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Fibroblasts are present throughout the body and function to maintain tissue homeostasis. Recent studies have identified diverse fibroblast subsets in healthy and injured tissues1,2, but the origins and functional roles of injury-induced fibroblast lineages remain unclear. Here we show that lung-specialized alveolar fibroblasts take on multiple molecular states with distinct roles in facilitating responses to fibrotic lung injury. We generate a genetic tool that uniquely targets alveolar fibroblasts to demonstrate their role in providing niches for alveolar stem cells in homeostasis and show that loss of this niche leads to exaggerated responses to acute lung injury. Lineage tracing identifies alveolar fibroblasts as the dominant origin for multiple emergent fibroblast subsets sequentially driven by inflammatory and pro-fibrotic signals after injury. We identify similar, but not completely identical, fibroblast lineages in human pulmonary fibrosis. TGFβ negatively regulates an inflammatory fibroblast subset that emerges early after injury and stimulates the differentiation into fibrotic fibroblasts to elicit intra-alveolar fibrosis. Blocking the induction of fibrotic fibroblasts in the alveolar fibroblast lineage abrogates fibrosis but exacerbates lung inflammation. These results demonstrate the multifaceted roles of the alveolar fibroblast lineage in maintaining normal alveolar homeostasis and orchestrating sequential responses to lung injury.

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGF-β1 signaling inhibitor epigallocatechin gallate (EGCG) to interstitial lung disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA-Seq on spare tissue. Biopsies from untreated patients showed higher fibroblast TGF-β1 signaling compared with nondisease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGF-β1 signaling and several proinflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzled-related protein 2 (sFRP2), an unrecognized TGF-β1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision-cut lung slices (PCLSs) from nondiseased donors, we found TGF-β1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature cytokeratin 5+ (Krt5+) basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGF-β1 signaling in ILD and the therapeutic potential of EGCG in reducing idiopathic pulmonary fibrosis-related (IPF-related) transcriptional changes and identify TGF-β1/noncanonical Wnt pathway crosstalk via sFRP2 as a mechanism for dysfunctional epithelial signaling in IPF/ILD.

Aging and cellular senescence are increasingly recognized as key contributors to pulmonary fibrosis. However, our understanding in the context of scleroderma-associated interstitial lung disease (SSc-ILD) is limited. To investigate, we leveraged previously established lung aging- and cell-specific senescence signatures to determine their presence and potential relevance to SSc-ILD. We performed a gene expression meta-analysis of lung tissues from 38 SSc-ILD and 18 healthy controls and found that markers (GDF15, COMP, and CDKN2A) and pathways (p53) of senescence were significantly increased in SSc-ILD. When probing the established aging and cellular senescence signatures, we found that epithelial and fibroblast senescence signatures had a 3.6- and 3.7-fold enrichment, respectively, in the lung tissue of SSc-ILD and that lung aging genes (CDKN2A, FRZB, PDE1A, and NAPI12) were increased in SSc-ILD. These signatures were also enriched in SSc skin and associated with degree of skin involvement (limited vs. diffuse cutaneous). To further support these findings, we examined telomere length (TL), a surrogate for aging, in the lung tissue and found that, independent of age, SSc-ILD had significantly shorter telomeres than controls in type II alveolar cells in the lung. TL in SSc-ILD was comparable to idiopathic pulmonary fibrosis, a disease of known aberrant aging. Taken together, this study provides novel insight into the possible mechanistic effects of accelerated aging and aberrant cellular senescence in SSc-ILD pathogenesis.

