Fibrosis In Systemic Sclerosis Research Articles

Laboratory strategy for autoantibodies testing as a diagnostic marker of pulmonary fibrosis in systemic sclerosis: A preliminary study prior to cohort registry of systemic sclerosis in West Java Indonesia

Background: Interstitial lung disease (ILD) is a complication often found in patients with Systemic Sclerosis (SSc). This usually manifests as pulmonary fibrosis and is the leading cause of death in SSc patients. Antinuclear antibodies indirect immunofluorescence (ANA-IIF) test and specific autoantibodies testing could be employed as a diagnostic marker to detect pulmonary fibrosis, but the high cost of these tests often burdens patients in developing countries. This study aims to compare ANA-IIF and an alternative, possibly inexpensive method to detect pulmonary fibrosis in SSc. Methods: This is a preliminary study prior to the cohort systemic sclerosis registry conducted in Rheumatology Outpatient Clinic and Clinical Pathology Department at Dr. Hasan Sadikin General Hospital, Bandung, from January 1, 2018 to December 31, 2020. Two contrastive laboratory testing strategies were followed to confirm pulmonary fibrosis in 61 SSc patients. The first strategy was to perform antinuclear antibodies indirect immunofluorescence (ANA-IIF) test followed by specific autoantibodies detection [anti-topoisomerase-I (ATA), Anti-Th/To, SSc Profile, Immunoblot]. The alternative strategy included conducting the tests for SSc autoantibodies in the reverse order. Results: A total of 42 out of 61 (68.8%) subjects were diagnosed with pulmonary fibrosis by high-resolution computed tomography (HRCT) scan. The first strategy detected dominant specific pulmonary fibrosis autoantibodies in subjects with pulmonary fibrosis: homogeneous nucleolar pattern was dominant (n = 25, 59.52%), followed by nucleolar pattern (n = 17, 40.48%). Of all the subjects with a homogeneous nucleolar pattern, ATA was detected in 15 subjects (60%), the combination of ATA and Th/To in 9 subjects (36%), and Ro52 in one subject (4%). The second strategy yielded positive autoantibodies results in 29 out of 42 (69.05%) subjects, ATA in 20 subjects (47.61%), and Th/To in 9 patients (21.44%), but failed to detect any autoantibodies specific to SSc-ILD in 13 subjects (30.95%). Conclusion: The first strategy [ANA-IIF followed specific autoantibodies testing (ATA, Th/To, SSc profile and immunoblot)] is more likely to detect specific autoantibodies toward ILD in SSc patients.

Distinct Metalloproteinase Expression and Functions in Systemic Sclerosis and Fibrosis: What We Know and the Potential for Intervention.

Systemic sclerosis (SSc) is a chronic debilitating idiopathic disorder, characterized by deposition of excessive extracellular matrix (ECM) proteins such as collagen which leads to fibrosis of the skin and other internal organs. During normal tissue repair and remodeling, the accumulation and turnover of ECM proteins are tightly regulated by the interaction of matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinases (TIMPs). SSc is associated with dysregulation of the activity of these proteolytic and inhibitory proteins within the tissue microenvironment, tipping the balance toward fibrosis. The resultant ECM accumulation further perpetuates tissue stiffness and decreased function, contributing to poor clinical outcomes. Understanding the expression and function of these endogenous enzymes and inhibitors within specific tissues is therefore critical to the development of therapies for SSc. This brief review describes recent advances in our understanding of the functions and mechanisms of ECM remodeling by metalloproteinases and their inhibitors in the skin and lungs affected in SSc. It highlights recent progress on potential candidates for intervention and therapeutic approaches for treating SSc fibrosis.

Expression and Pathogenic Analysis of Integrin Family Genes in Systemic Sclerosis

