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Rationale: In the United States (U.S.), ambulatory oxygen is recommended for patients with idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) who experience symptomatic exertional hypoxemia. Ambulatory oxygen need is often determined by submaximal hall walk testing; however, this may fail to accurately characterize exertional hypoxemia in some patients., Objectives: Assess for differences in ambulatory oxygen needs between IPF and COPD patients who completed a ramped treadmill protocol exercise test (RTPET) and correlate oxygen flow rates determined at highest level exertion with lung function and exercise parameters. Oxygen "need" is defined as flow rate needed to maintain oxygen saturation >90% in patients who desaturate to <88%., Methods: We conducted a retrospective review of RTPET results for IPF and COPD patients who also recently completed spirometry. The RTPET has three phases: rest, submaximal usual pace walking at 0% treadmill grade for 3 minutes, and highest level walking at the UP walk speed with increasing treadmill grade by 2% every 2 minutes. IPF patients were part of a clinical registry while COPD patients were identified based on diagnosis coding and spirometry (FEV1/FVC <0.70). The RTPET for both groups was completed based on a pulmonologist's referral., Measurements and Main Results: We included 329 IPF and 2,343 COPD patients. A greater proportion of IPF patients required ambulatory oxygen to maintain saturation >90%. After adjusting for demographic covariates and exercise parameters, IPF patients required higher ambulatory oxygen flow rates compared to COPD subjects with similar DLCO values. Of patients who did not require oxygen with submaximal usual pace testing, 49% with IPF and 24% with COPD required oxygen at highest level exertion., Conclusions: The RTPET identified higher oxygen flow needs at highest level exertion in IPF versus COPD patients; however, in both diseases, there was a significant proportion of patients who were only found to have exertional desaturation at highest level exertion. Current oxygen policies and reliance on submaximal testing may fail to meet the needs of patients with IPF and COPD. Further studies are needed to determine if oxygen prescriptions targeting highest level desaturation improve clinical outcomes, symptoms, or quality-of-life.

Background: The Myositis Interstitial Lung Disease Nintedanib Trial (MINT) is a hybrid trial, which is enrolling patients both at local sites and remotely via a decentralised site. The trial will investigate the efficacy and safety of nintedanib in patients with progressive myositis-associated interstitial lung disease (MA-ILD)., Methods/design: MINT is an exploratory, prospective randomised placebo-controlled trial. Eligible patients will have myositis and evidence of fibrosing ILD on high-resolution computed tomography (HRCT), be taking standard of care medications for myositis, and meet criteria for ILD progression within the prior 24 months based on decline in FVC, worsened fibrosis on HRCT, and/or worsened dyspnoea. Patients will be randomised 1:1 to receive nintedanib 150 mg twice daily or placebo for 12 weeks then open-label nintedanib for 12 weeks. Patients will be enrolled at local sites and a decentralised site. Most study visits will be completed remotely using telemedicine or digital health technologies. The primary endpoint is the change in Living with Pulmonary Fibrosis (L-PF) questionnaire dyspnoea domain score at week 12. Other endpoints include changes in other L-PF questionnaire domains, lung function, imaging, and physical activity, and assessment of adverse events. Data collected using remote versus clinic enrolment, and using home versus clinic spirometry, will be compared., Discussion: MINT is an innovative, hybrid trial that will evaluate the effects of nintedanib on symptoms, quality of life, and ILD progression in patients with progressive MA-ILD and provide valuable information on the utility of decentralised recruitment and remote data collection in clinical trials., Trial Registration: Clinicaltrials.gov NCT05799755 (date of registration: 05/04/2023)., (© 2024. The Author(s).)

Rationale: Particulate matter with an aerodynamic diameter ⩽2.5 μm is associated with adverse outcomes in fibrotic interstitial lung disease (fILD), but the impact of ultrafine particulates (UFPs; aerodynamic diameter ⩽100 nm) remains unknown. Objective: To evaluate UFP associations with clinical outcomes in fILD. Methods: We conducted a multicenter, prospective cohort study enrolling patients with fILD from the University of Pittsburgh Dorothy P. and Richard P. Simmons Center and the Pulmonary Fibrosis Foundation Patient Registry (PFF-PR). Using a national-scale UFP model, we linked exposures using three approaches in the Simmons cohort (residential address geocoordinates, ZIP code centroid geocoordinates, and ZIP code average) and two in the PFF-PR for which only five-digit ZIP code was available (ZIP code centroid and ZIP code average). We tested UFP associations with transplantation-free survival using multivariable Cox proportional-hazards models, baseline percentage predicted FVC and Dl CO using multivariable linear regressions, and decline in FVC and Dl CO using linear mixed models adjusting for age, sex, smoking, race, socioeconomic status, site, particulate matter with an aerodynamic diameter ⩽2.5, and nitrogen dioxide. Measurements and Main Results: Annual mean outdoor UFP concentrations for 2017 were estimated for 1,416 Simmons and 1,919 PFF-PR patients. Increased UFP concentration was associated with transplantation-free survival in fully adjusted Simmons residential address models (hazard ratio, 1.08 per 1,000 particles/cm 3 [95% confidence interval, 1.01-1.15]; P  = 0.02) but not PFF-PR models, which used less precise linkage approaches. Higher UFP exposure was associated with lower baseline FVC and more rapid FVC decline in the Simmons registry. Conclusions: Increased UFP exposure was associated with transplantation-free survival and lung function in the cohort with precise residential location linkage. This work highlights the need for more robust regulatory networks to study the health effects of UFPs nationwide.

Background: Acute exacerbations of idiopathic pulmonary fibrosis (AE-IPF) affect a significant proportion of patients with IPF. There are limited data to inform therapeutic strategies for AE-IPF, despite its high mortality. We discuss the rationale and design of STRIVE-IPF, a randomized, multi-center, open-label Phase IIb clinical trial to determine the efficacy of combined therapeutic plasma exchange (TPE), rituximab, and intravenous immunoglobulin (IVIG), in comparison to treatment as usual (TAU), among patients with acute IPF exacerbations., Methods: The STRIVE-IPF trial will randomize 51 patients among five sites in the United States. The inclusion criteria have been designed to select a study population with AE-IPF, as defined by American Thoracic Society criteria, while excluding patients with an alternative cause for a respiratory decompensation. The primary endpoint of this trial is six-month survival. Secondary endpoints include supplement oxygen requirement and six-minute walk distance which will be assessed immediately prior to treatment and after completion of therapy on day 19, as well as at periodic subsequent visits., Discussion: The experimental AE-IPF therapy proposed in this clinical trial was adapted from treatment regimens used in other antibody-mediated diseases. The regimen is initiated with TPE, which is expected to rapidly reduce circulating autoantibodies, followed by rituximab to reduce B-cells and finally IVIG, which likely has multiple effects, including affecting feedback inhibition of residual B-cells by Fc receptor occupancy. We have reported potential benefits of this experimental therapy for AE-IPF in previous anecdotal reports. This clinical trial has the potential to profoundly affect current paradigms and treatment approaches to patients with AE-IPF., Trial Registration: ClinicalTrials.gov identifier: NCT03286556., (© 2024. The Author(s).)