BackgroundStudies suggest a harmful pharmacogenomic interaction exists between short leukocyte telomere length (LTL) and immunosuppressants in idiopathic pulmonary fibrosis (IPF). It remains unknown if a similar interaction exists in non-IPF interstitial lung disease (ILD).MethodsA retrospective, multicentre cohort analysis was performed in fibrotic hypersensitivity pneumonitis (fHP), unclassifiable ILD (uILD) and connective tissue disease (CTD)-ILD patients from five centres. LTL was measured by quantitative PCR for discovery and replication cohorts and expressed as age-adjusted percentiles of normal. Inverse probability of treatment weights based on propensity scores were used to assess the association between mycophenolate or azathioprine exposure and age-adjusted LTL on 2-year transplant-free survival using weighted Cox proportional hazards regression incorporating time-dependent immunosuppressant exposure.ResultsThe discovery and replication cohorts included 613 and 325 patients, respectively. In total, 40% of patients were exposed to immunosuppression and 22% had LTL

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGF- [beta]1 signaling inhibitor epigallocatechin gallate (EGCG) to interstitial lung disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA-Seq on spare tissue. Biopsies from untreated patients showed higher fibroblast TGF-[beta]1 signaling compared with nondisease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGF- [beta]1 signaling and several proinflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzledrelated protein 2 (sFRP2), an unrecognized TGF-[beta]1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision-cut lung slices (PCLSs) from nondiseased donors, we found TGF-[beta]1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature cytokeratin 5+ (Krt5+) basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGF-[beta]1 signaling in ILD and the therapeutic potential of EGCG in reducing idiopathic pulmonary fibrosis-related (IPF-related) transcriptional changes and identify TGF-[beta]1/noncanonical Wnt pathway crosstalk via sFRP2 as a mechanism for dysfunctional epithelial signaling in IPF/ILD., Introduction Fibrotic interstitial lung diseases (ILDs), such as idiopathic pulmonary fibrosis (IPF), cause respiratory failure through progressive replacement of lung parenchyma with nonfunctional fibrotic tissue. IPF is widely thought to [...]

Primary graft dysfunction (PGD) is a major risk factor for chronic lung allograft dysfunction (CLAD) following lung transplantation, but the mechanisms linking these pathologies are poorly understood. We hypothesized that the replicative stress induced by PGD would lead to erosion of telomeres, and that this telomere dysfunction could potentiate CLAD. In a longitudinal cohort of 72 lung transplant recipients with >6 years median follow-up time, we assessed tissue telomere length, PGD grade, and freedom from CLAD. Epithelial telomere length and fibrosis-associated gene expression were assessed on endobronchial biopsies taken at 2 - 4 weeks post-transplant by TeloFISH assay and nanoString digital RNA counting. Negative-binomial mixed-effects and Cox-proportional hazards models accounted for TeloFISH staining batch effects and subject characteristics including donor age. Increasing grade of PGD severity was associated with shorter airway epithelial telomere lengths (P = 0.01). Transcriptomic analysis of fibrosis-associated genes showed alteration in fibrotic pathways in airway tissue recovering from PGD, while telomere dysfunction was associated with inflammation and impaired remodeling. Shorter tissue telomere length was in turn associated with increased CLAD risk, with a hazard ratio of 1.89 (95% CI 1.16 - 3.06) per standard deviation decrease in airway telomere length, after adjusting for subject characteristics. PGD may accelerate telomere dysfunction, potentiating immune responses and dysregulated repair. Epithelial cell telomere dysfunction may represent one of several mechanisms linking PGD to CLAD.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. Objectives: To identify and validate molecular determinants of IPF survival. Methods: A staged genome-wide association study was performed using paired genomic and survival data. Stage I cases were drawn from centers across the United States and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplantation-free survival (TFS). Stage I variants with nominal significance (P

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.

