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Tissue availability remains an important limitation of single-cell genomic technologies for investigating cellular heterogeneity in human health and disease. BAL represents a minimally invasive approach to assessing an individual's lung cellular environment for diagnosis and research. However, the lack of high-quality, healthy lung reference data is a major obstacle to using single-cell approaches to study a plethora of lung diseases. Here, we performed single-cell RNA sequencing on over 40,000 cells isolated from the BAL of four healthy volunteers. Of the six cell types or lineages we identified, macrophages were consistently the most numerous across individuals. Our analysis confirmed the expression of marker genes defining cell types despite background signals because of the ambient RNA found in many single-cell studies. We assessed the variability of gene expression across macrophages and defined a distinct subpopulation of cells expressing a set of genes associated with Macrophage Inflammatory Protein 1 (MIP-1). RNA in situ hybridization and reanalysis of published lung single-cell data validated the presence of this macrophage subpopulation. Thus, our study characterizes lung macrophage heterogeneity in healthy individuals and provides a valuable resource for future studies to understand the lung environment in health and disease.
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Proteínas Inflamatórias de Macrófagos , Macrófagos , Humanos , Proteínas Inflamatórias de Macrófagos/genética , Líquido da Lavagem Broncoalveolar , Voluntários Saudáveis , RNARESUMO
OBJECTIVES: Symptoms of people who have SSc are heterogeneous and difficult to address clinically. Because diverse symptoms often co-occur and may share common underlying mechanisms, identifying symptoms that cluster together may better target treatment approaches. We sought to identify and characterize patient subgroups based on symptom experience. METHODS: An exploratory hierarchical agglomerative cluster analysis was conducted to identify subgroups from a large SSc cohort from a single US academic medical centre. Patient-reported symptoms of pain interference, fatigue, sleep disturbance, dyspnoea, depression and anxiety were used for clustering. A multivariate analysis of variance (MANOVA) was used to examine the relative contribution of each variable across subgroups. Analyses of variance were performed to determine participant characteristics based on subgroup assignment. Presence of symptom clusters were tallied within subgroup. RESULTS: Participants (n = 587; 84% female, 41% diffuse cutaneous subtype, 59% early disease) divided into three subgroups via cluster analysis based on symptom severity: (i) no/minimal, (ii) mild, and (iii) moderate. Participants in mild and moderate symptoms subgroups had similar disease severity, but different symptom presentation. In the mild symptoms subgroup, pain, fatigue and sleep disturbance was the main symptom cluster. Participants in the moderate symptoms subgroup were characterized by co-occurring pain, fatigue, sleep disturbance, depression and anxiety. CONCLUSION: Identification of distinct symptom clusters, particularly among SSc patients who experience mild and moderate symptoms, suggests potential differences in treatment approach and in mechanisms underlying symptom experience that require further study.
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Escleroderma Sistêmico , Transtornos do Sono-Vigília , Humanos , Feminino , Masculino , Síndrome , Dor/etiologia , Fadiga/diagnóstico , Ansiedade/etiologia , Transtornos do Sono-Vigília/complicações , Escleroderma Sistêmico/complicações , Análise por Conglomerados , Depressão/etiologia , Depressão/diagnóstico , Qualidade de VidaRESUMO
OBJECTIVE: The pathogenesis of calcinosis cutis, a disabling complication of SSc, is poorly understood and effective treatments are lacking. Inorganic pyrophosphate (PPi) is a key regulator of ectopic mineralization, and its deficiency has been implicated in ectopic mineralization disorders. We therefore sought to test the hypothesis that SSc may be associated with reduced circulating PPi, which might play a pathogenic role in calcinosis cutis. METHODS: Subjects with SSc and age-matched controls without SSc were recruited from the outpatient rheumatology clinics at Rutgers and Northwestern Universities (US cohort), and from the Universities of Szeged and Debrecen (Hungarian cohort). Calcinosis cutis was confirmed by direct palpation, by imaging or both. Plasma PPi levels were determined in platelet-free plasma using ATP sulfurylase to convert PPi into ATP in the presence of excess adenosine 5' phosphosulfate. RESULTS: Eighty-one patients with SSc (40 diffuse cutaneous, and 41 limited cutaneous SSc) in the US cohort and 45 patients with SSc (19 diffuse cutaneous and 26 limited cutaneous SSc) in the Hungarian cohort were enrolled. Calcinosis was frequently detected (40% of US and 46% of the Hungarian cohort). Plasma PPi levels were significantly reduced in both SSc cohorts with and without calcinosis (US: P = 0.003; Hungarian: P < 0.001). CONCLUSIONS: Circulating PPi are significantly reduced in SSc patients with or without calcinosis. Reduced PPi may be important in the pathophysiology of calcinosis and contribute to tissue damage with chronic SSc. Administering PPi may be a therapeutic strategy and larger clinical studies are planned to confirm our findings.