Rationale: Recent data suggest that the localisation of airway epithelial cells in the distal lung in idiopathic pulmonary fibrosis (IPF) may drive pathology. We set out to discover whether chemokines expressed in these ectopic airway epithelial cells may contribute to the pathogenesis of IPF., Methods: We analysed whole lung and single-cell transcriptomic data obtained from patients with IPF. In addition, we measured chemokine levels in blood, bronchoalveolar lavage (BAL) of IPF patients and air-liquid interface cultures. We employed ex vivo donor and IPF lung fibroblasts and an animal model of pulmonary fibrosis to test the effects of chemokine signalling on fibroblast function., Results: By analysis of whole-lung transcriptomics, protein and BAL, we discovered that CXCL6 (a member of the interleukin-8 family) was increased in patients with IPF. Elevated CXCL6 levels in the BAL of two cohorts of patients with IPF were associated with poor survival (hazard ratio of death or progression 1.89, 95% CI 1.16-3.08; n=179, p=0.01). By immunostaining and single-cell RNA sequencing, CXCL6 was detected in secretory cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased collagen I levels in donor and IPF fibroblasts 4.4-fold and 1.7-fold, respectively. Both silencing of and chemical inhibition of CXCR1/2 blocked the effects of CXCL6 on collagen, while overexpression of CXCR2 increased collagen I levels 4.5-fold in IPF fibroblasts., Conclusions: CXCL6 is expressed in ectopic airway epithelial cells. Elevated levels of CXCL6 are associated with IPF mortality. CXCL6-driven collagen synthesis represents a functional consequence of ectopic localisation of airway epithelial cells in IPF., Competing Interests: Conflict of interest: N. Kaminski is a scientific founder at Thyron, and served as a consultant to Biogen Idec, Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Theravance, LifeMax, Three Lake Partners, Optikira, AstraZeneca, RohBar, Veracyte, Augmanity, CSL Behring, Galapagos and Thyron over the last 3 years, reports equity in Pliant and Thyron, and grants from Veracyte, Boehringer Ingelheim and BMS, and non-financial support from MiRagen and AstraZeneca. R. Lafyatis reports grants from Bristol Meyer Squib, Corbus, Formation, Moderna, Regeneron, Pfizer and Kiniksa, outside the submitted work, and served as a consultant with Bristol Meyers Squibb, Formation, Sanofi, Boehringer Ingelheim, Merck and Genentech/Roche. A. Prasse reports personal fees and non-financial support from Boehringer Ingelheim and Roche, personal fees from Novartis, AstraZeneca, Amgen, Pliant and Nitto Denko, outside the submitted work. The remaining authors have no potential conflicts of interest to disclose., (Copyright ©The authors 2024. For reproduction rights and permissions contact permissions@ersnet.org.)

Patients with interstitial lung diseases (ILD) often have hypoxemia at rest and/or with exertion, for which supplemental oxygen is commonly prescribed. The number of patients with ILD who require supplemental oxygen is unknown, although estimates suggest it could be as much as 40%; many of these patients may require high-flow support (>4 L/min). Despite its frequent use, there is limited evidence for the impact of supplemental oxygen on clinical outcomes in ILD, with recommendations for its use primarily based on older studies in patients with chronic obstructive pulmonary disease. Oxygen use in ILD is rarely included as an outcome in clinical trials. Available evidence suggests that supplemental oxygen in ILD may improve quality of life and some exercise parameters in patients whose hypoxemia is a limiting factor; however, oxygen therapy also places new burdens and barriers on some patients that may counter its beneficial effects. The cost of supplemental oxygen in ILD is also unknown but likely represents a significant portion of overall healthcare costs in these patients. Current Centers for Medicare and Medicaid reimbursement policies provide only a modest increase in payment for high oxygen flows, which may negatively impact access to oxygen services and equipment for some patients with ILD. Future studies should examine clinical and quality-of-life outcomes for oxygen use in ILD. In the meantime, given the current limited evidence for supplemental oxygen and considering cost factors and other barriers, providers should take a patient-focused approach when considering supplemental oxygen prescriptions in patients with ILD.

High-resolution computed tomography (HRCT) imaging is critical for diagnostic evaluation of Idiopathic Pulmonary Fibrosis (IPF). However, several other interstitial lung diseases (ILDs) often exhibit radiologic pattern similar to IPF on HRCT making the diagnosis of the disease difficult. Therefore, biomarkers that distinguish IPF from other ILDs can be a valuable aid in diagnosis. Using mass spectrometry, we performed proteomic analysis of plasma extracellular vesicles (EVs) in patients diagnosed with IPF, chronic hypersensitivity pneumonitis, nonspecific interstitial pneumonitis, and healthy subjects. A five-protein signature was identified by lasso regression and was validated in an independent cohort using ELISA. The five-protein signature derived from mass spectrometry data showed an area under the receiver operating characteristic curve of 0.915 (95%CI: 0.819-1.011) and 0.958 (95%CI: 0.882-1.034) for differentiating IPF from other ILDs and from healthy subjects, respectively. Stepwise backwards elimination yielded a model with 3 and 2 proteins for discriminating IPF from other ILDs and healthy subjects, respectively, without compromising diagnostic accuracy. In summary, we discovered and validated EV protein biomarkers for differential diagnosis of IPF in independent cohorts. Interestingly, the biomarker panel could also distinguish IPF and healthy subjects with high accuracy. The biomarkers need to be evaluated in large prospective cohorts to establish their clinical utility., Competing Interests: A.K.P. is employed with Izon Science US Ltd. The company has no role in design of the study and acquisition of experimental data and interpretation. All other authors have no conflict of interests., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise., Methods: We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1- overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells., Results: TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log 2 fold change (FC) 8.909, adjusted p-value 1.82×10 -35 ) and control fibroblasts (log 2 FC 8.975, adjusted p-value 3.72×10 -28 ). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log 2 FC 3.136, adjusted p-value 1.41×10 - 24 ), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts., Conclusions: Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases., Competing Interests: Conflict of interest: R. Lafyatis reports the following conflicts of interest outside the scope of work of this manuscript: R. Lafyatis has served as a consultant for Pfizer, Bristol Myers Squibb, Boehringer Ingelheim, Formation, Sanofi, Boehringer-Mannheim, Merck and Genentech/Roche, and holds or recently had research grants from Corbus, Formation, Moderna, Regeneron, Pfizer and Kiniksa, and holds equity in Thirona. All other authors report no relevant conflicts of interest., (Copyright ©The authors 2023. For reproduction rights and permissions contact permissions@ersnet.org.)

Rationale: Idiopathic pulmonary fibrosis (IPF) is a rare, irreversible, and progressive disease of the lungs. Common genetic variants, in addition to nongenetic factors, have been consistently associated with IPF. Rare variants identified by candidate gene, family-based, and exome studies have also been reported to associate with IPF. However, the extent to which rare variants, genome-wide, may contribute to the risk of IPF remains unknown. Objectives: We used whole-genome sequencing to investigate the role of rare variants, genome-wide, on IPF risk. Methods: As part of the Trans-Omics for Precision Medicine Program, we sequenced 2,180 cases of IPF. Association testing focused on the aggregated effect of rare variants (minor allele frequency ⩽0.01) within genes or regions. We also identified individual rare variants that are influential within genes and estimated the heritability of IPF on the basis of rare and common variants. Measurements and Main Results: Rare variants in both TERT and RTEL1 were significantly associated with IPF. A single rare variant in each of the TERT and RTEL1 genes was found to consistently influence the aggregated test statistics. There was no significant evidence of association with other previously reported rare variants. The SNP heritability of IPF was estimated to be 32% (SE = 3%). Conclusions: Rare variants within the TERT and RTEL1 genes and well-established common variants have the largest contribution to IPF risk overall. Efforts in risk profiling or the development of therapies for IPF that focus on TERT , RTEL1 , common variants, and environmental risk factors are likely to have the largest impact on this complex disease.

