Single-cell epigenomic dysregulation of Systemic Sclerosis fibroblasts via CREB1/EGR1 axis in self-assembled human skin equivalents.

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis, internal organ involvement and vascular dropout. We previously developed and phenotypically characterized an in vitro 3D skin-like tissue model of SSc, and now analyze the transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) characteristics of this model at single-cell resolution. SSc 3D skin-like tissues were fabricated using autologous fibroblasts, macrophages, and plasma from SSc patients or healthy control (HC) donors. SSc tissues displayed increased dermal thickness and contractility, as well as increased α-SMA staining. Single-cell transcriptomic and epigenomic analyses identified keratinocytes, macrophages, and five populations of fibroblasts (labeled FB1 - 5). Notably, FB1 APOE-expressing fibroblasts were 12-fold enriched in SSc tissues and were characterized by high EGR1 motif accessibility. Pseudotime analysis suggests that FB1 fibroblasts differentiate from a TGF-ß1-responsive fibroblast population and ligand-receptor analysis indicates that the FB1 fibroblasts are active in macrophage crosstalk via soluble ligands including FGF2 and APP. These findings provide characterization of the 3D skin-like model at single cell resolution and establish that it recapitulates subsets of fibroblasts and macrophage phenotypes observed in skin biopsies.

CDDO-Methyl Ester Inhibits BRAF Inhibitor Resistance and Remodels the Myeloid Compartment in BRAF-mutant Melanoma.

Approximately 50% of advanced melanomas harbor activating BRAF V600E mutations that are sensitive to BRAF inhibition. However, the duration of the response to BRAF inhibitors (BRAFi) has been limited due to the development of acquired resistance, which is preceded by recruitment of immunosuppressive myeloid cells and regulatory T cells (T regs ). While the addition of MAPK/ERK kinase 1 inhibitors (MEKi) prolongs therapeutic response to BRAF inhibition, most patients still develop resistance. Using a Braf V600E/+ /Pten -/- graft mouse model of melanoma, we now show that the addition of the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (C-Me) to the BRAFi vemurafenib analog PLX4720 at resistance significantly reduces tumor burden. Dual treatment remodels the BRAFi resistant-tumor microenvironment (TME), reducing infiltration of T regs and tumor associated macrophages (TAMs), and attenuates immunosuppressive cytokine production. For the first time, we characterize myeloid populations using scRNA-seq in BRAFi-resistant tumors and demonstrate that restoration of therapeutic response is associated with significant changes in immune-activated myeloid subset representation. Collectively, these studies suggest that C-Me inhibits acquired resistance to BRAFi. Use of C-Me in combination with other therapies may both inhibit melanoma growth and enhance therapeutic responsiveness more broadly.

Advances in biological and targeted therapies for systemic sclerosis.

INTRODUCTION: Systemic sclerosis (SSc) is a severe, and often life-threatening, autoimmune disease, which causes inflammation and fibrosis of the skin and internal organs. There are currently limited effective therapeutic options for patients with SSc. There are recently completed and ongoing phase 2 and 3 studies looking at biologic therapies for SSc that target the underlying pathogenesis of the disease. AREAS COVERED: The purpose of this review is to describe completed and ongoing trials of different biologic therapies for the treatment of SSc. This review discusses biologic therapy directed at multiple pathways that are believed to contribute to inflammation and fibrosis in SSc including T cell, B cell, direct cytokines, and JAK signaling. Data presented is based on authors' expertise of completed and ongoing trials. EXPERT OPINION: Tocilizumab and rituximab have supporting data to advocate for use in early SSc. Data from tocilizumab showed preservation of forced vital capacity (FVC) and beneficial effects on global composite measure. Recent data from different trials with rituximab in SSc (with and without interstitial lung disease) show beneficial effects on skin and FVC with good tolerability. We highlight the molecular heterogeneity in early SSc phenotype and the need to account for this in future trials.

CXCL9 Links Skin Inflammation and Fibrosis through CXCR3-Dependent Upregulation of Col1a1 in Fibroblasts.

