Structural basis of chronic beryllium disease: linking allergic hypersensitivity and autoimmunity.

T-cell-mediated hypersensitivity to metal cations is common in humans. How the T cell antigen receptor (TCR) recognizes these cations bound to a major histocompatibility complex (MHC) protein and self-peptide is unknown. Individuals carrying the MHCII allele, HLA-DP2, are at risk for chronic beryllium disease (CBD), a debilitating inflammatory lung condition caused by the reaction of CD4 T cells to inhaled beryllium. Here, we show that the T cell ligand is created when a Be(2+) cation becomes buried in an HLA-DP2/peptide complex, where it is coordinated by both MHC and peptide acidic amino acids. Surprisingly, the TCR does not interact with the Be(2+) itself, but rather with surface changes induced by the firmly bound Be(2+) and an accompanying Na(+) cation. Thus, CBD, by creating a new antigen by indirectly modifying the structure of preexisting self MHC-peptide complex, lies on the border between allergic hypersensitivity and autoimmunity.

Innate and Adaptive Immunity in Noninfectious Granulomatous Lung Disease.

Sarcoidosis and chronic beryllium disease are noninfectious lung diseases that are characterized by the presence of noncaseating granulomatous inflammation. Chronic beryllium disease is caused by occupational exposure to beryllium containing particles, whereas the etiology of sarcoidosis is not known. Genetic susceptibility for both diseases is associated with particular MHC class II alleles, and CD4+ T cells are implicated in their pathogenesis. The innate immune system plays a critical role in the initiation of pathogenic CD4+ T cell responses as well as the transition to active lung disease and disease progression. In this review, we highlight recent insights into Ag recognition in chronic beryllium disease and sarcoidosis. In addition, we discuss the current understanding of the dynamic interactions between the innate and adaptive immune systems and their impact on disease pathogenesis.

Multisystemic Beryllium Disease: An Exceptional Case Revealed by a Urinary Tract Granulomatosis.

Chronic beryllium disease (CBD), or berylliosis, is an interstitial lung disease caused by the chronic inhalation of finely particulate beryllium, frequently mistaken for sarcoidosis. It is rarely associated with skin nodular lesions, asymptomatic granulomatous hepatitis or calcium nephrolithiasis. To date, it has never been reported as a diffused multi-organ granulomatous disease. A 60-year-old Pakistani man, a former excavation worker with ancient history of suspected sarcoidosis, underwent a left nephroureterectomy for suspected papillary kidney carcinoma. The histopathological analysis showed a benign non-necrotic granulomatous infiltration of the renal pelvis and ureter. Six months later, he suffered from two consecutive episodes of acute kidney failure. Bladder biopsies found similar noncaseous granulomatosis and kidney biopsies showed interstitial nephritis. Known for suspected asthma, sleep apnea, and usual interstitial pneumonia, the patient would regularly consult for episodes of pyrexia, chills, nocturnal coughing, and wheezing. As kidney function gradually worsened, he ultimately started hemodialysis and was transferred to our facility. A positive blood beryllium lymphocyte proliferation test confirmed the diagnosis of CBD. This original report is the first description of multi-organ berylliosis with diffused urothelial granulomatosis and pseudo-tumor. The patient's pulmonary disease is minimal compared with renal and urinary tract involvement, eventually responsible for end-stage kidney disease. Berylliosis usually responds to glucocorticoids. This case report highlights the importance of evoking the diagnosis of CBD in the presence of any granulomatosis, even extra-thoracic, especially if associated with pulmonary symptoms, however atypical.

Hydrogen sulfide alleviates beryllium sulfate-induced ferroptosis and ferritinophagy in 16HBE cells.