Objectives: Emerging evidence shows that integrin members are involved in inflammation and fibrosis in systemic sclerosis (SSc). This study aimed at evaluating the expression of integrin family genes in the skin tissue from SSc patients and exploring the potential pathogenic mechanism.Methods: We utilized the public datasets of SSc skin tissue from the Gene Expression Omnibus (GEO) database to analyze the expression and clinical significance of integrin family genes in SSc. The expression of integrin members in skin tissue was also assessed by immunohistochemistry. In addition, functional enrichment and pathway analysis were conducted.Results: Compared with healthy controls, the mRNA and protein levels of ITGA5, ITGB2, and ITGB5 were upregulated in the skin of SSc patients. Further analysis indicated that the mRNA expression levels of ITGA5, ITGB2, and ITGB5 were positively correlated with modified Rodnan skin thickness score (mRSS). Functional enrichment and pathway analysis showed that integrin members may play multiple roles in the pathogenesis of SSc. Among them, ITGA5, ITGB2, and ITGB5 might synergistically promote SSc through affecting extracellular matrix (ECM) turnover, ECM–receptor interaction, focal adhesion, and leukocyte trans-endothelial migration, while ITGA5 and ITGB5 also might affect angiogenesis and endothelial cell function. In addition, ITGA5, ITGB2, and ITGA5 were associated with different pathways, respectively. ITGA5 was uniquely enriched for actin organization, while ITGB5 was for TGF-β signaling and ITGB2 for immune cell activation.Conclusion: Our results implied that the abnormal expression of integrin family genes including ITGA5, ITGB2, and ITGB5 may participate in multiple pathological processes in SSc. Further investigations are required for confirming this speculation.

TGFβ promotes low IL10-producing ILC2 with profibrotic ability involved in skin fibrosis in systemic sclerosis

ObjectiveInnate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin...

Myofibroblast transcriptome indicates SFRP2hi fibroblast progenitors in systemic sclerosis skin

Skin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells. The number of myofibroblasts in SSc skin correlates with the modified Rodnan skin score, the most widely used clinical measure of skin disease severity. Murine fibrosis models indicate that myofibroblasts can arise from a variety of different cell types, but their origin in SSc skin has remained uncertain. Utilizing single cell RNA-sequencing, we define different dermal fibroblast populations and transcriptome changes, comparing SSc to healthy dermal fibroblasts. Here, we show that SSc dermal myofibroblasts arise in two steps from an SFRP2hi/DPP4-expressing progenitor fibroblast population. In the first step, SSc fibroblasts show globally upregulated expression of transcriptome markers, such as PRSS23 and THBS1. A subset of these cells shows markers indicating that they are proliferating. Only a fraction of SFRP2hi SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. Bioinformatics analysis of the SSc fibroblast transcriptomes implicated upstream transcription factors, including FOSL2, RUNX1, STAT1, FOXP1, IRF7 and CREB3L1, as well as SMAD3, driving SSc myofibroblast differentiation.

Sildenafil Counteracts the In Vitro Activation of CXCL-9, CXCL-10 and CXCL-11/CXCR3 Axis Induced by Reactive Oxygen Species in Scleroderma Fibroblasts.

Simple SummarySystemic sclerosis (SSc) is a complex autoimmune disease characterized by different clinical features and a high risk of mortality due to multi-organ fibrosis. Oxidative stress plays a key role in SSc pathogenesis, and an altered redox state could be responsible for abnormal inflammatory condition, tissue damage and fibrosis. Different pro-inflammatory mediators (cytokines and chemokines) occur from the earlier to the last stage of this disease, but their relationship with clinical findings is still unclear. To this purpose, studies are still required which work to find biomarkers mirroring the disease progression and potential pharmacological targets improving treatment response. In this study, we demonstrated that the vasoactive drug sildenafil, a phosphodiesterase type 5 inhibitor commonly used to treat pulmonary hypertension, reduces the expression and secretion of pro-inflammatory chemokines in dermal fibroblasts isolated from SSc patients exposed to reactive oxygen species. We demonstrated that this effect relies on the ability of sildenafil to interfere with the pro-inflammatory pathways involved in the expression of these molecules and its capacity to counteract the plasma membrane translocation of their specific receptor. These results sustain clinical studies to consider the use of sildenafil in preventing tissue damage and fibrosis in systemic sclerosis by targeting essential biomarkers of disease progression and reducing the oxidative-stress-induced inflammation.Oxidative stress plays a key role in systemic sclerosis (SSc) pathogenesis, and an altered redox homeostasis might be responsible for abnormal inflammatory status, fibrosis and tissue damage extension. In this study, we explored the effect of the phosphodiesterase type 5 inhibitor sildenafil in modulating the activation of the CXCL-9, -10, -11/CXCR3 axis, which is fundamental in the perpetuation of inflammation in different autoimmune diseases, in the cell culture of SSc human dermal fibroblasts exposed to a pro-oxidant environment. We observed that sildenafil significantly reduced gene expression and release of CXCL-9, -10 and -11, inhibited the CXCR3 action and suppressed the activation of STAT1-, JNK- and p38MAPK pathways. This in vitro study on dermal fibroblasts supports clinical studies to consider the efficacy of sildenafil in preventing tissue damage and fibrosis in SSc by targeting central biomarkers of disease progression, vascular injuries and fibrosis and reducing the pro-inflammatory activation induced by oxidative stress.

