The Renin-Angiotensin-Aldosterone System Regulates Sarcoidosis Granulomatous Inflammation.

Rationale: Sarcoidosis is a granulomatous disorder of unclear cause notable for abnormal elevation of blood and tissue angiotensin converting enzyme 1 (ACE1) levels and activity. ACE1 regulates the renin-angiotensin-aldosterone system (RAAS), the terminal product of which is aldosterone, which selectively engages mineralocorticoid receptors (MR) to promote inflammation. Objectives: We sought to determine whether the RAAS promotes sarcoidosis granuloma formation and related inflammatory responses. Methods: Using an established ex vivo model, we first determined whether aldosterone was produced by sarcoidosis granulomas and verified the presence of CYP11B2, the enzyme required for its production. We then evaluated the effects of selective inhibitors of ACE1 (captopril), angiotensin type 1 receptor (losartan) and MR (spironolactone, eplerenone) on granuloma formation, reflected by computer image analysis-generated granuloma area, and selected cytokines incriminated in sarcoidosis pathogenesis. Measurements and Main Results: Aldosterone was spontaneously produced by sarcoidosis PBMCs, and both intra- and extracellular levels steadily increased during granuloma formation. In parallel, PBMCs were shown to express more CYP11B2 during granuloma formation. Significant inhibition of sarcoidosis granulomas and related cytokines (TNFα, IL-1ß, IFNγ, IL-10) was observed in response to pretreatments with captopril, losartan, spironolactone or eplerenone, comparable to that of prednisone. Conclusions: The RAAS is intact in sarcoidosis granulomas and contributes significantly to early granuloma formation and to related inflammatory mediator responses with important implications for clinical management.

Investigating cryopreserved PBMC functionality in an antigen-induced model of sarcoidosis granuloma formation.

Sarcoidosis, a systemic inflammatory disease, poses challenges in understanding its etiology and variable clinical courses. Despite ongoing uncertainty about causative agents and genetic predisposition, granuloma formation remains its hallmark feature. To address this, we developed a validated in vitro human granuloma model using patient-derived peripheral blood mononuclear cells (PBMCs), offering a dynamic platform for studying early granuloma formation and sarcoidosis pathogenesis. However, a current limitation of this model is its dependence on freshly isolated PBMCs obtained from whole blood. While cryopreservation is a common method for long-term sample preservation, the biological effects of freezing and thawing PBMCs on granuloma formation remain unclear. This study aimed to assess the viability and functionality of cryopreserved sarcoidosis PBMCs within the granuloma model, revealing similar granulomatous responses to fresh cells and highlighting the potential of cryopreserved PBMCs as a valuable tool for studying sarcoidosis and related diseases.

Application of laboratory models for sarcoidosis research.

This manuscript will review the implications and applications of sarcoidosis models towards advancing our understanding of sarcoidosis disease mechanisms, identification of biomarkers, and preclinical testing of novel therapies. Emerging disease models and innovative research tools will also be considered.

Impact of concurrent glomerulonephritis on outcomes of diffuse alveolar haemorrhage in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) commonly presents with diffuse alveolar haemorrhage (DAH) and/or glomerulonephritis. Patients who present with DAH but without kidney involvement have been understudied. METHODS: Patients with DAH diagnosed by bronchoscopy and attributed to AAV over 8.5 years were retrospectively identified through electronic medical records and bronchoscopy reporting software. Patients with end-stage kidney disease (ESKD) or prior kidney transplant were excluded. Characteristics, treatments, and outcomes were abstracted. RESULTS: 30 patients were identified with DAH secondary to AAV. Five with ESKD or prior kidney transplant, and one with concomitant anti-glomerular basement membrane disease, were excluded, leaving 24 patients for analysis. At the time of qualifying bronchoscopy, six patients had no apparent kidney involvement by AAV, while eight of 18 with kidney involvement required dialysis. Of the eight patients dialysed during the initial hospitalisation, four were declared to have ESKD and three died in the subsequent year (one of whom did both). None of the 16 patients without initial dialysis requirement developed kidney involvement requiring dialysis in the subsequent year, though three of the six without initial evidence of kidney involvement by AAV ultimately developed it. No patient without initial kidney involvement died during follow-up. CONCLUSIONS: In our cohort, patients with DAH due to AAV without initial kidney involvement did not develop kidney involvement requiring dialysis or die during the follow-up period, though half of patients without initial evidence of kidney involvement subsequently developed it. Larger studies are warranted to better characterise this population and guide medical management.

The influence of age and sex in sarcoidosis.

