaling in tissues from patients with fibrotic SSc, and we examined the impact of MD2 targeting using a potentially novel small molecule. Levels of MD2 and TLR4, and a TLR4-responsive gene signature, were enhanced in SSc skin biopsies. We developed a small molecule that selectively blocks MD2, which is uniquely required for TLR4 signaling. Targeting MD2/TLR4 abrogated inducible and constitutive myofibroblast transformation and matrix remodeling in fibroblast monolayers, as well as in 3-D scleroderma skin equivalents and human skin explants. Moreover, the selective TLR4 inhibitor prevented organ fibrosis in several preclinical disease models and mouse strains, and it reversed preexisting fibrosis. Fibroblast-specific deletion of TLR4 in mice afforded substantial protection from skin and lung fibrosis. By comparing experimentally generated fibroblast TLR4 gene signatures with SSc skin biopsy gene expression datasets, we identified a subset of SSc patients displaying an activated TLR4 signature. Together, results from these human and mouse studies implicate MD2/TLR4-dependent fibroblast activation as a key driver of persistent organ fibrosis. The results suggest that SSc patients with high TLR4 activity might show optimal therapeutic response to selective inhibitors of MD2/TLR4 complex formation.

The non-neuronal cyclin-dependent kinase 5 is a fibrotic mediator potentially implicated in systemic sclerosis and a novel therapeutic target.

The mechanisms underlying persistent fibroblast activation and myofibroblast phenoconversion in underlying multi-organ fibrosis in systemic sclerosis (SSc) remain incompletely understood, hindering effective therapies to slow or reverse the process. Cyclin-dependent kinase 5 (CDK5) is a pleiotropic member of the CDK family originally identified in neuronal cells. In contrast to other CDKs, CDK5 activity depends on its CDK5R1 subunit p35. Here we demonstrate that expression of p35 and CDK5 activity are induced by TGF-ß in fibroblasts and adipocytic cell types. Levels of p35 are markedly elevated in both SSc skin biopsies and explanted SSc fibroblasts, as well as in fibrotic skin in mice. Ectopic p35 and CDK5 suppressed adipogenic markers while stimulating collagen production and myofibroblast markers, whereas RNAi-mediated CDK5 knockdown abrogated TGF-β fibrotic responses in a Smad-independent manner. Pharmacological inhibitors of CDK5 likewise prevented and reversed TGF-β responses in fibroblast monolayers and in ex vivo human skin organ cultures, ameliorated collagen overproduction in SSc fibroblasts, and prevented and reversed skin fibrosis in two distinct mouse models of SSc. Together, these results reveal a previously unrecognized key function for p35/CDK5 as a mediator of mesenchymal cell fibrotic responses. The results suggest a potential pathogenic role for elevated p35 expression and CDK5 activity in SSc, and raise the possibility that their selective pharmacological targeting might represent a novel treatment approach in fibrosis.

Adiponectin is an endogenous anti-fibrotic mediator and therapeutic target

Skin fibrosis in systemic sclerosis (SSc) is accompanied by attrition of dermal white adipose tissue (dWAT) and reduced levels of circulating adiponectin. Since adiponectin has potent regulatory effects on fibroblasts, we sought to assess adiponectin signaling in SSc skin biopsies, and evaluate fibrosis in mice with adiponectin gain- and loss-of-function mutations. Furthermore, we investigated the effects and mechanism of action of agonist peptides targeting adiponectin receptors in vitro and in vivo. We found that adiponectin pathway activity was significantly reduced in a subset of SSc skin biopsies. Mice lacking adiponectin mounted an exaggerated dermal fibrotic response, while transgenic mice with constitutively elevated adiponectin showed selective dWAT expansion and protection from skin and peritoneal fibrosis. Adiponectin receptor agonists abrogated ex vivo fibrotic responses in explanted normal and SSc fibroblasts and in 3D human skin equivalents, in part by attenuating focal adhesion complex assembly, and prevented and reversed experimentally-induced organ fibrosis in mice. These results implicate aberrant adiponectin pathway activity in skin fibrosis, identifying a novel function for this pleiotropic adipokine in regulation of tissue remodeling. Restoring adiponectin signaling in SSc patients therefore might represent an innovative pharmacological strategy for intractable organ fibrosis.