POS0431 FIBROBLASTS ARE NOT JUST FIBROBLASTS - CLEAR DIFFERENCES IN ECM PRODUCTION BETWEEN DERMAL AND LUNG FIBROBLASTS IN RESPONSE TO GROWTH FACTORS

Background:Many systemic sclerosis (SSc) patients develop lung fibrosis, which contribute significantly to increased mortality1. Activated and proliferating fibroblasts are responsible for the excessive extracellular matrix (ECM) formation and stiffening of the connective tissue leading to skin and lung fibrosis. There is currently no effective treatment for the fibrosis in SSc and there is therefore a medical need for further understanding the pathogenesis of fibrosis. Fibrosis is associated with different growth factors, including tumor growth factor beta 1 (TGF-β1) and platelet derived growth factor-ab (PDGF-ab)2.Objectives:We investigated how stimulation with TGF-β1 and PDGF-ab affected the migration capacity and the ECM production using translational biomarkers of type I, III and VI collagens in healthy human dermal and lung fibroblasts.Methods:The fibroblasts were grown in DMEM media containing 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid for up to 12 days. The cells were stimulated with TGF-β1 [0.04-1 nM] or PDGF-ab [3 nM] at treatment initiation and changed twice a week. Non-stimulated fibroblasts (w/o) were used as control. A wound was induced by scratching the cells at day 1 after treatment initiation and the migration was followed for 2 days. Type I, III and VI collagen formation (PRO-C1, PRO-C3 and PRO-C6, respectively) were evaluated by validated ELISAs (Nordic Bioscience) in supernatant from day 0, 4, 8 and 12. Statistical analysis included 2-way ANOVA and Dunnett’s test.Results:The PDGF-ab stimulated dermal fibroblasts migrated significantly more than the non-stimulated (p<0.0001) and TGF-β1 stimulated (p<0.001) dermal fibroblasts 48 hours after the scratch (migration app. 70%, 30% and 30% respectively). There was no difference between the migration of the non-stimulated, TGF-β1 and PDGF-ab stimulated lung fibroblasts after 48 hours, as all migrated to approximately 70%.TGF-β1 stimulation led to a significant increase in type I collagen formation (PRO-C1) in both dermal and lung fibroblasts from day 4 and onwards compared to w/o (p<0.0001). TGF-β1 also lead to a significant increase in type III collagen formation (PRO-C3) from day 8 in lung fibroblasts compared to w/o (p<0.0001). PDGF-ab stimulation led to a significant increase in type III collagen formation in dermal fibroblasts from day 8 compared to w/o (p<0.0001). PDGF-ab stimulation led to a significant increase in type VI collagen formation (PRO-C6) in both dermal and lung fibroblasts from day 4 and onwards compared to w/o (p<0.0001).Conclusion:PDGF-ab increased the migration activity of the dermal fibroblasts, where the lung fibroblasts had a general high migration activity. The dermal and lung fibroblasts showcase the same ECM production within both type I and type VI collagen formation. The two fibroblasts types did however react opposite each other regarding the type III collagen formation: the dermal fibroblasts responded to PDGF-ab stimulation, where the lung fibroblasts responded to the TGF-β1 stimulation. The clear differences in the ECM production between the dermal and lung fibroblasts can be important in the search for an effective treatment for fibrosis in SSc and related lung fibrosis.

Fli1 deficiency suppresses RALDH1 activity of dermal dendritic cells and related induction of regulatory T cells: a possible role in scleroderma