Beryllium sulfate (BeSO4 ) can result to lung injuries, such as leading to lipid peroxidation and autophagy, and the treatment of beryllium disease has not been well improved. Ferroptosis is a regulated cell death process driven by iron-dependent and lipid peroxidation, while ferritinophagy is a process mediated by nuclear receptor coactivator 4 (NCOA4), combined with ferritin heavy chain 1 (FTH1) degradation and release Fe2+ , which regulated intracellular iron metabolism and ferroptosis. Hydrogen sulfide (H2 S) has the effects of antioxidant, antiautophagy, and antiferroptosis. This study aimed to investigate the effect of H2 S on BeSO4 -induced ferroptosis and ferritinophagy in 16HBE cells and the underlying mechanism. In this study, BeSO4 -induced 16HBE cell injury model was established based on cellular level and pretreated with deferoxamine (DFO, a ferroptosis inhibitor), sodium hydrosulfide (NaHS, a H2 S donor), or NCOA4 siRNA and, subsequently, performed to detect the levels of lipid peroxidation and Fe2+ and the biomarkers of ferroptosis and ferritinophagy. More importantly, our research found that DFO, NaHS, or NCOA4 siRNA alleviated BeSO4 -induced ferroptosis and ferritinophagy by decreasing the accumulation of Fe2+ and lipid peroxides. Furthermore, the relationship between ferroptosis, ferritinophagy, H2 S, and beryllium disease is not well defined; therefore, our research is innovative. Overall, our results provided a new theoretical basis for the prevention and treatment of beryllium disease and suggested that the application of H2 S, blocking ferroptosis, and ferritinophagy may be a potential therapeutic direction for the prevention and treatment of beryllium disease.

Multiomic Signatures of Chronic Beryllium Disease Bronchoalveolar Lavage Cells Relate to T-Cell Function and Innate Immunity.

Chronic beryllium disease (CBD) is a Th1 granulomatous lung disease preceded by sensitization to beryllium (BeS). We profiled the methylome, transcriptome, and selected proteins in the lung to identify molecular signatures and networks associated with BeS and CBD. BAL cell DNA and RNA were profiled using microarrays from CBD (n = 30), BeS (n = 30), and control subjects (n = 12). BAL fluid proteins were measured using Olink Immune Response Panel proteins from CBD (n = 22) and BeS (n = 22) subjects. Linear models identified features associated with CBD, adjusting for covariation and batch effects. Multiomic integration methods identified correlated features between datasets. We identified 1,546 differentially expressed genes in CBD versus control subjects and 204 in CBD versus BeS. Of the 101 shared transcripts, 24 have significant cis relationships between gene expression and DNA methylation, assessed using expression quantitative trait methylation analysis, including genes not previously identified in CBD. A multiomic model of top DNA methylation and gene expression features demonstrated that the first component separated CBD from other samples and the second component separated control subjects from remaining samples. The top features on component one were enriched for T-lymphocyte function, and the top features on component two were enriched for innate immune signaling. We identified six differentially abundant proteins in CBD versus BeS, with two (SIT1 and SH2D1A) selected as important RNA features in the multiomic model. Our integrated analysis of DNA methylation, gene expression, and proteins in the lung identified multiomic signatures of CBD that differentiated it from BeS and control subjects.

HLA-DPB1 E69 genotype and exposure in beryllium sensitisation and disease.

OBJECTIVES: Human leukocyte antigen-DP beta 1 (HLA-DPB1) with a glutamic acid at the 69th position of the ß chain (E69) genotype and inhalational beryllium exposure individually contribute to risk of chronic beryllium disease (CBD) and beryllium sensitisation (BeS) in exposed individuals. This retrospective nested case-control study assessed the contribution of genetics and exposure in the development of BeS and CBD. METHODS: Workers with BeS (n=444), CBD (n=449) and beryllium-exposed controls (n=890) were enrolled from studies conducted at nuclear weapons and primary beryllium manufacturing facilities. Lifetime-average beryllium exposure estimates were based on workers' job questionnaires and historical and industrial hygienist exposure estimates, blinded to genotype and case status. Genotyping was performed using sequence-specific primer-PCR. Logistic regression models were developed allowing for over-dispersion, adjusting for workforce, race, sex and ethnicity. RESULTS: Having no E69 alleles was associated with lower odds of both CBD and BeS; every additional E69 allele increased odds for CBD and BeS. Increasing exposure was associated with lower odds of BeS. CBD was not associated with exposure as compared to controls, yet the per cent of individuals with CBD versus BeS increased with increasing exposure. No evidence of a gene-by-exposure interaction was found for CBD or BeS. CONCLUSIONS: Risk of CBD increases with E69 allele frequency and increasing exposure, although no gene by environment interaction was found. A decreased risk of BeS with increasing exposure and lack of exposure response in CBD cases may be due to the limitations of reconstructed exposure estimates. Although reducing exposure may not prevent BeS, it may reduce CBD and the associated health effects, especially in those carrying E69 alleles.

