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Abstract Introduction The lung is the most commonly affected organ in sarcoidosis, present in over 90% of cases. However, cardiac involvement may also occur and can significantly complicate patient outcomes. We propose to explore further. Methods Adults with isolated pulmonary sarcoidosis and combined pulmonary-cardiac sarcoidosis were identified using ICD-10 codes from the National Inpatient Sample (2016-2022). Patients with any additional organ involvement beyond lung or heart were excluded to ensure distinct comparison groups. We compared age, sex, race, insurance type, hospital characteristics, comorbidities, and healthcare burden between groups. Results Over the seven-year study period, we identified 154,125 hospitalizations of patients with either isolated pulmonary or combined pulmonary-cardiac sarcoidosis. Of these, 2.41% (n = 3,715) were combined pulmonary-cardiac cases, while the remainder were isolated pulmonary sarcoidosis. Patients with combined pulmonary-cardiac sarcoidosis were significantly younger(mean age: 58.2 vs. 62.0 years, p < 0.001), but had a higher mean Charlson Comorbidity Index (CCI) score (3.49 vs. 2.77, p < 0.001) compared to those with isolated pulmonary disease. Multiple demographic differences were found: isolated pulmonary cases were predominantly female(59.5%), with a higher proportion of White patients(46.7%), whereas combined cases were predominantly male(60.4%), with a higher proportion of Black patients(53.0%). Both groups were primarily covered by Medicare(58.1% of isolated vs. 48.1% of combined), with a higher proportion of combined pulmonary-cardiac sarcoidosis cases treated at urban teaching centers(92.3% vs. 78.9%, p < 0.001). Patients with combined pulmonary-cardiac sarcoidosis demonstrated a higher prevalence of multiple comorbidities, including chronic kidney disease, prior myocardial infarction, complete atrioventricular block, obstructive sleep apnea, congestive heart failure, peripheral vascular disease, and valvular disease. Conversely, the prevalence of chronic obstructive pulmonary disease was lower in this group. Hospitalization burden was substantially greater among patients with combined pulmonary-cardiac sarcoidosis (Charlson Comorbidity Index 2.77±0.01 vs. 3.49±0.07), evidenced by longer mean length of stay (6.1±0.05 vs. 7.6±0.32 days) and significantly higher total hospital charges ($82,987±1,121 vs. $122,460±6,991) (Table 1). Conclusions Combined pulmonary-cardiac sarcoidosis affected younger patients with greater comorbidity and hospitalization burden, with notable racial disparities. As cardiac sarcoidosis is frequently underdiagnosed, systematic cardiac screening protocols in high-risk sarcoidosis patients are essential for earlier detection and improved management. This abstract is funded by: None

Abstract Introduction/Rationale Hepatic sarcoidosis (HS) is a rare manifestation of sarcoidosis occurring in 6 to 11% of patients with sarcoidosis. Little is known about the clinical characteristics and overall outcome of patients who present with HS. Two recent single center studies from Europe and the U.S. outlined the clinical characteristics, outcomes, and therapeutic interventions in these patients. Our study aims to add to this body of knowledge; in particular, highlighting the clinical presentation, therapeutic interventions, comorbid disease burden, and overall outcomes in patients with HS presenting to a large tertiary care sarcoidosis center of excellence in the U.S. Methods We performed a retrospective review of all patients with hepatic sarcoidosis (HS) seen at the Sarcoidosis Center of Excellence at East Carolina University (ECU) between 2000 and 2025. Data including demographics, clinical manifestations, diagnostic tests, treatments and outcomes, were extracted. Results A total of 794 patients with sarcoidosis were identified. Twenty-one (21) patients had over 50% of missing data and were excluded. Of the 773 patients included in the study, 62 patients (8%) had hepatic sarcoidosis. Of these, 73% were female and 79% were African American. The mean age at diagnosis was 40 years. Thirty-nine percent (39%) of patients underwent a liver biopsy and majority (64%) had a Scadding CXR I or II at diagnosis. There was no significant difference in the age (at diagnosis), race, gender, BMI, smoking status, or pulmonary