Racial variability in immune responses only partially explains differential systemic sclerosis disease severity.

OBJECTIVE: To understand if autoantibodies account for racial variation in disease severity, we compared autoantibody distribution and associated phenotype between self-identified black and white systemic sclerosis (SSc) patients. METHODS: 803 black and 2178 white SSc patients had systematic testing for autoantibodies using Euroimmun (centromere (ACA), RNA-polymerase III (POLR3), Scl70, PM/Scl, NOR90, Th/To, Ku, U3RNP and Ro52) and commercial ELISA (U1RNP). In this observational study, logistic regression was performed to assess the association between self-identified race and outcomes, adjusting for autoantibodies. To estimate whether the effect of race was mediated by autoantibody status, race coefficients from multivariate models including and excluding autoantibodies were compared. RESULTS: Anti-Scl70, anti-U1RNP, anti-U3RNP, anti-Th/To, anti-Ku and anti-NOR90 were more common in the black cohort than in the white cohort, which was enriched for ACA, anti-POLR3 and anti-PM/Scl. Black individuals had a higher prevalence of severe Raynaud's, skin, lung, gastrointestinal and renal disease whereas white individuals had a higher prevalence of severe heart and muscle disease. Adjusting for autoantibodies decreased the effect of race on outcome for telangiectasias, forced vital capacity <70%, pulmonary hypertension and severe lung, heart, muscle and gastrointestinal disease by 11%-44% and increased the association between race and renal crisis and severe kidney disease by 37%-52%. CONCLUSIONS: This study is the largest systematic analysis of autoantibody responses in a geographically diverse population of black SSc patients. Black and white individuals with SSc have distinct autoantibody profiles. Autoantibodies explain only a fraction of the effect of race on clinical outcomes, suggesting other factors contribute to disparate outcomes between these groups.

HLA and autoantibodies define scleroderma subtypes and risk in African and European Americans and suggest a role for molecular mimicry.

Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examined HLA associations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk, and the HLA-DRB1*08:04 allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and HLA-DRB1*07:01 alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13:01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 allele frequency in multiple populations was observed (r = 0.98, P = 3 × 10-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/ß allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.

Peripheral blood leucocyte telomere length is associated with progression of interstitial lung disease in systemic sclerosis.

BACKGROUND: Peripheral blood leucocyte telomere length (PBL-TL) is associated with outcomes in patients with idiopathic pulmonary fibrosis. Whether PBL-TL is associated with progression of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is unknown. METHODS: A retrospective observational cohort study was performed using prospectively collected data from 213 patients with SSc followed at the University of California San Francisco (UCSF) Scleroderma Center. PBL-TL was measured by quantitative PCR of DNA isolated from peripheral blood. Associations between PBL-TL and pulmonary function test trends in patients with SSc-ILD were assessed by longitudinal analysis using Generalised Linear Mixed Models. Findings were validated in a cohort of 61 patients with SSc-ILD enrolled in the Stanford University Scleroderma Center database. RESULTS: Patients with UCSF SSc with ILD were found to have shorter PBL-TL compared with those without ILD (6554±671 base pairs (bp) vs 6782±698 bp, p=0.01). Shorter PBL-TL was associated with the presence of ILD (adjusted OR 2.1 per 1000 bp TL decrease, 95% CI [1.25 to 3.70], p=0.006). PBL-TL was shorter in patients with SSc-ILD lacking SSc-specific autoantibodies compared with seropositive subjects (6237±647 bp vs 6651±653 bp, p=0.004). Shorter PBL-TL was associated with increased risk for lung function deterioration with an average of 67 mL greater loss in per year for every 1000 bp decrease in PBL-TL in the combined SSc-ILD cohorts (longitudinal analysis, adjusted model: 95% CI -104 mL to -33 mL, p<0.001). CONCLUSIONS: These findings suggest that telomere dysfunction may be associated with SSc-ILD progression and that PBL-TL measurement may be useful for stratifying risk for SSc-ILD progression.