BackgroundAldehyde dehydrogenase 1 family member A1 (RALDH1)-producing dermal dendritic cells (DCs), a conventional DC subset regulating skin fibrosis, are decreased in the involved skin of patients with systemic sclerosis (SSc). In this study, we investigated the contribution of Fli1 deficiency, a potential predisposing factor of SSc, to the phenotypical alteration of RALDH1-producing dermal DCs by using SSc model mice and SSc skin samples.MethodsBleomycin (BLM)-induced skin fibrosis was generated with Fli1+/− and wild-type mice. The proportions of DC and CD4+ T cell subsets were determined by flow cytometry in the dermis of BLM-treated mice. Fli1 expression in dermal DCs was evaluated by immunofluorescence with skin samples of SSc and healthy control subjects.ResultsRALDH activity of dermal DCs was significantly decreased in BLM-treated Fli1+/− mice compared with BLM-treated wild-type mice, whereas the proportion of CD103−CD11b− dermal DCs, a major DC subset producing RALDH1 in response to BLM injection, was comparable between groups. Relevant to this finding, the proportion of regulatory T cells (Tregs) in the dermis was decreased in BLM-treated Fli1+/− mice relative to BLM-treated wild-type mice, while the proportions of Th1, Th2 and Th17 cells were unaltered. In the involved skin of SSc patients, Fli1 was downregulated in CD11c+ cells, including dermal DCs.ConclusionsFli1 deficiency inhibits RALDH1 activity of CD103−CD11b− dermal DCs and related induction of Tregs in BLM-treated mice. Considering Fli1 reduction in SSc dermal DCs, Fli1deficiency may impair the dermal DC-Treg system, contributing to the development of skin fibrosis in SSc.

Rational repurposing of tocilizumab for treatment of lung fibrosis in systemic sclerosis.

Rational repurposing of tocilizumab for treatment of lung fibrosis in systemic sclerosis.

Modulation of microRNome by Human Cytomegalovirus and Human Herpesvirus 6 Infection in Human Dermal Fibroblasts: Possible Significance in the Induction of Fibrosis in Systemic Sclerosis.

Human cytomegalovirus (HCMV) and Human herpesvirus 6 (HHV-6) have been reportedly suggested as triggers of the onset and/or progression of systemic sclerosis (SSc), a severe autoimmune disorder characterized by multi-organ fibrosis. The etiology and pathogenesis of SSc are still largely unknown but virological and immunological observations support a role for these beta-herpesviruses, and we recently observed a direct impact of HCMV and HHV-6 infection on the expression of cell factors associated with fibrosis at the cell level. Since miRNA expression has been found profoundly deregulated at the tissue level, here we aimed to investigate the impact on cell microRNome (miRNome) of HCMV and HHV-6 infection in in vitro infected primary human dermal fibroblasts, which represent one of the main SSc target cells. The analysis, performed by Taqman arrays detecting and quantifying 754 microRNAs (miRNAs), showed that both herpesviruses significantly modulated miRNA expression in infected cells, with evident early and late effects and deep modulation (>10 fold) of >40 miRNAs at each time post infection, including those previously recognized for their key function in fibrosis. The correlation between these in vitro results with in vivo observations is strongly suggestive of a role of HCMV and/or HHV-6 in the multistep pathogenesis of fibrosis in SSc and in the induction of fibrosis-signaling pathways finally leading to tissue fibrosis. The identification of specific miRNAs may open the way to their use as biomarkers for SSc diagnosis, assessment of disease progression and possible antifibrotic therapies.

P149 The intracellular chloride channel 4 (CLIC4) plays an important role in systemic sclerosis fibroblast activation

Abstract Background/Aims The intracellular chloride ion channel CLIC4 mediates the activation of cancer associated fibroblasts. Interestingly, systemic sclerosis (SSc) fibroblasts display a number of similar properties to cancer associated fibroblasts. Tissue fibrosis in SSc is driven by active fibroblasts (myofibroblasts). Therefore in this study we investigated the role of CLIC4 in SSc fibroblast activation. Methods Dermal fibroblasts were obtained from full thickness skin biopsies from SSc patients (early-diffuse). RNA and protein were collected from the fibroblasts and CLIC4 transcript and protein levels were assessed by qPCR and western blot. SSc patient fibroblasts were treated with the chloride ion channel inhibitors NPPB and IAA-94. Results CLIC4 was found to be expressed at significantly higher levels in SSc patients fibroblasts compared to healthy controls, at both the transcript (3.7 fold) and protein (1.7 fold) levels. Inhibition of the TGF-β signalling pathway led to reduced CLIC4 expression in SSc fibroblasts, confirming this pathway as the main driver of CLIC4 expression. Finally, treatment of SSc fibroblasts with small molecule inhibitors that target the channel led to reduced expression of the myofibroblast markers collagen type 1 and alpha-smooth muscle actin, suggesting a direct role for CLIC4 in SSc associated skin fibrosis. Conclusion We have identified a novel role for CLIC4 in SSc myofibroblast activation, which further strengthen the similarities between SSc fibroblasts and cancer associated fibroblasts. Furthermore this study highlights this channel as a novel target for therapeutic intervention. Disclosure C. Wasson: None. R. Ross: None. R. Morton: None. J. Mankouri: None. F. Del Galdo: None.

