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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).

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.

Sarcoidosis likely results from the exposure of a genetically susceptible subject to an environmental agent, possibly an infectious one. Mycobacterial and propionibacterial organisms are the most commonly implicated potential etiologic agents. Propionibacterium acnes is the only microorganism, however, found in sarcoid lesions by bacterial culture. To evaluate the pathogenic role of this indigenous bacterium, we screened for the bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger-factor protein (TIG antibody). We examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, and corresponding control samples from 275 patients with non-sarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients. Immunohistochemistry with PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. In nongranulomatous areas, small round bodies detected by PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some non-sarcoid patients. Large-spheroidal acid-fast bodies, Hamazaki–Wesenberg bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these Hamazaki–Wesenberg bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body. The high frequency and specificity of P. acnes, detected by PAB antibody within sarcoid granulomas, indicates that this indigenous bacterium might be the cause of granuloma formation in many sarcoid patients.

Sarcoidosis is a granulomatous disease of unknown etiology, although M. tuberculosis may play a role in the pathogenesis. The traditional view holds that inflammation in sarcoidosis is compartmentalized to involved organs. To determine whether whole blood gene expression signatures reflect inflammatory pathways in the lung in sarcoidosis and whether these signatures overlap with tuberculosis. We analyzed transcriptomic data from blood and lung biopsies in sarcoidosis and compared these profiles with blood transcriptomic data from tuberculosis and other diseases. Applying machine learning algorithms to blood gene expression data, we built a classifier that distinguished sarcoidosis from health in derivation and validation cohorts (92% sensitivity, 92% specificity). The most discriminative genes were confirmed by quantitative PCR and correlated with disease severity. Transcript profiles significantly induced in blood overlapped with those in lung biopsies and identified shared dominant inflammatory pathways (e.g., Type-I/II interferons). Sarcoidosis and tuberculosis shared more overlap in blood gene expression compared with other diseases using the 86-gene signature reported to be specific for tuberculosis and the sarcoidosis signature presented herein, although reapplication of machine learning algorithms could identify genes specific for sarcoidosis. These data indicate that blood transcriptome analysis provides a noninvasive method for identifying inflammatory pathways in sarcoidosis, that these pathways may be leveraged to complement more invasive procedures for diagnosis or assessment of disease severity, and that sarcoidosis and tuberculosis share overlap in gene regulation of specific inflammatory pathways.

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).

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas “multifocal” granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40–60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68 , CD14 , ITGAM , ITGAX , and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.

Background and aimsSarcoidosis is characterised by a compartmentalisation of CD4+ T helper 1 (Th1) lymphocytes and activated macrophages in involved organs, including the lung. Recently, Th17 effector CD4+ T cells have been claimed to be involved in the pathogenesis of granuloma formation. The objective of this study was to investigate the involvement of Th17 cells in the pathogenesis of sarcoidosis.MethodsPeripheral and pulmonary Th17 cells were evaluated by flow cytometry, real-time PCR, immunohistochemistry analyses and functional assays in patients with sarcoidosis in different phases of the disease and in control subjects.ResultsTh17 cells were detected both in the peripheral blood (4.72±2.27% of CD4+ T cells) and in the bronchoalveolar lavage (BAL) (8.81±2.25% of CD4+ T lymphocytes) of patients with sarcoidosis and T cell alveolitis. Immunohistochemical analysis of lung and lymph node specimens showed that interleukin 17 (IL-17)+/CD4+ T cells infiltrate sarcoid tissues surrounding the central core of the granuloma. IL-17 was expressed by macrophages infiltrating sarcoid tissue and/or forming the granuloma core (7.88±2.40% of alveolar macrophages). Analysis of some lung specimens highlighted the persistence of IL-17+/CD4+ T cells in relapsed patients and their absence in the recovered cases. Migratory assays demonstrated the ability of the Th17 cell to respond to the chemotactic stimulus CCL20—that is, the CCR6 ligand (74.8±8.5 vs 7.6±2.8 migrating BAL lymphocytes/high-powered field, with and without CCL20, respectively).ConclusionsTh17 cells participate in the alveolitic/granuloma phase and also to the progression towards the fibrotic phase of the disease. The recruitment of this cell subset may be driven by CCL20 chemokine.