Autoantibodies targeting telomere-associated proteins in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune fibrotic disease affecting multiple tissues including the lung. A subset of patients with SSc with lung disease exhibit short telomeres in circulating lymphocytes, but the mechanisms underlying this observation are unclear. METHODS: Sera from the Johns Hopkins and University of California, San Francisco (UCSF) Scleroderma Centers were screened for autoantibodies targeting telomerase and the shelterin proteins using immunoprecipitation and ELISA. We determined the relationship between autoantibodies targeting the shelterin protein TERF1 and telomere length in peripheral leucocytes measured by qPCR and flow cytometry and fluorescent in situ hybridisation (Flow-FISH). We also explored clinical associations of these autoantibodies. RESULTS: In a subset of patients with SSc, we identified autoantibodies targeting telomerase and the shelterin proteins that were rarely present in rheumatoid arthritis, myositis and healthy controls. TERF1 autoantibodies were present in 40/442 (9.0%) patients with SSc and were associated with severe lung disease (OR 2.4, p=0.04, Fisher's exact test) and short lymphocyte telomere length. 6/6 (100%) patients with TERF1 autoantibodies in the Hopkins cohort and 14/18 (78%) patients in the UCSF cohort had a shorter telomere length in lymphocytes or leukocytes, respectively, relative to the expected age-adjusted telomere length. TERF1 autoantibodies were present in 11/152 (7.2%) patients with idiopathic pulmonary fibrosis (IPF), a fibrotic lung disease believed to be mediated by telomere dysfunction. CONCLUSIONS: Autoantibodies targeting telomere-associated proteins in a subset of patients with SSc are associated with short lymphocyte telomere length and lung disease. The specificity of these autoantibodies for SSc and IPF suggests that telomere dysfunction may have a distinct role in the pathogenesis of SSc and pulmonary fibrosis.

Longitudinal assessment of interstitial lung disease in single lung transplant recipients with scleroderma.

OBJECTIVE: To investigate the natural history of fibrotic lung disease in recipients of a single lung transplant for scleroderma-associated interstitial lung disease (ILD). METHODS: Global ILD (including ground glass, nodular opacities and fibrosis) was categorized into severity quintiles on first and last post-transplant CT scans, and percent fibrosis by manual contouring was also determined, in nine single lung transplant recipients. Quantitative mean lung densities and volumes for the native and allograft lungs were also acquired. RESULTS: In the native lung, global ILD severity quintile worsened in two cases and percent fibrosis worsened in four cases (range 5-28%). In the lung allograft, one case each developed mild, moderate and severe ILD; of these, new fibrotic ILD (involving <10% of lung) occurred in two cases and acute cellular rejection occurred in one. The average change in native lung density over time was +2.2 Hounsfield Units per year and lung volume +1.4 ml per year, whereas the allograft lung density changed by -5.5 Hounsfield Units per year and total volume +27 ml per year (P = 0.011 and P = 0.039 for native vs allograft density and volume comparisons, respectively). CONCLUSIONS: While the course of ILD in the native and transplanted lungs varied in this series, these cases illustrate that disease progression is common in the native lung, suggesting that either the immune process continues to target autoantigens or ongoing fibrotic pathways are active in the native lung. Mild lung disease may occur in the allograft after several years due to either allograft rejection or recurrent mild ILD.

Heart-lung transplantation: A viable option for connective tissue diseases.

BACKGROUND: While lung transplantation (LTx) has been effective for connective tissue disease (CTD) patients with pulmonary involvement, outcomes for heart-lung transplantation (HLTx) are less defined. The aim of this study is to evaluate HLTx in CTD patients utilizing the UNOS database. METHODS: HLTx patients with CTD (HLTx-CTD) were compared to both LTx patients with CTD (LTx-CTD) and HLTx patients with all other indications (HLTx-OI) from 1999 to 2018. Primary outcome was 1- and 5-year graft survival. Secondary outcomes included freedom from first-year rejection and outcomes prior to transplant discharge. RESULTS: 1143/29 323 adults received first-time HLTx or LTx for CTD. Seventeen were HLTx-CTD (3.3% of total HLTx) and 1126 were LTx-CTD (3.9% of total LTx). There were 492 HLTx-OI. Transplant hemodynamic values including cardiac output, pulmonary capillary wedge pressure, and calculated pulmonary vascular resistance were significantly worse for HLTx-CTD vs LTx-CTD (4.2 vs 5.4 L/min, P = .005; 14 vs 10 mm Hg, P = .009; 439 vs 267 dynes, P = .007, respectively). Cardiac status 1 was more common for HLTx-CTD vs HLTx-OI (94% vs 56%, P < .001). HLTx-CTD 1 and 5-year graft survival was similar compared to LTx-CTD and HLTx-OI. CONCLUSION: HLTx-CTD is a valid option for carefully selected patients with CTD cardiac and pulmonary involvement with similar morbidity and mortality compared to LTx-CTD and HLTx-OI.