ImportancePulmonary fibrosis (PF) is characterized by progressive scarring of lung tissue and poor survival. Racial and ethnic minority populations face the greatest risk of morbidity and mortality from disparities impacting respiratory health, but the pattern of age at clinically relevant outcomes across diverse racial and ethnic populations with PF is unknown.ObjectiveTo compare the age at PF-related outcomes and the heterogeneity in survival patterns among Hispanic, non-Hispanic Black, and non-Hispanic White participants.Design, setting, and participantsThis cohort study included adult patients with a PF diagnosis and used data from prospective clinical registries: the Pulmonary Fibrosis Foundation Registry (PFFR) for the primary cohort and registries from 4 geographically distinct tertiary hospitals in the US for the external multicenter validation (EMV) cohort. Patients were followed between January 2003 and April 2021.ExposuresRace and ethnicity comparisons between Black, Hispanic, and White participants with PF.Main outcomes and measuresAge and sex distribution of participants were measured at the time of study enrollment. All-cause mortality and age at PF diagnosis, hospitalization, lung transplant, and death were assessed in participants over 14 389 person-years. Differences between racial and ethnic groups were compared using Wilcoxon rank sum tests, Bartlett 1-way analysis of variance, and χ2 tests, and crude mortality rates and rate ratios were assessed across racial and ethnic categories using Cox proportional hazards regression models.ResultsIn total, 4792 participants with PF were assessed (mean [SD] age, 66.1 [11.2] years; 2779 [58.0%] male; 488 [10.2%] Black, 319 [6.7%] Hispanic, and 3985 [83.2%] White); 1904 were in the PFFR and 2888 in the EMV cohort. Black patients with PF were consistently younger than White patients (mean [SD] age at baseline, 57.9 [12.0] vs 68.6 [9.6] years; P

Pulmonary fibrosis (PF) is characterized by profound scarring and poor survival. We investigated the association of leukocyte telomere length (LTL) with chronological age and mortality across racially diverse PF cohorts. LTL measurements among participants with PF stratified by race/ethnicity were assessed in relation to age and all-cause mortality, and compared to controls. Generalized linear models were used to evaluate the age-LTL relationship, Cox proportional hazards models were used for hazard ratio estimation, and the Cochran-Armitage test was used to assess quartiles of LTL. Standardized LTL shortened with increasing chronological age; this association in controls was strengthened in PF (R = -0.28; P

Alveolar formation increases the surface area for gas exchange. A molecular understanding of alveologenesis remains incomplete. Here, we show that the autonomic nerve and alveolar myofibroblast form a functional unit in mice. Myofibroblasts secrete neurotrophins to promote neurite extension/survival, whereas neurotransmitters released from autonomic terminals are necessary for myofibroblast proliferation and migration, a key step in alveologenesis. This establishes a functional link between autonomic innervation and alveolar formation. We also discover that planar cell polarity (PCP) signaling employs a Wnt-Fz/Ror-Vangl cascade to regulate the cytoskeleton and neurotransmitter trafficking/release from the terminals of autonomic nerves. This represents a new aspect of PCP signaling in conferring cellular properties. Together, these studies offer molecular insight into how autonomic activity controls alveolar formation. Our work also illustrates the fundamental principle of how two tissues (e.g., nerves and lungs) interact to build alveoli at the organismal level.

BackgroundProgressive fibrosing interstitial lung disease (ILD) is characterised by parenchymal scar formation, leading to high morbidity and mortality. The ability to predict this phenotype remains elusive. We conducted a proteomic analysis to identify novel plasma biomarkers of progressive fibrosing ILD and developed a proteomic signature to predict this phenotype.MethodsRelative plasma concentrations for 368 biomarkers were determined with use of a semi-quantitative, targeted proteomic platform in patients with connective tissue disease-associated ILD, chronic hypersensitivity pneumonitis, or unclassifiable ILD who provided research blood draws at the University of California (discovery cohort) and the University of Texas (validation cohort). Univariable logistic regression was used to identify individual biomarkers associated with 1-year ILD progression, defined as death, lung transplant, or 10% or greater relative forced vital capacity (FVC) decline. A proteomic signature of progressive fibrosing ILD was then derived with use of machine learning in the University of California cohort and validated in the University of Texas cohort.FindingsThe discovery cohort comprised 385 patients (mean age 63·6 years, 59% female) and the validation cohort comprised 204 patients (mean age 60·7 years, 61% female). 31 biomarkers were associated with progressive fibrosing ILD in the discovery cohort, with 17 maintaining an association in the validation cohort. Validated biomarkers showed a consistent association with progressive fibrosing ILD irrespective of ILD clinical diagnosis. A proteomic signature comprising 12 biomarkers was derived by machine learning and validated in the University of Texas cohort, in which it had a sensitivity of 0·90 and corresponding negative predictive value of 0·91, suggesting that approximately 10% of patients with a low-risk proteomic signature would experience ILD progression in the year after blood draw. Those with a low-risk proteomic signature experienced an FVC change of +85·7 mL (95% CI 6·9 to 164·4) and those with a high-risk signature experienced an FVC change of -227·1 mL (-286·7 to -167·5). A theoretical clinical trial restricted to patients with a high-risk proteomic signature would require 80% fewer patients than one designed without regard to proteomic signature.Interpretation17 plasma biomarkers of progressive fibrosing ILD were identified and showed consistent associations across ILD subtypes. A proteomic signature of progressive fibrosing ILD could enrich clinical trial cohorts and avoid the need for antecedent progression when defining progressive fibrosing ILD for clinical trial enrolment.FundingNational Heart Lung and Blood Institute.

