Molecular Mechanism in the Development of Pulmonary Fibrosis in Patients with Sarcoidosis.

Sarcoidosis is a multisystemic disease of unknown etiology characterized by the formation of granulomas in various organs, especially lung and mediastinal hilar lymph nodes. The clinical course and manifestations are unpredictable: spontaneous remission can occur in approximately two thirds of patients; up to 20% of patients have chronic course of the lung disease (called advanced pulmonary sarcoidosis, APS) resulting in progressive loss of lung function, sometimes life-threatening that can lead to respiratory failure and death. The immunopathology mechanism leading from granuloma formation to the fibrosis in APS still remains elusive. Recent studies have provided new insights into the genetic factors and immune components involved in the clinical manifestation of the disease. In this review we aim to summarize the clinical-prognostic characteristics and molecular pathways which are believed to be associated with the development of APS.

Comparative Analysis of FCGR Gene Polymorphism in Pulmonary Sarcoidosis and Tuberculosis.

The clinical outcome of sarcoidosis (SA) is very similar to tuberculosis (TB); however, they are treated differently and should not be confused. In search for their biomarkers, we have previously revealed changes in the phagocytic activity of monocytes in sarcoidosis and tuberculosis. On these monocytes we found a higher expression of receptors for the Fc fragment of immunoglobulin G (FcγR) in SA and TB patients vs. healthy controls. FcγRs are responsible for the binding of immune complexes (ICs) to initiate an (auto)immune response and for ICs clearance. Surprisingly, our SA patients had a high blood level of ICs, despite the abundant presence of FcγRs. It pointed to FcγR disfunction, presumably caused by the polymorphism of their (FCGR) genes. Therefore, we present here an analysis of the occurrence of FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B variants in Caucasian SA and TB patients, and healthy individuals with the use of polymerase chain reaction (PCR) and real-time PCR. The presented data point to a possibility of supporting the differential diagnosis of SA and TB by analyzing FCGR2C, FCGR3A and FCGR3B polymorphism, while for severe stages of SA also by studying FCGR2A variants. Additionally, the genotyping of FCGR2A and FCGR3B might serve as a marker of SA progression.

Immune mapping of human tuberculosis and sarcoidosis lung granulomas.

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.

Comparative Analysis of Gene Expression in Fibroblastic Foci in Patients with Idiopathic Pulmonary Fibrosis and Pulmonary Sarcoidosis.

BACKGROUND: Fibroblastic foci (FF) are characteristic features of usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) and one cardinal feature thought to represent a key mechanism of pathogenesis. Hence, FF have a high impact on UIP/IPF diagnosis in current guidelines. However, although less frequent, these histomorphological hallmarks also occur in other fibrotic pulmonary diseases. Currently, there is therefore a gap in knowledge regarding the underlying molecular similarities and differences of FF in different disease entities. METHODS: In this work, we analyzed the compartment-specific gene expression profiles of FF in IPF and sarcoidosis in order to elucidate similarities and differences as well as shared pathomechanisms. For this purpose, we used laser capture microdissection, mRNA and protein expression analysis. Biological pathway analysis was performed using two different gene expression databases. As control samples, we used healthy lung tissue that was donated but not used for lung transplantation. RESULTS: Based on Holm Bonferroni corrected expression data, mRNA expression analysis revealed a significantly altered expression signature for 136 out of 760 genes compared to healthy controls while half of these showed a similar regulation in both groups. Immunostaining of selected markers from each group corroborated these results. However, when comparing all differentially expressed genes with the fdr-based expression data, only 2 of these genes were differentially expressed between sarcoidosis and IPF compared to controls, i.e., calcium transport protein 1 (CAT1) and SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), both in the sarcoidosis group. Direct comparison of sarcoidosis and IPF did not show any differentially regulated genes independent from the statistical methodology. Biological pathway analysis revealed a number of fibrosis-related pathways pronounced in IPF without differences in the regulatory direction. CONCLUSIONS: These results demonstrate that FF of end-stage IPF and sarcoidosis lungs, although different in initiation, are similar in gene and protein expression, encouraging further studies on the use of antifibrotic agents in sarcoidosis.