Lung Transplantation in Patients With Systemic Sclerosis.

PURPOSE OF REVIEW: The purpose of this study was to highlight recent findings on evaluation for lung transplantation and outcomes after transplantation in patients with systemic sclerosis (scleroderma). RECENT FINDINGS: The recognition that extra-pulmonary disease manifestations can seriously compromise post-transplant outcomes has changed the way patients are screened for lung transplant candidacy. Reluctance to transplant subjects affected by scleroderma has been driven by the complexity and multisystem nature of this disease. Multiple recent reports provide convincing findings that scleroderma patients undergoing lung transplantation have similar outcomes as those with other non-rheumatologic pulmonary conditions, even when significant esophageal dysmotility is present. New evidence supports the notion that scleroderma should not be a contraindication for referral to lung transplant. Future studies are needed to improve risk stratification, to define protocols for screening and management of extra-pulmonary complications, and to optimize immunosuppression before and after transplant.

A methodology for exploring biomarker--phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations.

BACKGROUND: This work seeks to develop a methodology for identifying reliable biomarkers of disease activity, progression and outcome through the identification of significant associations between high-throughput flow cytometry (FC) data and interstitial lung disease (ILD) - a systemic sclerosis (SSc, or scleroderma) clinical phenotype which is the leading cause of morbidity and mortality in SSc. A specific aim of the work involves developing a clinically useful screening tool that could yield accurate assessments of disease state such as the risk or presence of SSc-ILD, the activity of lung involvement and the likelihood to respond to therapeutic intervention. Ultimately this instrument could facilitate a refined stratification of SSc patients into clinically relevant subsets at the time of diagnosis and subsequently during the course of the disease and thus help in preventing bad outcomes from disease progression or unnecessary treatment side effects. The methods utilized in the work involve: (1) clinical and peripheral blood flow cytometry data (Immune Response In Scleroderma, IRIS) from consented patients followed at the Johns Hopkins Scleroderma Center. (2) machine learning (Conditional Random Forests - CRF) coupled with Gene Set Enrichment Analysis (GSEA) to identify subsets of FC variables that are highly effective in classifying ILD patients; and (3) stochastic simulation to design, train and validate ILD risk screening tools. RESULTS: Our hybrid analysis approach (CRF-GSEA) proved successful in predicting SSc patient ILD status with a high degree of success (>82% correct classification in validation; 79 patients in the training data set, 40 patients in the validation data set). CONCLUSIONS: IRIS flow cytometry data provides useful information in assessing the ILD status of SSc patients. Our new approach combining Conditional Random Forests and Gene Set Enrichment Analysis was successful in identifying a subset of flow cytometry variables to create a screening tool that proved effective in correctly identifying ILD patients in the training and validation data sets. From a somewhat broader perspective, the identification of subsets of flow cytometry variables that exhibit coordinated movement (i.e., multi-variable up or down regulation) may lead to insights into possible effector pathways and thereby improve the state of knowledge of systemic sclerosis pathogenesis.

The degree of skin involvement identifies distinct lung disease outcomes and survival in systemic sclerosis.