function when patients with HS were compared to the rest of the cohort (n = 711) (Table 1). CXR Scadding stage at presentation was also similar except that 5% of non-HS patients had a stage IV CXR at presentation compared to 0 patients with HS (Table 1). Evaluation for other organ involvement revealed that patients with HS were more likely to have splenic, Calcium-Vit D dysregulation, extra-thoracic lymphadenopathy, and bone/bone marrow involvement than non-HS patients (Table 2/Figure 1). Treatment patterns including use of methotrexate and infliximab were identical except that only 5% of patients were treated with ursodiol. This was unexpectedly low (Table 3). Outcomes including fatigue at any point during the follow-up and mortality are presented in Table 4. 10.4% of non-HS patients died versus 4.9% of HS patients. No patients in our cohort underwent a liver transplant. Conclusion Hepatic sarcoidosis was found in 8% of patients and was associated with multi-organ disease. Patients received similar medications, including methotrexate, as patients without hepatic sarcoidosis. This abstract is funded by: None

Abstract Rationale Systemic sclerosis (SSc) is a rare, chronic autoimmune disease characterized by vasculopathy and fibrosis of the skin and internal organs. Among its manifestations, interstitial lung disease (ILD) is the leading cause of mortality, yet its clinical course is variable, ranging from stable to progressive pulmonary fibrosis (PPF). The 2022 ATS/ERS/JRS/ALAT guidelines defined progressive PPF as a phenotype of non- Idiopathic Pulmonary Fibrosis (IPF) - ILDs that behave similarly to IPF. Although a subset of SSc-ILD patients develop PPF, biomarkers predicting progression remain lacking. We and others have shown that endothelial dysfunction contributes to the progression of fibrotic lung disease, and that the pulmonary vasculature, continuously exposed to the bloodstream, is influenced by soluble circulating factors, including circulating extracellular vesicles (cEVs), which carry bioactive cargo capable of modulating endothelial behavior. Here, we investigated whether cEVs from SSc patients with distinct fibrotic phenotypes differentially influence human lung endothelial cells (ECs), promoting a pro-fibrotic endothelial phenotype. Methods plasma samples were collected from five groups: 1) Healthy controls (n = 4); 2) SSc-noILD (no high-resolution-CT (HCRT( or functional evidence of ILD at time of enrollment, n = 4); 3) SSc-ILD (HCRT and functional evidence of ILD without meeting the PPF criteria at time of sampling, n = 3); 4) SSc-PPF (meeting the PPF criteria, plasma collected prior to anti-fibrotic therapy, n = 4); 5) IPF (plasma collected prior to anti-fibrotic therapy, n = 3). cEVs were isolated using size-exclusion-chromatography and characterized using Nanoparticle Tracking Analysis (NTA). Human lung ECs were treated with cEVs from the five groups (2x109 particles/ml, 48 hours), followed by qPCR for markers selected for their established involvement in endothelial dysfunction during lung fibrotic progression. Results NTA showed no significant differences in plasma cEV concentrations across the five groups. Human lung ECs exposed to cEVs from SSc or SSc-ILD patients exhibited no significant gene expression changes compared to healthy controls. In contrast, cEVs from SSc-PPF patients markedly downregulated endothelial identity (PECAM1, ERG, TEK), barrier integrity (CDH13, S1PR1), and basement membrane (COL4A1, HSPG2) genes, similarly to changes induced by cEVs from IPF patients. Conclusion cEVs from SSc-PPF patients induce pro-fibrotic transcriptional changes in lung ECs resembling those induced by IPF cEVs and distinct from SSc-ILD cEVs, suggesting that EVs carry cargo that both reflect and influence fibrosis progression. These findings highlight the potential of cEVs as biomarkers for identifying SSC-ILD patients at risk of progression and highlight the clinical relevance of future studies aimed at characterizing their cargo. This abstract is funded by: James Depauw Grant for Pilot Projects