Morphea is characterized by initial inflammation followed by fibrosis of the skin and soft tissue. Despite its substantial morbidity, the pathogenesis of morphea is poorly studied. Previous work showed that CXCR3 ligands CXCL9 and CXCL10 are highly upregulated in the sera and lesional skin of patients with morphea. We found that an early inflammatory subcutaneous bleomycin mouse model of dermal fibrosis mirrors the clinical, histological, and immune dysregulation observed in human morphea. We used this model to examine the role of the CXCR3 chemokine axis in the pathogenesis of cutaneous fibrosis. Using the REX3 (Reporting the Expression of CXCR3 ligands) mice, we characterized which cells produce CXCR3 ligands over time. We found that fibroblasts contribute the bulk of CXCL9-RFP and CXCL10-BFP by percentage, whereas macrophages produce high amounts on a per-cell basis. To determine whether these chemokines are mechanistically involved in pathogenesis, we treated Cxcl9-, Cxcl10-, or Cxcr3-deficient mice with bleomycin and found that fibrosis is dependent on CXCL9 and CXCR3. Addition of recombinant CXCL9 but not CXCL10 to cultured mouse fibroblasts induced Col1a1 mRNA expression, indicating that the chemokine itself contributes to fibrosis. Taken together, our studies provide evidence that CXCL9 and its receptor CXCR3 are functionally required for inflammatory fibrosis.

Clinical and Molecular Findings After Autologous Stem Cell Transplantation or Cyclophosphamide for Scleroderma: Handling Missing Longitudinal Data.

OBJECTIVE: Among individuals with systemic sclerosis (SSc) randomized to cyclophosphamide (CYC) (n = 34) or hematopoietic stem cell transplantation (HSCT) (n = 33), we examined longitudinal trends of clinical, pulmonary function, and quality of life measures while accounting for the influence of early failures on treatment comparisons. METHODS: Assuming that data were missing at random, mixed-effects regression models were used to estimate longitudinal trends for clinical measures when comparing treatment groups. Results were compared to observed means and to longitudinal trends estimated from shared parameter models, assuming that data were missing not at random. Longitudinal trends for SSc intrinsic molecular subsets defined by baseline gene expression signatures (normal-like, inflammatory, and fibroproliferative signatures) were also studied. RESULTS: Available observed means for pulmonary function tests appeared to improve over time in both arms. However, after accounting for participant loss, forced vital capacity in HSCT recipients increased by 0.77 percentage points/year but worsened by -3.70/year for CYC (P = 0.004). Similar results were found for diffusing capacity for carbon monoxide and quality of life indicators. Results for both analytic models were consistent. HSCT recipients in the inflammatory (n = 20) and fibroproliferative (n = 20) subsets had superior long-term trends compared to CYC for pulmonary and quality of life measures. HSCT was also superior for modified Rodnan skin thickness scores in the fibroproliferative subset. For the normal-like subset (n = 22), superiority of HSCT was less apparent. CONCLUSION: Longitudinal trends estimated from 2 statistical models affirm the efficacy of HSCT over CYC in severe SSc. Failure to account for early loss of participants may distort estimated clinical trends over the long term.

Human dermal fibroblast-derived exosomes induce macrophage activation in systemic sclerosis.

OBJECTIVES: Prior work demonstrates that co-cultured macrophages and fibroblasts from patients with SSc engage in reciprocal activation. However, the mechanism by which these cell types communicate and contribute to fibrosis and inflammation in SSc is unknown. METHODS: Fibroblasts were isolated from skin biopsies obtained from 7 SSc patients or 6 healthy age and gender-matched control subjects following written informed consent. Human donor-derived macrophages were cultured with exosomes isolated from control or SSc fibroblasts for an additional 48 h. Macrophages were immunophenotyped using flow cytometry, qRT-PCR and multiplex. For mutual activation studies, exosome-activated macrophages were co-cultured with SSc or healthy fibroblasts using Transwells. RESULTS: Macrophages activated with dermal fibroblast-derived exosomes from SSc patients upregulated surface expression of CD163, CD206, MHC Class II and CD16 and secreted increased levels of IL-6, IL-10, IL-12p40 and TNF compared with macrophages incubated with healthy control fibroblasts (n = 7, P < 0.05). Exosome-stimulated macrophages and SSc fibroblasts engaged in reciprocal activation, as production of collagen and fibronectin was significantly increased in SSc fibroblasts receiving signals from SSc exosome-stimulated macrophages (n = 7, P < 0.05). CONCLUSION: In this work, we demonstrate for the first time that human SSc dermal fibroblasts mediate macrophage activation through exosomes. Our findings suggest that macrophages and fibroblasts engage in cross-talk in SSc skin, resulting in mutual activation, inflammation, and extracellular matrix (ECM) deposition. Collectively, these studies implicate macrophages and fibroblasts as cooperative mediators of fibrosis in SSc and suggest therapeutic targeting of both cell types may provide maximal benefit in ameliorating disease in SSc patients.