OBJECTIVE: To determine whether the pattern of skin involvement can predict clinical features, risk of restrictive lung disease (RLD) and survival in a large scleroderma (SSc) cohort. METHODS: Demographic and clinical data collected over 30 years from 2205 patients with SSc were retrospectively analysed after subdividing subjects into four subtypes based on pattern of skin fibrosis: type 0 (no skin involvement), type 1 (limited to metacarpophalangeal joints), type 2 (distal to elbows/knees) and type 3 (proximal to elbows/knees). Clinical features associated with skin subsets were identified by regression analyses. Kaplan-Meier and Cox proportional hazards models were used to compare time to RLD and survival across subtypes. RESULTS: The presence and severity of RLD were positively associated with skin subtype (p<0.001). RLD prevalence incrementally ranged from 51.9% in type 0 to 76.7% in type 3 (p<0.001). Type 2 SSc exhibited a distinct phenotype with intermediate risk for RLD relative to type 1 (higher, p<0.001) and type 3 (lower, p<0.001) and a unique autoantibody profile, with a prevalence of anticentromere antibodies lower than type 1 (28.9% vs 44.1%, p=0.001) and of anti-topoisomerase I antibodies similar to type 3 (32.8% vs 28.7%, p=0.38). These autoantibodies were also found to be significant negative (OR=0.33, p<0.001) and positive (OR=1.6, p=0.01) predictors of RLD risk, respectively. Mortality was also intermediate in type 2 patients relative to type 3 (p=0.0003) and type 1 (p=0.066). CONCLUSIONS: These data suggest that the current classification subdividing SSc into limited and diffuse cutaneous subtypes misclassifies an intermediate group of patients exhibiting unique autoantibody profile, disease course and clinical outcomes.

A PD-1highCD4+ T Cell Population With a Cytotoxic Phenotype is Associated With Interstitial Lung Disease in Systemic Sclerosis.

OBJECTIVE: T cells contribute to tissue injury in systemic sclerosis (SSc), yet the specific T cell subsets expanded in patients with SSc remain incompletely defined. Here we evaluated specific phenotypes and functions of peripheral helper T (Tph) and follicular helper T (Tfh) cells, which have been implicated in autoantibody production, and assessed their associations with clinical features in a well-characterized cohort of patients with SSc. METHODS: Mass cytometry of T cells from peripheral blood mononuclear cells of patients with SSc and controls were evaluated using t-distributed stochastic neighbor embedding visualization, biaxial gating, and marker expression levels. Findings were validated with flow cytometry and in vitro assays. RESULTS: The frequencies of PD-1highCXCR5+ Tfh cells and PD-1highCXCR5- Tph cells were similar in patients with SSc and controls. t-distributed stochastic neighbor embedding visualization (tSNE) revealed distinct populations within the PD-1highCXCR5- cells distinguished by expression of HLA-DR and inducible costimulator (ICOS). Among PD-1highCXCR5- cells, only the HLA-DR+ICOS- cell population was expanded in patients with SSc. Cytometric and RNA sequencing analyses indicated that these cells expressed cytotoxic rather than B cell helper features. HLA-DR+ICOS- PD-1highCXCR5- cells were less potent in inducing B cell plasmablast differentiation and antibody production than comparator T helper cell populations. HLA-DR+ICOS-PD-1highCXCR5- cells were significantly associated with the presence and severity of interstitial lung disease among patients with SSc. CONCLUSION: Among PD-1highCXCR5- T cells, a subset of HLA-DR+ICOS- cells with cytotoxic features is specifically expanded in patients with SSc and is significantly associated with interstitial lung disease severity. This potential cytotoxicity appearing in the CD4 T cell population can be evaluated as a prognostic disease biomarker in patients with SSc.

Protein biomarkers of disease progression in patients with systemic sclerosis associated interstitial lung disease.