Beryllium disease among construction trade workers at Department of Energy nuclear sites: A follow-up.

BACKGROUND: Construction workers at U.S. Department of Energy (DOE) nuclear weapons facilities are screened to identify DOE-related occupational illnesses, including beryllium sensitization (BeS) and chronic beryllium disease (CBD). The study objectives were to estimate beryllium disease risks and the CBD claims acceptance rate in the energy workers' benefits program. METHODS: Workers diagnosed with BeS via beryllium lymphocyte proliferation test (BeLPT) included in screening examinations were interviewed about subsequent diagnosis of CBD. We estimated the proportion who developed CBD based on the ratio of CBD cases, based on self-reported compensation claim status, to all workers with BeS interviewed. We used stratified analyses to explore trends in disease frequency by age, race, sex, DOE employment duration, site, trade group, and cigarette smoking history. RESULTS: Between 1998 and 2020, 21,854 workers received a BeLPT; 262 (1.20%) had BeS (two abnormals or one abnormal plus one borderline test); 212 (0.97%) had a single abnormal BeLPT. Of 177 BeS workers interviewed, 35 (19.8%) reported an accepted CBD compensation claim. The claims acceptance rate among BeS workers increased with years of DOE employment, from 8.4% with <5 years to 33.3% for >25 or more years. Five of 68 interviewed workers with a single positive BeLPT reported CBD claim acceptance; an additional CBD case was confirmed by chart review (8.8%). CONCLUSIONS: Years of DOE work predict the risk of developing CBD among those sensitized and getting a claim for CBD accepted. Ongoing surveillance and increased awareness of the risk of beryllium exposure and CBD as an occupational disease among construction workers are needed.

[Sarcoidosis and berylliosis]. / Sarkoidose und Berylliose.

Sarcoidosis and berylliosis (chronic beryllium disease, CBD) are granulomatous diseases and are phenocopies which cannot be differentiated based on the clinical presentation. Whereas for sarcoidosis the eliciting agent is unknown, for berylliosis an exposure to beryllium (mostly as occupational exposure) can be confirmed that therefore induces a sensitization against beryllium. The diagnosis is generally made in patients with a typical clinical presentation, the histological proof of a non-necrotizing granuloma and the exclusion of other diseases causing granulomas. In most cases, granulomas can be detected in the lungs and/or (intrathoracic) lymph nodes. The proof of sensitization to beryllium for the differential diagnosis can be performed with a so-called beryllium lymphocyte proliferation test in peripheral mononuclear blood cells or cells from a bronchoalveolar lavage. The objectives of treatment are avoidance of functional organ impairment and symptom control. Immunosuppressive therapy (initially mostly with corticosteroids) and supportive measures can prove beneficial; however, in many cases clinical observation can be sufficient because of stable disease or spontaneous resolution. In addition, further beryllium exposure must be avoided, which mostly necessitates a change of the workplace.

Analysis of single nucleotide polymorphisms in chronic beryllium disease.

Sarcoidosis and chronic beryllium disease (CBD) are phenocopies, however the latter one has a clear trigger factor that is beryllium exposure. This study analyses single nucleotide polymorphisms (SNPs) in a large cohort for beryllium-exposed persons. SNPs were chosen for their relevance in sarcoidosis. Even though one of largest cohorts of beryllium-exposed persons was analysed, no statistically relevant association between any SNP and CBD could be verified. Notably, some SNPs exhibit inverse OR for beryllium sensitization and CBD with nominally statistical significance, which allows hypothesizing about pathophysiological role of genes for the disease triggering and development.