Endothelial-to-mesenchymal transition in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by significant vascular alterations and multi-organ fibrosis. Microvascular alterations are the first event of SSc and injured endothelial cells (ECs) may transdifferentiate towards myofibroblasts, the cells responsible for fibrosis and collagen deposition. This process is identified as endothelial-to-mesenchymal transition (EndMT), and understanding of its development is pivotal to identify early pathogenetic events and new therapeutic targets for SSc. In this review, we have highlighted the molecular mechanisms of EndMT and summarize the evidence of the role played by EndMT during the development of progressive fibrosis in SSc, also exploring the possible therapeutic role of its inhibition.

Clinical and Prognostic Characteristics of "Disease-specific" Autoantibodies in Systemic Scleroderma

Systemic sclerosis (SSc) is a connective tissue disease of autoimmune nature characterized by the triad of vascular injury, autoimmunity (cellular and humoral) and tissue fibrosis. Autoantibodies do not seem to be simply epiphenomena, but are involved in disease pathogenesis. It is believed that the SSc-specific autoantibodies are responsible both for amplifying immune response and targeting cell types that are relevant in the pathophysiology of SSc. In the current understanding of the pathogenesis of SSc, the leading role is assigned to the vascular lesion of the microvasculature arising against the background of immunity dysregulation and the intensification of fibrosis processes associated with the action of various growth factors and cytokines. In SSc, as in other systemic autoimmune diseases, chronic B-lymphocytic activation is observed, which results in a loss of tolerance to self antigens. Circulation of a wide range of autoantibodies (antibodies) is a characteristic feature of SSc. It is possible that autoantigens in SSc are released during ischemia-reperfusion tissue damage that occurs against the background of vasospastic vascular reactions characteristic of this disease. In the process of B-cell presentation of autoantigens through costimulatory surface molecules, activation of T-lymphocytes occurs, followed by clonal expansion of T-cell subpopulations, which are autoreactive towards endothelium and fibroblasts. It is believed that B cells play an important role in the initial mechanisms of fibrosis in SSc, and chronic activation of B cells is directly related to the development of sclerodermic fibrosis through the production of antibodies and fibrogenic cytokines. Despite many years of efforts, a complete concept explaining the role of antibodies in the pathogenesis of SSc has not yet been created. At the same time, a huge amount of information has been accumulated on the diagnostic and prognostic significance of circulating antibodies, the importance of which for the supervision of the patient can hardly be overestimated.

Inhibition of skin fibrosis in systemic sclerosis by botulinum toxin B via the suppression of oxidative stress.

Oxidative stress has been reported to play an important role in the pathogenesis of skin fibrosis in systemic sclerosis (SSc). We previously identified that botulinum toxin (BTX) injection suppresses pressure ulcer formation in a cutaneous ischemia-reperfusion injury mouse model by regulation of oxidative stress. However, the therapeutic possibility of BTX administration for preventing skin fibrosis in SSc is unclear. The objective of this study was to investigate the effect of BTX-B on skin fibrosis in a murine model of SSc and determine the underlying mechanism. We found that BTX-B injection significantly reduced dermal thickness and inflammatory cell infiltration in bleomycin-induced skin fibrosis lesion in mice. We also identified that the oxidative stress signal detected through bioluminescence in OKD48 mice after bleomycin injection in the skin was significantly decreased by BTX-B. Additionally, mRNA levels of oxidative stress associated factors (NOX2, HO-1, Trx2) were significantly decreased by BTX-B. Apoptotic cells in the lesional skin of bleomycin-treated mice were significantly reduced by BTX-B. Oxidant-induced intracellular accumulation of reactive oxygen species in SSc fibroblasts was also inhibited by BTX-B. In conclusion, BTX-B might improve bleomycin-induced skin fibrosis via the suppression of oxidative stress and inflammatory cells in the skin. BTX-B injection may have a therapeutic effect on skin fibrosis in SSc.

Serum vasohibin-1 levels: A potential marker of dermal and pulmonary fibrosis in systemic sclerosis.