Abstract Introduction: Observational data suggest that the 19-gauge (G) needle for endobronchial ultrasound (EBUS)-guided transbronchial needle aspiration (TBNA) offers a higher diagnostic yield than the 22-G needle in sarcoidosis. No randomized trial has compared the yield of the two needles. Methods: We randomized consecutive subjects with suspected sarcoidosis and enlarged thoracic lymph nodes to undergo EBUS-TBNA with either the 19-G or the 22-G needle. We compared the study groups for diagnostic sensitivity (primary outcome) assessed by the yield of granulomas in subjects finally diagnosed with sarcoidosis. We also compared the sample adequacy, difficulty performing the needle puncture assessed on a visual analog scale (VAS), the subject’s cough intensity on an operator-rated VAS, and procedure-related complications (secondary outcomes). Results: We randomized 150 (mean age, 43.0 years; 55% women) subjects and diagnosed sarcoidosis in 116 subjects. The diagnostic sensitivity of the 19-G needle (45/60, 75.0%) was not higher (p = 0.52) than the 22-G needle (39/56, 69.6%). We obtained adequate aspirates in 90.0% and 85.7% of subjects in the respective groups (p = 0.48). The operators had greater difficulty puncturing lymph nodes with the 19-G needle (p = 0.03), while the operator-assessed cough intensity was similar in the groups (p = 0.41). Transient hypoxemia was the only complication encountered during EBUS-TBNA (two subjects in either group). Conclusion: We did not find the 19-G needle superior to the 22-G in diagnostic sensitivity, specimen adequacy, or safety of EBUS-TBNA in sarcoidosis. Puncturing the lymph nodes was more difficult with the 19-G needle.

Background Sarcoidosis is a heterogeneous granulomatous disease with no accurate biomarkers of disease progression. Therefore, we profiled and integrated the DNA methylome, mRNAs, and microRNAs to identify molecular changes associated with sarcoidosis and disease progression that might illuminate underlying mechanisms of disease and potential biomarkers. Methods Bronchoalveolar lavage cells from 64 sarcoidosis subjects and 16 healthy controls were used. DNA methylation was profiled on Illumina HumanMethylationEPIC arrays, mRNA by RNA-sequencing, and miRNAs by small RNA-sequencing. Linear models were fit to test for effect of sarcoidosis diagnosis and progression phenotype, adjusting for age, sex, smoking, and principal components of the data. We built a supervised multi-omics model using a subset of features from each dataset. Results We identified 1,459 CpGs, 64 mRNAs, and five miRNAs associated with sarcoidosis versus controls and four mRNAs associated with disease progression. Our integrated model emphasized the prominence of the PI3K/AKT1 pathway, which is important in T cell and mTOR function. Novel immune related genes and miRNAs including LYST , RGS14 , SLFN12L , and hsa-miR-199b-5p, distinguished sarcoidosis from controls. Our integrated model also demonstrated differential expression/methylation of IL20RB , ABCC11 , SFSWAP , AGBL4 , miR-146a-3p, and miR-378b between non-progressive and progressive sarcoidosis. Conclusions Leveraging the DNA methylome, transcriptome, and miRNA-sequencing in sarcoidosis BAL cells, we detected widespread molecular changes associated with disease, many which are involved in immune response. These molecules may serve as diagnostic/prognostic biomarkers and/or drug targets, although future testing is required for confirmation.

The critical innate immune mechanisms that regulate granulomatous inflammation in sarcoidosis are unknown. Because the granuloma-inducing component of sarcoidosis tissues has physicochemical properties similar to those of amyloid fibrils, we hypothesized that host proteins capable of forming poorly soluble aggregates or amyloid regulate inflammation in sarcoidosis. To determine the role of the amyloid precursor protein, serum amyloid A, as an innate regulator of granulomatous inflammation in sarcoidosis. Serum amyloid A expression was determined by immunohistochemistry in sarcoidosis and control tissues and by ELISA. The effect of serum amyloid A on nuclear factor (NF)-kappaB induction, cytokine expression, and Toll-like receptor-2 stimulation was determined with transformed human cell lines and bronchoalveolar lavage cells from patients with sarcoidosis. The effects of serum amyloid A on regulating helper T cell type 1 (Th1) granulomatous inflammation were determined in experimental models of sarcoidosis, using Mycobacterium tuberculosis catalase-peroxidase. We found that the intensity of expression and distribution of serum amyloid A within sarcoidosis granulomas was unlike that in many other granulomatous diseases. Serum amyloid A localized to macrophages and giant cells within sarcoidosis granulomas but correlated with CD3(+) lymphocytes, linking expression to local Th1 responses. Serum amyloid A activated NF-kappaB in Toll-like receptor-2-expressing human cell lines; regulated experimental Th1-mediated granulomatous inflammation through IFN-gamma, tumor necrosis factor, IL-10, and Toll-like receptor-2; and stimulated production of tumor necrosis factor, IL-10, and IL-18 in lung cells from patients with sarcoidosis, effects inhibited by blocking Toll-like receptor-2. Serum amyloid A is a constituent and innate regulator of granulomatous inflammation in sarcoidosis through Toll-like receptor-2, providing a mechanism for chronic disease and new therapeutic targets.