Single Cell Transcriptomics Implicate Novel Monocyte and T Cell Immune Dysregulation in Sarcoidosis

Sarcoidosis is a systemic inflammatory disease characterized by infiltration of immune cells into granulomas. Previous gene expression studies using heterogeneous cell mixtures lack insight into cell-type-specific immune dysregulation. We performed the first single-cell RNA-sequencing study of sarcoidosis in peripheral immune cells in 48 patients and controls. Following unbiased clustering, differentially expressed genes were identified for 18 cell types and bioinformatically assessed for function and pathway enrichment. Our results reveal persistent activation of circulating classical monocytes with subsequent upregulation of trafficking molecules. Specifically, classical monocytes upregulated distinct markers of activation including adhesion molecules, pattern recognition receptors, and chemokine receptors, as well as enrichment of immunoregulatory pathways HMGB1, mTOR, and ephrin receptor signaling. Predictive modeling implicated TGFβ and mTOR signaling as drivers of persistent monocyte activation. Additionally, sarcoidosis T cell subsets displayed patterns of dysregulation. CD4 naïve T cells were enriched for markers of apoptosis and Th17/T(reg) differentiation, while effector T cells showed enrichment of anergy-related pathways. Differentially expressed genes in regulatory T cells suggested dysfunctional p53, cell death, and TNFR2 signaling. Using more sensitive technology and more precise units of measure, we identify cell-type specific, novel inflammatory and regulatory pathways. Based on our findings, we suggest a novel model involving four convergent arms of dysregulation: persistent hyperactivation of innate and adaptive immunity via classical monocytes and CD4 naïve T cells, regulatory T cell dysfunction, and effector T cell anergy. We further our understanding of the immunopathology of sarcoidosis and point to novel therapeutic targets

Local and Systemic CD4 +

Investigation of the Th1 immune response in sarcoidosis CD4+ T cells has revealed reduced proliferative capacity and cytokine expression upon TCR stimulation. In other disease models, such cellular dysfunction has been associated with a step-wise, progressive loss of T cell function that results from chronic antigenic stimulation. T cell exhaustion is defined by decreased cytokine production upon TCR activation, decreased proliferation, increased expression of inhibitory cell surface receptors, and increased susceptibility to apoptosis. We characterized sarcoidosis CD4+ T cell immune function in systemic and local environments among subjects undergoing disease progression compared to those experiencing disease resolution. Spontaneous and TCR-stimulated Th1 cytokine expression and proliferation assays were performed in 53 sarcoidosis subjects and 30 healthy controls. PD-1 expression and apoptosis were assessed by flow cytometry. Compared to healthy controls, sarcoidosis CD4+ T cells demonstrated reductions in Th1 cytokine expression, proliferative capacity (p<0.05), enhanced apoptosis (p<0.01), and increased PD-1 expression (p<0.001). BAL-derived CD4+ T cells also demonstrated multiple facets of T cell exhaustion (p<0.05). Reversal of CD4+ T cell exhaustion was observed in subjects undergoing spontaneous resolution (p<0.05). Sarcoidosis CD4+ T cells exhibit loss of cellular function during progressive disease that follows the archetype of T cell exhaustion

Risk Indicators of Sarcoidosis Evolution-Unified Protocol (RISE-UP): protocol for a multi-centre, longitudinal, observational study to identify clinical features that are predictive of sarcoidosis progression

IntroductionSarcoidosis is a pulmonary and systemic granulomatous disease with a wide range of potential outcomes, from spontaneous resolution to end-stage organ damage and death. Currently, clinicians have no easy-to-use risk stratification tools for important clinical outcomes in sarcoidosis, such as progressive lung disease. This study will address two clinical practice needs: (1) development of a risk calculator that provides an estimate of the likelihood of pulmonary progression in sarcoidosis patients during the follow-up period and (2) determine the optimal interval for serial clinical monitoring (eg, 6, 12, 18 months) using these risk prediction tools.Methods and analysisThe Risk Indicators of Sarcoidosis Evolution-Unified Protocol study is a National Institutes of Health-sponsored, longitudinal observational study of adults with pulmonary sarcoidosis who will be enrolled at five US tertiary care centres. Participants will be evaluated at approximately 6-month intervals for up to 60 months with collection of lung function, blood samples and clinical data. The target sample size is 557 and the primary objective is to determine which clinical features measured during a routine clinic visit carry the most prognostic information for predicting clinical progression of pulmonary sarcoidosis over the follow-up period. The primary outcome measure will be quantified by a clinically meaningful change in forced vital capacity, forced expiratory volume in 1 s or diffusing capacity of the lung for carbon monoxide. The secondary objective is to determine if blood biomarkers measured during a routine clinic visit can improve the risk assessment modelling for progression of pulmonary sarcoidosis over the follow-up period.Ethics and disseminationThe study protocol has been approved by the Institutional Review Boards at each centre and the reliance Institutional Review Board overseeing the study (WCG, Protocol #20222400). Participants will provide informed consent prior to enrolment. Results will be disseminated via publication in a relevant peer-reviewed journal.Trial registration numberNCT05567133

