BACKGROUND:Accurate diagnosis of interstitial lung disease remains challenging. Multidisciplinary discussion is the current gold standard; however, it is resource-intensive and relies on expert opinion. Biologically based markers of disease are needed. RESEARCH QUESTION:Can measurements of circulating proteins aid in the differentiation of idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (FHP), and would such proteins reflect differences in immune function? STUDY DESIGN AND METHODS:We measured circulating matrix metalloproteinases in patients enrolled in the Pulmonary Fibrosis Foundation Patient Registry and a multisite interstitial lung disease cohort with either IPF or FHP using a high-throughput proteomic platform (Olink Explore 3072). Single-variable logistic regression was used to evaluate each protein's ability to differentiate IPF and FHP. Proteins with an area under the receiver operating characteristic curve > 0.70 were included in multivariable logistic regression. Gene ontology, KEGG, and Gene Set Enrichment Analysis of whole-blood messenger RNA revealed relationships between matrix metalloproteinase 12 (MMP12) abundance and transcriptional expression. RESULTS:MMP12 was significantly elevated in IPF compared with FHP (IPF, 1.43 ± 0.9; FHP, 0.69 ± 0.9; P < .001). MMP12 improved differentiation by increasing the area under the receiver operating characteristic curve from 0.81 to 0.85 when added to a model developed from clinical, lung function, and radiologic data (OR, 2.16; 95% CI, 1.44-3.25; P < .001). Gene Set Enrichment Analysis comparing patients with high (≥ median) vs low (< median) MMP12 found enrichment in interleukin, granulocyte-monocyte colony-stimulating factor, and CD28 costimulation pathways. Gene ontology showed enrichment in biological processes related to host response to external stimuli, whereas KEGG revealed enrichment in T-cell differentiation and viral signaling. INTERPRETATION:Our results show that semiquantitative MMP12 measurement improves the differentiation of IPF and FHP compared with clinical, lung function, and radiologic variables. Elevated MMP12 was associated with enrichment of interleukin/granulocyte-monocyte colony-stimulating factor signaling and T-cell regulation pathways.

Rheumatoid arthritis-associated autoantibodies have been associated with interstitial lung abnormalities (ILA) among community-dwelling adults. We aimed to determine whether greater numbers of positive anti-citrullinated protein antibodies (ACPAs) are associated with ILA in community-dwelling adults. We performed a period cross-sectional study. We measured 29 ACPAs via Bio-Plex bead array (2003–2005) and ILA on full-lung CTs (2010–2012) in 868 participants enrolled in the Multi-Ethnic Study of Atherosclerosis. A positive ACPA was defined as titer > 75th percentile. We used generalized additive and logistic regression models to examine associations between number of positive ACPA and ILA and tested for effect modification by race/ethnicity. In an adjusted model, each 1 unit increase in log-transformed number of positive ACPA was associated with a 17

Progressive interstitial lung disease (ILD) leads to declining lung function and death. New therapies to treat ILD are urgently needed. Here we performed a secondary analysis of proteomic data from ten ILD cohorts across the United States, Canada, and United Kingdom. Causal mediation analysis was used to estimate the effect of plasma proteins previously linked to organ fibrosis in mechanistic studies (exposure) on survival (outcome) through lung function decline (mediator). Of 102 proteins tested in a discovery cohort (n = 1963), 47 were mediated by declining lung function. Of these 47 proteins, 7 showed sustained mediation in an independent validation cohort (n = 1172). Proteins with the strongest mediated effect were amphiregulin and integrin beta six. Sensitivity analysis showed that results were robust to unmeasured confounding. Here we provide epidemiological evidence implicating seven proteins as potentially causal of progressive ILD. These findings build upon mechanistic studies showing a causal link between these proteins and organ fibrosis, supporting their prioritization for therapeutic consideration.