Genomic biomarkers in chronic beryllium disease and sarcoidosis.

Background Previous gene expression studies have identified genes IFNγ, TNFα, RNase 3, CXCL9, and CD55 as potential biomarkers for sarcoidosis and/or chronic beryllium disease (CBD). We hypothesized that differential expression of these genes could function as diagnostic biomarkers for sarcoidosis and CBD, and prognostic biomarkers for sarcoidosis. Study Design/Methods We performed RT-qPCR on whole blood samples from CBD (n = 132), beryllium sensitized (BeS) (n = 109), and sarcoidosis (n = 99) cases and non-diseased controls (n = 97) to determine differential expression of target genes. We then performed logistic regression modeling and generated ROC curves to determine which genes could most accurately differentiate: 1) CBD versus sarcoidosis 2) CBD versus BeS 3) sarcoidosis versus controls 4) non-progressive versus progressive sarcoidosis. Results CD55 and TNFα were significantly upregulated, while CXCL9 was significantly downregulated in CBD compared to sarcoidosis (p < 0.05). The ROC curve from the logistic regression model demonstrated high discriminatory ability of the combination of CD55, TNFα, and CXCL9 to distinguish between CBD and sarcoidosis with an AUC of 0.98. CD55 and TNFα were significantly downregulated in sarcoidosis compared to controls (p < 0.05). The ROC curve from the model showed a reasonable discriminatory ability of CD55 and TNFα to distinguish between sarcoidosis and controls with an AUC of 0.86. There was no combination of genes that could accurately differentiate between CBD and BeS or sarcoidosis phenotypes. Interpretation CD55, TNFα and CXCL9 expression levels can accurately differentiate between CBD and sarcoidosis, while CD55 and TNFα expression levels can accurately differentiate sarcoidosis and controls.

Transcriptomics of bronchoalveolar lavage cells identifies new molecular endotypes of sarcoidosis.

BACKGROUND: Sarcoidosis is a multisystem granulomatous disease of unknown origin with a variable and often unpredictable course and pattern of organ involvement. In this study we sought to identify specific bronchoalveolar lavage (BAL) cell gene expression patterns indicative of distinct disease phenotypic traits. METHODS: RNA sequencing by Ion Torrent Proton was performed on BAL cells obtained from 215 well-characterised patients with pulmonary sarcoidosis enrolled in the multicentre Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Weighted gene co-expression network analysis and nonparametric statistics were used to analyse genome-wide BAL transcriptome. Validation of results was performed using a microarray expression dataset of an independent sarcoidosis cohort (Freiburg, Germany; n=50). RESULTS: Our supervised analysis found associations between distinct transcriptional programmes and major pulmonary phenotypic manifestations of sarcoidosis including T-helper type 1 (Th1) and Th17 pathways associated with hilar lymphadenopathy, transforming growth factor-ß1 (TGFB1) and mechanistic target of rapamycin (MTOR) signalling with parenchymal involvement, and interleukin (IL)-7 and IL-2 with airway involvement. Our unsupervised analysis revealed gene modules that uncovered four potential sarcoidosis endotypes including hilar lymphadenopathy with increased acute T-cell immune response; extraocular organ involvement with PI3K activation pathways; chronic and multiorgan disease with increased immune response pathways; and multiorgan involvement, with increased IL-1 and IL-18 immune and inflammatory responses. We validated the occurrence of these endotypes using gene expression, pulmonary function tests and cell differentials from Freiburg. CONCLUSION: Taken together, our results identify BAL gene expression programmes that characterise major pulmonary sarcoidosis phenotypes and suggest the presence of distinct disease molecular endotypes.

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.

Matrix Metalloproteinase-12 Is Required for Granuloma Progression.

