Viktor Martyanov

Introduction: Previously, we discovered similar esophageal gene expression patterns in patients with systemic sclerosis and eosinophilic esophagitis where eosinophil/mast cell–targeted therapies are beneficial. Because systemic sclerosis and eosinophilic esophagitis patients experience similar esophageal symptoms, we hypothesized that eosinophil/mast cell–directed therapy may potentially benefit systemic sclerosis patients. Herein, we determine the association between esophageal mast cell quantities, gene expression, and clinical parameters in order to identify systemic sclerosis patients who may benefit from eosinophil/mast cell–directed therapy. Methods: Esophageal biopsies from systemic sclerosis patients and healthy participants were stained for tryptase, a mast cell marker, and associations with relevant clinical parameters including 24-h esophageal pH testing were assessed. Intra-epithelial mast cell density was quantified by semi-automated microscopy. Microarray data were utilized for functional and gene set enrichment analyses and to identify intrinsic subset assignment, a systemic sclerosis molecular classification system that includes inflammatory, proliferative, limited, and normal-like subsets. Results: Esophageal biopsies from 40 systemic sclerosis patients (39 receiving proton pump inhibition) and eleven healthy participants were studied. Mast cell numbers in both the upper esophagus ( r _s = 0.638, p = 0.004) and the entire (upper + lower) esophagus ( r _s = 0.562, p = 0.019) significantly correlated with acid exposure time percentage. The inflammatory, fibroproliferative, and normal-like intrinsic subset originally defined in skin biopsies were identified in esophageal biopsies. Although esophageal mast cell numbers in systemic sclerosis patients and healthy participants were similar, gene expression for mast cell–related pathways showed significant upregulation in the inflammatory intrinsic subset of systemic sclerosis patients compared to patients classified as proliferative or normal-like. Discussion: Esophageal mast cell numbers are heterogeneous in systemic sclerosis patients and may correlate with acid exposure. Patients with inflammatory intrinsic subset profiles in the esophagus demonstrate more tryptase staining. Mast cell–targeted therapy may be a useful therapeutic approach in systemic sclerosis patients belonging to the inflammatory intrinsic subset, but additional studies are warranted.

Objective: To assess the safety and efficacy of lenabasum in diffuse cutaneous systemic sclerosis (dcSSc).Methods: A randomized, double‐blind, placebo‐controlled, phase II study was conducted at 9 SSc clinics in the US. Adults with dcSSc of ≤6 years’ duration who were receiving stable standard‐of‐care treatment were randomized to receive lenabasum (n = 27) or placebo (n = 15). Lenabasum doses were 5 mg once daily, 20 mg once daily, or 20 mg twice daily for 4 weeks, followed by 20 mg twice daily for 8 weeks. Safety and efficacy were assessed at weeks 4, 8, 12, and 16.Results: Adverse events (AEs) occurred in 63% of the lenabasum group and 60% of the placebo group, with no serious AEs related to lenabasum. Compared to placebo, lenabasum treatment was associated with greater improvement in the American College of Rheumatology Combined Response Index in diffuse cutaneous Systemic Sclerosis (CRISS) score and other efficacy outcome measures that assessed overall disease, skin involvement, and patient‐reported function. The median CRISS score increased in the lenabasum group during the study, reaching 0.33, versus 0.00 in the placebo group, at week 16 (P = 0.07 by 2‐sided mixed‐effects model repeated‐measures analysis). Gene expression in inflammation and fibrosis pathways was reduced, and inflammation and fibrosis were improved on histologic evaluation of skin biopsy specimens, in the lenabasum group compared to the placebo group (all P ≤ 0.05).Conclusion: Despite a short trial duration in a small number of patients in this phase II study in dcSSc, our findings indicate that lenabasum improves efficacy outcomes and underlying disease pathology with a favorable safety profile.