BackgroundTelomere dysfunction is associated with idiopathic pulmonary fibrosis (IPF) and worse outcomes following lung transplantation. Telomere dysfunction may impair immunity by upregulating p53 and arresting proliferation, but its influence on allograft-specific immune responses is unknown. We hypothesized that subjects undergoing lung transplantation for IPF would have impaired T cell proliferation to donor antigens.MethodsWe analyzed peripheral blood mononuclear cells (PBMC) from 14 IPF lung transplant recipients and 12 age-matched non-IPF subjects, before and 2 years after transplantation, as well as PBMC from 9 non-transplant controls. We quantified T cell proliferation and cytokine secretion to donor antigens. Associations between PBMC telomere length, measured by quantitative PCR, and T cell proliferation to alloantigens were evaluated with generalized estimating equation models.ResultsIPF subjects demonstrated impaired CD8+ T cell proliferation to donor antigens pre-transplant (p < 0.05). IL-2, IL-7, and IL-15 cytokine stimulation restored T cell proliferation, while p53 upregulation blocked proliferation. IPF subjects had shorter PBMC telomere lengths than non-IPF subjects (p < 0.001), and short PBMC telomere length was associated with impaired CD8+ T cell proliferation to alloantigens (p = 0.002).ConclusionsIPF as an indication for lung transplant is associated with short PBMC telomere length and impaired T cell responses to donor antigens. However, the rescue of proliferation following cytokine exposure suggests that alloimmune anergy could be overcome. Telomere length may inform immunosuppression strategies for IPF recipients.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of [p16.sup.Ink4a+] cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic [p16.sup.Ink4a+] fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted [p16.sup.INK4a-hi] human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced [p16.sup.INK4a+] fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where [p16.sup.INK4a+] cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases., Introduction Hayflick's original description of a stereotyped proliferative arrest for primary cells in culture as 'senescence at the cellular level' (1) gave way to geroscience that seeks to unravel the [...]

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive, fatal fibrotic interstitial lung disease. Few circulating biomarkers have been identified to have causal effects on IPF.MethodsTo identify candidate IPF-influencing circulating proteins, we undertook an efficient screen of circulating proteins by applying a two-sample Mendelian randomisation (MR) approach with existing publicly available data. For instruments, we used genetic determinants of circulating proteins which reside cis to the encoded gene (cis-single nucleotide polymorphisms (SNPs)), identified by two genome-wide association studies (GWASs) in European individuals (3301 and 3200 subjects). We then applied MR methods to test if the levels of these circulating proteins influenced IPF susceptibility in the largest IPF GWAS (2668 cases and 8591 controls). We validated the MR results using colocalisation analyses to ensure that both the circulating proteins and IPF shared a common genetic signal.ResultsMR analyses of 834 proteins found that a 1 sd increase in circulating galactoside 3(4)-l-fucosyltransferase (FUT3) and α-(1,3)-fucosyltransferase 5 (FUT5) was associated with a reduced risk of IPF (OR 0.81, 95% CI 0.74-0.88; p=6.3×10-7 and OR 0.76, 95% CI 0.68-0.86; p=1.1×10-5, respectively). Sensitivity analyses including multiple cis-SNPs provided similar estimates both for FUT3 (inverse variance weighted (IVW) OR 0.84, 95% CI 0.78-0.91; p=9.8×10-6 and MR-Egger OR 0.69, 95% CI 0.50-0.97; p=0.03) and FUT5 (IVW OR 0.84, 95% CI 0.77-0.92; p=1.4×10-4 and MR-Egger OR 0.59, 95% CI 0.38-0.90; p=0.01). FUT3 and FUT5 signals colocalised with IPF signals, with posterior probabilities of a shared genetic signal of 99.9% and 97.7%, respectively. Further transcriptomic investigations supported the protective effects of FUT3 for IPF.ConclusionsAn efficient MR scan of 834 circulating proteins provided evidence that genetically increased circulating FUT3 level is associated with reduced risk of IPF.