Vasohibin-1 (VASH-1) is a potent anti-angiogenic factor mainly produced by endothelial cells. In addition, VASH-1 prevents TGF-β-dependent activation of renal fibroblasts. Since systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and fibrosis of multiple organs, VASH-1 may be involved in the development of this disease. In this study, we investigated the potential role of VASH-1 in SSc by evaluating the clinical correlation between serum VASH-1 levels and the expression of VASH-1 in SSc-involved skin. Serum VASH-1 levels were higher in SSc patients, especially those with diffuse cutaneous involvement, than in healthy controls and positively correlated with skin score. Furthermore, SSc patients with interstitial lung disease had significantly elevated levels of serum VASH-1 as compared to those without. Importantly, serum VASH-1 levels correlated inversely with both the percentage of predicted vital capacity and the percentage of predicted diffusion lung capacity for carbon monoxide and positively with serum KL-6 levels, but not serum surfactant protein D levels. In SSc-involved skin, VASH1 mRNA was remarkably upregulated compared with healthy control skin, but the major source of VASH-1 was not clear. Fli1 deficiency, a predisposing factor inducing SSc-like endothelial properties, did not affect VASH-1 expression in human dermal microvascular endothelial cells. Collectively, these results suggest that VASH-1 upregulation in the skin and sera is linked to dermal and pulmonary fibrotic changes in SSc, while the contribution of VASH-1 to SSc vasculopathy seems to be limited.

Downregulated IRF8 in Monocytes and Macrophages of Patients with Systemic Sclerosis May Aggravate the Fibrotic Phenotype

Monocytes and macrophages may be involved in the pathogenesis of systemic sclerosis (SSc); however, the etiology and regulation of monocyte and macrophage function in SSc remain unknown. IRF8 is a transcriptional regulator that is essential for the differentiation and function of monocytes and macrophages and thus may be involved in the regulation of macrophage phenotypes in SSc fibrosis. In this study, we measured IRF8 levels in circulating monocytes of 26 patients with SSc (diffuse cutaneous SSc, n= 11; limited cutaneous SSc, n= 15) and 14 healthy controls. IRF8 levels were significantly suppressed in monocytes of patients with diffuse cutaneous SSc and correlated negatively with the modified Rodnan total skin thickness score. Next, we assessed expression levels of cell surface markers, cytokine profiles, and components of extracellular matrix in IRF8-silenced monocyte-derived macrophages. IRF8-silenced monocyte-derived macrophages displayed an M2 phenotype and significantly upregulated mRNA and protein levels of profibrotic factors and extracellular matrix components. Finally, we assessed skin fibrosis in myeloid cell-specific IRF8 conditional knockout (Irf8flox/flox; Lyz2Cre/+) mice and found upregulated mRNA levels of extracellular matrix components and increased bleomycin-induced skin fibrosis. In conclusion, altered IRF8 regulation in monocytes and macrophages may be involved in SSc pathogenesis.

TGFβ-1 Induced Cross-Linking of the Extracellular Matrix of Primary Human Dermal Fibroblasts.

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFβ signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFβ contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFβ-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFβ-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFβ-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFβ-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFβ signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.

Circulating collagen neo-epitopes and their role in the prediction of fibrosis in patients with systemic sclerosis: a multicentre cohort study