The emerging role of inorganic elements as potential antigens in sarcoidosis.

PURPOSE OF REVIEW: Previous studies mainly described a role for organic agents as possible triggers for sarcoidosis. In this review, we address recent studies suggesting a possible role for inorganic elements, such as metals or silica in sarcoidosis pathogenesis. RECENT FINDINGS: Several epidemiological papers suggest that inorganic agents, either by environmental exposures or occupational activities, could trigger sarcoidosis. Association between inorganics and sarcoidosis is also described in several recently published case reports and studies demonstrating immunological sensitization to inorganic agents in sarcoidosis patients.Studies comparing chronic beryllium disease (CBD) and sarcoidosis suggest that although antigenic triggers may differ, underlying processes may be comparable.Besides the fact that a growing number of studies show a possible role for inorganic triggers, it is also suggested that inorganic triggered sarcoidosis may result in a more severe phenotype, including pulmonary fibrosis. SUMMARY: We can use the knowledge already gained on CBD pathogenesis to conduct further research into role of inorganics, such as metals and silica as antigens in sarcoidosis. Given the importance of a lymphocyte proliferation test (LPT) in diagnosing CBD, it seems obvious to also implement this test in the diagnostic work-up of sarcoidosis to identify patients with an inorganic antigenic trigger of their disease.

Age-associated B Cells Appear in Patients with Granulomatous Lung Diseases.

Rationale: A subpopulation of B cells (age-associated B cells [ABCs]) is increased in mice and humans with infections or autoimmune diseases. Because depletion of these cells might be valuable in patients with certain lung diseases, the goal was to find out if ABC-like cells were at elevated levels in such patients.Objectives: To measure ABC-like cell percentages in patients with lung granulomatous diseases.Methods: Peripheral blood and BAL cells from patients with sarcoidosis, beryllium sensitivity, or hypersensitivity pneumonitis and healthy subjects were analyzed for the percentage of B cells that were ABC-like, defined by expression of CD11c, low levels of CD21, FcRL 1-5 (Fc receptor-like protein 1-5) expression, and, in some cases, T-bet.Measurements and Main Results: ABC-like cells in blood were at low percentages in healthy subjects and higher percentages in patients with sarcoidosis as well as at high percentages among BAL cells of patients with sarcoidosis, beryllium disease, and hypersensitivity pneumonitis. Treatment of patients with sarcoidosis led to reduced percentages of ABC-like cells in blood.Conclusions: Increased levels of ABC-like cells in patients with sarcoidosis may be useful in diagnosis. The increase in percentage of ABC-like cells in patients with lung granulomatous diseases and decrease in treated patients suggests that depletion of these cells may be valuable.

Involvement of Dendritic Cells and Th17 Cells in Induced Tertiary Lymphoid Structures in a Chronic Beryllium Disease Mouse Model.

OBJECTIVE: To study airway pathophysiology and the role of dendritic cells (DCs) and IL-17 receptor (IL-17R) signals in a mouse model for CBD. METHODS: Here, we present a CBD mouse model in which mice were exposed to beryllium during three weeks. We also exposed IL-17R-deficient mice and mice in which DCs were depleted. RESULTS: Eight weeks after the initial beryllium exposure, an inflammatory response was detected in the lungs. Mice displayed inflammation of the lower airways that included focal dense infiltrates, granuloma-like foci, and tertiary lymphoid structure (TLS) containing T cells, B cells, and germinal centers. Alveolar cell analysis showed significantly increased numbers of CD4+ T cells expressing IFNγ, IL-17, or both cytokines. The pathogenic role of IL-17R signals was demonstrated in IL-17R-deficient mice, which had strongly reduced lung inflammation and TLS development following beryllium exposure. In CBD mice, pulmonary DC subsets including CD103+ conventional DCs (cDCs), CD11b+ cDCs, and monocyte-derived DCs (moDCs) were also prominently increased. We used diphtheria toxin receptor-mediated targeted cell ablation to conditionally deplete DCs and found that DCs are essential for the maintenance of TLS in CBD. Furthermore, the presence of antinuclear autoantibodies in the serum of CBD mice showed that CBD had characteristics of autoimmune disease. CONCLUSIONS: We generated a translational model of sarcoidosis driven by beryllium and show that DCs and IL-17R signals play a pathophysiological role in CBD development as well as in established CBD in vivo.