Alveolar formation requires coordinated movement and interaction between alveolar epithelial cells, mesenchymal myofibroblasts, and endothelial cells/pericytes to produce secondary septa. These processes rely on the acquisition of distinct cellular properties to enable ligand secretion for cell-cell signaling and initiate morphogenesis through cellular contraction, cell migration, and cell shape change. In this study, we showed that mitochondrial activity and distribution play a key role in bestowing cellular functions on both alveolar epithelial cells and mesenchymal myofibroblasts for generating secondary septa to form alveoli in mice. These results suggest that mitochondrial function is tightly regulated to empower cellular machineries in a spatially specific manner. Indeed, such regulation via mitochondria is required for secretion of ligands, such as platelet-derived growth factor, from alveolar epithelial cells to influence myofibroblast proliferation and contraction/migration. Moreover, mitochondrial function enables myofibroblast contraction/migration during alveolar formation. Together, these findings yield novel mechanistic insights into how mitochondria regulate pivotal steps of alveologenesis. They highlight selective utilization of energy in cells and diverse energy demands in different cellular processes during development. Our work serves as a paradigm for studying how mitochondria control tissue patterning.

BackgroundPeripheral blood leucocyte telomere length (PBL-TL) is associated with outcomes in patients with idiopathic pulmonary fibrosis. Whether PBL-TL is associated with progression of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is unknown.MethodsA retrospective observational cohort study was performed using prospectively collected data from 213 patients with SSc followed at the University of California San Francisco (UCSF) Scleroderma Center. PBL-TL was measured by quantitative PCR of DNA isolated from peripheral blood. Associations between PBL-TL and pulmonary function test trends in patients with SSc-ILD were assessed by longitudinal analysis using Generalised Linear Mixed Models. Findings were validated in a cohort of 61 patients with SSc-ILD enrolled in the Stanford University Scleroderma Center database.ResultsPatients with UCSF SSc with ILD were found to have shorter PBL-TL compared with those without ILD (6554±671 base pairs (bp) vs 6782±698 bp, p=0.01). Shorter PBL-TL was associated with the presence of ILD (adjusted OR 2.1 per 1000 bp TL decrease, 95% CI [1.25 to 3.70], p=0.006). PBL-TL was shorter in patients with SSc-ILD lacking SSc-specific autoantibodies compared with seropositive subjects (6237±647 bp vs 6651±653 bp, p=0.004). Shorter PBL-TL was associated with increased risk for lung function deterioration with an average of 67 mL greater loss in per year for every 1000 bp decrease in PBL-TL in the combined SSc-ILD cohorts (longitudinal analysis, adjusted model: 95% CI -104 mL to -33 mL, p

Rationaleαv integrins, key regulators of transforming growth factor-β activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvβ6) and fibroblasts (αvβ1) in fibrotic lungs.ObjectivesWe evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis.MethodsSelective αvβ6 and αvβ1, dual αvβ6/αvβ1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-β cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-β signaling. Bleomycin-challenged mice treated with dual αvβ6/αvβ1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation.Measurements and main resultsInhibition of integrins αvβ6 and αvβ1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvβ6/αvβ1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone.ConclusionsIn the fibrotic lung, dual inhibition of integrins αvβ6 and αvβ1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-β.