Rationale: Rheumatoid arthritis (RA) has been implicated in interstitial lung disease, as the majority of studies have comprised patients with known RA. However, it remains unclear whether an underlying risk for RA in combination with genetic risk for pulmonary fibrosis is associated with radiological markers of early lung injury and fibrosis in broader population samples. Objective: We sought to determine whether genetic and serological biomarkers of RA risk in combination with the MUC5B (rs35705950) risk allele (T) are associated with interstitial lung abnormalities (ILAs) on computed tomography scans. Methods: Associations of RA-risk HLA-DRB1 alleles ( * 04:01 , * 04:08 , * 04:05 , * 04:04 , and * 10:01 ) and serum RA autoantibodies with ILA in the Multi-Ethnic Study of Atherosclerosis (MESA; n = 4,018) and COPDGene (n = 5,963) cohorts were modeled using logistic regression and adjusted for age, sex, self-reported race and ethnicity, smoking history, body mass index, and principal components of genetic ancestry. Results: The prevalence of an RA-risk HLA-DRB1 allele was 16.5% and 21.9% in the MESA and COPDGene cohorts, respectively. ILA was present in 3.9% and 11% of the MESA and COPDGene cohorts, respectively. An RA-risk HLA-DRB1 allele was not significantly associated with ILA in the MESA and COPDGene cohorts. In the MESA cohort, higher serum levels of immunoglobulin (Ig)A rheumatoid factor (RF) and anticyclic citrullinated peptide were associated with odds ratios for ILA of 1.20 (95% confidence interval [CI] = 1.07-1.35) and 1.19 (95% CI = 1.04-1.38), respectively. Among smokers without baseline ILA, per doubling of IgM RF was associated with an odds ratio for ILA 10 years later of 1.25 (95% CI = 1.08-1.44). Associations were not significantly different by MUC5B risk allele status. Conclusions: RA-related HLA-DRB1 alleles were not associated with ILA, whereas higher serum levels of IgM RF among smokers without baseline ILA were associated with subsequent ILA.

Rationale: Rheumatoid arthritis (RA) has been implicated in interstitial lung disease, as the majority of studies have comprised patients with known RA. However, it remains unclear whether an underlying risk for RA in combination with genetic risk for pulmonary fibrosis is associated with radiological markers of early lung injury and fibrosis in broader population samples. Objective: We sought to determine whether genetic and serological biomarkers of RA risk in combination with the MUC5B (rs35705950) risk allele (T) are associated with interstitial lung abnormalities (ILAs) on computed tomography scans. Methods: Associations of RA-risk HLA-DRB1 alleles (*04:01, *04:08, *04:05, *04:04, and *10:01) and serum RA autoantibodies with ILA in the Multi-Ethnic Study of Atherosclerosis (MESA; n = 4,018) and COPDGene (n = 5,963) cohorts were modeled using logistic regression and adjusted for age, sex, self-reported race and ethnicity, smoking history, body mass index, and principal components of genetic ancestry. Results: The prevalence of an RA-risk HLA-DRB1 allele was 16.5% and 21.9% in the MESA and COPDGene cohorts, respectively. ILA was present in 3.9% and 11% of the MESA and COPDGene cohorts, respectively. An RA-risk HLA-DRB1 allele was not significantly associated with ILA in the MESA and COPDGene cohorts. In the MESA cohort, higher serum levels of immunoglobulin (Ig)A rheumatoid factor (RF) and anticyclic citrullinated peptide were associated with odds ratios for ILA of 1.20 (95% confidence interval [CI] = 1.07-1.35) and 1.19 (95% CI = 1.04-1.38), respectively. Among smokers without baseline ILA, per doubling of IgM RF was associated with an odds ratio for ILA 10 years later of 1.25 (95% CI = 1.08-1.44). Associations were not significantly different by MUC5B risk allele status. Conclusions: RA-related HLA-DRB1 alleles were not associated with ILA, whereas higher serum levels of IgM RF among smokers without baseline ILA were associated with subsequent ILA.