Genetic, Immunologic, and Environmental Basis of Sarcoidosis

Sarcoidosis is a multisystem disease with tremendous heterogeneity in disease manifestations, severity, and clinical course that varies among different ethnic and racial groups. To better understand this disease and to improve the outcomes of patients, a National Heart, Lung, and Blood Institute workshop was convened to assess the current state of knowledge, gaps, and research needs across the clinical, genetic, environmental, and immunologic arenas. We also explored to what extent the interplay of the genetic, environmental, and immunologic factors could explain the different phenotypes and outcomes of patients with sarcoidosis, including the chronic phenotypes that have the greatest healthcare burden. The potential use of current genetic, epigenetic, and immunologic tools along with study approaches that integrate environmental exposures and precise clinical phenotyping were also explored. Finally, we made expert panel-based consensus recommendations for research approaches and priorities to improve our understanding of the effect of these factors on the health outcomes in sarcoidosis

Cellular Recognition of Mycobacterium tuberculosis ESAT-6 and KatG Peptides in Systemic Sarcoidosis

Sarcoidosis is an enigmatic disease with a pathology similar to that of tuberculosis. We detected Th-1 immune responses to Mycobacterium tuberculosis ESAT-6 and KatG peptides from peripheral blood mononuclear cells from 15/26 sarcoidosis, 1/24 purified-protein-derivative-negative (PPD−) (P < 0.0001, Fisher's exact test), and 7/8 PPD-positive (PPD+) subjects (P = 0.21). This finding provides immunologic links between mycobacteria and systemic sarcoidosis

Development of a Sarcoidosis Murine Lung Granuloma Model Using Mycobacterium Superoxide Dismutase A Peptide

Sarcoidosis is characterized by noncaseating granulomas containing CD4+ T cells with a Th1 immunophenotype. Although the causative antigens remain unknown, independent studies noted molecular and immunologic evidence of mycobacterial virulence factors in sarcoidosis specimens. A major limiting factor in discovering new insights into the pathogenesis of sarcoidosis is the lack of an animal model. Using a distinct superoxide dismutase A peptide (sodA) associated with sarcoidosis granulomas, we developed a pulmonary model of sarcoidosis granulomatous inflammation. Mice were sensitized by a subcutaneous injection of sodA, incorporated in incomplete Freund's adjuvant (IFA). Control subjects consisted of mice with no sensitization (ConNS), sensitized with IFA only (ConIFA), or with Schistosoma mansoni eggs. Fourteen days later, sensitized mice were challenged by tail-vein injection of naked beads, covalently coupled to sodA peptides or to schistosome egg antigens (SEA). Histologic analysis revealed hilar lymphadenopathy and noncaseating granulomas in the lungs of sodA-treated or SEA-treated mice. Flow cytometry of bronchoalveolar lavage (BAL) demonstrated CD4+ T-cell responses against sodA peptide in the sodA-sensitized mice only. Cytometric bead analysis revealed significant differences in IL-2 and IFN-γ secretion in the BAL fluid of sodA-treated mice, compared with mice that received SEA or naked beads (P = 0.008, Wilcoxon rank sum test). ConNS and ConIFA mice demonstrated no significant formation of granuloma, and no Th1 immunophenotype. The use of microbial peptides distinct for sarcoidosis reveals a histologic and immunologic profile in the murine model that correlates well with those profiles noted in human sarcoidosis, providing the framework to investigate the molecular basis for the progression or resolution of sarcoidosis