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Calcinose/sangue , Calcinose/etiologia , Difosfatos/sangue , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/complicações , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeAssuntos
Doenças Pulmonares Intersticiais/genética , Escleroderma Sistêmico/genética , Homeostase do Telômero/genética , Encurtamento do Telômero/genética , Telômero/genética , Estudos de Coortes , Feminino , Granulócitos/citologia , Humanos , Doenças Pulmonares Intersticiais/epidemiologia , Doenças Pulmonares Intersticiais/fisiopatologia , Linfócitos/citologia , Masculino , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Escleroderma Sistêmico/epidemiologia , Escleroderma Sistêmico/fisiopatologia , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Reino UnidoRESUMO
BACKGROUND: To characterize sleep disturbance in patients with established rheumatoid arthritis (RA) and explore the relationship between sleep and mechanisms of central nervous system pain regulation. METHODS: Forty-eight RA participants completed wrist-worn actigraphy monitoring and daily sleep diaries for 14 days to assess sleep-wake parameters. Participants underwent quantitative sensory testing to assess pressure pain thresholds, temporal summation, and conditioned pain modulation. Data were analyzed using descriptive statistics, Spearman's correlation, and multivariable median regression analyses. RESULTS: Median actigraphy and sleep diary derived sleep duration was 7.6 h (interquartile range (IQR) 7.0, 8.2) and 7.1 h (IQR 6.7, 7.6), respectively. Actigraphy based sleep fragmentation (rho = 0.34), wake after sleep onset (rho = 0.36), and sleep efficiency (rho = -0.32) were each related to higher temporal summation values in unadjusted analyses, but these relationships did not persist after controlling for age, body mass index, disease duration, and swollen joint count. No significant relationships were observed between sleep with pressure pain thresholds and conditioned pain modulation. CONCLUSION: Actigraphy and sleep diary monitoring are well tolerated in established RA patients. Future investigations should include both subjective and objective assessments, as they may provide information relating to different components and mechanisms.
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OBJECTIVE: While interstitial lung disease (ILD) is the leading cause of morbidity and mortality in systemic sclerosis (SSc), there remains a paucity of predictive markers to assess disease progression. We previously demonstrated that adipose tissue metabolism and adipokine homeostasis is dysregulated in SSc. The present study was undertaken to determine the association and predictive ability of the novel adipokine C1q/tumor necrosis factor-related protein 9 (CTRP9) for SSc-associated ILD. METHODS: We performed a retrospective longitudinal study utilizing the Northwestern Scleroderma Program Patient Registry and Biorepository. Serum levels of CTRP9 were measured in 110 SSc patients at baseline, and demographic, clinical, and pulmonary function test data were collected in 12-month intervals to 48 months. Longitudinal trajectory of forced vital capacity percent predicted (FVC%) was used as a primary outcome measure. We utilized a mixed model to compare trajectories of lung function by CTRP9 groups and performed latent trajectory analysis to accommodate for heterogeneity. RESULTS: In cross-sectional analysis, elevated circulating CTRP9 was associated with significantly lower FVC% at baseline (72% ± 17 versus 80% ± 18; P = 0.02) and 48 months (68 ± 19 versus 84 ± 18; P = 0.001). In mixed model analysis, high CTRP9 was associated with worse lung function but not with a different trajectory (P = 0.23). In contrast, low CTRP9 identified patients with stability of lung disease with reasonable accuracy (sensitivity 73%). Latent trajectory analysis confirmed the association of lower CTRP9 with higher FVC%. CONCLUSION: Higher circulating CTRP9 associated with worse pulmonary function, while low CTRP9 identified patients with lung disease stability over time. These findings suggest that CTRP9 may be a potential biomarker in SSc-associated ILD.