Ubiquitination-mediated protein degradation is associated with the development of pulmonary fibrosis. We and others have shown that Nedd4L plays anti-inflammatory and anti-fibrotic roles by targeting lysophosphatidic acid receptor 1 (LPAR1), p-Smad2/3, and β-catenin, and other molecules for their degradation in lung epithelial cells and fibroblasts. However, the molecular regulation of Nedd4L expression in lung fibroblasts has not been studied. In this study, we find that Nedd4L levels are significantly suppressed in lung myofibroblasts in IPF patients and in experimental pulmonary fibrosis, and in TGF-β1-treated lung fibroblasts. Nedd4L knockdown promotes TGF-β1-mediated phosphorylation of Smad2/3 and lung myofibroblast differentiation. Mechanistically, Nedd4L targets TGF-β receptor II (TβRII), the first key enzyme of TGF-β1-mediated signaling, for its ubiquitination and degradation. Further, we show that inhibition of transcriptional factor E2F rescues Nedd4L levels and mitigates experimental pulmonary fibrosis. Together, our data reveal insight into mechanisms by which E2F-mediated Nedd4L suppression contributes to the pathogenesis of lung fibrosis. This study provides evidence showing that upregulation of Nedd4L is a potential therapeutic strategy to treat fibrotic disorders including lung fibrosis., Competing Interests: Disclosures D.J.K. reports collaborative research funding from Regeneron Pharmaceuticals in pulmonary hypertension, which is unrelated to this article. None of the other authors has any conflicts of interest, financial or otherwise, to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease (ILD) whose outcomes are worsened with air pollution exposures. DNA methylation (DNAm) patterns are altered in lungs and blood from patients with IPF, but the relationship between air pollution exposures and DNAm patterns in IPF remains unexplored. This study aimed to evaluate the association of PM 2.5 and constituent components with global DNAm in patients with IPF. Patients enrolled in either the University of Pittsburgh Simmons Center for ILD Registry (Simmons) or the U.S.-wide Pulmonary Fibrosis Foundation (PFF) Patient Registry with peripheral blood DNA samples were included. The averages of monthly exposures to PM 2.5 and constituents over 1-year and 3-months pre-blood collection were matched to patient residential coordinates using satellite-derived hybrid models. Global DNAm percentage (%5 mC) was determined using the ELISA-based MethylFlash assay. Associations of pollutants with %5 mC were assessed using beta-regression, Cox models for mortality, and linear regression for baseline lung function. Mediation proportion was determined for models where pollutant-mortality and pollutant-%5 mC associations were significant. Inclusion criteria were met by 313 Simmons and 746 PFF patients with IPF. Higher PM 2.5 3-month exposures prior to blood collection were associated with higher %5 mC in Simmons (β = 0.02, 95%CI 0.0003-0.05, p = 0.047), with trends in the same direction in the 1-year period in both cohorts. Higher exposures to sulfate, nitrate, ammonium, and black carbon constituents were associated with higher %5 mC in multiple models. Percent 5 mC was not associated with IPF mortality or lung function, but was found to mediate between 2 and 5% of the associations of PM 2.5 , sulfate, and ammonium with mortality. In conclusion, we found that higher global DNAm is a novel biomarker for increased PM 2.5 and anthropogenic constituent exposure in patients with IPF. Mechanistic research is needed to determine if DNAm has pathogenic relevance in mediating associations between pollutants and mortality in IPF., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gillian C. Goobie reports financial support was provided by Pulmonary Fibrosis Foundation. Gillian C. Goobie reports a relationship with Boehringer Ingelheim Corp USA that includes: funding grants. Kathleen O. Lindell reports a relationship with Boehringer Ingelheim Corp USA that includes: funding grants. Daniel J. Kass reports a relationship with Boehringer Ingelheim Corp USA that includes: funding grants. S. Mehdi Nouraie reports a relationship with Boehringer Ingelheim Corp USA that includes: funding grants. Christopher J. Ryerson reports a relationship with Boehringer Ingelheim Canada Ltd that includes: funding grants. Christopher J. Ryerson reports a relationship with Hoffmann-La Roche Limited that includes:. Kerri A. Johannson reports a relationship with Boehringer Ingelheim Canada Ltd that includes: consulting or advisory and funding grants. Kerri A. Johannson reports a relationship with Three Lakes Foundation that includes: consulting or advisory and funding grants. Kerri A. Johannson reports a relationship with Hoffmann-La Roche Limited that includes: consulting or advisory. James P. Fabisiak reports a relationship with Heinz Endowments that includes: funding grants. Daniel J. Kass reports a relationship with National Institutes of Health that includes: funding grants. Yingze Zhang reports a relationship with National Institutes of Health that includes: funding grants., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Importance: Particulate matter 2.5 μm or less in diameter (PM2.5) is associated with adverse outcomes for patients with idiopathic pulmonary fibrosis, but its association with other fibrotic interstitial lung diseases (fILDs) and the association of PM2.5 composition with adverse outcomes remain unclear., Objective: To investigate the association of PM2.5 exposure with mortality and lung function among patients with fILD., Design, Setting, and Participants: In this multicenter, international, prospective cohort study, patients were enrolled in the Simmons Center for Interstitial Lung Disease Registry at the University of Pittsburgh in Pittsburgh, Pennsylvania; 42 sites of the Pulmonary Fibrosis Foundation Registry; and 8 sites of the Canadian Registry for Pulmonary Fibrosis. A total of 6683 patients with fILD were included (Simmons, 1424; Pulmonary Fibrosis Foundation, 1870; and Canadian Registry for Pulmonary Fibrosis, 3389). Data were analyzed from June 1, 2021, to August 2, 2022., Exposures: Exposure to PM2.5 and its constituents was estimated with hybrid models, combining satellite-derived aerosol optical depth with chemical transport models and ground-based PM2.5 measurements., Main Outcomes and Measures: Multivariable linear regression was used to test associations of exposures 5 years before enrollment with baseline forced vital capacity and diffusion capacity for carbon monoxide. Multivariable Cox models were used to test associations of exposure in the 5 years before censoring with mortality, and linear mixed models were used to test associations of exposure with a decrease in lung function. Multiconstituent analyses were performed with quantile-based g-computation. Cohort effect estimates were meta-analyzed. Models were adjusted for age, sex, smoking history, race, a socioeconomic variable, and site (only for Pulmonary Fibrosis Foundation and Canadian Registry for Pulmonary Fibrosis cohorts)., Results: Median follow-up across the 3 cohorts was 2.9 years (IQR, 1.5-4.5 years), with death for 28% of patients and lung transplant for 10% of patients. Of the 6683 patients in the cohort, 3653 were men (55%), 205 were Black (3.1%), and 5609 were White (84.0%). Median (IQR) age at enrollment across all cohorts was 66 (58-73) years. A PM2.5 exposure of 8 μg/m3 or more was associated with a hazard ratio for mortality of 4.40 (95% CI, 3.51-5.51) in the Simmons cohort, 1.71 (95% CI, 1.32-2.21) in the Pulmonary Fibrosis Foundation cohort, and 1.45 (95% CI, 1.18-1.79) in the Canadian Registry for Pulmonary Fibrosis cohort. Increasing exposure to sulfate, nitrate, and ammonium PM2.5 constituents was associated with increased mortality across all cohorts, and multiconstituent models demonstrated that these constituents tended to be associated with the most adverse outcomes with regard to mortality and baseline lung function. Meta-analyses revealed consistent associations of exposure to sulfate and ammonium with mortality and with the rate of decrease in forced vital capacity and diffusion capacity of carbon monoxide and an association of increasing levels of PM2.5 multiconstituent mixture with all outcomes., Conclusions and Relevance: This cohort study found that exposure to PM2.5 was associated with baseline severity, disease progression, and mortality among patients with fILD and that sulfate, ammonium, and nitrate constituents were associated with the most harm, highlighting the need for reductions in human-derived sources of pollution.