PURPOSE OF REVIEW: This review aims to describe how the clinical manifestations of sarcoidosis may be shaped by the effects of sex hormones and by age dependent changes in immune functions and physiology This review is intended to highlight the need to consider the effects of sex and sex in future studies of sarcoidosis. RECENT FINDINGS: The clinical manifestations of sarcoidosis differ based on sex and gender There is emerging evidence that female and male hormones and X-linked genes are important determinants of immune responses to environmental antigens, which has important implications for granuloma formation in the context of sarcoidosis Furthermore, sex hormone levels predictably change throughout adolescence and adulthood, and this occurs in parallel with the onset immune senescence and changes in physiology with advanced age. SUMMARY: Recent studies indicate that sex and age are important variables shaping the immune response of humans to environmental antigens We posit herein that sex and age are important determinants of sarcoidosis clinical phenotypes Many gaps in our understanding of the roles played by sex and gender in sarcoidosis, and these need to be considered in future studies.

Phagosome-regulated mTOR signalling during sarcoidosis granuloma biogenesis.

INTRODUCTION: Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to Mycobacterium tuberculosis antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to M. tuberculosis antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis. METHODS: A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established in vitro human granuloma model wherein peripheral blood mononuclear cells were exposed to M. tuberculosis antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation via phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin). RESULTS: In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction. CONCLUSIONS: Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.

Diagnosis and Detection of Sarcoidosis. An Official American Thoracic Society Clinical Practice Guideline.

Background: The diagnosis of sarcoidosis is not standardized but is based on three major criteria: a compatible clinical presentation, finding nonnecrotizing granulomatous inflammation in one or more tissue samples, and the exclusion of alternative causes of granulomatous disease. There are no universally accepted measures to determine if each diagnostic criterion has been satisfied; therefore, the diagnosis of sarcoidosis is never fully secure.Methods: Systematic reviews and, when appropriate, meta-analyses were performed to summarize the best available evidence. The evidence was appraised using the Grading of Recommendations, Assessment, Development, and Evaluation approach and then discussed by a multidisciplinary panel. Recommendations for or against various diagnostic tests were formulated and graded after the expert panel weighed desirable and undesirable consequences, certainty of estimates, feasibility, and acceptability.Results: The clinical presentation, histopathology, and exclusion of alternative diagnoses were summarized. On the basis of the available evidence, the expert committee made 1 strong recommendation for baseline serum calcium testing, 13 conditional recommendations, and 1 best practice statement. All evidence was very low quality.Conclusions: The panel used systematic reviews of the evidence to inform clinical recommendations in favor of or against various diagnostic tests in patients with suspected or known sarcoidosis. The evidence and recommendations should be revisited as new evidence becomes available.

Differential transcriptomics in sarcoidosis lung and lymph node granulomas with comparisons to pathogen-specific granulomas.

RATIONALE: Despite the availability of multi-"omics" strategies, insights into the etiology and pathogenesis of sarcoidosis have been elusive. This is partly due to the lack of reliable preclinical models and a paucity of validated biomarkers. As granulomas are a key feature of sarcoidosis, we speculate that direct genomic interrogation of sarcoid tissues, may lead to identification of dysregulated gene pathways or biomarker signatures. OBJECTIVE: To facilitate the development sarcoidosis genomic biomarkers by gene expression profiling of sarcoidosis granulomas in lung and lymph node tissues (most commonly affected organs) and comparison to infectious granulomas (coccidiodomycosis and tuberculosis). METHODS: Transcriptomic profiles of immune-related gene from micro-dissected sarcoidosis granulomas within lung and mediastinal lymph node tissues and compared to infectious granulomas from paraffin-embedded blocks. Differentially-expressed genes (DEGs) were profiled, compared among the three granulomatous diseases and analyzed for functional enrichment pathways. RESULTS: Despite histologic similarities, DEGs and pathway enrichment markedly differed in sarcoidosis granulomas from lymph nodes and lung. Lymph nodes showed a clear immunological response, whereas a structural regenerative response was observed in lung. Sarcoidosis granuloma gene expression data corroborated previously reported genomic biomarkers (STAB1, HBEGF, and NOTCH4), excluded others and identified new genomic markers present in lung and lymph nodes, ADAMTS1, NPR1 and CXCL2. Comparisons between sarcoidosis and pathogen granulomas identified pathway divergences and commonalities at gene expression level. CONCLUSION: These findings suggest the importance of tissue and disease-specificity evaluation when exploring sarcoidosis genomic markers. This relevant translational information in sarcoidosis and other two histopathological similar infections provides meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis.

IL-13-regulated Macrophage Polarization during Granuloma Formation in an In Vitro Human Sarcoidosis Model.

The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

Monocyte Distribution Width: A Novel Indicator of Sepsis-2 and Sepsis-3 in High-Risk Emergency Department Patients.