Clinical Phenotypes of Patients With Systemic Sclerosis With Distinct Molecular Signatures in Skin.

OBJECTIVE: Systemic sclerosis (SSc) patients are classified according to degree of skin fibrosis (limited and diffuse cutaneous [lc and dc]) and serum autoantibodies. We undertook the present multicenter study to determine whether intrinsic subset (IS) classification based upon skin gene expression yields additional valuable clinical information. METHODS: SSc patients and healthy participants (HPs) were classified into Normal-like, Limited, Fibroproliferative, and Inflammatory ISs using a previously trained classifier. Clinical data were obtained (serum autoantibodies, pulmonary function testing, modified Rodnan skin thickness scores [mRSS], and high-resolution chest computed tomography [HRCT]). Statistical analyses were performed to compare patients classified by IS, traditional cutaneous classification, and serum autoantibodies. RESULTS: A total of 223 participants (165 SSc [115 dcSSc and 50 lcSSc] and 58 HPs) were classified. Inflammatory IS patients had higher mRSS (22.1 ± 9.9; P < 0.001) than other ISs and dcSSc patients (19.4 ± 9.4; P = 0.05) despite similar disease duration (median [interquartile range] months 14.9 [19.9] vs. 18.4 [31.6]; P = 0.48). In multivariable modeling, no significant association between mRSS and RNA polymerase III (P = 0.07) or anti-topoisomerase I (Scl-70) (P = 0.09) was found. Radiographic interstitial lung disease (ILD) was more prevalent in Fibroproliferative IS compared with other ISs (91%; P = 0.04) with similar prevalence between lcSSc and dcSSc (67% vs. 76%; P = 0.73). Positive Scl-70 antibody was the strongest ILD predictor (P < 0.001). Interestingly, all lcSSc/Fibroproliferative patients demonstrated radiographic ILD. CONCLUSIONS: Classification by IS identifies patients with distinct clinical phenotypes versus traditional cutaneous or autoantibody classification. IS classification identifies subgroups of SSc patients with more radiographic ILD (Fibroproliferative), higher mRSS (Inflammatory), and milder phenotype (Normal-like) and may provide additional clinically useful information to current SSc classification systems.

Machine-learning classification identifies patients with early systemic sclerosis as abatacept responders via CD28 pathway modulation.

Here, the efficacy of abatacept in patients with early diffuse systemic sclerosis (dcSSc) was analyzed to test the hypothesis that patients in the inflammatory intrinsic subset would show the most significant clinical improvement. Eighty-four participants with dcSSc were randomized to receive abatacept or placebo for 12 months. RNA-Seq was performed on 233 skin paired biopsies at baseline and at 3 and 6 months. Improvement was defined as a 5-point or more than 20% change in modified Rodnan skin score (mRSS) between baseline and 12 months. Samples were assigned to intrinsic gene expression subsets (inflammatory, fibroproliferative, or normal-like subsets). In the abatacept arm, change in mRSS was most pronounced for the inflammatory and normal-like subsets relative to the placebo subset. Gene expression for participants on placebo remained in the original molecular subset, whereas inflammatory participants treated with abatacept had gene expression that moved toward the normal-like subset. The Costimulation of the CD28 Family Reactome Pathway decreased in patients who improved on abatacept and was specific to the inflammatory subset. Patients in the inflammatory subset had elevation of the Costimulation of the CD28 Family pathway at baseline relative to that of participants in the fibroproliferative and normal-like subsets. There was a correlation between improved ΔmRSS and baseline expression of the Costimulation of the CD28 Family pathway. This study provides an example of precision medicine in systemic sclerosis clinical trials.