Abstract A population of pathologic fibroblasts has been identified in several human fibrotic lung diseases, including IPF. These cells, marked by high expression of collagen triple-helix repeat containing 1 (CTHRC1), display an invasive phenotype and aggressively deposit extracellular matrix. The goal of this study was to identify transcriptional regulators of the CTHRC1+ pathologic fibroblast state. We analyzed published single-cell RNA sequencing data of IPF and control lungs to predict transcription factor activity in CTHRC1+ fibroblasts, which led us to hypothesize that transcription factors in the nuclear factor of activated T-cells (NFAT) family may contribute to the pathologic fibroblast state. To test whether NFAT family members promote and/or maintain pathologic fibroblasts, we silenced each cytosol-localized NFAT (NFATc1-4) in lung fibroblasts from IPF donors (passage 3-6, N = 5 donors) and measured expression of key NFAT target genes related to fibrosis using qPCR. NFATC1 silencing significantly reduced expression of CTHRC1, SERPINE1, FN1, HAS2, and TNC, whereas other computationally predicted regulators showed little effect. Our computational analysis also predicted high activator protein 1 (AP-1) activity in pathologic fibroblasts, which interestingly has been found to act in concert with NFATs to promote an effector response in T-cells. Thus, we hypothesized that NFAT:AP-1 signaling may be involved in lung fibroblast activation. To test this hypothesis, we employed a small molecule inhibitor of NFAT:AP-1 complex formation on DNA (“Compound 10”, 25-50 µM). NFAT:AP-1 inhibition significantly reduced expression of fibrillar collagens COL1A1 and COL3A1, as well as CTHRC1 in primary IPF lung fibroblasts. Additionally, NFAT:AP-1 inhibition prevented TGF-β-mediated increase of several fibrosis-related NFATc1 targets. Aside from CTHRC1, NFAT:AP-1 inhibition led to decrease in a different set of NFATc1 targets than NFATc1 silencing, suggesting a potentially complex relationship between NFATc1 and AP-1 in fibroblast gene regulation. Finally, in mouse precision cut lung slices (PCLS) stimulated with a fibrosis-inducing cocktail ex vivo, NFAT:AP-1 inhibition prevented increased expression of Col1a1 and Cthrc1. Taken together, these studies suggest that NFATc1, together with AP-1, is involved in fibrogenic activation of pathological fibroblasts. Ongoing and future experiments will determine the roles of these signaling mechanisms in matrix deposition and contraction, as well as fibroblast invasion using human fibroblasts and PCLS models. Furthermore, we aim to determine how mechanical forces and soluble factors encountered in IPF lungs promote NFAT signaling in fibroblasts. This abstract is funded by: NHLBI, Pulmonary Fibrosis Foundation

Abstract Pulmonary hyalinizing granuloma (PHG) is a rare lung disease that usually presents as nodules or masses and can be mistaken for malignancy or infection on imaging. Its etiology remains unclear, but it is frequently associated with autoimmune diseases, chronic inflammation, or prior infection. Given its nonspecific radiographic features PHG presents a diagnostic challenge, especially in patients with connective tissue disorders. A 64-year-old woman with long-standing Sjögren’s syndrome and interstitial lung disease, presented with a 20-year history of progressive dyspnea, morning joint stiffness, xerostomia, and xerophthalmia. Serologic testing revealed elevated ANA (1:320, speckled), positive anti-SS-A/Ro antibodies, rheumatoid factor (90 IU/mL), and anti-NXP-2 antibody, with normal IgG4 levels. Pulmonary function testing confirmed severe restrictive impairment and markedly reduced diffusion capacity. CT dating back at least 4 years demonstrated a usual interstitial pneumonia (UIP) pattern with a straight edge sign, the four corners sign and anterior predominance, consistent with connective tissue disease-associated ILD (CTD-ILD). Four years prior, lung biopsy showed pattern consistent with Nonspecific Interstitial Pneumonia. In the year leading up to her death, she was found to have Pseudomonas pneumonia within the cystic lung at the left base. We suspect chronic infection-related inflammation caused bilateral disease progression, with imaging now revealing thick consolidative opacities in both the lung apices and bases. PET-CT showed diffuse hypermetabolic activity in consolidative areas. Due to severe functional impairment, she received a bilateral lung transplant but died from surgery-related complications. Gross examination of explanted lungs revealed multiple firm, coalescent nodules within areas of fibrosis. Histopathologic analysis showed dense, lamellar, keloid-like hyaline collagen arranged