The role of constitutional genetic defects in idiopathic pulmonary fibrosis (IPF) is increasingly appreciated. Monogenic disorders associated with IPF affect two pathways: telomere maintenance, accounting for approximately 10% of all patients with IPF, and surfactant biology, responsible for 1%-3% of cases and often co-occurring with lung cancer. We examined the prevalence of rare variants in five surfactant-related genes, SFTPA1, SFPTA2, SFTPC, ABCA3, and NKX2-1, that were previously linked to lung disease in whole genome sequencing data from 431 patients with IPF. We identified functionally deleterious rare variants in SFTPA2 with a prevalence of 1.3% in individuals with and without a family history of IPF. All individuals had no personal history of lung cancer, but substantial bronchiolar metaplasia was noted on lung explants and biopsies. Five patients had novel missense variants in NKX2-1, but the contribution to disease is unclear. In general, patients were younger and had longer telomeres compared with the majority of patients with IPF suggesting that these features may be useful for identifying this subset of patients in the clinic. These data suggest that SFTPA2 variants may be more common in unselected IPF cohorts and may manifest in the absence of personal/family history of lung cancer or IPF., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Background: This multicentre, international, prospective cohort study evaluated whether patients with pulmonary sarcoidosis living in neighbourhoods with greater material and social disadvantage experience worse clinical outcomes., Methods: The area deprivation index and the Canadian Index of Multiple Deprivation evaluate neighbourhood-level disadvantage in the US and Canada, with higher scores reflecting greater disadvantage. Multivariable linear regression evaluated associations of disadvantage with baseline forced vital capacity (FVC) or diffusing capacity of the lung for carbon monoxide ( D LCO ) and linear mixed effects models for associations with rate of FVC or D LCO decline, and competing hazards models were used for survival analyses in the US cohort, evaluating competing outcomes of death or lung transplantation. Adjustments were made for age at diagnosis, sex, race and smoking history., Results: We included 477 US and 122 Canadian patients with sarcoidosis. Higher disadvantage was not associated with survival or baseline FVC. The highest disadvantage quartile was associated with lower baseline D LCO in the US cohort (β = -6.80, 95% CI -13.16 to -0.44, p=0.04), with similar findings in the Canadian cohort (β = -7.47, 95% CI -20.28 to 5.33, p=0.25); with more rapid decline in FVC and D LCO in the US cohort (FVC β = -0.40, 95% CI -0.70 to -0.11, p=0.007; D LCO β = -0.59, 95% CI -0.95 to -0.23, p=0.001); and with more rapid FVC decline in the Canadian cohort (FVC β = -0.80, 95% CI -1.37 to -0.24, p=0.003)., Conclusion: Patients with sarcoidosis living in high disadvantage neighbourhoods experience worse baseline lung function and more rapid lung function decline, highlighting the need for better understanding of how neighbourhood-level factors impact individual patient outcomes., Competing Interests: Conflict of interest: G.C. Goobie receives research funding and support through the Pulmonary Fibrosis Foundation Scholars Award Program and the University of British Columbia Clinician Investigator Program. C.J. Ryerson, D. Assayag and M.R.J. Kolb report personal fees and grants from Boehringer Ingelheim and Hoffman La Roche outside the submitted work. K.A. Johannson reports personal fees, nonfinancial support and other support from Boehringer Ingelheim and the Three Lakes Foundation, personal fees from Hoffman-La Roche Ltd, and grants from the University Hospital Foundation, the University of Calgary Cumming School of Medicine, and the Pulmonary Fibrosis Society of Calgary. V. Marcoux reports grants and personal fees from Boehringer Ingelheim Canada, Hoffman La-Roche Ltd and AstraZeneca, and grants from the University of Saskatchewan and the Royal University Hospital Foundation. M.R.J. Kolb also reports personal fees and grants from GSK, Gilead, Actelion, Respivert, Genoa, Alkermes, Pharmaxis, Prometric, Indalo and Third Pole. H. Manganas reports grants from Hoffman-La Roche Ltd, Galapagos and BMS, and personal fees and research grants from Boehringer Ingelheim. D. Assayag also reports personal fees and grants from Novartis. J.H. Fisher reports grants from the Canadian Pulmonary Fibrosis Foundation, and personal fees from Boehringer Ingelheim and AstraZeneca, outside of the submitted work. D.J. Kass is supported in part by AR060780, HL133232, UL1 TR001857 and AR076024 (NIH grants), Boehringer Ingelheim grants, and receives collaborative research funding from Regeneron Pharmaceuticals, outside of the submitted work. Y. Zhang is supported in part by AR076024 (NIH grant). S. Keil, E. Schikowski, N. Khalil, X. Chen, K.F. Gibson and K.O. Lindell report no competing interests. S.M. Nouraie receives grant support from Boehringer Ingelheim USA., (Copyright ©The authors 2022.)

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease. Heme oxygenase-1 (HMOX1/HO-1) is an enzyme that catalyzes the degradation of heme. The role of HO-1 in the pathogenesis of IPF has been studied; however, the molecular regulation of HO-1 and its role in IPF are still unclear. In this study, we found that HO-1 protein levels significantly increased in lung myofibroblasts in IPF patients and in lungs in a murine model of bleomycin-induced lung fibrosis. In addition, we observed that administration of a E2F transcription factor inhibitor elevated HO-1 mRNA and protein levels in lung fibroblasts. Downregulation of E2F2 by siRNA transfection increased HO-1 mRNA and protein levels, while overexpression of E2F2 reduced HO-1 levels. However, overexpression of E2F2 did not alter hemin-induced HO-1 protein levels. Furthermore, modulation of HO-1 levels regulated TGF-β1-induced myofibroblast differentiation without altering the phosphorylation of Smad2/3 in lung fibroblast cells. Moreover, the phosphorylation of protein kinase B (Akt) was significantly upregulated in HO-1-depleted lung fibroblast cells. In summary, this study demonstrated that E2F2 regulates the baseline expression of HO-1, but has no effect on modulating HO-1 expression by hemin. Finally, elevated HO-1 expression contributes to the TGF-β1-induced lung myofibroblast differentiation through the activation of the serine/threonine kinase AKT pathway. Overall, our findings suggest that targeting E2F2/HO-1 might be a new therapeutic strategy to treat fibrotic diseases such as IPF.

Accumulation of excessive extracellular matrix (ECM) components from lung fibroblasts is a feature of systemic sclerosis-associated interstitial lung disease (SSc-ILD), and there is increasing evidence that innate immune signaling pathways contribute to these processes. Toll-like receptors (TLRs) are innate immune sensors activated by danger signals derived from pathogens or host molecular patterns. Several damage-associated molecular pattern (DAMP) molecules are elevated in SSc-ILD plasma, including ligands that activate TLR9, an innate immune sensor recently implicated in driving profibrotic responses in fibroblasts. Fibronectin and the isoform fibronectin-extra domain A (FN-EDA) are prominent in pathological extracellular matrix accumulation, but mechanisms promoting FN-EDA accumulation are only partially understood. Here, we show that TLR9 activation increases FN-EDA accumulation in MRC5 and SSc-ILD fibroblasts, but that this effect is independent of changes in FN-EDA gene transcription. Rather, we describe a novel mechanism where TLR9 activation inhibits FN-EDA turnover via reduced FN-EDA ubiquitination. TLR9 ligand ODN2006 reduces ubiquitinated FN-EDA destined for lysosomal degradation, an effect abrogated with TLR9 knockdown or inhibition. Taken together, these results provide rationale for disrupting the TLR9 signaling axis or FN-EDA degradation pathways to reduce FN-EDA accumulation in SSc-ILD fibroblasts. More broadly, enhancing intracellular degradation of ECM components through TLR9 inhibition or enhanced ECM turnover could be a novel strategy to attenuate pathogenic ECM accumulation in SSc-ILD.

Rationale: Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers. Objectives: We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable. Methods: Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort. Measurements and Main Results: In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4 + lymphopenia predominated, with lower CD4 + /CD8 + ratios in severe COVID-19 compared with patients with mild disease ( P  < 0.0001). In severe disease, immunodominant CD4 + T-cell responses to Spike-1 (S1) produced increased in vitro TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4 + TNF-α + T-cell responses inversely correlated with absolute CD4 + counts from patients with severe COVID-19 ( n  = 76; R  = -0.797; P  < 0.0001). In vitro TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4 + T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 ( P  < 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4 + cells with infliximab treatment. We also evaluated BAL and lung explant CD4 + T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells. Conclusions: Together, our findings show CD4 + dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.