Matrix metalloproteinase-7 level is associated with worse baseline pulmonary function and worse transplant-free survival in scleroderma interstitial lung disease https://bit.ly/3zZhja7

BackgroundThe major risk factor for idiopathic pulmonary fibrosis (IPF), MUC5B rs35705950, was found to be associated with rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Whilst the MUC5B rs35705950 T risk allele has been associated with better survival in IPF, its impact on RA-ILD prognosis remains to be determined. Our objective was to explore the influence of MUC5B rs35705950 on survival and progression in RA-ILD.MethodsThrough an international retrospective observational study, patients with RA-ILD were genotyped for the MUC5B rs35705950 variant and consecutive pulmonary function tests (PFTs) findings were collected. Longitudinal data up to a 10-year follow-up were considered and analyzed using mixed regression models. Proportional hazards and joint proportional hazards models were used to analyze the association of baseline and longitudinal variables with lung transplant-free survival. Significant progression of RA-ILD was defined as at least an absolute or relative 10% decline of forced vital capacity at 2 years from baseline.ResultsOut of 321 registered patients, 261 were included in the study: 139 women (53.3%), median age at RA-ILD diagnosis 65 years (interquartile range [IQR] 57 to 71), 151 ever smokers (59.2%). Median follow-up was 3.5 years (IQR 1.3 to 6.6). Mortality rate was 32% (95%CI 19 to 42) at 10 years. The MUC5B rs35705950 variant did not impact lung transplant-free survival (HR for the T risk allele carriers=1.26; 95%CI 0.61 to 2.62; P=0.53). Decline in pulmonary function at 2 years was not influenced by MUC5B rs35705950 (OR=0.95; 95%CI 0.44 to 2.05; P=0.89), irrespective of the HRCT pattern.ConclusionIn this study, the MUC5B rs35705950 promoter variant did not influence transplant- free survival or decline in pulmonary function in patients with RA-ILD.

Fibrotic interstitial lung disease (ILD) represents a large group of pulmonary disorders that are often progressive and associated with high morbidity and early mortality. Important advancements in the past 10 years have enabled a better understanding, characterisation, and treatment of these diseases. This Series paper summarises the current approach to treatment of fibrotic ILDs, both pharmacological and non-pharmacological, including recent discoveries and practice-changing clinical trials. We further outline controversies and challenges, with discussion of evolving concepts and future research directions.

Rationale: Sarcopenia is associated with disability and death. The optimal definition and clinical relevance of sarcopenia in lung transplantation remain unknown. Objectives: To assess the construct and predictive validity of sarcopenia definitions in lung transplant candidates. Methods: In a multicenter prospective cohort of 424 lung transplant candidates, we evaluated limited (muscle mass only) and expanded (muscle mass and quality) sarcopenia definitions from the European Working Group on Sarcopenia in Older People 2 (EWGSOP2), the Foundation for the National Institutes of Health (FNIH), and a cohort-specific distribution-based lowest quartile definition. We assessed construct validity using associations with conceptually related factors. We evaluated the relationship between sarcopenia and frailty using generalized additive models. We also evaluated associations between sarcopenia definitions and key pretransplant outcomes, including disability (quantified by the Lung Transplant Valued Life Activities scale [range, 0-3; higher scores = worse disability; minimally important difference, 0.3]) and waitlist delisting/death, by multivariate linear and Cox regression, respectively. Results: Sarcopenia prevalence ranged from 6% to13% by definition used. The limited EWGSOP2 definition demonstrated the highest construct validity, followed by the expanded EWGSOP2 definition and both limited and expanded FNIH and lowest quartile definitions. Sarcopenia exhibited a linear association with the risk of frailty. The EWGSOP2 and expanded lowest quartile definitions were associated with disability, ranging from 0.20 to 0.25 higher Lung Transplant Valued Life Activities scores. Sarcopenia was associated with increased risk of waitlist delisting or death by the limited and expanded lowest quartile definitions (hazard ratio [HR], 3.8; 95% confidence interval [CI], 1.4-9.9 and HR, 3.5; 95% CI, 1.1-11.0, respectively) and the EWGSOP2 limited definition (HR, 2.8; 95% CI, 0.9-8.6) but not with the three other candidate definitions. Conclusions: The prevalence and validity of sarcopenia vary by definition; the EWGSOP2 limited definition exhibited the broadest validity in lung transplant candidates. The linear relationship between low muscle mass and frailty highlights sarcopenia's contribution to frailty and also questions the clinical utility of a sarcopenia cut-point in advanced lung disease. The associations between sarcopenia and important pretransplant outcomes support further investigation into using body composition for candidate risk stratification.