Rationale: Oral microbiota associate with diseases of the mouth and serve as a source of lung microbiota. However, the role of oral microbiota in lung disease is unknown. Objectives: To determine associations between oral microbiota and disease severity and death in idiopathic pulmonary fibrosis (IPF). Methods: We analyzed 16S rRNA gene and shotgun metagenomic sequencing data of buccal swabs from 511 patients with IPF in the multicenter CleanUP-IPF (Study of Clinical Efficacy of Antimicrobial Therapy Strategy Using Pragmatic Design in IPF) trial. Buccal swabs were collected from usual care and antimicrobial cohorts. Microbiome data were correlated with measures of disease severity using principal component analysis and linear regression models. Associations between the buccal microbiome and mortality were determined using Cox additive models, Kaplan-Meier analysis, and Cox proportional hazards models. Measurements and Main Results: Greater buccal microbial diversity associated with lower FVC at baseline (mean difference, -3.60; 95% confidence interval [CI], -5.92 to -1.29% predicted FVC per 1-unit increment). The buccal proportion of Streptococcus correlated positively with FVC (mean difference, 0.80; 95% CI, 0.16 to 1.43% predicted per 10% increase) (n = 490). Greater microbial diversity was associated with an increased risk of death (hazard ratio, 1.73; 95% CI, 1.03-2.90), whereas a greater proportion of Streptococcus was associated with a reduced risk of death (HR, 0.85; 95% CI, 0.73 to 0.99). The Streptococcus genus was mainly composed of Streptococcus mitis species. Conclusions: Increasing buccal microbial diversity predicts disease severity and death in IPF. The oral commensal S. mitis spp associates with preserved lung function and improved survival.

Background:The clinical course of idiopathic pulmonary fibrosis (IPF) is highly variable and unpredictable, with multiple genetic variants influencing IPF outcomes. Notably, rare pathogenic variants in telomere-related genes are associated with poorer clinical outcomes in these patients. Here we assessed whether rare qualifying variants (QVs) in monogenic adult-onset pulmonary fibrosis (PF) genes are associated with IPF survival. Using polygenic risk scores (PRS), we also evaluated the influence of common IPF risk variants in individuals carrying these QVs. Methods:We identified QVs in telomere and non-telomere genes linked to monogenic PF forms using whole-genome sequences (WGS) from 888 Pulmonary Fibrosis Foundation Patient Registry (PFFPR) individuals. We also derived a PRS for IPF (PRS-IPF) from 19 previously published common sentinel IPF variants. Using regression models, we then examined the mutual relationships of QVs and PRS-IPF and their association with survival. Validation of results was sought in WGS from an independent IPF study (PROFILE, n=472), and results from the two cohorts were meta-analyzed. Results:Carriers of QVs in monogenic adult-onset PF genes, representing nearly 1 out of 6 IPF patients, were associated with lower PRS-IPF (Odds Ratio [OR]: 1.79; 95% Confidence Interval [CI]: 1.15-2.81; p=0.010) and shorter survival (Hazard Ratio [HR]: 1.53; 95% CI: 1.12-2.10; p=7.3×10-3). Notably, carriers of pathogenic variants at telomere genes showed the strongest association with survival (HR: 1.76; 95% CI: 1.13-2.76; p=0.013). The meta-analysis of the results showed a consistent direction of effect across both cohorts. Conclusions:We revealed the opposite effects of QVs and PRS-IPF on IPF survival. Thus, a distinct IPF molecular subtype might be defined by QVs in monogenic adult-onset PF genes. Assessing the carrier status for QVs and modelling PRS-IPF promises to further contribute to predicting disease progression among IPF patients.

"Mapping the Proteomic Landscape of Radiological Lung Abnormalities." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp.

Recent genetic and genomic advancements have elucidated the complex etiology of idiopathic pulmonary fibrosis (IPF) and other progressive fibrotic interstitial lung diseases (ILDs), emphasizing the contribution of heritable factors. This state-of-the-art review synthesizes evidence on significant genetic contributors to pulmonary fibrosis (PF), including rare genetic variants and common SNPs. The MUC5B promoter variant is unusual, a common SNP that markedly elevates the risk of early and established PF. We address the utility of genetic variation in enhancing understanding of disease pathogenesis and clinical phenotypes, improving disease definitions, and informing prognosis and treatment response. Critical research gaps are highlighted, particularly the underrepresentation of non- European ancestries in PF genetic studies and the exploration of PF phenotypes beyond usual interstitial pneumonia/IPF. We discuss the role of telomere length, often critically short in PF, and its link to progression and mortality, underscoring the genetic complexity involving telomere biology genes ( TERT , TERC) ) and others like SFTPC and MUC5B. . In addition, we address the potential of gene-by-environment interactions to modulate disease manifestation, advocating for precision medicine in PF. Insights from gene expression profiling studies and multiomic analyses highlight the promise for understanding disease pathogenesis and offer new approaches to clinical care, therapeutic drug development, and biomarker discovery. Finally, we discuss the ethical, legal, and social implications of genomic research and therapies in PF, stressing the need for sound practices and informed clinical genetic discussions. Looking forward, we advocate for comprehensive genetic testing panels and polygenic risk scores to improve the management of PF and related ILDs across diverse populations.