Systemic sclerosis is a rare connective tissue disease; and interstitial lung disease (SSc-ILD) is associated with significant morbidity and mortality. There are no clinical, radiologic features, nor biomarkers that identify the specific time when patients are at risk for progression at which the benefits from treatment outweigh the risks. Our study aimed to identify blood protein biomarkers associated with progression of interstitial lung disease in patients with SSc-ILD using an unbiased, high-throughput approach. We classified SSc-ILD as progressive or stable based on change in forced vital capacity over 12 months or less. We profiled serum proteins by quantitative mass spectrometry and analyzed the association between protein levels and progression of SSc-ILD via logistic regression. The proteins associated with at a p value of < 0.1 were queried in the ingenuity pathway analysis (IPA) software to identify interaction networks, signaling, and metabolic pathways. Through principal component analysis, the relationship between the top 10 principal components and progression was evaluated. Unsupervised hierarchical clustering with heatmapping was done to define unique groups. The cohort consisted of 72 patients, 32 with progressive SSc-ILD and 40 with stable disease with similar baseline characteristics. Of a total of 794 proteins, 29 were associated with disease progression. After adjusting for multiple testing, these associations did not remain significant. IPA identified five upstream regulators that targeted proteins associated with progression, as well as a canonical pathway with a higher signal in the progression group. Principal component analysis showed that the ten components with the highest Eigenvalues represented 41% of the variability of the sample. Unsupervised clustering analysis revealed no significant heterogeneity between the subjects. We identified 29 proteins associated with progressive SSc-ILD. While these associations did not remain significant after accounting for multiple testing, some of these proteins are part of pathways relevant to autoimmunity and fibrogenesis. Limitations included a small sample size and a proportion of immunosuppressant use in the cohort, which could have altered the expression of inflammatory and immunologic proteins. Future directions include a targeted evaluation of these proteins in another SSc-ILD cohort or application of this study design to a treatment naïve population.

Oxidative stress promotes fibrosis in systemic sclerosis through stabilization of a kinase-phosphatase complex.

Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.

Long-term experience of mycophenolate mofetil for treatment of diffuse cutaneous systemic sclerosis.

BACKGROUND: Immunosuppressive therapy may potentially alter the natural disease course of scleroderma. There have been reports of using mycophenolate mofetil (MMF) for the treatment of scleroderma skin disease. OBJECTIVE: To analyse the experience of using MMF for the treatment of active diffuse cutaneous scleroderma. METHODS: The authors compared the change in mean modified Rodnan skin scores (mRSS) in an MMF cohort at baseline with scores at 3, 6, 9 and 12 months and with those of historical controls from a pooled analysis of three multicentre randomised clinical trials of recombinant human relaxin, d-penicillamine and oral bovine type I collagen. RESULTS: Improvement in mRSS after treatment with MMF compared with baseline was seen as early as 3 months and continued through the 12-month follow-up. The mRSS of the MMF cohort was not different from that of the historical controls at 6 months (MMF -3.05 ± 7.4 vs relaxin -4.83 ± 6.99, p=0.059), but was significantly lower at 12 months (MMF -7.59 ± 10.1 vs d-penicillamine -2.47 ± 8.6, p<0.001; collagen -3.4 ± 7.12, p=0.002). General and muscle severity scores and quality of life measures also improved compared with baseline. Pulmonary function remained stable. CONCLUSIONS: MMF may benefit skin disease in patients with diffuse scleroderma, but prospective studies are required to determine its role.

Antibodies recognising sulfated carbohydrates are prevalent in systemic sclerosis and associated with pulmonary vascular disease.

BACKGROUND: Glycosylation represents an important modification that regulates biological processes in tissues relevant for disease pathogenesis in systemic sclerosis (SSc), including the endothelium and extracellular matrix. Whether patients with SSc develop antibodies to carbohydrates is not known. OBJECTIVES: To determine the prevalence and clinical phenotype associated with serum IgG antibodies recognising distinct glycans in patients with SSc. METHODS: Pooled serum samples from patients with SSc and controls were screened for the presence of specific anticarbohydrate antibodies using a novel array containing over 300 glycans. Antibody titres to 4-sulfated N-acetyl-lactosamine (4S-LacNAc, (4OSO3)Galß1-4GlcNAc) were determined in 181 individual serum samples from patients with SSc by ELISA and associated with disease phenotype. RESULTS: 4S-LacNAc was identified as a target in pooled SSc serum. Anti-4S-LAcNAc antibodies were detected in 27/181 patients with SSc (14.9%) compared with 1/40 healthy controls (2.5%). Sulfation at position C4 of galactose (4S-LacNAc) was found to be critical for immunogenicity. Anti-4S-LacNAc antibody-positive patients with SSc had a higher prevalence of pulmonary hypertension by echocardiography than anti-4S-LacNAc-negative patients (15/27 (55.7%) vs 49/154 (31.8%), p=0.02) with an OR of 2.6 (95% CI 1.1 to 6.3). Anti-4S-LacNAc-positive patients accounted for 23.4% of all patients with pulmonary hypertension. CONCLUSION: Serum from patients with SSc contains IgG antibodies targeting distinct sulfated carbohydrates. The presence of anti-4S-LacNAc antibodies is associated with a high prevalence of pulmonary hypertension. These results suggest that specific post-translational carbohydrate modifications may act as important immunogens in SSc and may contribute to disease pathogenesis.