A genomic meta-analysis of clinical variables and their association with intrinsic molecular subsets in systemic sclerosis.

OBJECTIVES: Four intrinsic molecular subsets (inflammatory, fibroproliferative, limited, normal-like) have previously been identified in SSc and are characterized by unique gene expression signatures and pathways. The intrinsic subsets have been linked to improvement with specific therapies. Here, we investigated associations between baseline demographics and intrinsic molecular subsets in a meta-analysis of published datasets. METHODS: Publicly available gene expression data from skin biopsies of 311 SSc patients measured by DNA microarray were classified into the intrinsic molecular subsets. RNA-sequencing data from 84 participants from the ASSET trial were used as a validation cohort. Baseline clinical demographics and intrinsic molecular subsets were tested for statistically significant associations. RESULTS: Males were more likely to be classified in the fibroproliferative subset (P = 0.0046). SSc patients who identified as African American/Black were 2.5 times more likely to be classified as fibroproliferative compared with White/Caucasian patients (P = 0.0378). ASSET participants sera positive for anti-RNA pol I and RNA pol III autoantibodies were enriched in the inflammatory subset (P = 5.8 × 10-5, P = 9.3 × 10-5, respectively), while anti-Scl-70 was enriched in the fibroproliferative subset. Mean modified Rodnan Skin Score (mRSS) was statistically higher in the inflammatory and fibroproliferative subsets compared with normal-like (P = 0.0027). The average disease duration for inflammatory subset was less than fibroproliferative and normal-like intrinsic subsets (P = 8.8 × 10-4). CONCLUSIONS: We identified multiple statistically significant differences in baseline demographics between the intrinsic subsets that may represent underlying features of disease pathogenesis (e.g. chronological stages of fibrosis) and have implications for treatments that are more likely to work in certain SSc populations.

Self-Assembled Human Skin Equivalents Model Macrophage Activation of Cutaneous Fibrogenesis in Systemic Sclerosis.

OBJECTIVE: The development of precision therapeutics for systemic sclerosis (SSc) has been hindered by the lack of models that accurately mimic the disease in vitro. This study was undertaken to design and test a self-assembled skin equivalent (saSE) system that recapitulates the cross-talk between macrophages and fibroblasts in cutaneous fibrosis. METHODS: SSc-derived dermal fibroblasts (SScDFs) and normal dermal fibroblasts (NDFs) were cultured with CD14+ monocytes from SSc patients or healthy controls to allow de novo stroma formation. Monocyte donor-matched plasma was introduced at week 3 prior to seeding keratinocytes to produce saSE with a stratified epithelium. Tissue was characterized by immunohistochemical staining, atomic force microscopy, enzyme-linked immunosorbent assay, and quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Stroma synthesized de novo from NDFs and SScDFs supported a fully stratified epithelium to form saSE. A thicker and stiffer dermis was generated by saSE with SScDFs, and more interleukin-6 and transforming growth factor ß (TGFß) was secreted by saSE with SScDFs compared to saSE with NDFs, regardless of the inclusion of monocytes. Tissue with SSc monocytes and plasma had amplified dermal thickness and stiffness relative to control tissue. Viable CD163+ macrophages were found within the stroma of saSE 5 weeks after seeding. Additionally, SSc saSE contained greater numbers of CD163+ and CD206+ macrophages compared to control saSE. TGFß blockade inhibited stromal stiffness to a greater extent in SSc saSE compared to control saSE. CONCLUSION: These data suggest reciprocal activation between macrophages and fibroblasts that increases tissue thickness and stiffness, which is dependent in part on TGFß activation. The saSE system may serve as a platform for preclinical therapeutic testing and for molecular characterization of SSc skin pathology through recapitulation of the interactions between macrophages and fibroblasts.