concentrically around vessels with perivascular lymphoplasmacytic infiltrates, these findings were consistent with PHG. Special stains were negative for amyloid, iron, and infectious organisms. This case demonstrates the potential consequences of uncontrolled inflammation in patients with ILD and emphasizes the importance of considering empirical treatment due to the elevated risk of significant complications in advanced lung disease. In patients with Sjögren’s syndrome and progressive ILD, the coexistence of fibrotic and nodular lesions can obscure the distinction between progressive fibrosis, neoplasia, and rare entities such as PHG. Although corticosteroids and immunosuppressive agents may stabilize disease in select cases, no standardized therapy exists. Lung transplantation remains the only curative option in end-stage disease. Pulmonary hyalinizing granuloma should be considered in patients with autoimmune disorders and atypical pleural thickening or nodular consolidations. This abstract is funded by: None

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosing interstitial lung disease that continues to have high morbidity and mortality. There are only 3 approved medications, (nintedanib, pirfenidone and nerandomilast) which marginally slow FVC decline and only nerandomilast having some mortality benefit. All three have no effect on quality of life (exacerbations and hospitalizations). There is an urgent need to find more effective treatment options. Since IPF is predominantly a disease of the elderly with a peak prevalence in those 65 and older, it is common for these individuals to have other comorbidities such as Hyperlipidemia (HLD). Interestingly, in a prior small study of IPF patients, there is evidence to suggest clinical benefit from ezetimibe, a medication used to treat HLD. This study aims to further evaluate the clinical benefits of ezetimibe in patients with IPF in a large national cohort. Methods We conducted this study using the Optum Labs Data Warehouse (OLDW). The OLDW contains de-identified administrative claims data, including medical and pharmacy claims and enrollment records. This study used claims data from commercial, Medicare Advantage (MA), and Medicare Fee-for-service (FFS) enrollees between October 1st, 2014, and December 31st, 2021. Diagnosis of IPF was defined using ICD-9 and ICD-10 codes. Patients without a diagnosis of IPF were excluded. P < 0.05 was set to determine statistical significance. Two matched patient cohorts depending on ezetimibe use were defined. Primary outcome was all-cause mortality, and secondary outcome was hospitalizations. Results We identified 7724 patients with IPF. After adjusting for age, age group, gender, race, health plan, year of IPF diagnosis, smoking, diabetes, statin treatment, and antifibrotic treatment, two cohorts were identified and matched, patients treated with ezetimibe (n = 3862) and patients not treated with ezetimibe (n = 3862). Among the patients with IPF treated with ezetimibe, ezetimibe use was associated with a reduction in all-cause mortality (hazard ratio [95% CI]: 0.704 [0.660 - 0.752], p-value: <0.001) and hospitalizations(hazard ratio [95% CI]: 0.907 [0.857 - 0.959], p-value: 0.001) compared to patients not treated with ezetimibe. Conclusion In patients with IPF, treatment with ezetimibe may be associated with improved clinical outcomes. Further studies are necessary to confirm benefits, assess anti-fibrotic effects, and inform implementation in management of IPF. As IPF remains a disease with limited treatment options, repurposing drugs continues to offer an avenue to find affordable, safe and already approved medications for use. This abstract is funded by: None

Abstract Rationale Interstitial lung abnormalities (ILA) are abnormal findings on chest CT scans that may represent early-stages of interstitial lung disease. The presence of fibrosis (traction bronchiectasis and/or honeycombing) identifies those with ILA at an increased risk for mortality, however, determining who will progress remains a challenge, and the role of blood-based biomarkers in ILA risk-stratification is unknown. Methods Data from four prospective cohort studies were used to identify associations between plasma proteins measured with DNA aptamers (SomaScan) and ILA, fibrosis, and mortality. The Genetic Epidemiology of COPD Study (COPDGene) was used to identify protein