BACKGROUND: Anti fi brotics are effective in slowing FVC decline in idiopathic pulmonary fi brosis (IPF). However, whether anti fi brotic type is differentially associated with FVC decline remains inconclusive. RESEARCH QUESTION: Are there signi fi cant differences in 12-month FVC decline between pirfenidone and nintedanib? STUDY DESIGN AND METHODS: A post hoc analysis was performed using the Clinical Ef fi cacy of Antimicrobial Therapy Strategy Using Pragmatic Design in IPF (CleanUP-IPF) trial (No. NCT02759120). Participants who reported using pirfenidone or nintedanib on enrollment into the trial were in the primary analysis. Spirometry was scheduled at baseline and the 12and 24-month study visits. Linear mixed-effects models with random intercept and slope were used to examine changes in FVC over time. Models were adjusted for age, sex, smoking history, coronary artery disease history, baseline FVC, and 12-month spline term. Survival and nonelective respiratory hospitalization by anti fi brotic type were determined using Cox regression models with adjustment for age, sex, smoking history, coronary artery disease history, and baseline FVC and diffusing capacity for carbon monoxide. RESULTS: Out of the 513 participants with IPF randomized in the CleanUP-IPF trial, 407 reported using pirfenidone (n = 264, 65%) or nintedanib (n = 143, 35%). The pirfenidone group had more participants with a history of coronary artery disease than the nintedanib group (34.1% vs 20.3%, respectively). Patients treated with nintedanib had a higher 12-month visit FVC than patients treated with pirfenidone (mean difference, 106 mL; 95% CI, 34-178). This difference was attenuated at the 24-month study visit. There were no signi fi cant differences in overall survival and nonelective respiratory hospitalization between the pirfenidone- and nintedanib-treated groups. INTERPRETATION: Patients with IPF who used nintedanib had a slower 12-month FVC decline than pirfenidone in a post hoc analysis of a clinical trial. CHEST 2024; 165(5):1163-1173

Rationale: Idiopathic pulmonary fibrosis (IPF) causes irreversible fibrosis of the lung parenchyma. Although antifibrotic therapy can slow IPF progression, treatment response is variable. There exists a critical need to develop a precision medicine approach to IPF. Objectives: To identify and validate biologically driven molecular endotypes of IPF. Methods: Latent class analysis (LCA) was independently performed in prospectively recruited discovery (n = 875) and validation (n = 347) cohorts. Twenty-five plasma biomarkers associated with fibrogenesis served as class-defining variables. The association between molecular endotype and 4-year transplant-free survival was tested using multivariable Cox regression adjusted for baseline confounders. Endotype-dependent differential treatment response to future antifibrotic exposure was then assessed in a pooled cohort of patients naive to antifibrotic therapy at the time of biomarker measurement (n = 555). Measurements and Main Results: LCA independently identified two latent classes in both cohorts (P < 0.0001). WFDC2 (WAP four-disulfide core domain protein 2) was the most important determinant of class membership across cohorts. Membership in class 2 was characterized by higher biomarker concentrations and a higher risk of death or transplant (discovery, hazard ratio [HR], 2.02; 95% confidence interval [CI], 1.64-2.48; P < 0.001; validation, HR, 1.95; 95% CI, 1.34-2.82; P < 0.001). In pooled analysis, significant heterogeneity in treatment effect was observed between endotypes (P = 0.030 for interaction), with a favorable antifibrotic response in class 2 (HR, 0.64; 95% CI, 0.45-0.93; P = 0.018) but not in class 1 (HR, 1.19; 95% CI, 0.77-1.84; P = 0.422). Conclusions: In this multicohort study, we identified two novel molecular endotypes of IPF with divergent clinical outcomes and responses to antifibrotic therapy. Pending further validation, these endotypes could enable a precision medicine approach for future IPF clinical trials.