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Doenças Pulmonares Intersticiais , Escleroderma Sistêmico , Humanos , Estudos Retrospectivos , Estudos Longitudinais , Estudos Transversais , Pulmão , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/complicações , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/diagnóstico , Capacidade VitalRESUMO
OBJECTIVE: Systemic sclerosis (SSc) is characterized by immune activation, vasculopathy, and unresolving fibrosis in the skin, lungs, and other organs. We performed RNA-sequencing analysis on skin biopsy samples and peripheral blood mononuclear cells (PBMCs) from SSc patients and unaffected controls to better understand the pathogenesis of SSc. METHODS: We analyzed these data 1) to test for case/control differences and 2) to identify genes whose expression levels correlate with SSc severity as measured by local skin score, modified Rodnan skin thickness score (MRSS), forced vital capacity (FVC), or diffusing capacity for carbon monoxide (DLco). RESULTS: We found that PBMCs from SSc patients showed a strong type I interferon signature. This signal was found to be replicated in the skin, with additional signals for increased extracellular matrix (ECM) genes, classical complement pathway activation, and the presence of B cells. Notably, we observed a marked decrease in the expression of SPAG17, a cilia component, in SSc skin. We identified genes that correlated with the MRSS, DLco, and FVC in SSc PBMCs and skin using weighted gene coexpression network analysis. These genes were largely distinct from the case/control differentially expressed genes. In PBMCs, type I interferon signatures negatively correlated with the DLco. In SSc skin, ECM gene expression positively correlated with the MRSS. Network analysis of SSc skin genes that correlated with clinical features identified the noncoding RNAs SOX9-AS1 and ROCR, both near the SOX9 locus, as highly connected, "hub-like" genes in the network. CONCLUSION: These results identify noncoding RNAs and SPAG17 as novel factors potentially implicated in the pathogenesis of SSc.
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Proteínas dos Microtúbulos , Fatores de Transcrição SOX9 , Escleroderma Sistêmico , Humanos , Cílios/metabolismo , Cílios/patologia , Interferon Tipo I , Leucócitos Mononucleares/metabolismo , RNA não Traduzido/genética , Pele/patologia , Fatores de Transcrição SOX9/genética , Proteínas dos Microtúbulos/genéticaRESUMO
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.
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Doenças Pulmonares Intersticiais , Escleroderma Sistêmico , Humanos , Fibrose , Doenças Pulmonares Intersticiais/diagnóstico por imagem , Doenças Pulmonares Intersticiais/patologia , Pele/diagnóstico por imagem , Pele/patologia , Autoanticorpos , FenótipoRESUMO
Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.
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Miofibroblastos , Escleroderma Sistêmico , Animais , Humanos , Camundongos , Células Cultivadas , Colágeno/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Fibrose , Proteínas dos Microtúbulos/metabolismo , Miofibroblastos/patologia , Escleroderma Sistêmico/patologia , Pele/patologiaRESUMO
OBJECTIVE: Patients with diffuse cutaneous systemic sclerosis (dcSSc) display a complex clinical phenotype. Transcriptional profiling of whole blood or tissue from patients are affected by changes in cellular composition that drive gene expression and an inability to detect minority cell populations. We undertook this study to focus on the 2 main subtypes of circulating monocytes, classical monocytes (CMs) and nonclassical monocytes (NCMs) as a biomarker of SSc disease severity. METHODS: SSc patients were recruited from the Prospective Registry for Early Systemic Sclerosis. Clinical data were collected, as well as peripheral blood for isolation of CMs and NCMs. Age-, sex-, and race-matched healthy volunteers were recruited as controls. Bulk macrophages were isolated from the skin in a separate cohort. All samples were assayed by RNA sequencing (RNA-seq). RESULTS: We used an unbiased approach to cluster patients into 3 groups (groups A-C) based on the transcriptional signatures of CMs relative to controls. Each group maintained their characteristic transcriptional signature in NCMs. Genes up-regulated in group C demonstrated the highest expression compared to the other groups in SSc skin macrophages, relative to controls. Patients from groups B and C exhibited worse lung function than group A, although there was no difference in SSc skin disease at baseline, relative to controls. We validated our approach by applying our group classifications to published bulk monocyte RNA-seq data from SSc patients, and we found that patients without skin disease were most likely to be classified as group A. CONCLUSION: We are the first to show that transcriptional signatures of CMs and NCMs can be used to unbiasedly stratify SSc patients and correlate with disease activity outcome measures.