associations, and findings were replicated by meta-analysis of the Age, Gene/Environment Susceptibility-Reykjavik study (AGES-Reykjavik), SubPopulations and InteRmediate Outcome Measures in COPD Study (SPIROMICS), and Framingham Heart Study (FHS). Protein associations with ILA and with fibrotic ILA compared to non-fibrotic ILA were performed using multivariable logistic regression; among those with ILA, Cox proportional hazards models were used to identify protein associations with mortality. Models were adjusted for age, gender, race, BMI, smoking status, pack-year smoking history, and for mortality, COPD. Benjamini-Hochberg false discovery rate p-values <0.05 were considered significant. Results 524 (10.7%) of 4,899 COPDGene participants had ILA, of whom 57 (10.9%) had fibrosis; of 8,146 AGES-Reykjavik, SPIROMICS and FHS participants included in the meta-analysis, 666 (8.2%) had ILA, of whom 198 (29.7%) had fibrosis. In COPDGene, 230 proteins were associated with ILA; the most significant of which were SFTPD (adjusted odds ratio per standard deviation [SD] of protein [OR]=2.21, p = 3.7E-41) and SFTPB (OR = 1.82, p = 7.9E-37). In the meta-analysis, 159 of the ILA-associated proteins were replicated. Of the 230 ILA-associated proteins in COPDGene, 9 were associated with fibrosis, and 5 of these 9 proteins were associated with mortality among those with ILA. The 5 proteins associated with all three outcomes were WFDC2 (ORILA=1.90, p = 2.6E-34; ORFibrosis=2.00, p = 0.0001; adjusted hazards of mortality per SD of protein [HR]=1.59, p = 3.4E-05), GDF15 (ORILA=1.67, p = 3.6E-23; ORFibrosis=1.78, p = 0.0006; HR = 1.63, p = 1.4E-06), SCGB3A1 (ORILA=1.52, p = 4.5e-14; ORFibrosis=1.80, p = 0.0002; HR = 1.25, p = 0.02), TREM1 (ORILA=1.44, p = 1.4e-14; ORFibrosis=1.62, p = 0.001; HR = 1.25, p = 0.02) ICAM5 (ORILA=1.42, p = 1.2e-11; ORFibrosis=1.79, p = 0.0001; HR = 1.31, p = 0.002). In the meta-analysis, 6 of the 9 fibrosis-associated proteins and all 5 ILA mortality-associated proteins replicated. Conclusion This represents the first plasma proteomic screen demonstrating reproducible biomarker associations not only with ILA presence, but also with measures of severity and adverse outcomes. These results provide compelling targets for further study, including their prospective evaluation as clinical biomarkers for early disease identification and prognostication. This abstract is funded by: Multiple Sources

Abstract Background Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity in India. Newly available 2025 ambient PM2.5 snapshots and expanding Health Management Information System (HMIS) reporting to examine how current exposure and TB burden map to COPD disease burden. Methods We combined publicly reported January-May 2025 PM2.5 monthly averages from the CREA/CPCB snapshots (state capital/city aggregates) with state-level COPD DALY estimates from published India state-level disease burden reports (PHFI/IHME GBD summaries) and GBD TB incidence, where available. We harmonized state names and matched capital/city PM2.5 to their states to create a state-level table. Spearman rank correlation assessed associations between mean PM2.5 (Jan-May 2025) and COPD DALYs per 100,000; TB incidence was examined as a secondary covariate. (See Methods note below for data provenance and limitations.) Results 2025 PM2.5 snapshots indicate widespread exceedance of WHO and national guidelines, with northern/industrial states showing the highest monthly means (e.g., Delhi >150 µg/m³ in Jan 2025 on CREA snapshots). In an illustrative state-level aggregation using CREA’s Jan-May 2025 capital-city PM2.5 summaries merged with public GBD/PHFI COPD DALY estimates, PM2.5 and COPD DALYs were strongly positively associated (Spearman ρ ≈ 0.99, p < 0.001 using the illustrative aggregation provided). TB incidence showed a moderate positive correlation with COPD DALYs in this aggregation (Spearman ρ ≈ 0.39, p ≈ 0.16). The accompanying figure (two panels) visualizes state COPD DALY burden and the PM2.5-DALY relationship; the CSV shows the aggregated values used. Limitations Ecological aggregation may overstate the strength of observed associations due to unmeasured confounders (e.g., age, smoking, urbanization). Individual-level causality cannot be inferred; further cohort and longitudinal studies are warranted. Policy Implications These findings underscore the urgency of PM2.5 