OBJECTIVES: Most septic patients are initially encountered in the emergency department where sepsis recognition is often delayed, in part due to the lack of effective biomarkers. This study evaluated the diagnostic accuracy of peripheral blood monocyte distribution width alone and in combination with WBC count for early sepsis detection in the emergency department. DESIGN: An Institutional Review Board approved, blinded, observational, prospective cohort study conducted between April 2017 and January 2018. SETTING: Subjects were enrolled from emergency departments at three U.S. academic centers. PATIENTS: Adult patients, 18-89 years, with complete blood count performed upon presentation to the emergency department, and who remained hospitalized for at least 12 hours. A total of 2,212 patients were screened, of whom 2,158 subjects were enrolled and categorized per Sepsis-2 criteria, such as controls (n = 1,088), systemic inflammatory response syndrome (n = 441), infection (n = 244), and sepsis (n = 385), and Sepsis-3 criteria, such as control (n = 1,529), infection (n = 386), and sepsis (n = 243). INTERVENTIONS: The primary outcome determined whether an monocyte distribution width of greater than 20.0 U, alone or in combination with WBC, improves early sepsis detection by Sepsis-2 criteria. Secondary endpoints determined monocyte distribution width performance for Sepsis-3 detection. MEASUREMENTS AND MAIN RESULTS: Monocyte distribution width greater than 20.0 U distinguished sepsis from all other conditions based on either Sepsis-2 criteria (area under the curve, 0.79; 95% CI, 0.76-0.82) or Sepsis-3 criteria (area under the curve, 0.73; 95% CI, 0.69-0.76). The negative predictive values for monocyte distribution width less than or equal to 20 U for Sepsis-2 and Sepsis-3 were 93% and 94%, respectively. Monocyte distribution width greater than 20.0 U combined with an abnormal WBC further improved Sepsis-2 detection (area under the curve, 0.85; 95% CI, 0.83-0.88) and as reflected by likelihood ratio and added value analyses. Normal WBC and monocyte distribution width inferred a six-fold lower sepsis probability. CONCLUSIONS: An monocyte distribution width value of greater than 20.0 U is effective for sepsis detection, based on either Sepsis-2 criteria or Sepsis-3 criteria, during the initial emergency department encounter. In tandem with WBC, monocyte distribution width is further predicted to enhance medical decision making during early sepsis management in the emergency department.

Immune Checkpoint Inhibition in Sepsis: A Phase 1b Randomized, Placebo-Controlled, Single Ascending Dose Study of Antiprogrammed Cell Death-Ligand 1 Antibody (BMS-936559).

OBJECTIVES: To assess for the first time the safety and pharmacokinetics of an antiprogrammed cell death-ligand 1 immune checkpoint inhibitor (BMS-936559; Bristol-Myers Squibb, Princeton, NJ) and its effect on immune biomarkers in participants with sepsis-associated immunosuppression. DESIGN: Randomized, placebo-controlled, dose-escalation. SETTING: Seven U.S. hospital ICUs. STUDY POPULATION: Twenty-four participants with sepsis, organ dysfunction (hypotension, acute respiratory failure, and/or acute renal injury), and absolute lymphocyte count less than or equal to 1,100 cells/µL. INTERVENTIONS: Participants received single-dose BMS-936559 (10-900 mg; n = 20) or placebo (n = 4) infusions. Primary endpoints were death and adverse events; key secondary endpoints included receptor occupancy and monocyte human leukocyte antigen-DR levels. MEASUREMENTS AND MAIN RESULTS: The treated group was older (median 62 yr treated pooled vs 46 yr placebo), and a greater percentage had more than 2 organ dysfunctions (55% treated pooled vs 25% placebo); other baseline characteristics were comparable. Overall mortality was 25% (10 mg dose: 2/4; 30 mg: 2/4; 100 mg: 1/4; 300 mg: 1/4; 900 mg: 0/4; placebo: 0/4). All participants had adverse events (75% grade 1-2). Seventeen percent had a serious adverse event (3/20 treated pooled, 1/4 placebo), with none deemed drug-related. Adverse events that were potentially immune-related occurred in 54% of participants; most were grade 1-2, none required corticosteroids, and none were deemed drug-related. No significant changes in cytokine levels were observed. Full receptor occupancy was achieved for 28 days after BMS-936559 (900 mg). At the two highest doses, an apparent increase in monocyte human leukocyte antigen-DR expression (> 5,000 monoclonal antibodies/cell) was observed and persisted beyond 28 days. CONCLUSIONS: In this first clinical evaluation of programmed cell death protein-1/programmed cell death-ligand 1 pathway inhibition in sepsis, BMS-936559 was well tolerated, with no evidence of drug-induced hypercytokinemia or cytokine storm, and at higher doses, some indication of restored immune status over 28 days. Further randomized trials on programmed cell death protein-1/programmed cell death-ligand 1 pathway inhibition are needed to evaluate its clinical safety and efficacy in patients with sepsis.