Background: Idiopathic pulmonary fibrosis (IPF) is the most common indication for lung transplantation in North America and variants in telomere-maintenance genes are the most common identifiable cause of IPF. We reasoned that younger IPF patients are more likely to undergo lung transplantation and we hypothesized that lung transplant recipients would be enriched for individuals with telomere-mediated disease due to the earlier onset and more severe disease in these patients., Methods: Individuals with IPF who underwent lung transplantation or were evaluated in an interstitial lung disease specialty clinic who did not undergo lung transplantation were examined. Genetic evaluation was completed via whole genome sequencing (WGS) of 426 individuals and targeted sequencing for 5 individuals. Rare variants in genes previously associated with IPF were classified using the American College of Medical Genetics guidelines. Telomere length from WGS data was measured using TelSeq software. Patient characteristics were collected via medical record review., Results: Of 431 individuals, 149 underwent lung transplantation for IPF. The median age of diagnosis of transplanted vs non-transplanted individuals was significantly younger (60 years vs 70 years, respectively, p<0.0001). IPF lung transplant recipients (IPF-LTRs) were twice as likely to have telomere-related rare variants compared to non-transplanted individuals (24% vs 12%, respectively, p=0.0013). IPF-LTRs had shorter telomeres than non-transplanted IPF patients (p=0.0028) and >85% had telomeres below the age-adjusted mean. Post-transplant survival and CLAD were similar amongst IPF-LTRs with rare variants in telomere-maintenance genes compared to those without, as well as in those with short telomeres versus longer telomeres., Conclusions: There is an enrichment for telomere-maintenance gene variants and short telomeres among IPF-LTRs. However, transplant outcomes of survival and CLAD do not differ by gene variants or telomere length within IPF-LTRs. Our findings support individual with telomere-mediated disease should not be excluded from lung transplantation and focusing research efforts on therapies directed toward individuals with short-telomere mediated disease., Competing Interests: Conflict of interest This project was supported by National Heart, Lung, and Blood Institute Grant R01HL135062 (JA), R01 HL133184 (JM), UPMC Immune Transplant and Therapy Center, UPMC Genomics Research Center, Kathleen Ford Fund for Lung Transplant Research, The Richard P. Simmons Chair for Interstitial Lung Disease, and the Simmons Genetics Research Fund., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Fibrotic interstitial lung disease (fILD) is a group of pathologic entities characterized by scarring of the lungs and high morbidity and mortality. Research investigating how socioeconomic and residential factors impact outcomes in patients with fILD is lacking. Objectives: To determine the association between neighborhood-level disadvantage and presentation severity, disease progression, lung transplantation, and mortality in patients with fILD from the United States and Canada. Methods: We performed a multicenter, international, prospective cohort study of 4,729 patients with fILD from one U.S. and eight Canadian ILD registry sites. Neighborhood-level disadvantage was measured by the area deprivation index in the United States and the Canadian Index of Multiple Deprivation in Canada. Measurements and Main Results: In the U.S. but not in the Canadian cohort, patients with fILD living in neighborhoods with the greatest disadvantage (top quartile) experience the highest risk of mortality (hazard ratio = 1.51, P  = 0.002), and in subgroups of patients with idiopathic pulmonary fibrosis, the top quartile of disadvantage experienced the lowest odds of lung transplantation (odds ratio = 0.46, P  = 0.04). Greater disadvantage was associated with reduced baseline DL CO in both cohorts, but it was not associated with baseline FVC or FVC or DL CO decline in either cohort. Conclusions: Patients with fILD who live in areas with greater neighborhood-level disadvantage in the United States experience higher mortality, and patients with idiopathic pulmonary fibrosis experience lower odds of lung transplantation. These disparities are not seen in Canadian patients, which may indicate differences in access to care between the United States and Canada.

Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxin's antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Patients with idiopathic pulmonary fibrosis (IPF) and their caregivers experience stress, symptom burden, poor quality of life, and inadequate preparedness for end-of-life (EOL) care planning as the disease progresses. The hypothesis for this study was that the early introduction of palliative care in the course of IPF would improve knowledge and preparation for EOL, patient-reported outcomes, and advance care planning in patients with IPF and their caregivers. Objectives: We sought to determine the feasibility, acceptability, and efficacy of a nurse-led early palliative care intervention entitled "A Program of SUPPORT" (Symptom management, Understanding the disease, Pulmonary rehabilitation, Palliative care, Oxygen therapy, Research participation, and Transplantation) in patients with IPF and their caregivers. Methods: Patients with IPF (diagnosed in the year previous to their initial center visit) from the University of Pittsburgh Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease at University of Pittsburgh Medical Center-together with their caregivers-were randomized to receive the intervention "A Program of SUPPORT" or usual care. This included a total of three research visits aligned with their clinic visit over a period of 6 to 8 months. We measured feasibility, acceptability, and efficacy of this intervention. Results: A total of 136 patient/caregiver dyads were eligible, and a total of 76 dyads were enrolled and participated. Participants were predominately White males >65 years old. Thirteen percent did not have an identified caregiver. Feasibility was limited; 56% of eligible dyads were enrolled. Eligible dyads (24%) were interested in participating but too fatigued to stay after their clinic visit. There was high attrition (20% of participants died before the study was completed). "A Program of SUPPORT" was acceptable to participants. Efficacy demonstrated a significant improvement in caregiver's knowledge, disease preparedness, and confidence in caring for the patient as well as an improvement in knowledge and advance care planning completion in patient participants. Conclusions: Patients with IPF and their caregivers have unmet needs regarding knowledge of their disease, self-management strategies, and preparedness for EOL planning. This nurse-led intervention demonstrated acceptability and efficacy in knowledge and advance care planning completion in patients and in knowledge, disease preparedness, and confidence in caregivers. Future research should identify additional strategies, including telemedicine resources to reach additional patients and their caregivers earlier in their disease course. Clinical trial registered with clinicaltrials.gov (NCT02929017).

TOLLIP (Toll-interacting protein) is an intracellular adaptor protein with diverse actions throughout the body. In a context- and cell type-specific manner, TOLLIP can function as an inhibitor of inflammation and endoplasmic-reticulum stress, an activator of autophagy, or a critical regulator of intracellular vacuole trafficking. The distinct functions of this protein have been linked to innate immune responses and lung epithelial-cell apoptosis. TOLLIP genetic variants have been associated with a variety of chronic lung diseases, including idiopathic pulmonary fibrosis, asthma, and primary graft dysfunction after lung transplantation, and with infections, such as tuberculosis, Legionella pneumonia, and respiratory viruses. TOLLIP exists in a delicate homeostatic balance, with both positive and negative effects on the trajectory of pulmonary diseases. This translational review summarizes the genetic and molecular associations that link TOLLIP to the development and progression of noninfectious and infectious pulmonary diseases. We highlight current limitations of in vitro and in vivo models in assessing the role of TOLLIP in these conditions, and we describe future approaches that will enable a more nuanced exploration of the role of TOLLIP in pulmonary conditions. There has been a surge in recent research evaluating the role of this protein in human diseases, but critical mechanistic pathways require further exploration. By understanding its biologic functions in disease-specific contexts, we will be able to determine whether TOLLIP can be therapeutically modulated to treat pulmonary diseases.