The Utility of Plasma Vascular Biomarkers in Systemic Sclerosis: A Prospective Longitudinal Analysis.

OBJECTIVE: In cross-sectional studies, pulmonary hypertension (PH) and ischemic digital lesions are 2 scleroderma vascular outcomes associated with abnormalities in biomarkers of angiogenesis. The clinical usefulness of these biomarkers is unknown, in part due to lack of data on longitudinal measurement. This prospective longitudinal study was undertaken to evaluate vascular biomarker measurements in patients with systemic sclerosis (SSc) over time. METHODS: We conducted a prospective cohort study of 300 patients with SSc who were followed up for at least a 5-year period and lacked evidence of PH and/or active ischemic digital lesions at enrollment. Levels of hepatocyte growth factor (HGF), soluble Flt-1 (sFlt-1), soluble endoglin, endostatin, and placental growth factor (PLGF) were obtained at multiple time points and assessed for their ability to predict the development of PH/ischemic digital lesions. Hazard ratios (HRs) with 95% confidence intervals (95% CIs) were calculated. RESULTS: Forty-six patients (15%) developed PH and 69 patients (23%) developed an ischemic digital lesion. In time-to-event analyses, the following 3 biomarkers measured at cohort entry were found to be significantly associated with the development of PH: HGF (HR 1.99 [95% CI 1.24-3.17], P = 0.004), sFlt-1 (HR 3.04 [95% CI 1.29-7.14], P = 0.011), and PLGF (HR 2.74 [95% CI 1.32-5.69], P = 0.007). As time approaching PH diagnosis decreased, there was no corresponding increase in any biomarker level. Upon converting each continuous vascular biomarker into a binary variable, a dose-response relationship was observed for the number of elevated biomarkers at cohort entry and the risk of developing PH. With each additional elevated biomarker at cohort entry, there was a 78% increase in the risk of developing PH (HR 1.78 [95% CI 1.2-2.6], P = 0.004). CONCLUSION: These data suggest that molecules involved in angiogenesis reflect vascular perturbation, and that elevations in these biomarkers at first encounter can indicate patients who are at risk of PH development.

Clinical and Molecular Phenotyping in Scleromyxedema Pretreatment and Posttreatment With Intravenous Immunoglobulin.

OBJECTIVE: Scleromyxedema (SMX) is a rare systemic sclerosis mimic that often responds to intravenous immunoglobulin (IVIG) therapy, yet the resulting clinical and biochemical changes have not been well characterized. To better understand the pathogenesis of the disease and the efficacy of IVIG, we sought to explore whether IVIG would introduce a measurable biologic effect corresponding with clinical improvement. METHODS: Fifteen patients with SMX were recruited for the study. Clinical information and peripheral blood mononuclear cells for flow cytometry were obtained immediately before and again 1-2 weeks after patients received IVIG therapy. Ten patients also underwent skin biopsies for gene expression analysis both before and after IVIG therapy. Clinical data included measures of skin involvement (modification of the modified Rodnan skin thickness score [MMRSS] and percentage of body surface area) and several patient-reported outcome measures assessing patients' skin. RESULTS: Posttreatment, the average MMRSS score decreased from mean ± SD 13.6 ± 2.6 to 10.3 ± 1.9; P = 0.003. There were also significant improvements in skin flexibility (mean ± SD 5.4 ± 0.8 to 3.2 ± 0.7; P = 0.003) and softening (mean ± SD 4.9 ± 0.9 to 2.6 ± 0.6; P = 0.022). Baseline levels of Tc17 cells (CD8+CCR6+CXCR3+CCR4-) correlated with the extent of skin involvement as measured by MMRSS pretreatment (r = 0.69, P = 0.012) and decreased after IVIG therapy (mean ± SD 3.4% ± 3.2% to 1.3% ± 1.7%; P = 0.008). Posttreatment analysis of RNA in skin tissue revealed a decrease in gene expression of transforming growth factor ß (TGFß) cytokines as well as several interferon-inducible proteins. CONCLUSION: This open-label study further supports the evidence that patients with SMX respond both objectively and subjectively to IVIG therapy. Biologic studies suggest a role for T lymphocytes in the pathogenesis of the disease and reveal the potential significance of TGFß and interferon pathways.