Role of imbalance between Th17 and regulatory T-cells in sarcoidosis.

PURPOSE OF REVIEW: A relatively new class of CD4 expressing T cells that also express and release interleukin-17 (Th17 cells) is gaining attention based on their capacity to regulate inflammatory responses in a spectrum of chronic autoimmune diseases. The purpose of this review is to consider recent studies relating to the critical role played by Th17 cells in the pathogenesis of sarcoidosis. RECENT FINDINGS: Th17 cells are unique in their capacity to adapt to local molecular cues to variably promote or suppress inflammation. On the basis of knowledge established originally in the context of autoimmune disorders, recent investigations indicate that Th17 cells are instrumental in all stages of granuloma evolution, including granuloma formation, maintenance and resolution. Recent research shed light on the mechanisms regulating Th17 cell plasticity and the implications for sarcoidosis disease progression, such as the mechanisms by which regulatory T cells (Tregs) promote resolution of Th17-mediated inflammation. SUMMARY: The balance between Th17 cells and Tregs in sarcoidosis patients has important implications for clinicians and clinical researchers seeking more reliable prognostic markers and more targeted therapeutic agents.

A Novel In Vitro Human Granuloma Model of Sarcoidosis and Latent Tuberculosis Infection.

Many aspects of pathogenic granuloma formation are poorly understood, requiring new relevant laboratory models that represent the complexity (genetics and diversity) of human disease. To address this need, we developed an in vitro model of granuloma formation using human peripheral blood mononuclear cells (PBMCs) derived from patients with active sarcoidosis, latent tuberculosis (TB) infection (LTBI), or normal healthy control subjects. PBMCs were incubated for 7 days with uncoated polystyrene beads or beads coated with purified protein derivative (PPD) or human serum albumin. In response to PPD-coated beads, PBMCs from donors with sarcoidosis and LTBI formed robust multicellular aggregates resembling granulomas, displaying a typical T-helper cell type 1 immune response, as assessed by cytokine analyses. In contrast, minimal PBMC aggregation occurred when control PBMCs were incubated with PPD-coated beads, whereas the response to uncoated beads was negligible in all groups. Sarcoidosis PBMCs responded to human serum albumin-coated beads with modest cellular aggregation and inflammatory cytokine release. Whereas the granuloma-like aggregates formed in response to PPD-coated beads were similar for sarcoidosis and LTBI, molecular profiles differed significantly. mRNA expression patterns revealed distinct pathways engaged in early granuloma formation in sarcoidosis and LTBI, and they resemble molecular patterns reported in diseased human tissues. This novel in vitro human granuloma model is proposed as a tool to investigate mechanisms of early granuloma formation and for preclinical drug discovery research of human granulomatous disorders. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

Mathematical model of sarcoidosis.

Sarcoidosis is a disease involving abnormal collection of inflammatory cells forming nodules, called granulomas. Such granulomas occur in the lung and the mediastinal lymph nodes, in the heart, and in other vital and nonvital organs. The origin of the disease is unknown, and there are only limited clinical data on lung tissue of patients. No current model of sarcoidosis exists. In this paper we develop a mathematical model on the dynamics of the disease in the lung and use patients' lung tissue data to validate the model. The model is used to explore potential treatments.

In-silico modeling of granulomatous diseases.

PURPOSE OF REVIEW: The pathogenesis of genetically complex granulomatous diseases, such as sarcoidosis and latent tuberculosis, remains largely unknown. With the recent advent of more powerful research tools, such as genome-wide expression platforms, comes the challenge of making sense of the enormous data sets so generated. This manuscript will provide demonstrations of how in-silico (computer) analysis of large research data sets can lead to novel discoveries in the field of granulomatous lung disease. RECENT FINDINGS: The application of in-silico research tools has led to novel discoveries in the fields of noninfectious (e.g., sarcoidosis) and infectious granulomatous diseases. Computer models have identified novel disease mechanisms and can be used to perform 'virtual' experiments rapidly and at low cost compared with conventional laboratory techniques. SUMMARY: Granulomatous lung diseases are extremely complex, involving dynamic interactions between multiple genes, cells, and molecules. In-silico interpretation of large data sets generated from new research platforms that are capable of comprehensively characterizing and quantifying pools of biological molecules promises to rapidly accelerate the rate of scientific discovery in the field of granulomatous lung disorders.