Transforming growth factor-β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2-anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade.

BackgroundThe failure of immune surveillance to remove senescent cells drive age-related diseases. Here, we target an endogenous immune surveillance mechanism that can promote elimination of senescent cells and reverse disease progression.MethodsWe identify a class of lipid-activated T cells, invariant natural killer T cells (iNKTs) are involved in the removal of pathologic senescent cells. We use two disease models in which senescent cells accumulate to test whether activation of iNKT cells was sufficient to eliminate senescent cells in vivo.FindingsSenescent preadipocytes accumulate in white adipose tissue of chronic high-fat diet (HFD) fed mice, and activation of iNKT cells with the prototypical glycolipid antigen alpha-galactosylceramide (αGalCer) led to a reduction of these cells with improved glucose control. Similarly, senescent cells accumulate within the lungs of mice injured by inhalational bleomycin, and αGalCer-induced activation of iNKT cells greatly limited this accumulation, decreased the lung fibrosis and improved survival. Furthermore, co-culture experiments showed that the preferential cytotoxic activity of iNKT cells to senescent cells is conserved in human cells.ConclusionsThese results uncover a senolytic capacity of tissue-resident iNKT cells and pave the way for anti-senescence therapies that target these cells and their mechanism of activation.

ObjectivesSystemic sclerosis (SSc) is an autoimmune fibrotic disease affecting multiple tissues including the lung. A subset of patients with SSc with lung disease exhibit short telomeres in circulating lymphocytes, but the mechanisms underlying this observation are unclear.MethodsSera from the Johns Hopkins and University of California, San Francisco (UCSF) Scleroderma Centers were screened for autoantibodies targeting telomerase and the shelterin proteins using immunoprecipitation and ELISA. We determined the relationship between autoantibodies targeting the shelterin protein TERF1 and telomere length in peripheral leucocytes measured by qPCR and flow cytometry and fluorescent in situ hybridisation (Flow-FISH). We also explored clinical associations of these autoantibodies.ResultsIn a subset of patients with SSc, we identified autoantibodies targeting telomerase and the shelterin proteins that were rarely present in rheumatoid arthritis, myositis and healthy controls. TERF1 autoantibodies were present in 40/442 (9.0%) patients with SSc and were associated with severe lung disease (OR 2.4, p=0.04, Fisher's exact test) and short lymphocyte telomere length. 6/6 (100%) patients with TERF1 autoantibodies in the Hopkins cohort and 14/18 (78%) patients in the UCSF cohort had a shorter telomere length in lymphocytes or leukocytes, respectively, relative to the expected age-adjusted telomere length. TERF1 autoantibodies were present in 11/152 (7.2%) patients with idiopathic pulmonary fibrosis (IPF), a fibrotic lung disease believed to be mediated by telomere dysfunction.ConclusionsAutoantibodies targeting telomere-associated proteins in a subset of patients with SSc are associated with short lymphocyte telomere length and lung disease. The specificity of these autoantibodies for SSc and IPF suggests that telomere dysfunction may have a distinct role in the pathogenesis of SSc and pulmonary fibrosis.

We recently identified epigallocatechin gallate (EGCG), a trihydroxyphenolic compound, as a dual inhibitor of lysyl oxidase-like2 and transforming growth factor-β1 (TGFβ1) receptor kinase that when given orally to patients with idiopathic pulmonary fibrosis (IPF) reversed profibrotic biomarkers in their diagnostic biopsies. Here, we extend these findings to advanced pulmonary fibrosis using cultured precision-cut lung slices from explants of patients with IPF undergoing transplantation. During these experiments, we were surprised to discover that not only did EGCG attenuate TGFβ1 signalling and new collagen accumulation but also activated matrix metalloproteinase-dependent collagen I turnover, raising the possibility of slow fibrosis resolution with continued treatment.