Rationale: Distinguishing connective tissue disease-associated interstitial lung disease (CTD-ILD) from idiopathic pulmonary fibrosis (IPF) can be clinically challenging. Objectives: To identify proteins that separate and classify patients with CTD-ILD and those with IPF. Methods: Four registries with 1,247 patients with IPF and 352 patients with CTD-ILD were included in analyses. Plasma samples were subjected to high-throughput proteomics assays. Protein features were prioritized using recursive feature elimination to construct a proteomic classifier. Multiple machine learning models, including support vector machine, LASSO (least absolute shrinkage and selection operator) regression, random forest, and imbalanced Random Forest, were trained and tested in independent cohorts. The validated models were used to classify each case iteratively in external datasets. Measurements and Main Results: A classifier with 37 proteins (proteomic classifier 37 [PC37]) was enriched in the biological process of bronchiole development and smooth muscle proliferation and immune responses. Four machine learning models used PC37 with sex and age score to generate continuous classification values. Receiver operating characteristic curve analyses of these scores demonstrated consistent areas under the curve of 0.85-0.90 in the test cohort and 0.94-0.96 in the single-sample dataset. Binary classification demonstrated 78.6-80.4% sensitivity and 76-84.4% specificity in the test cohort and 93.5-96.1% sensitivity and 69.5-77.6% specificity in the single-sample classification dataset. Composite analysis of all machine learning models confirmed 78.2% (194 of 248) accuracy in the test cohort and 82.9% (208 of 251) in the single-sample classification dataset. Conclusions: Multiple machine learning models trained with large cohort proteomic datasets consistently distinguished CTD-ILD from IPF. Many of the identified proteins are involved in immune pathways. We further developed a novel approach for single-sample classification, which could facilitate honing the differential diagnosis of ILD in challenging cases and improve clinical decision making.

This study investigates correlates of anti-S1 antibody response following COVID-19 vaccination in a U.S. population-based meta-cohort of adults participating in longstanding NIH-funded cohort studies. Anti-S1 antibodies were measured from dried blood spots collected between February 2021-August 2022 using Luminex-based microsphere immunoassays. Of 6245 participants, mean age was 73 years (range, 21-100), 58% were female, and 76% were non-Hispanic White. Nearly 52% of participants received the BNT162b2 vaccine and 48% received the mRNA-1273 vaccine. Lower anti-S1 antibody levels are associated with age of 65 years or older, male sex, higher body mass index, smoking, diabetes, COPD and receipt of BNT16b2 vaccine (vs mRNA-1273). Participants with a prior infection, particularly those with a history of hospitalized illness, have higher anti-S1 antibody levels. These results suggest that adults with certain socio-demographic and clinical characteristics may have less robust antibody responses to COVID-19 vaccination and could be prioritized for more frequent re-vaccination.

Rationale: Accelerated biological aging has been implicated in the development of interstitial lung disease (ILD) and other diseases of aging but remains poorly understood. Objectives: To identify plasma proteins that mediate the relationship between chronological age and survival association in patients with ILD. Methods: Causal mediation analysis was performed to identify plasma proteins that mediated the chronological age-survival relationship in an idiopathic pulmonary fibrosis discovery cohort. Proteins mediating this relationship after adjustment for false discovery were advanced for testing in an independent ILD validation cohort and explored in a chronic obstructive pulmonary disease cohort. A proteomic-based measure of biological age was constructed and survival analysis performed, assessing the impact of biological age and peripheral blood telomere length on the chronological age-survival relationship. Measurements and Main Results: Twenty-two proteins mediated the chronological age-survival relationship after adjustment for false discovery in the idiopathic pulmonary fibrosis discovery cohort (n = 874), with 19 remaining significant mediators of this relationship in the ILD validation cohort (n = 983) and one mediating this relationship in the chronic obstructive pulmonary disease cohort. Latent transforming growth factor-β binding protein 2 and ectodysplasin A2 receptor showed the strongest mediation across cohorts. A proteomic measure of biological age completely attenuated the chronological age-survival association and better discriminated survival than chronological age. Results were robust to adjustment for peripheral blood telomere length, which did not mediate the chronological age-survival relationship. Conclusions: Molecular measures of aging completely mediate the relationship between chronological age and survival, suggesting that chronological age has no direct effect on ILD survival.