mitigation in high-burden states and advocate scaling up systematic post-TB spirometry, alongside strengthened COPD surveillance via HMIS. Actionable priorities include strict industrial emission controls, targeted clean air campaigns in megacities, and regional COPD-TB risk screenings. Conclusions State-level 2025 PM2.5 exposure and TB burden map onto regions of higher COPD burden, suggesting that current air-quality conditions and TB sequelae remain important, interacting drivers of COPD morbidity in India. These late-breaking snapshots support urgent policies to reduce PM2.5 and implement systematic post-TB spirometry and strengthened COPD surveillance through HMIS. This abstract is funded by: None

Abstract Rationale Mismatch of airway to lung size has been implicated in chronic obstructive pulmonary disease (COPD) pathophysiology, whether arising from disproportioned growth or from acquired pathologies over the life course. Lower airway-to-lung ratio (ALR), assessed reproducibly via computed tomography (CT) as the mean airway lumen diameters (trachea-to-subsegments) divided by the cubed root of lung volume, is associated with COPD risk and mortality (all-cause, respiratory, cardiovascular, lung cancer). We sought to characterize the airway biology associated with lower ALR by profiling bronchial gene expression across current and former smokers. Methods We leveraged clinical, radiologic, and bronchial epithelial RNA sequencing data from the SPIROMICS (SubPopulations and Intermediate Outcomes Measures in COPD Study) Phase 1 Bronchoscopy Sub-study (n = 161). To analyze transcriptional differential expression (DE) between participants based on CT-assessed ALR, we utilized DESeq2 and then identified impacted pathways by Ingenuity Pathway Analysis (IPA). Generating cell-type transcriptional signatures from lung and blood single cell sequencing atlases, we also assessed association of ALR with differences in airway cellular composition. Results Across all ever-smokers, after adjustment for age, sex, height, COPD status, smoking status, and pack-years, n = 1,324 genes from airway epithelial samples exhibited DE at a false discovery rate (FDR) <0.2. Per IPA, enriched pathways associated with lower ALR included neutrophilic degranulation and macrophage activity. In cell type analysis, lower ALR was associated with higher signatures of macrophages, neutrophils, dendritic cells, and B cells, and lower ciliated and club cell signatures. When we repeated DE analysis using the same covariate adjustments plus an interaction term for ALR and smoking, n = 713 DE genes were identified at FDR <0.2; the association of DE with lower ALR was overwhelmingly in current rather than former smokers (3699 vs. 2 genes). By pathway and cell type analyses, the association between lower ALR and heightened inflammation (greater macrophage and neutrophil signaling) and epithelial dysregulation (downregulated ciliated and club cell function) was primarily restricted to current smokers. Conclusions In adult ever-smokers, lower ALR is associated with transcriptomic evidence of heightened neutrophil- and macrophage-driven airway inflammation and reduced ciliated and club cell expression, associations that were most evident among current smokers. Whether these cross-sectional patterns, which suggest impaired mucociliary defense and a diminished capacity to clear inhaled particulates, are the result of pre-existing reduced ALR, its cause, or both, will require further investigation. This abstract is funded by: SPIROMICS was supported by contracts from the NIH/NHLBI (HHSN268200900013C, HHSN268200900014C, HHSN268200900015C, HHSN268200900016C, HHSN268200900017C, HHSN268200900018C, HHSN268200900019C, HHSN268200900020C, 75N92024D00012), grants from the NIH/NHLBI (U01HL137880, U24HL141762, R01HL182622, R01HL144718, and R01HL093081), and supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from Amgen; AstraZeneca/MedImmune; Bayer; Bellerophon Therapeutics; Boehringer-Ingelheim Pharmaceuticals, Inc.; Bristol Myers Squibb; Chiesi Farmaceutici S.p.A.; Forest Research Institute, Inc.; Genentech; GlaxoSmithKline; Grifols Therapeutics, Inc.; Ikaria, Inc.; MGC Diagnostics; Novartis Pharmaceuticals Corporation; Nycomed GmbH; Polarean; ProterixBio; Regeneron Pharmaceuticals, Inc.; Sanofi; Sunovion; Takeda Pharmaceutical Company; Theravance Biopharma; Verona; and Mylan/Viatris.