Background: Pentraxin 3 (PTX3) influences innate immunity and inflammation, host defence, the complement cascade and angiogenesis. PTX3 expression in lung and blood of subjects with tobacco exposure, and its potential relationship with disease pattern and clinical outcome are poorly understood., Methods: Using independent platforms and cohorts, we identified associations of PTX3 gene expression in lung tissue and plasma from current and former tobacco smokers (with and without chronic obstructive pulmonary disease, COPD) to disease phenotypes including quantitative CT determined emphysema, lung function, symptoms and survival. Two putative regulatory variants of the PTX3 gene were examined for association with COPD manifestations. The relationship between plasma PTX3 and hyaluronic acid levels was further examined., Results: PTX3 gene expression in lung tissue was directly correlated with emphysema severity (p<0.0001). Circulating levels of PTX3 were inversely correlated with FEV 1 (p=0.006), and positively associated with emphysema severity (p=0.004) and mortality (p=0.008). Two PTX3 gene regulatory variants were associated with a lower risk for emphysema and expiratory airflow obstruction, and plasma levels of PTX3 and hyaluronic acid were related., Conclusions: These data show strong and overlapping associations of lung and blood PTX3 levels, and PTX3 regulatory gene variants, with the severity of airflow obstruction, emphysema and mortality among smokers. These findings have potential implications regarding the pathogenesis of smoking-related lung diseases and warrant further exploration for the use of PTX3 as a predictive biomarker., Competing Interests: Competing interests: NK reports personal fees from Biogen Idec, Boehringer Ingelheim, Third Rock, Pliant, Samumed, uMedii, Indaloo, Theravance, LifeMax, Three Lake Partners, RohBar; Non-financial support from Miragen and a research grant from Veracyte. NK has a patent New Therapies in Pulmonary Fibrosis and a patent on Peripheral Blood Gene Expression that is licensed to Biotech. All are outside this work., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Lung microbiota profiles in patients with early idiopathic pulmonary fibrosis (IPF) have been associated with disease progression; however, the topographic heterogeneity of lung microbiota and their roles in advanced IPF are unknown., Methods: We performed a retrospective, case-control study of explanted lung tissue obtained at the time of lung transplantation or rapid autopsy from patients with IPF and other chronic lung diseases (connective tissue disease-associated interstitial lung disease (CTD-ILD), cystic fibrosis (CF), COPD and donor lungs unsuitable for transplant from Center for Organ Recovery and Education (CORE)). We sampled subpleural tissue and airway-based specimens (bronchial washings and airway tissue) and quantified bacterial load and profiled communities by amplification and sequencing of the 16S rRNA gene., Findings: Explants from 62 patients with IPF, 15 patients with CTD-ILD, 20 patients with CF, 20 patients with COPD and 20 CORE patients were included. Airway-based samples had higher bacterial load compared with distal parenchymal tissue. IPF basilar tissue had much lower bacterial load compared with CF and CORE lungs (p<0.001). No microbial community differences were found between parenchymal tissue samples from different IPF lobes. Dirichlet multinomial models revealed an IPF cluster (29%) with distinct composition, high bacterial load and low alpha diversity, exhibiting higher odds for acute exacerbation or death., Interpretation: IPF explants had low biomass in the distal parenchyma of all three lobes with higher bacterial load in the airways. The discovery of a distinct subgroup of patients with IPF with higher bacterial load and worse clinical outcomes supports investigation of personalised medicine approaches for microbiome-targeted interventions., Competing Interests: Competing interests: BJMcV is a consultant for The VeraMedica Institute and receives research funding from Bayer Pharmaceuticals. GDK has received research funding from Karius. The other authors have no conflicts of interest to declare., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Cellular senescence due to telomere dysfunction has been hypothesized to play a role in age-associated diseases including idiopathic pulmonary fibrosis (IPF). It has been postulated that paracrine mediators originating from senescent alveolar epithelia signal to surrounding mesenchymal cells and contribute to disease pathogenesis. However, murine models of telomere-induced alveolar epithelial senescence fail to display the canonical senescence-associated secretory phenotype (SASP) that is observed in senescent human cells. In an effort to understand human-specific responses to telomere dysfunction, we modeled telomere dysfunction-induced senescence in a human alveolar epithelial cell line. We hypothesized that this system would enable us to probe for differences in transcriptional and proteomic senescence pathways in vitro and to identify novel secreted protein (secretome) changes that potentially contribute to the pathogenesis of IPF. Following induction of telomere dysfunction, a robust senescence phenotype was observed. RNA-seq analysis of the senescent cells revealed the SASP and comparisons to previous murine data highlighted differences in response to telomere dysfunction. We conducted a proteomic analysis of the senescent cells using a novel biotin ligase capable of labeling secreted proteins. Candidate biomarkers selected from our transcriptional and secretome data were then evaluated in IPF and control patient plasma. Four novel proteins were found to be differentially expressed between the patient groups: stanniocalcin-1, contactin-1, tenascin C, and total inhibin. Our data show that human telomere-induced, alveolar epithelial senescence results in a transcriptional SASP that is distinct from that seen in analogous murine cells. Our findings suggest that studies in animal models should be carefully validated given the possibility of species-specific responses to telomere dysfunction. We also describe a pragmatic approach for the study of the consequences of telomere-induced alveolar epithelial cell senescence in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sullivan, Jiang, Hinchie, Roth, Bahudhanapati, Nouraie, Liu, McDyer, Mallampalli, Zhang, Kass, Finkel and Alder.)

Smad4 plays a central role in the regulation of extracellular matrix (ECM) protein expression and cell differentiation; however, the molecular regulation of Smad4 protein stability by a deubiquitinase has not been reported. In the current study, we reveal that a deubiquitinase USP13 stabilizes Smad4, ultimately modulating ECM protein expression in lung fibroblast cells. USP13 was increased in primary adult lung fibroblasts isolated from bleomycin-challenged mice and transforming growth factor (TGF)-β1-treated primary mouse lung fibroblasts. In a bleomycin-induced murine model of lung fibrosis, USP13-deficient mice showed reduced ECM levels such as fibronectin (FN) and collagen compared with wild-type mice. The reductions in both protein levels and mRNA expression of ECM were observed in the isolated lung fibroblasts from USP13-deficient mice, suggesting that downregulation of USP13 reduces ECM levels through inhibiting its transcription. To investigate the molecular mechanisms by which USP13 modulates ECM expression, we focused on the role of USP13 on Smad4 expression. Overexpression of USP13 increased FN and Smad4 protein levels in lung fibroblasts, while downregulation of USP13 reduced Smad4 protein levels, without altering Smad4 mRNA expression, suggesting that USP13 regulates Smad4 protein stability. Knockdown of USP13 decreased Smad4 half-life and promoted Smad4 ubiquitination. Both Smad4 and USP13 were co-localized in the cytoplasm in treated cell, and co-translocated into the nucleus in response to TGF-β1. The results indicate that USP13 promotes ECM expression by stabilizing Smad4 in lung fibroblasts and plays a role in the maintenance of the extracellular matrix in lungs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is characterized by altered epithelial cell phenotypes, which are associated with myofibroblast accumulation in the lung. Atypical alveolar epithelial cells in IPF express molecular markers of airway epithelium. Polymorphisms within and around Toll interacting protein (TOLLIP) are associated with the susceptibility to IPF and mortality. However, the functional role of TOLLIP in IPF is unknown. Using lung tissues from IPF and control subjects, we showed that expression of TOLLIP gene in the lung parenchyma is globally lower in IPF compared to controls. Lung cells expressing significant levels of TOLLIP include macrophages, alveolar type II, and basal cells. TOLLIP protein expression is lower in the parenchyma of IPF lungs but is expressed in the atypical epithelial cells of the distal fibrotic regions. Using overexpression and silencing approaches, we demonstrate that TOLLIP protects cells from bleomycin-induced apoptosis using primary bronchial epithelial cells and BEAS-2B cells. The protective effects are mediated by reducing mitochondrial reactive oxygen species (ROS) levels and upregulating autophagy. Therefore, global downregulation of the TOLLIP gene in IPF lungs may predispose injured lung epithelial cells to apoptosis and to the development of IPF., (© 2020 Federation of American Societies for Experimental Biology.)