Objective: Genome‐wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte‐derived macrophages and assessed their interaction with SSc fibroblasts.Methods: Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age‐ and sex‐matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates.Results: The gene expression signature associated with blood‐derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin‐6, and transforming growth factor β under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05).Conclusion: These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

There are no effective treatments or validated clinical response markers in systemic sclerosis (SSc). We assessed imaging biomarkers and performed gene expression profiling in a single-arm open-label clinical trial of tyrosine kinase inhibitor dasatinib in patients with SSc-associated interstitial lung disease (SSc-ILD). Primary objectives were safety and pharmacokinetics. Secondary outcomes included clinical assessments, quantitative high-resolution computed tomography (HRCT) of the chest, serum biomarker assays and skin biopsy-based gene expression subset assignments. Clinical response was defined as decrease of >5 or >20% from baseline in the modified Rodnan Skin Score (MRSS). Pulmonary function was assessed at baseline and day 169. Dasatinib was well-tolerated in 31 patients receiving drug for a median of nine months. No significant changes in clinical assessments or serum biomarkers were seen at six months. By quantitative HRCT, 65% of patients showed no progression of lung fibrosis, and 39% showed no progression of total ILD. Among 12 subjects with available baseline and post-treatment skin biopsies, three were improvers and nine were non-improvers. Improvers mapped to the fibroproliferative or normal-like subsets, while seven out of nine non-improvers were in the inflammatory subset (p = 0.0455). Improvers showed stability in forced vital capacity (FVC) and diffusing capacity for carbon monoxide (D_LCO), while both measures showed a decline in non-improvers (p = 0.1289 and p = 0.0195, respectively). Inflammatory gene expression subset was associated with higher baseline HRCT score (p = 0.0556). Non-improvers showed significant increase in lung fibrosis (p = 0.0313). In patients with SSc-ILD dasatinib treatment was associated with acceptable safety profile but no significant clinical efficacy. Patients in the inflammatory gene expression subset showed increase in skin fibrosis, decreasing pulmonary function and worsening lung fibrosis during the study. These findings suggest that target tissue-specific gene expression analyses can help match patients and therapeutic interventions in heterogeneous diseases such as SSc, and quantitative HRCT is useful for assessing clinical outcomes. Clinicaltrials.gov NCT00764309

Systemic sclerosis (SSc; scleroderma) is an uncommon autoimmune rheumatic disease characterised by autoimmunity, vasculopathy and fibrosis. Gene expression profiling distinguishes scleroderma from normal skin, and can detect different subsets of disease, with potential to identify prognostic biomarkers of organ involvement or response to therapy. We have performed gene expression profiling in skin samples from patients with limited cutaneous SSc (lcSSc). Total RNA was extracted from clinically uninvolved skin biopsies of 15 patients with lcSSc and 8 healthy controls (HC). Gene expression profiling was performed on a DNA oligonucleotide microarray chip. Differentially expressed genes (DEG) were identified using significance analysis of microarrays (SAM). Functional enrichment analysis of gene signatures was done via g:Profiler. There were 218 DEG between lcSSc and HC samples (false discovery rate <10%): 181/218 DEG were upregulated in lcSSc samples. Hierarchical clustering of DEG suggested the presence of two separate groups of lcSSc samples: “limited 1” and “limited 2”. The limited-1 group (13 samples, 10 unique patients) showed upregulation of genes involved in cell adhesion, cardiovascular system (CVS) development, extracellular matrix and immune and inflammatory response. The CVS development signature was of particular interest as its genes showed very strong enrichment in response to wounding, response to transforming growth factor (TGF)-β and kinase cascade. Neither limited-2 samples (six samples, five unique patients) nor HC samples showed functional enrichment. There were no significant differences in demographic or clinical parameters between these two groups. These results were confirmed using a second independent cohort. Our study suggests the presence of molecular subsets in lcSSc based on gene expression profiling of biopsies from uninvolved skin. This may reflect important differences in pathogenesis within these patient groups. We identify differential expression of a subset of genes that relate to CVS and are enriched in fibrotic signalling. This may shed light on mechanisms of vascular disease in SSc. The enrichment in profibrotic profile suggests that dysregulated gene expression may contribute to vasculopathy and fibrosis in different disease subsets.

Background: Limited cutaneous SSc (lcSSc) is characterized by less extensive skin fibrosis but patients can develop major internal organ complications and vascular manifestations. Gene expression analysis of SSc biopsies has been used to define molecular subsets of the disease and provide mechanistic insight into pathobiology. Clinically uninvolved skin in the diffuse cutaneous subset has closely replicated gene expression signatures compared with biopsies from clinically involved skin and a number of vasoactive genes have been identified this way. We have investigated the gene expression abnormalities in the more prevalent lcSSc subset through detailed transcriptional analysis of skin biopsies taken from uninvolved forearm skin.