Definition of Naturally Processed Peptides Reveals Convergent Presentation of Autoantigenic Topoisomerase I Epitopes in Scleroderma.

OBJECTIVE: Autoimmune responses to DNA topoisomerase I (topo I) are found in a subset of scleroderma patients who are at high risk for interstitial lung disease (ILD) and mortality. Anti-topo I antibodies (ATAs) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted topo I-specific CD4+ T cells is associated with the presence, severity, and progression of ILD. Although this strongly implicates the presentation of topo I peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of topo I has not been studied. METHODS: We developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of topo I presented by monocyte-derived dendritic cells (mo-DCs) from 6 ATA-positive patients with scleroderma. Mo-DCs were pulsed with topo I protein, HLA-DR-peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients. RESULTS: We found that a common set of 10 topo I epitopes was presented by Mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptide-binding motifs within the HLA-DRß chains of ATA-positive patients and a subset of topo I epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% of ATA-positive patients (8 of 11), and the number of epitopes recognized correlated with ILD severity (P = 0.025). CONCLUSION: These findings mechanistically implicate the presentation of a convergent set of topo I epitopes in the development of scleroderma.

Digital ulcers: should debridement be a standard of care in systemic sclerosis?

Digital ulcers are a serious, recurrent complication in patients with systemic sclerosis. They are often slow to heal and exquisitely painful. Local wound care, such as debridement of the wound bed, is an essential component in the management of digital ulcers in systemic sclerosis. However, digital ulcer debridement is not a standard of care, and there is substantial international variation in the use of this approach. In this Viewpoint, we discuss the assessment of the wound bed and different methods of debridement using the model of tissue management, infection and inflammation, moisture control, and wound edge or epidermal advancement, known as TIME. We highlight the challenges in standard practice and the need for research into local wound care for this type of ulceration, before suggesting a potential roadmap to develop a standardised approach to support ulcer debridement in systemic sclerosis. Debridement might be the missing component in optimising the management of digital ulcers and we propose that the approach should be rigorously investigated as a standard of care in this common complication of systemic sclerosis.

Regulatory T cells in skin are uniquely poised to suppress profibrotic immune responses.

At the center of fibrosing diseases is the aberrant activation of tissue fibroblasts. The cellular and molecular mechanisms of how the immune system augments fibroblast activation have been described; however, little is known about how the immune system controls fibroblast function in tissues. Here, we identify regulatory T cells (Tregs) as important regulators of fibroblast activation in skin. Bulk cell and single-cell analysis of Tregs in murine skin and lungs revealed that Tregs in skin are transcriptionally distinct and skewed toward T helper 2 (TH2) differentiation. When compared with Tregs in lung, skin Tregs preferentially expressed high levels of GATA3, the master TH2 transcription factor. Genes regulated by GATA3 were highly enriched in skin "TH2 Treg" subsets. In functional experiments, Treg depletion resulted in a preferential increase in TH2 cytokine production in skin. Both acute depletion and chronic reduction of Tregs resulted in spontaneous skin fibroblast activation, profibrotic gene expression, and dermal fibrosis, all of which were exacerbated in a bleomycin-induced murine model of skin sclerosis. Lineage-specific deletion of Gata3 in Tregs resulted in an exacerbation of TH2 cytokine secretion that was preferential to skin, resulting in enhanced fibroblast activation and dermal fibrosis. Together, we demonstrate that Tregs play a critical role in regulating fibroblast activation in skin and do so by expressing a unique tissue-restricted transcriptional program that is mediated, at least in part, by GATA3.