Background: Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by granuloma formation. Mechanisms for chronic persistence of granulomas are unknown. Matrix Metalloproteinase-12 (MMP12) degrades extracellular matrix elastin and enables infiltration of immune cells responsible for inflammation and granuloma formation. Previous studies report increased MMP12 in sarcoidosis patients and association between MMP12 expression and disease severity. We also observed elevated MMP12 in our multiwall carbon nanotube (MWCNT) murine model of granulomatous inflammation. Here we hypothesized that MMP12 is important to acute and late phases of granuloma pathogenesis. To test this hypothesis, we analyzed granulomatous and inflammatory responses of Mmp12 knock-out (KO) mice at 10 (acute) and 60 days (late) after MWCNT instillation. Methods: C57BL/6 (wildtype) and Mmp12 KO mice underwent oropharyngeal instillation of MWCNT. Lungs were harvested at 3, 10, 20, and 60 days post instillation for evaluation of MMP12 expression and granulomatous changes. Bronchoalveolar lavage (BAL) cells were analyzed 60 days after MWCNT instillation for expression of mediators thought to play a role in sarcoid granulomatosis: peroxisome proliferator-activated receptor-gamma (PPARγ), interferon-gamma (IFN-γ), and CCL2 (MCP-1). Results: Pulmonary granuloma appearance at 10 days after MWCNT instillation showed no differences between wildtype and Mmp12 KO mice. In contrast, by 60 days after MWCNT instillation, Mmp12 KO mice revealed markedly attenuated granuloma formation together with elevated PPARγ and reduced IFNγ expression in BAL cells compared to wildtype. Unexpectedly, Mmp12 KO mice further demonstrated increased alveolar macrophages with increased CCL2 at 60 days. Conclusions: The striking reduction of granuloma formation at day 60 in Mmp12 KO mice suggests that MMP12 is required to maintain chronic granuloma pathophysiology. The increased PPARγ and decreased IFNγ findings suggest that these mediators also may be involved since previous studies have shown that PPARγ suppresses IFNγ and PPARγ deficiency amplifies granuloma formation. Interestingly, a role of MMP12 in granuloma resolution is also suggested by increases in both macrophage influx and CCL2. Overall, our results strongly implicate MMP12 as a key factor in granuloma persistence and as a possible therapeutic target in chronic pulmonary sarcoidosis.

Current Sarcoidosis Models and the Importance of Focusing on the Granuloma.

The inability to effectively model sarcoidosis in the laboratory or in animals continues to hinder the discovery and translation of new, targeted treatments. The granuloma is the signature pathological hallmark of sarcoidosis, yet there are significant knowledge gaps that exist with regard to how granulomas form. Significant progress toward improved therapeutic and prognostic strategies in sarcoidosis hinges on tractable experimental models that recapitulate the process of granuloma formation in sarcoidosis and allow for mechanistic insights into the molecular events involved. Through its inherent representation of the complex genetics underpinning immune cell dysregulation in sarcoidosis, a recently developed in vitro human granuloma model holds promise in providing detailed mechanistic insight into sarcoidosis-specific disease regulating pathways at play during early stages of granuloma formation. The purpose of this review is to critically evaluate current sarcoidosis models and assess their potential to progress the field toward the goal of improved therapies in this disease. We conclude with the potential integrated use of preclinical models to accelerate progress toward identifying and testing new drugs and drug combinations that can be rapidly brought to clinical trials.

[Whole exome sequencing and analysis of a Chinese family with familial pulmonary sarcoidosis].

Objective: To analyze the clinical features and the results of the whole exome sequencing (WES) of a Chinese family containing both pulmonary sarcoidosis patients and healthy members, and to find potent genes and variants that may be involved in the pathogenesis of sarcoidosis. Methods: Three patients with pulmonary sarcoidosis and 1 healthy member was included from a Chinese Han family in the north of China diagnosed in November 2016, which characterized as 2 consecutive generations including 2 males and 1 female, aged from 23 to 69 years old. The proband is Ⅱ-6. Pulmonary sarcoidosis was diagnosed by clinical features, imaging and pathological findings, and clinical data such as family history were collected. Whole blood samples were taken and WES (Illumina NovaSeq S2) was performed. The pathogenicity analysis and gene annotation analysis were performed by ExAC, SIFT, Polyphenv2, Metascape databases. Results: It was found that 27 genes were highly pathogenic in the database filtering result. After gene annotation analysis, we found that ZC3H12A gene can negatively regulate the differentiation of Th17 cells, which may be involved in the onset of pulmonary sarcoidosis. Sanger sequencing confirmed the c.1361 A>G variant in 3 sarcoidosis patients but normal in healthy member. Conclusions: In patients with familial pulmonary sarcoidosis, the genetic background could regulate immune response which is one of the pathogenic mechanisms of sarcoidosis. The whole exome test and gene ontology analysis showed that Ⅱ-2, Ⅱ-6 and Ⅲ-1 pulmonary sarcoidosis patients in this family were all shared the same variant on ZC3H12A gene, which played a pivotal role in differentiation of Th17 cells and is a potent pathogenesis gene in this Chinese pulmonary sarcoidosis family.