SARS-CoV-2 Impairs Dendritic Cells and Regulates DC-SIGN Gene Expression in Tissues.

The current spreading coronavirus SARS-CoV-2 is highly infectious and pathogenic. In this study, we screened the gene expression of three host receptors (ACE2, DC-SIGN and L-SIGN) of SARS coronaviruses and dendritic cells (DCs) status in bulk and single cell transcriptomic datasets of upper airway, lung or blood of COVID-19 patients and healthy controls. In COVID-19 patients, DC-SIGN gene expression was interestingly decreased in lung DCs but increased in blood DCs. Within DCs, conventional DCs (cDCs) were depleted while plasmacytoid DCs (pDCs) were augmented in the lungs of mild COVID-19. In severe cases, we identified augmented types of immature DCs (CD22+ or ANXA1+ DCs) with MHCII downregulation. In this study, our observation indicates that DCs in severe cases stimulate innate immune responses but fail to specifically present SARS-CoV-2. It provides insights into the profound modulation of DC function in severe COVID-19.

A Case of Recalcitrant Linear Morphea Responding to Subcutaneous Abatacept.

INTRODUCTION: Linear morphea is an inflammatory condition that is often treated with systemic glucocorticoids and methotrexate, with mycophenolate mofetil being used as an alternative agent. However, there are few published reports on beneficial effect of abatacept for refractory disease. We present a case of a woman in her 30s who presented with linear morphea on her scalp, with a notable response following the addition of subcutaneous abatacept. METHODS: Computational analysis was performed comparing the immune cell scores of skin biopsies from 5 morphea skin biopsies from 3 unique patients and 15 healthy control skin biopsies. P < 0.05 was considered statistically significant. RESULTS: Immune cell scores demonstrated a statistically significant enrichment of activated CD4 memory T cells, M1 macrophages, monocytes, and memory B cells comparing skin biopsies of morphea vs healthy controls (p < 0.05 for all). DISCUSSION: Abatacept may be considered for recalcitrant cases of morphea. Our computational analysis supports a well-designed study to assess abatacept as first line therapy.

Mast Cell Activation in the Systemic Sclerosis Esophagus.

INTRODUCTION: Previously, we discovered similar esophageal gene expression patterns in patients with systemic sclerosis (SSc) and eosinophilic esophagitis (EoE) where eosinophil/mast cell-targeted therapies are beneficial. Because SSc and EoE patients experience similar esophageal symptoms, we hypothesized that eosinophil/mast cell-directed therapy may potentially benefit SSc patients. Herein, we determine the association between esophageal mast cell quantities, gene expression and clinical parameters in order to identify SSc patients who may benefit from eosinophil/mast cell-directed therapy. METHODS: Esophageal biopsies from SSc patients and healthy participants were stained for tryptase, a mast cell marker, and associations with relevant clinical parameters including 24h 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 (IS) assignment, an SSc molecular classification system that includes inflammatory, proliferative, limited and normal-like subsets. RESULTS: Esophageal biopsies from 40 SSc patients (39 receiving proton pump inhibition) and eleven healthy participants were studied. Mast cell numbers in both the upper esophagus (rs = 0.638, p = 0.004) and the entire (upper + lower) esophagus (rs = 0.562, p = 0.019) significantly correlated with acid exposure time percentage. The inflammatory, fibroproliferative, and normal-like ISs originally defined in skin biopsies were identified in esophageal biopsies. Although esophageal mast cell numbers in SSc patients and healthy participants were similar, gene expression for mast cell-related pathways showed significant upregulation in the inflammatory IS of SSc patients compared to patients classified as proliferative or normal-like. DISCUSSION: Esophageal mast cell numbers are heterogeneous in SSc patients and may correlate with acid exposure. Patients with inflammatory IS profiles in the esophagus demonstrate more tryptase staining. Mast cell targeted therapy may be a useful therapeutic approach in SSc patients belonging to the inflammatory IS, but additional studies are warranted.

Lymphocyte subset abnormalities in early diffuse cutaneous systemic sclerosis.