Abstract Rationale Eosinophilic airway inflammation in COPD is associated with more frequent exacerbations,lower lung function,and corticosteroid responsiveness.DNA methylation captures information about genetic factors and environmental exposures, but is under-studied in eosinophilic COPD.We hypothesized that sputum cell methylation patterns reflect genomic regulation of inflammatory endotypes in COPD by DNA sequence variation. Methods We analyzed DNA previously extracted from induced sputum cells for a prior study(PMID38261629) for which sufficient quantity DNA remained for methylation arrays.For this analysis,we focused on n = 132 SPIROMICS participants with ≥20 pack-years smoking history.Samples were stratified into tertiles based on sputum eosinophil proportions,previously determined from sputum cell differential analysis by the centralized sputum analysis core for SPIROMICS(Univ of North Carolina).We assessed proportion of DNA methylation (β values) spanning 936,866 CpG sites using the Illumina Infinium Methylation EPICv2.0 BeadChip Array,which we transformed into M values(meffil R package). To identify differentially methylated regions (DMRs)(FDR<0.05 in minimum of 2 CpG sites per gene region),we contrasted CpG β values between the high-eosinophil and low-eosinophil tertiles(N = 44 each),using the DMRcate package.We tested for enrichment of genes overlapping with significant DMRs using Enrichr (GWAS Catalog 2025 gene set library).To identify genetic variants associated with DNA methylation levels,we performed Methylation Quantitative Trait Loci (meQTL) analysis using TensorQTL.For each CpG site,we tested associations with SNPs within a ± 500 kb window (cis-meQTL).The linear regression model included age,sex,BMI,smoking status,smoking pack-years,study site,inhaled corticosteroid use, the first two genetic principal components, cell-type proportions (macrophages/monocytes, neutrophils, squamous epithelial cells, lymphocytes), and eosinophil tertile . Significant meQTLs were identified at an FDR<0.05 using Benjamini-Hochberg correction. Results A total of 532 eosinophil-associated DMRs (FDR < 0.05) were identified between the high- and low-eosinophil groups,encompassing 446 unique genes.The top enriched pathways related to counts of eosinophils (adjusted p = 0. 00002464), lymphocytes (adjusted p = 0.0001860), and the sum of eosinophils-plus-basophils (adjusted p = 0.0001860).We identified 18,432 significant SNP-CpG/meQTL pairs (FDR < 0.05),suggesting extensive genetic influence on airway DNA methylation patterns.Among the n = 521 eosinophil-associated CpGs that overlapped with meQTLs,rs55687040 (TENT2,a gene involved in mRNA and miRNA processing) is a GWAS locus for FEV1/FVC ratio (PMID: 26634245), suggesting possible genetic control of airway eosinophilic methylation signatures. Conclusions Our findings disclose distinct DNA methylation signatures associated with sputum eosinophil proportion in COPD.A subset of these CpGs are regulated by genetic variants,suggesting an integrated genetic-epigenetic mechanism underlying airway eosinophilic inflammation with the potential to influence COPD risk and severity. This abstract is funded by: SPIROMICS was supported by contracts from the NIH/NHLBI (HHSN268200900013C, HHSN268200900014C, HHSN268200900015C, HHSN268200900016C, HHSN268200900017C, HHSN268200900018C, HHSN268200900019C, HHSN268200900020C), grants from the NIH/NHLBI (U01 HL137880, U24 HL141762, R01 HL182622, and R01 HL144718), and supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from Amgen; AstraZeneca/MedImmune; Bayer; Bellerophon Therapeutics; Boehringer-Ingelheim Pharmaceuticals, Inc.; Chiesi Farmaceutici S.p.A.; Forest Research Institute, Inc.; Genentech; GlaxoSmithKline; Grifols Therapeutics, Inc.; Ikaria, Inc.; MGC Diagnostics; Novartis Pharmaceuticals Corporation; Nycomed GmbH; Polarean; ProterixBio; Regeneron Pharmaceuticals, Inc.; Sanofi; Sunovion; Takeda Pharmaceutical Company; and Theravance Biopharma and Mylan/Viatris.