Association of TGF-ß3 and ANXA11 with pulmonary sarcoidosis in Greek population.

BACKGROUND: In sarcoidosis, the direction and intensity of immunological reactions involved in disease pathophysiology is affected by variation in the genes coding for effector and regulatory molecules with immune functions. This study, therefore, investigates polymorphic variants in genes involved in inflammation, immune reactions, and granuloma formation in context of their plausible association with sarcoidosis, with specific focus on Greek population. METHODS: A total of 18 single-nucleotide polymorphisms (SNPs) were genotyped in Greek patients with pulmonary sarcoidosis (n = 103) and in healthy Greek control subjects (n = 100) using multiplexed MassARRAY (MassARRAY ®) iPLEX assay based on MALDI-TOF mass spectrometry. RESULTS: TGF-ß3 rs3917200*G variant was associated with sarcoidosis (OR: 3.04 [95% CI: 1.98-4.69], p = 2.76*10-7). Further, ANXA11 rs1049550*A variant was associated with sarcoidosis (OR: 0.59 [0.39-0.89], p = 0.01). CONCLUSIONS: This first study of genetic variation of immune-related genes in Greek patients with sarcoidosis brings to attention a novel disease 'susceptibility' factor: TGF-ß3 rs3917200*G allele. It also confirms previously reported 'protective' association between sarcoidosis and functional variant ANXA11 rs1049550*A. Further work is required to validate these findings and to expand investigation of their plausible relationship with clinical course of the disease.

Clinical phenotyping: role in treatment decisions in sarcoidosis.

A variety of phenotypic categorisations have been developed for sarcoidosis. Phenotyping has been used for genetics studies and to guide treatment selection. The authors participated in a Delphi expert consensus panel to develop a proposed phenotype categorisation and treatment recommendations for pulmonary sarcoidosis patients. Panellists reached consensus that asymptomatic patients with normal pulmonary function and adenopathy alone or normal chest imaging do not require therapy, while symptomatic patients with impaired pulmonary function or infiltrates should be treated. The panel did not reach consensus on asymptomatic patients with abnormal chest imaging or reduced pulmonary function, or symptomatic patients with normal chest imaging and pulmonary function. The proposed phenotype categories and associated treatment recommendations are asymptomatic (no therapy), acute (disease duration <1-2 years, apparently self-limited, corticosteroids), chronic (antimetabolites and other second-line therapies) and advanced (biologics). Some clinical settings, such as dyspnoea/hypoxaemia at rest, severely impaired or rapidly decreasing pulmonary function tests, and severe cardiac, neurologic, ocular or renal involvement warrant immediate therapy.

CXCL9 and CXCL10 are differentially associated with systemic organ involvement and pulmonary disease severity in sarcoidosis.