BACKGROUND: Abnormalities in lymphocyte surface markers and functions have been described in systemic sclerosis (SSc), but conflicting results abound, and these studies often examined patients with heterogeneous disease duration, severity, clinical phenotype, and concurrent immunosuppressive agents. We studied a clinically homogeneous group of early diffuse cutaneous SSc patients not exposed to immunosuppressive drugs who were enrolled in a clinical trial and compared their immune parameters to healthy control subjects. METHODS: Lymphocyte subsets were enumerated by multi-parameter flow cytometry of peripheral blood mononuclear cells at baseline visit. Production of the cytokines IL-4 and IL-17 was measured by intracellular flow cytometry following T cell activation. RESULTS: SSc patients had increased percentages of CD4+ T cells but lower percentages of CD8+ T cells versus controls. The CD28-negative population was expanded in SSc, in the CD4 subset. Striking expansion of CD319+ T cells was noted among the CD4+ cells, in which they were barely detectable in healthy subjects. Frequencies of IL-4 producing cells did not differ between SSc and controls, but expansion of IL-17 producing cells was observed in SSc. A higher proportion of CD319+ cells produced cytokines, compared to other CD4+ cells. Numbers of activated T cells, regulatory T cells, and B cells were similar in SSc and control groups. Circulating follicular helper but not peripheral helper T cells were slightly expanded in SSc. CONCLUSION: In this carefully selected group of early diffuse cutaneous SSc patients, analysis of immune cell parameters has identified abnormalities that likely reflect disease pathogenesis and that are candidate biomarkers for sub-classification and targeted treatment. The CD4+CD319+ (SLAM-F7+) cells are cytotoxic and oligoclonal, were recently shown to be a dominant T cell population in perivascular lymphocytic infiltrates in SSc skin, actively secrete cytokines, and are emerging as a target for novel treatments of SSc.

Machine learning integration of scleroderma histology and gene expression identifies fibroblast polarisation as a hallmark of clinical severity and improvement.

OBJECTIVE: We sought to determine histologic and gene expression features of clinical improvement in early diffuse cutaneous systemic sclerosis (dcSSc; scleroderma). METHODS: Fifty-eight forearm biopsies were evaluated from 26 individuals with dcSSc in two clinical trials. Histologic/immunophenotypic assessments of global severity, alpha-smooth muscle actin (aSMA), CD34, collagen, inflammatory infiltrate, follicles and thickness were compared with gene expression and clinical data. Support vector machine learning was performed using scleroderma gene expression subset (normal-like, fibroproliferative, inflammatory) as classifiers and histology scores as inputs. Comparison of w-vector mean absolute weights was used to identify histologic features most predictive of gene expression subset. We then tested for differential gene expression according to histologic severity and compared those with clinical improvement (according to the Combined Response Index in Systemic Sclerosis). RESULTS: aSMA was highest and CD34 lowest in samples with highest local Modified Rodnan Skin Score. CD34 and aSMA changed significantly from baseline to 52 weeks in clinical improvers. CD34 and aSMA were the strongest predictors of gene expression subset, with highest CD34 staining in the normal-like subset (p<0.001) and highest aSMA staining in the inflammatory subset (p=0.016). Analysis of gene expression according to CD34 and aSMA binarised scores identified a 47-gene fibroblast polarisation signature that decreases over time only in improvers (vs non-improvers). Pathway analysis of these genes identified gene expression signatures of inflammatory fibroblasts. CONCLUSION: CD34 and aSMA stains describe distinct fibroblast polarisation states, are associated with gene expression subsets and clinical assessments, and may be useful biomarkers of clinical severity and improvement in dcSSc.

Molecular "omic" signatures in systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by immunologic, vascular, and extracellular matrix abnormalities. Variation in the proportion and/or timing of activation in the deregulated molecular pathways that underlie SSc may explain the observed clinical heterogeneity in terms of disease phenotype and treatment response. In recent years, SSc research has generated massive amounts of "omics" level data. In this review, we discuss the body of "omics" level work in SSc and how each layer provides unique insight to our understanding of SSc. We posit that effective integration of genomic, transcriptomic, metagenomic, and epigenomic data is an important step toward precision medicine and is vital to the identification of effective therapeutic options for patients with SSc.