TLR9 and IL-1R1 Promote Mobilization of Pulmonary Dendritic Cells during Beryllium Sensitization.

Metal-induced hypersensitivity is driven by dendritic cells (DCs) that migrate from the site of exposure to the lymph nodes, upregulate costimulatory molecules, and initiate metal-specific CD4+ T cell responses. Chronic beryllium disease (CBD), a life-threatening metal-induced hypersensitivity, is driven by beryllium-specific CD4+ Th1 cells that expand in the lung-draining lymph nodes (LDLNs) after beryllium exposure (sensitization phase) and are recruited back to the lung, where they orchestrate granulomatous lung disease (elicitation phase). To understand more about how beryllium exposures impact DC function during sensitization, we examined the early events in the lung and LDLNs after pulmonary exposure to different physiochemical forms of beryllium. Exposure to soluble or crystalline forms of beryllium induced alveolar macrophage death/release of IL-1α and DNA, enhanced migration of CD80hi DCs to the LDLNs, and sensitized HLA-DP2 transgenic mice after single low-dose exposures, whereas exposures to insoluble particulate forms beryllium did not. IL-1α and DNA released by alveolar macrophages upregulated CD80 on immature BMDC via IL-1R1 and TLR9, respectively. Intrapulmonary exposure of mice to IL-1R and TLR9 agonists without beryllium was sufficient to drive accumulation of CD80hi DCs in the LDLNs, whereas blocking both pathways prevented accumulation of CD80hi DCs in the LDLNs of beryllium-exposed mice. Thus, in contrast to particulate forms of beryllium, which are poor sensitizers, soluble or crystalline forms of beryllium promote death of alveolar macrophages and their release of IL-1α and DNA, which act as damage-associated molecular pattern molecules to enhance DC function during beryllium sensitization.

Transplantation for work-related lung disease in the USA.

OBJECTIVES: Work-related lung diseases (WRLDs) are entirely preventable. To assess the impact of WRLDs on the US transplant system, we identified adult lung transplant recipients with a WRLD diagnosis specified at the time of transplant to describe demographic, payer and clinical characteristics of these patients and to assess post-transplant survival. METHODS: Using US registry data from 1991 to 2018, we identified lung transplant recipients with WRLDs including coal workers' pneumoconiosis, silicosis, asbestosis, metal pneumoconiosis and berylliosis. RESULTS: The frequency of WRLD-associated transplants has increased over time. Among 230 lung transplants for WRLD, a majority were performed since 2009; 79 were for coal workers' pneumoconiosis and 78 were for silicosis. Patients with coal workers' pneumoconiosis were predominantly from West Virginia (n=31), Kentucky (n=23) or Virginia (n=10). States with the highest number of patients with silicosis transplant were Pennsylvania (n=12) and West Virginia (n=8). Patients with metal pneumoconiosis and asbestosis had the lowest and highest mean age at transplant (48.8 and 62.1 years). Median post-transplant survival was 8.2 years for patients with asbestosis, 6.6 years for coal workers' pneumoconiosis and 7.8 years for silicosis. Risk of death among patients with silicosis, coal workers' pneumoconiosis and asbestosis did not differ when compared with patients with idiopathic pulmonary fibrosis. CONCLUSIONS: Lung transplants for WRLDs are increasingly common, indicating a need for primary prevention and surveillance in high-risk occupations. Collection of patient occupational history by the registry could enhance case identification and inform prevention strategies.

Health aspects of exposure to emissions from burning coal of high beryllium content: interactions with the immune system.