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Esclerodermia Difusa , Esclerodermia Localizada , Escleroderma Sistêmico , Humanos , Monócitos/metabolismo , Escleroderma Sistêmico/metabolismo , Esclerodermia Difusa/genética , Esclerodermia Difusa/diagnóstico , Macrófagos/metabolismo , Biomarcadores , Pele/metabolismoRESUMO
Monocytes are abundant immune cells that infiltrate inflamed organs. However, the majority of monocyte studies focus on circulating cells, rather than those in tissue. Here, we identify and characterize an intravascular synovial monocyte population resembling circulating non-classical monocytes and an extravascular tissue-resident monocyte-lineage cell (TR-MC) population distinct in surface marker and transcriptional profile from circulating monocytes, dendritic cells, and tissue macrophages that are conserved in rheumatoid arthritis (RA) patients. TR-MCs are independent of NR4A1 and CCR2, long lived, and embryonically derived. TR-MCs undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. Moreover, pathways that are activated in TR-MCs at the peak of arthritis overlap with those that are downregulated in LFA1-/- TR-MCs. These findings show a facet of mononuclear cell biology that could be imperative to understanding tissue-resident myeloid cell function in RA.
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Artrite Reumatoide , Monócitos , Humanos , Monócitos/metabolismo , Membrana Sinovial , Inflamação/metabolismoRESUMO
BACKGROUND: Although esophageal dysmotility is common in systemic sclerosis (SSc)/scleroderma, little is known regarding the pathophysiology of motor abnormalities driving reflux severity and dysphagia. This study aimed to assess primary and secondary peristalsis in SSc using a comprehensive esophageal motility assessment applying high-resolution manometry (HRM) and functional luminal imaging probe (FLIP) Panometry. METHODS: A total of 32 patients with scleroderma (28 female; ages 38-77; 20 limited SSc, 12 diffuse SSc) completed FLIP Panometry and HRM. Secondary peristalsis, i.e., contractile responses (CR), was classified on FLIP Panometry by pattern of contractility as normal (NCR), borderline (BCR), impaired/disordered (IDCR), or absent (ACR). Primary peristalsis on HRM was assessed according to the Chicago classification. RESULTS: The manometric diagnoses were 56% (n = 18) absent contractility, 22% (n = 7) ineffective esophageal motility (IEM), and 22% (n = 7) normal motility. Secondary peristalsis (CRs) included 38% (n = 12) ACR, 38% (n = 12) IDCR, 19% (n = 6) BCR, and 15% (n = 5) NCR. The median (IQR) esophagogastric junction (EGJ) distensibility index (DI) was 5.8 mm2 /mmHg (4.8-10.1) mm2 /mmHg; EGJ-DI was >8.0 mm2 /mmHg in 31%, and >2.0 mm2 /mmHg in 100% of patients. Among 18 patients with absent contractility on HRM, 11 had ACR, 5 had IDCR, and 2 had BCR. Among 7 patients with IEM, 1 had ACR, 5 had IDCR, and 1 NCR. All of the patients with normal peristalsis had NCR or BCR. CONCLUSIONS: This was the first study assessing combined HRM and FLIP Panometry in a cohort of SSc patients, which demonstrated heterogeneity in primary and secondary peristalsis. This complementary approach facilitates characterizing esophageal function in SSc, although future study to examine clinical outcomes remains necessary.
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Transtornos da Motilidade Esofágica , Escleroderma Sistêmico , Adulto , Idoso , Feminino , Humanos , Manometria/métodos , Pessoa de Meia-Idade , Peristaltismo , Escleroderma Sistêmico/complicaçõesRESUMO
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.
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Rationale: Fibrosis leads to failure of the skin, lungs, and other organs in systemic sclerosis; accounts for substantial morbidity and mortality; and lacks effective therapy. Myofibroblast activation underlies organ fibrosis, but the key extracellular cues driving persistence of the process remain incompletely characterized. Objectives: The objectives were to evaluate activation of the IL6/JAK/STAT axis associated with fibrosis in skin and lung biopsies from systemic sclerosis patients and effects of the Food and Drug Administration-approved JAK/STAT inhibitor, tofacitinib, on skin and lung fibrosis in animal models. Methods: Bioinformatic analysis showed that IL6/JAK/STAT3 and tofacitinib gene signatures were aberrant in biopsies from systemic sclerosis patients in four independent cohorts. The results were confirmed by JAK and STAT3 phosphorylation in both skin and lung biopsies from patients with systemic sclerosis. Furthermore, treatment of mice with the selective JAK inhibitor tofacitinib not only prevented bleomycin-induced skin and lung fibrosis but also reduced skin fibrosis in TSK1/+ mice. Conclusion: These findings implicate the JAK/STAT pathway in systemic sclerosis skin and lung fibrosis and identify tofacitinib as a potential antifibrotic agent for the treatment of systemic sclerosis and other fibrotic diseases.