BACKGROUND: Sarcoidosis is a granulomatous inflammatory disease with limited blood markers to predict outcomes. The interferon-gamma (IFN-γ)-inducible chemotactic cytokines (chemokines), CXCL9 and CXCL10, are both increased in sarcoidosis patients, yet they possess important molecular differences. Our study determined if serum chemokines correlated with different aspects of disease severity. METHODS: We measured CXCL9 and CXCL10 serum levels at initial study visits and longitudinally in sarcoidosis subjects using ELISA. We examined these chemokines' relationships with pulmonary and organ involvement outcomes, their gene expression, peripheral blood immune cell populations, and immunosuppression use. RESULTS: Higher CXCL10 levels negatively correlated with FVC, TLC, and DLCO at subjects' initial visit and when measured repeatedly over two years. CXCL10 also positively correlated with longitudinal respiratory symptom severity. Additionally, for every log10(CXCL10) increase, the risk of longitudinal pulmonary function decline increased 8.8 times over the 5-year study period (95% CI 1.6-50, p = 0.014, log10(CXCL0) range 0.84-2.7). In contrast, CXCL9 levels positively correlated with systemic organ involvement at initial study visit (1.5 additional organs involved for every log10(CXCL9) increase, 95% CI 1.1-2.0, p = 0.022, log10(CXCL9) range 1.3-3.3). CXCL10, not CXCL9, positively correlated with its own blood gene expression and monocyte level. Immunosuppressive treatment was associated with lower levels of both chemokines. CONCLUSIONS: In sarcoidosis subjects, serum CXCL9 levels correlated with systemic organ involvement and CXCL10 levels strongly correlated with respiratory outcomes, which may ultimately prove helpful in clinical management. These differing associations may be due to differences in cellular regulation and tissue origin.

mRNA expression profile of bronchoalveolar lavage fluid cells from patients with idiopathic pulmonary fibrosis and sarcoidosis.

BACKGROUND: Sarcoidosis and idiopathic pulmonary fibrosis (IPF) are two most frequent forms of interstitial lung diseases (ILDs). Cellular and biochemical composition of bronchoalveolar lavage fluid (BALf) was shown to reflect the fibrotic changes in the lung. However, the usefulness of BALf cellular profile evaluation in the diagnosis of ILDs is limited. The aim of the study was a multivariate, molecular analysis of BALf cells from IPF and sarcoidosis patients. METHODS: Transcriptomic measurements were carried out using Affymetrix Human Gene 2.1 ST ArrayStrip in 21 samples: 9 IPF and 12 sarcoidosis. The mRNA expression for the most significantly differentiating genes was evaluated by real-time PCR in 32 samples (11 IPF and 21 sarcoidosis). RESULTS: The number of genes differentially expressed between IPF and sarcoidosis groups was 4832 (13359 probesets). Cluster analysis indicated that sarcoidosis BALf cells are characterized by increased mRNA expression of genes associated with ribosome biogenesis. Clusters formed by genes with changed mRNA expression in IPF samples were implicated in the processes of cell adhesion and migration, metalloproteinase expression and negative regulation of cell proliferation. The GO analysis indicated that predominant biological processes associated with the differential mRNA gene expression of BALf cells were upregulation of neutrophils in IPF and lymphocytes in sarcoidosis. CONCLUSIONS: Analysis of BALf from sarcoidosis and IPF showed highly different mRNA profile of cells. The most important biological processes observed at the molecular level in BALf cells were associated with ribosome biogenesis and proteasome apparatus in sarcoidosis and neutrophilic dysfunction in IPF.

DNA Methylation Changes in Lung Immune Cells Are Associated with Granulomatous Lung Disease.

Epigenetic marks are likely to explain variability of response to antigen in granulomatous lung disease. The objective of this study was to identify DNA methylation and gene expression changes associated with chronic beryllium disease (CBD) and sarcoidosis in lung cells obtained by BAL. BAL cells from CBD (n = 8), beryllium-sensitized (n = 8), sarcoidosis (n = 8), and additional progressive sarcoidosis (n = 9) and remitting (n = 15) sarcoidosis were profiled on the Illumina 450k methylation and Affymetrix/Agilent gene expression microarrays. Statistical analyses were performed to identify DNA methylation and gene expression changes associated with CBD, sarcoidosis, and disease progression in sarcoidosis. DNA methylation array findings were validated by pyrosequencing. We identified 52,860 significant (P < 0.005 and q < 0.05) CpGs associated with CBD; 2,726 CpGs near 1,944 unique genes have greater than 25% methylation change. A total of 69% of differentially methylated genes are significantly (q < 0.05) differentially expressed in CBD, with many canonical inverse relationships of methylation and expression in genes critical to T-helper cell type 1 differentiation, chemokines and their receptors, and other genes involved in immunity. Testing of these CBD-associated CpGs in sarcoidosis reveals that methylation changes only approach significance, but are methylated in the same direction, suggesting similarities between the two diseases with more heterogeneity in sarcoidosis that limits power with the current sample size. Analysis of progressive versus remitting sarcoidosis identified 15,215 CpGs (P < 0.005 and q < 0.05), but only 801 of them have greater than 5% methylation change, demonstrating that DNA methylation marks of disease progression changes are more subtle. Our study highlights the significance of epigenetic marks in lung immune response in granulomatous lung disease.