Beryllium has an impact on the human health of professionally or non-occupationally exposed people. Current evidence suggests that beryllium acts as a hapten with limited antigenic properties and is presented by antigen presenting cells to CD4+ T cells, which possess specific antigen receptors. The immunological changes in humoral immunoreactivity were considered biomarkers of beryllium exposure. In the present, due to the development of immunologic knowledge, tests of cellular immunity have promising potential for further research in this field. The historical view of the immune response to beryllium in acute and/or chronic beryllium disease is an example of the development of the interaction between mechanisms of innate and adaptive (specific), humoral and cellular immunity. The authors emphasize the increasing importance of immunological aspects in the studies of health impacts of human exposure to environmental pollutants.

Pneumoconiosis with a Sarcoid-Like Reaction Other than Beryllium Exposure: A Case Report and Literature Review.

Background: Chronic beryllium disease (CBD) is a granulomatous disease that resembles sarcoidosis but is caused by beryllium. Clinical manifestations similar to those observed in CBD have occasionally been reported in exposure to dusts of other metals. However, reports describing the clinical, radiographic, and pathological findings in conditions other than beryllium-induced granulomatous lung diseases, and detailed information on mineralogical analyses of metal dusts, are limited. Case presentation: A 51-year-old Japanese man with rapidly progressing nodular shadows on chest radiography, and a 10-year occupation history of underground construction without beryllium exposure, was referred to our hospital. High-resolution computed tomography showed well-defined multiple centrilobular and perilobular nodules, and thickening of the intralobular septa in the middle and lower zones of both lungs. No extrathoracic manifestations were observed. Pathologically, the lung specimens showed 5-12 mm nodules with dust deposition and several non-necrotizing granulomas along the lymphatic routes. X-ray analytical electron microscopy of the same specimens revealed aluminum, iron, titanium, and silica deposition in the lung tissues. The patient stopped smoking and changed his occupation to avoid further dust exposure; the chest radiography shadows decreased 5 years later. Conclusion: The radiological appearances of CBD and sarcoidosis are similar, although mediastinal or hilar lymphadenopathy is less common in CBD and is usually seen in the presence of parenchymal opacities. Extrathoracic manifestations are also rare. Despite limited evidence, these findings are similar to those observed in pneumoconiosis with a sarcoid-like reaction due to exposure to dust other than of beryllium. Aluminum is frequently detected in patients with pneumoconiosis with a sarcoid-like reaction and is listed as an inorganic agent in the etiology of sarcoidosis. It was also detected in our patient and may have contributed to the etiology. Additionally, our case suggests that cessation of dust exposure may contribute to improvement under the aforementioned conditions.

Polymorphism of FCGR3A gene in chronic beryllium disease.

Previously we showed that alveolar macrophages (AMs) from patients with chronic beryllium disease (CBD) and beryllium sensitization (BeS) demonstrated significantly greater cell surface CD16 (encoded by the FCGR3A gene) than controls. We hypothesized that these differences were related to polymorphisms in the FCGR3A gene. This study was to determine the association between FCGR3A polymorphisms in CBD, BeS versus controls as well as clinical data, providing potential information about disease pathogenesis, risk, and activity. A total of 189 CBD/154 BeS/150 controls (92 Be-exposed non-diseased and 58 healthy controls) were included in this study. Sequence-specific primers polymerase chain reaction (PCR-SSP) was used to determine FCGR3A 158V/F polymorphisms. We found significantly higher frequencies of the 158V allele (OR: 1.60 (CI: 1.17-2.19), p = 0.004) and 158VV homozygotes (OR: 2.97 (CI: 1.48-5.97) p = 0.007) in CBD versus controls. No differences were found in the frequencies of FCGR3A alleles or genotypes between BeS versus controls and CBD versus BeS. Average changes in exercise testing maximum workload (Wlm), maximum oxygen consumption (VO2m), and diffusion capacity of carbon monoxide (DLCO) demonstrated greater decline over time in those CBD cases with the 158VV gene, modeled between 10 and 40 years from first beryllium exposure. The FCGR3A V158F polymorphism is associated with CBD compared to BeS and controls and may impact lung function in CBD.

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.