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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 ß 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.
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Fibroblastos/metabolismo , Macrófagos/imunologia , Escleroderma Sistêmico/genética , Pele/metabolismo , Adulto , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Diferenciação Celular , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Técnicas de Cocultura , Feminino , Fibrose/genética , Fibrose/imunologia , Fibrose/metabolismo , Antígenos HLA-DR/imunologia , Humanos , Imunofenotipagem , Interleucina-6/genética , Interleucina-6/imunologia , Lectinas Tipo C/imunologia , Leucócitos Mononucleares , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Pessoa de Meia-Idade , Monócitos/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/imunologia , Receptores de Superfície Celular/imunologia , Fator de Transcrição STAT3/metabolismo , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismo , Pele/patologia , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
BACKGROUND: There is an urgent need for biomarkers to better stratify patients with idiopathic pulmonary fibrosis by risk for lung transplantation allocation who have the same clinical presentation. We aimed to investigate whether a specific immune cell type from patients with idiopathic pulmonary fibrosis could identify those at higher risk of poor outcomes. We then sought to validate our findings using cytometry and electronic health records. METHODS: We first did a discovery analysis with transcriptome data from the Gene Expression Omnibus at the National Center for Biotechnology Information for 120 peripheral blood mononuclear cell (PBMC) samples of patients with idiopathic pulmonary fibrosis. We estimated percentages of 13 immune cell types using statistical deconvolution, and investigated the association of these cell types with transplant-free survival. We validated these results using PBMC samples from patients with idiopathic pulmonary fibrosis in two independent cohorts (COMET and Yale). COMET profiled monocyte counts in 45 patients with idiopathic pulmonary fibrosis from March 12, 2010, to March 10, 2011, using flow cytometry; we tested if increased monocyte count was associated with the primary outcome of disease progression. In the Yale cohort, 15 patients with idiopathic pulmonary fibrosis (with five healthy controls) were classed as high risk or low risk from April 28, 2014, to Aug 20, 2015, using a 52-gene signature, and we assessed whether monocyte percentage (measured by cytometry by time of flight) was higher in high-risk patients. We then examined complete blood count values in the electronic health records (EHR) of 45â068 patients with idiopathic pulmonary fibrosis, systemic sclerosis, hypertrophic cardiomyopathy, or myelofibrosis from Stanford (Jan 01, 2008, to Dec 31, 2015), Northwestern (Feb 15, 2001 to July 31, 2017), Vanderbilt (Jan 01, 2008, to Dec 31, 2016), and Optum Clinformatics DataMart (Jan 01, 2004, to Dec 31, 2016) cohorts, and examined whether absolute monocyte counts of 0·95 K/µL or greater were associated with all-cause mortality in these patients. FINDINGS: In the discovery analysis, estimated CD14+ classical monocyte percentages above the mean were associated with shorter transplant-free survival times (hazard ratio [HR] 1·82, 95% CI 1·05-3·14), whereas higher percentages of T cells and B cells were not (0·97, 0·59-1·66; and 0·78, 0·45-1·34 respectively). In two validation cohorts (COMET trial and the Yale cohort), patients with higher monocyte counts were at higher risk for poor outcomes (COMET Wilcoxon p=0·025; Yale Wilcoxon p=0·049). Monocyte counts of 0·95 K/µL or greater were associated with mortality after adjusting for forced vital capacity (HR 2·47, 95% CI 1·48-4·15; p=0·0063), and the gender, age, and physiology index (HR 2·06, 95% CI 1·22-3·47; p=0·0068) across the COMET, Stanford, and Northwestern datasets). Analysis of medical records of 7459 patients with idiopathic pulmonary fibrosis showed that patients with monocyte counts of 0·95 K/µL or greater were at increased risk of mortality with lung transplantation as a censoring event, after adjusting for age at diagnosis and sex (Stanford HR=2·30, 95% CI 0·94-5·63; Vanderbilt 1·52, 1·21-1·89; Optum 1·74, 1·33-2·27). Likewise, higher absolute monocyte count was associated with shortened survival in patients with hypertrophic cardiomyopathy across all three cohorts, and in patients with systemic sclerosis or myelofibrosis in two of the three cohorts. INTERPRETATION: Monocyte count could be incorporated into the clinical assessment of patients with idiopathic pulmonary fibrosis and other fibrotic disorders. Further investigation into the mechanistic role of monocytes in fibrosis might lead to insights that assist the development of new therapies. FUNDING: Bill & Melinda Gates Foundation, US National Institute of Allergy and Infectious Diseases, and US National Library of Medicine.