Idiopathic pulmonary fibrosis (IPF) is a lethal, medically refractory syndrome characterized by intrapulmonary accumulations of extracellular matrix (ECM) proteins produced by fibroblasts. Activation, clonal expansion, and differentiation of lymphocytes are also frequently present in IPF. Activated T cells are known to exert several effects that promote ECM production, but opposing homeostatic actions, wherein T cells can inhibit fibrosis, are less well understood. We found that CD27, a TNF receptor ubiquitously expressed on naive T cells, is downregulated on CD4 T cells of patients with IPF and that CD70, the sole ligand for CD27, is present on human pulmonary fibroblasts. We hypothesized that cognate engagements between lymphocyte CD27 and fibroblast CD70 could have functional consequences. Accordingly, a series of subsequent studies were conducted to examine the possible role of CD27-CD70 interactions in the regulation of fibrogenesis. Using IB, flow cytometry, RT-PCR, and kinomic assays, we found that fibroblast CD70 expression was inversely correlated with cell density and upregulated by TGF-β1 (transforming growth factor-β1). CD70 agonists, including T-cell-derived soluble CD27, markedly diminished fibroblast collagen and fibronectin synthesis, and these effects were potent enough to also inhibit profibrotic actions of TGF-β1 on ECM production in vitro and in two distinct ex vivo human skin models. CD70 activation was mediated by AKT (protein kinase B) and complex interconnected signaling pathways, and it was abated by prior CD70 knockdown. These results show that the CD70-CD27 axis modulates T-cell-fibroblast interactions and may be an important regulator of fibrosis and wound healing. Fibroblast CD70 could also be a novel target for specific mechanistically based antifibrosis treatments.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with a median survival of 3-4 years from time of initial diagnosis, similar to the time course of many malignancies. A hallmark of IPF is its unpredictable disease course, ranging from long periods of clinical stability to acute exacerbations with rapid decompensation. As the disease progresses, patients with chronic cough and progressive exertional dyspnea become oxygen dependent. They may experience significant distress due to concurrent depression, anxiety, and fatigue, which often lead to increased symptom burden and decreased quality of life. Despite these complications, palliative care is an underutilized, and often underappreciated, resource before end-of-life care in this population. While there is growing recognition about early palliative care in IPF, current data suggest referral patterns vary widely based on institutional practices. In addition to focusing on symptom management, there is emphasis on supplemental oxygen use, pulmonary rehabilitation, quality of life, and end-of-life care. Importantly, increased use of support groups and national foundation forums have served as venues for further disease education, communication, and advanced care planning outside of the hospital settings. The purpose of this review article is to discuss the clinical features of IPF, the role of palliative care in chronic disease management, current data supporting benefits of palliative care in IPF, its role in symptom management, and practices to help patients and their caregivers achieve their best quality of life.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder (EBV-PTLD) is a serious complication in lung transplant recipients (LTRs) associated with significant mortality. We performed a single-center retrospective study to evaluate the risks for PTLD in LTRs over a 7-year period. Of 611 evaluable LTRs, we identified 28 cases of PTLD, with an incidence of 4.6%. Kaplan-Meier analysis showed a decreased freedom from PTLD in idiopathic pulmonary fibrosis (IPF)-LTRs (P < .02). Using a multivariable Cox proportional hazards model, we found IPF (hazard ratio [HR] 3.51, 95% confidence interval [CI] 1.33-8.21, P = .01) and alemtuzumab induction therapy (HR 2.73, 95% CI 1.10-6.74, P = .03) as risk factors for PTLD, compared to EBV mismatch (HR: 34.43, 95% CI 15.57-76.09, P < .0001). Early PTLD (first year) was associated with alemtuzumab use (P = .04), whereas IPF was a predictor for late PTLD (after first year) (P = .002), after controlling for age and sex. Kaplan-Meier analysis revealed a shorter time to death from PTLD in IPF LTRs compared to other patients (P = .04). The use of alemtuzumab in EBV mismatch was found to particularly increase PTLD risk. Together, our findings identify IPF LTRs as a susceptible population for PTLD. Further studies are required to understand the mechanisms driving PTLD in IPF LTRs and develop strategies to mitigate risk., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Relaxin/relaxin family peptide receptor 1 (RXFP1) signaling is important for both normal physiology and disease. Strong preclinical evidence supports relaxin as a potent antifibrotic molecule. However, relaxin-based therapy failed in clinical trial in patients with systemic sclerosis. We and others have discovered that aberrant expression of RXFP1 may contribute to the abnormal relaxin/RXFP1 signaling in different diseases. Reduced RXFP1 expression and alternative splicing transcripts with potential functional consequences have been observed in fibrotic tissues. A relative decrease in RXFP1 expression in fibrotic tissues-specifically lung and skin-may explain a potential insensitivity to relaxin. In addition, receptor dimerization also plays important roles in relaxin/RXFP1 signaling., Methods: This review describes the tissue specific expression, characteristics of the splicing variants, and homo/heterodimerization of RXFP1 in both normal physiological function and human diseases. We discuss the potential implications of these molecular features for developing therapeutics to restore relaxin/RXFP1 signaling and to harness relaxin's potential antifibrotic effects., Results: Relaxin/RXFP1 signaling is important in both normal physiology and in human diseases. Reduced expression of RXFP1 in fibrotic lung and skin tissues surrenders both relaxin/RXFP1 signaling and their responsiveness to exogenous relaxin treatments. Alternative splicing and receptor dimerization are also important in regulating relaxin/RXFP1 signaling., Conclusions: Understanding the molecular mechanisms that drive aberrant expression of RXFP1 in disease and the functional roles of alternative splicing and receptor dimerization will provide insight into therapeutic targets that may restore the relaxin responsiveness of fibrotic tissues., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Systemic scleroderma (SSc) is an autoimmune disease that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive profibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three-quarters of individuals with SSc develop pulmonary fibrosis within 5 years, the main cause of SSc mortality. No approved medicines to manage lung SSc currently exist. Recent research suggests that profibrotic signaling by transforming growth factor β (TGF-β) is directly tied to SSc. Previous studies have also shown that ubiquitin E3 ligases potently control TGF-β signaling through targeted degradation of key regulatory proteins; however, the roles of these ligases in SSc-TGF-β signaling remain unclear. Here we utilized primary SSc patient lung cells for high-throughput screening of TGF-β signaling via high-content imaging of nuclear translocation of the profibrotic transcription factor SMAD family member 2/3 (SMAD2/3). We screened an RNAi library targeting ubiquitin E3 ligases and observed that knockdown of the E3 ligase Kelch-like protein 42 (KLHL42) impairs TGF-β-dependent profibrotic signaling. KLHL42 knockdown reduced fibrotic tissue production and decreased TGF-β-mediated SMAD activation. Using unbiased ubiquitin proteomics, we identified phosphatase 2 regulatory subunit B'ϵ (PPP2R5ϵ) as a KLHL42 substrate. Mechanistic experiments validated ubiquitin-mediated control of PPP2R5ϵ stability through KLHL42. PPP2R5ϵ knockdown exacerbated TGF-β-mediated profibrotic signaling, indicating a role of PPP2R5ϵ in SSc. Our findings indicate that the KLHL42-PPP2R5ϵ axis controls profibrotic signaling in SSc lung fibroblasts. We propose that future studies could investigate whether chemical inhibition of KLHL42 may ameliorate profibrotic signaling in SSc., (© 2020 Lear et al.)

Objective: Interstitial lung disease (ILD) is a frequent complication of rheumatoid arthritis (RA), occurring in up to 40% of patients during the course of their disease. Early diagnosis is critical, particularly given the shared clinicoepidemiologic features between advanced rheumatoid arthritis-associated ILD (RA-ILD) and idiopathic pulmonary fibrosis (IPF). This study was undertaken to define the molecular basis of this overlap through comparative profiling of serum proteins in RA-ILD and IPF., Methods: Multiplex enzyme-linked immunosorbent assays (ELISAs) were used to profile 45 protein biomarkers encompassing cytokines/chemokines, growth factors, and matrix metalloproteinases (MMPs) in sera obtained from RA patients with ILD and those without, individuals with IPF, and healthy controls. Levels of selected serum proteins were compared between patient subgroups using adjusted linear regression, principal component analysis (PCA), and least absolute shrinkage and selection operator (LASSO) modeling., Results: Multiplex ELISA-based assessment of sera from 2 independent cohorts (Veterans Affairs [VA] and Non-VA) revealed a number of non-overlapping biomarkers distinguishing RA-ILD from RA without ILD (RA-no ILD) in adjusted regression models. Parallel analysis of sera from IPF patients also yielded a discriminatory panel of protein markers in models adjusted for age/sex/smoking, which showed differential overlap with profiles linked to RA-ILD in the VA cohort versus the Non-VA cohort. PCA revealed several distinct functional groups of RA-ILD-associated markers that, in the VA cohort, encompassed proinflammatory cytokines/chemokines as well as 2 different subsets of MMPs. Finally, LASSO regression modeling in the Non-VA and VA cohorts revealed distinct biomarker combinations capable of discriminating RA-ILD from RA-no ILD., Conclusion: Comparative serum protein biomarker profiling represents a viable method for distinguishing RA-ILD from RA-no ILD and identifying population-specific mediators shared with IPF., (© 2019, American College of Rheumatology.)

Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, ultimately leading to organ scarring. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have recently emerged as pivotal drivers of mesenchymal cell activation in human fibrosis. Therapeutic strategies inhibiting YAP and TAZ have been hindered by the critical role that these proteins play in regeneration and homeostasis in different cell types. Here, we find that the Gα s -coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells relative to other resident cells of these organs. Agonism of DRD1 selectively inhibits YAP/TAZ function in mesenchymal cells and shifts their phenotype from profibrotic to fibrosis resolving, reversing in vitro extracellular matrix stiffening and in vivo tissue fibrosis in mouse models. Aromatic l-amino acid decarboxylase [DOPA decarboxylase (DDC)], the enzyme responsible for the final step in biosynthesis of dopamine, is decreased in the lungs of subjects with idiopathic pulmonary fibrosis, and its expression inversely correlates with disease severity, consistent with an endogenous protective role for dopamine signaling that is lost in pulmonary fibrosis. Together, these findings establish a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via DRD1 that reverses fibrosis in mice., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: The processes that result in progression of idiopathic pulmonary fibrosis (IPF) remain enigmatic. Moreover, the course of this disease can be highly variable and difficult to accurately predict. We hypothesized analyses of body mass index (BMI), a simple, routine clinical measure, may also have prognostic value in these patients, and might provide mechanistic insights. We investigated the associations of BMI changes with outcome, plasma adipokines, and adaptive immune activation among IPF patients., Methods: Data were analyzed in an IPF discovery cohort (n = 131) from the University of Pittsburgh, and findings confirmed in patients from the University of Alabama at Birmingham (n = 148). Plasma adipokines were measured by ELISA and T-cell phenotypes determined by flow cytometry., Results: Transplant-free one-year survivals in subjects with the greatest rates of BMI decrements, as percentages of initial BMI (>0.68%/month), were worse than among those with more stable BMI in both discovery (HR = 1.8, 95%CI = 1.1-3.2, p = 0.038) and replication cohorts (HR = 2.5, 95%CI = 1.2-5.2, p = 0.02), when adjusted for age, baseline BMI, and pulmonary function. BMI decrements >0.68%/month were also associated with greater mortality after later lung transplantations (HR = 4.6, 95%CI = 1.7-12.5, p = 0.003). Circulating leptin and adiponectin levels correlated with BMI, but neither adipokine was prognostic per se. BMI decrements were significantly associated with increased proportions of circulating end-differentiated (CD28null) CD4 T-cells (CD28%), a validated marker of repetitive T-cell activation and IPF prognoses., Conclusions: IPF patients with greatest BMI decrements had worse outcomes, and this effect persisted after lung transplantation. Weight loss in these patients is a harbinger of poor prognoses, and may reflect an underlying systemic process, such as adaptive immune activation., Competing Interests: The authors have declared that no competing interests exist.

Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology, and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells from mice with epithelial-specific telomere dysfunction identified the transforming growth factor-β family member, growth and differentiation factor 15 ( Gdf15 ), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single-cell RNA sequencing of human lungs identifies epithelial cells as the primary source of GDF15 , and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.

A comprehensive understanding of the changes in gene expression in cell types involved in idiopathic pulmonary fibrosis (IPF) will shed light on the mechanisms underlying the loss of alveolar epithelial cells and development of honeycomb cysts and fibroblastic foci. We sought to understand changes in IPF lung cell transcriptomes and gain insight into innate immune aspects of pathogenesis.We investigated IPF pathogenesis using single-cell RNA-sequencing of fresh lung explants, comparing human IPF fibrotic lower lobes reflecting late disease, upper lobes reflecting early disease and normal lungs.IPF lower lobes showed increased fibroblasts, and basal, ciliated, goblet and club cells, but decreased alveolar epithelial cells, and marked alterations in inflammatory cells. We found three discrete macrophage subpopulations in normal and fibrotic lungs, one expressing monocyte markers, one highly expressing FABP4 and INHBA (FABP4 hi ), and one highly expressing SPP1 and MERTK (SPP1 hi ). SPP1 hi macrophages in fibrotic lower lobes showed highly upregulated SPP1 and MERTK expression. Low-level local proliferation of SPP1 hi macrophages in normal lungs was strikingly increased in IPF lungs.Co-localisation and causal modelling supported the role for these highly proliferative SPP1 hi macrophages in activation of IPF myofibroblasts in lung fibrosis. These data suggest that SPP1 hi macrophages contribute importantly to lung fibrosis in IPF, and that therapeutic strategies targeting MERTK and macrophage proliferation may show promise for treatment of this disease., Competing Interests: Conflict of interest: C. Morse has nothing to disclose. Conflict of interest: T. Tabib has nothing to disclose. Conflict of interest: J. Sembrat has nothing to disclose. Conflict of interest: K. Buschur has nothing to disclose. Conflict of interest: H. Trejo Bittar has nothing to disclose. Conflict of interest: E. Valenzi has nothing to disclose. Conflict of interest: Y. Jiang has nothing to disclose. Conflict of interest: D.J. Kass reports grants from NIH, during the conduct of the study; grants from Regeneron, outside the submitted work. Conflict of interest: K. Gibson has nothing to disclose. Conflict of interest: W. Chen has nothing to disclose. Conflict of interest: A. Mora has nothing to disclose. Conflict of interest: P.V. Benos has nothing to disclose. Conflict of interest: M. Rojas has nothing to disclose. Conflict of interest: R. Lafyatis has nothing to disclose., (Copyright ©ERS 2019.)

Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases ( n  = 3,624) and control subjects ( n  = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1 ) individual common variants via logistic regression and 2 ) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele ( P  = 9.60 × 10 -295 ). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with an unpredictable course and a median survival of three to four years. This timeline challenges providers to approach diagnosis, oxygen therapy, rehabilitation, transplantation, and end-of-life discussions in limited encounters. There is currently no widely accepted guideline for determining when IPF patients should be referred to palliative care (PC). Objective: We sought to describe the patient and clinical factors associated with PC referral, as well as its impact on mortality and location of death. We also aimed to examine temporal trends in PC referral in this population. Materials and Methods: Patient data were retrospectively extracted from the health system repository of our specialty referral center for all new IPF patients evaluated between 2000 and 2016 ( n  = 828). Exclusion criteria included transplant recipients and patients who did not have IPF. Results: One hundred twelve (13.5%) IPF patients received formal PC referral. Recipients were older at diagnosis (72 years vs. 69 years, p  < 0.001), had higher frequency of Charlson Comorbidity Index ≥1 (55% vs. 42%, p  = 0.011), resided closer to our institution (16 miles vs. 54 miles, p  < 0.001), and had a higher number of total outpatient visits (7 vs. 4, p  < 0.001). PC was associated with less in-hospital death (44% vs. 60%, p  = 0.006) and more in-home and hospice death (56% vs. 40%, p  = 0.006). Conclusions: IPF patients referred to PC were older with more severe comorbidities, resided closer to our specialty referral center, and had more outpatient follow-up. This was associated with more in-home and hospice deaths. The patient-provider relationship and frequency of follow-up visits likely play important roles in the introduction of end-of-life discussions.