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).

Bronchoalveolar lavage characteristics correlate with HLA tag SNPs in patients with Löfgren's syndrome and other sarcoidosis.

Genetic susceptibility for sarcoidosis and Löfgren's syndrome (LS) has been associated with prognosis. Human leukocyte antigen (HLA)-DRB1*03 is over-represented in LS, and is associated with a good prognosis, whereas HLA-DRB1*15-positive patients have a more chronic course of sarcoidosis. These HLA-DRB1 types can be easily tagged by single nucleotide polymorphisms (SNPs). Our aim was to evaluate the association between these tag SNPs and bronchoalveolar lavage (BAL) characteristics. In 29 patients, both complete HLA-DRB1* locus genotyping and SNP tagging was performed in parallel. HLA-DRB1 type was inferred from the presence of *03 tag rs2040410 allele A and referred to as *03. HLA-DRB1*15 was inferred from the presence of tag SNP rs3135388 allele A and referred to as *15. For BAL analysis, 122 patients with LS and 165 patients with non-LS sarcoidosis were included. BAL lymphocyte subsets were analyzed by flow cytometry. The presence of tag SNPs completely corresponded with HLA-DRB1*03/*15 genotypes in all 29 patients in whom both HLA-DRB1* genotyping and SNP tagging was performed. In all patients together, *03+ /*15- patients showed a higher CD4+ /CD8+ ratio than *03- /*15+ (P = 0·004) and *03- /*15- (P = 0·001). LS patients with *03+ /*15- had a lower BAL lymphocyte count compared to *03- /*15+  patients (P = 0·011). Non-LS sarcoidosis patients with *03+ /*15- patients showed a decreased CD103+ CD4+ /CD4+ ratio compared to *03- /*15+  patients (P = 0·045) and *03- /*15- patients (P = 0·018). We found that HLA-DRB1*03 and HLA-DRB1*15 can be approximated by genotyping of tag SNPs and corresponds with the degree of lymphocytosis and cell phenotypes in BAL in both LS and non-LS sarcoidosis patients.

Cathepsin S, a new serum biomarker of sarcoidosis discovered by transcriptome analysis of alveolar macrophages.

BACKGROUND: Development of reliable new biomarkers remains crucial to improve diagnosis and assessing disease activity in sarcoidosis. The objective of this study was to seek such markers from the gene expression signature of alveolar macrophages by transcriptome analysis. METHODS: Pooled RNA extracted from alveolar macrophages from patients with active sarcoidosis and control patients was subjected to transcriptome analysis using microarrays. Expressed gene intensity in sarcoidosis relative to that in control was calculated. We measured serum cathepsin S (CTSS) concentrations in 89 healthy volunteers, 107 patients with sarcoidosis, 26 with interstitial pneumonia, 150 with pneumoconiosis, and 76 with pulmonary mycobacteriosis by the enzyme-linked immunosorbent assay. RESULTS: Among 12 genes with ratios higher than that of a housekeeping gene, we selected CTSS for scrutinizing protein expression in serum because of the feasibility of the protein assay. CTSS concentrations were significantly increased in sarcoidosis compared with not only controls but also all the other lung diseases. Receiver operating characteristics curve for sarcoidosis and parenchymal lung diseases revealed an area under the curve of 0.800 (95% confidence interval, 0.751-0.850; p=1.4 x 10-18) with 70% sensitivity and 78% specificity at a CTSS concentration of 15.5 ng/ml. A significant trend was identified between CTSS concentrations and the number of affected organs. Serum CTSS concentrations varied in parallel with clinical courses both spontaneously and in response to corticosteroid therapy. Epithelioid cells in granulomas were positive for immunohistochemical staining with CTSS. CONCLUSIONS: CTSS has the potential to be a useful biomarker in sarcoidosis.