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Fibrose Pulmonar Idiopática/sangue , Contagem de Leucócitos/estatística & dados numéricos , Leucócitos Mononucleares , Medição de Risco/métodos , Adulto , Biomarcadores/sangue , Feminino , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/cirurgia , Transplante de Pulmão , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Estudos RetrospectivosRESUMO
PURPOSE: To evaluate the utility of cardiac magnetic resonance (CMR) T1 mapping in early systemic sclerosis (SSc) and its association with skin score. METHODS: Twenty-four consecutive patients with early SSc referred for cardiovascular evaluation and 12 controls without SSc were evaluated. All patients underwent cine, T1 mapping, and late gadolinium enhanced (LGE) CMR imaging. T1 mapping indices were compared between SSc patients and controls (extracellular volume fraction [ECV], gadolinium partition coefficient [λ], pre-contrast T1, and post-contrast T1). The association between T1 mapping parameters and the modified Rodnan skin score (mRSS) was determined. RESULTS: There were no significant differences in cardiac structure/function between SSc patients and controls on cine imaging, and 8/24 (33%) SSc patients had evidence of LGE (i.e., focal myocardial fibrosis). Of the T1 mapping parameters (indices indicative of diffuse myocardial fibrosis), ECV differentiated SSc patients from controls the best, followed by λ, even when the eight SSc patients with LGE were excluded. ECV had a sensitivity and specificity of 75% and 75% for diffuse myocardial fibrosis (optimal abnormal cut-off value of >27% [area under ROC curve=0.85]). In the 16 patients without evidence of LGE, each of the 4 CMR T1 mapping parameters (ECV, λ, Pre-T1 and Post-T1) correlated with mRSS (R=0.51-0.65, P=0.007-0.043), indicating a correlation between SSc cardiac and skin fibrosis. CONCLUSIONS: The four T1 mapping indices are significantly correlated with mRSS in patients with early SSc. Quantification of diffuse myocardial fibrosis using ECV should be considered as a marker for cardiac involvement in SSc clinical studies.
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Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2.
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Imunossupressores/uso terapêutico , Ácido Micofenólico/uso terapêutico , Células Mieloides/efeitos dos fármacos , Escleroderma Sistêmico/tratamento farmacológico , Adulto , Biópsia , Estudos de Casos e Controles , Contagem de Células , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imunossupressores/farmacologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/farmacologia , Células Mieloides/imunologia , Estudos Prospectivos , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Pele/citologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Resultado do TratamentoRESUMO
OBJECTIVE: Currently, there are no reliable biomarkers for predicting therapeutic response in patients with rheumatoid arthritis (RA). The synovium may unlock critical information for determining efficacy, since a reduction in the numbers of sublining synovial macrophages remains the most reproducible biomarker. Thus, a clinically actionable method for the collection of synovial tissue, which can be analyzed using high-throughput strategies, must become a reality. This study was undertaken to assess the feasibility of utilizing synovial biopsies as a precision medicine-based approach for patients with RA. METHODS: Rheumatologists at 6 US academic sites were trained in minimally invasive ultrasound-guided synovial tissue biopsy. Biopsy specimens obtained from patients with RA and synovial tissue from patients with osteoarthritis (OA) were subjected to histologic analysis, fluorescence-activated cell sorting, and RNA sequencing (RNA-seq). An optimized protocol for digesting synovial tissue was developed to generate high-quality RNA-seq libraries from isolated macrophage populations. Associations were determined between macrophage transcriptional profiles and clinical parameters in RA patients. RESULTS: Patients with RA reported minimal adverse effects in response to synovial biopsy. Comparable RNA quality was observed from synovial tissue and isolated macrophages between patients with RA and patients with OA. Whole tissue samples from patients with RA demonstrated a high degree of transcriptional heterogeneity. In contrast, the transcriptional profile of isolated RA synovial macrophages highlighted different subpopulations of patients and identified 6 novel transcriptional modules that were associated with disease activity and therapy. CONCLUSION: Performance of synovial tissue biopsies by rheumatologists in the US is feasible and generates high-quality samples for research. Through the use of cutting-edge technologies to analyze synovial biopsy specimens in conjunction with corresponding clinical information, a precision medicine-based approach for patients with RA is attainable.