<h2>Summary</h2><h3>Background</h3> Extracellular matrix remodelling is a hallmark of systemic sclerosis. We evaluated extracellular matrix neo-epitopes as potential serum biomarkers for progression of fibrosis in systemic sclerosis. <h3>Methods</h3> We included patients meeting the 2013 American College of Rheumatology and European League Against Rheumatism criteria and healthy controls from a derivation and validation cohort. The primary outcome was progression of fibrosis at follow-up, defined as decline in percentage of predicted forced vital capacity of 10% or more in patients with interstitial lung disease or increase in modified Rodnan skin score of 25% or more and more than 5 points at a 1-year follow-up visit. Longitudinal assessment and biobanking followed European Scleroderma Trials and Research standards. Extracellular matrix-degradation (BGM, C3M, C4M, and C6M) and extracellular matrix-formation neo-epitopes (PRO-C1, PRO-C3, PRO-C4, PRO-C5, and PRO-C6) were measured in serum using validated ELISAs. <h3>Findings</h3> Between Aug 18, 2011, and Jan 19, 2015, 149 patients with systemic sclerosis (27 [18%] progressors and 122 [82%] non-progressors) and 29 healthy controls were included in the derivation cohort. Concentrations of type III and IV collagen neo-epitopes were higher in patients with systemic sclerosis compared with healthy controls and were significantly associated with systemic sclerosis in univariable logistic regression. Concentrations of degradation neo-epitopes of type III and IV collagens and their turnover ratios distinguished between progressors and non-progressors (C3M area under the curve 0·77 [95% CI 0·67–0·86], p<0·0001; PRO-C3:C3M 0·70 [0·59–0·80], p=0·0013; C4M 0·73 [0·63–0·82], p<0·0001; PRO-C4:C4M 0·75 [0·64–0·86], p<0·0001). 384 patients with systemic sclerosis (73 [19%] progressors) and 60 healthy controls were included in the multicentre validation cohort between April 17, 2003, and Jan 24, 2017. Analysis of the validation cohort confirmed that neo-epitopes of type III and IV collagens are changed in progressors. In a pooled analysis of both cohorts, the serum concentrations of formation neo-epitopes PRO-C3 and PRO-C4 and the turnover ratio of type IV collagen (PRO-C4:C4M) were higher in skin progressors. The turnover ratio of type IV collagen and PRO-C3 significantly predicted skin progression in a multivariable model adjusted for modified Rodnan skin score, sex, and age. <h3>Interpretation</h3> These data suggest that neo-epitopes of type III and IV collagens are promising biomarkers for the assessment and prediction of extracellular matrix remodelling in systemic sclerosis. They could be used in clinical practice to risk stratify patients at risk of progression of fibrosis. <h3>Funding</h3> None.

Expression of macrophage migration inhibitory factor and its receptor CD74 in systemic sclerosis.

Macrophage migration inhibitory factor (MIF) has been associated with the pathogenesis of several rheumatic diseases. In systemic sclerosis (SSc) it has been shown that MIF expression is dysregulated in serum and skin. However, the MIF receptor, CD74, has been poorly investigated and its potential role in the pathogenesis of SSc remains unknown. This study aimed to analyze mRNA, tissue, and serum expression of MIF and CD74 in patients with limited (lcSSc) and diffuse (dcSSc) systemic sclerosis. A case-control study in 20 SSc patients and 20 control subjects (CS) from southern México was conducted. MIF and CD74 mRNA expression levels were quantified by real-time PCR, MIF serum levels were measured by an ELISA kit, and MIF and its receptor CD74 were evaluated by immunohistochemistry of skin biopsies. MIF mRNA expression was significantly higher in CS than in SSc patients (p = 0.02), while CD74 showed no differences between patients and CS. MIF serum levels were similar between SSc patients and CS: dcSSc = 3.82 ng/ml, lcSSc = 3.57 ng/ml, and CS = 3.28 ng/ml. In skin biopsies of SSc, MIF and CD74 were enhanced in keratinocytes, while they showed decreased expression in endothelial cells. On the other hand, the staining of CD74 was high in fibroblasts of dcSSc patients. Our findings show MIF and CD74 deregulation at the transcriptional and translational levels in SSc, which might be associated with the proinflammatory process leading to tissue remodeling and excessive fibrosis in SSc.

Targeting CD38-dependent NAD+ metabolism to mitigate multiple organ fibrosis

SummaryThe processes underlying synchronous multiple organ fibrosis in systemic sclerosis (SSc) remain poorly understood. Age-related pathologies are associated with organismal decline in nicotinamide adenine dinucleotide (NAD+) that is due to dysregulation of NAD+ homeostasis and involves the NADase CD38. We now show that CD38 is upregulated in patients with diffuse cutaneous SSc, and CD38 levels in the skin associate with molecular fibrosis signatures, as well as clinical fibrosis scores, while expression of key NAD+-synthesizing enzymes is unaltered. Boosting NAD+ via genetic or pharmacological CD38 targeting or NAD+ precursor supplementation protected mice from skin, lung, and peritoneal fibrosis. In mechanistic experiments, CD38 was found to reduce NAD+ levels and sirtuin activity to augment cellular fibrotic responses, while inhibiting CD38 had the opposite effect. Thus, we identify CD38 upregulation and resulting disrupted NAD+ homeostasis as a fundamental mechanism driving fibrosis in SSc, suggesting that CD38 might represent a novel therapeutic target.