AimsIdiopathic pulmonary fibrosis (IPF) is a genetically mediated, age-associated, progressive form of pulmonary fibrosis characterised pathologically by a usual interstitial pneumonia (UIP) pattern of fibrosis. The UIP pattern is also found in pulmonary fibrosis attributable to clinical diagnoses other than IPF (non-IPF UIP), whose clinical course is similarly poor, suggesting common molecular drivers. This study investigates whether IPF and non-IPF UIP lungs similarly express markers of telomere dysfunction and senescence.Methods and resultsTo test whether patients with IPF and non-IPF UIP share molecular drivers, lung tissues from 169 IPF patients and 57 non-IPF UIP patients were histopathologically and molecularly compared. Histopathological changes in both IPF and non-IPF UIP patients included temporal heterogeneity, microscopic honeycombing, fibroblast foci, and dense collagen fibrosis. Non-IPF UIP lungs were more likely to have lymphocytic infiltration, non-caseating granulomas, airway-centred inflammation, or small airways disease. Telomeres were shorter in alveolar type II (AECII) cells of both IPF and non-IPF UIP lungs than in those of age-similar, unused donor, controls. Levels of molecular markers of senescence (p16 and p21) were elevated in lysates of IPF and non-IPF UIP lungs. Immunostaining localised expression of these proteins to AECII cells. The mucin 5B (MUC5B) gene promoter variant minor allele frequency was similar between IPF and non-IPF UIP patients, and MUC5B expression was similar in IPF and non-IPF UIP lungs.ConclusionsMolecular markers of telomere dysfunction and senescence are pathologically expressed in both IPF and non-IPF UIP lungs. These findings suggest that common molecular drivers may contribute to the pathogenesis of UIP-associated pulmonary fibrosis, regardless of the clinical diagnosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal fibrotic interstitial lung disease. Few circulating biomarkers have been identified to have causal effects on IPF.To identify candidate IPF-influencing circulating proteins, we undertook an efficient screen of circulating proteins by applying a two-sample Mendelian randomisation (MR) approach with existing publicly available data. For instruments we used genetic determinants of circulating proteins which reside cis to the encoded gene (cis-SNPs), identified by two genome-wide association studies (GWASs) in European individuals (3301 and 3200 subjects). We then applied MR methods to test if the levels of these circulating proteins influenced IPF susceptibility in the largest IPF GWAS (2668 cases and 8591 controls). We validated the MR results using colocalization analyses to ensure that both the circulating proteins and IPF shared a common genetic signal.MR analyses of 834 proteins found that a one sd increase in circulating FUT3 and FUT5 was associated with a reduced risk of IPF (OR: 0.81, 95%CI: 0.74-0.88, p=6.3×10-7, and OR: 0.76, 95%CI: 0.68-0.86, p=1.1×10-5). Sensitivity analyses including multiple-cis SNPs provided similar estimates both for FUT3 (inverse variance weighted [IVW] OR: 0.84, 95%CI: 0.78-0.91, p=9.8×10-6, MR-Egger OR: 0.69, 95%CI: 0.50-0.97, p=0.03) and FUT5 (IVW OR: 0.84, 95%CI: 0.77-0.92, p=1.4×10-4, MR-Egger OR: 0.59, 95%CI: 0.38-0.90, p=0.01) FUT3 and FUT5 signals colocalized with IPF signals, with posterior probabilities of a shared genetic signal of 99.9% and 97.7%. Further transcriptomic investigations supported the protective effects of FUT3 for IPF.An efficient MR scan of 834 circulating proteins provided evidence that genetically increased circulating FUT3 level is associated with reduced risk of IPF.

Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis-associated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging.

Despite many reports about pulmonary blood vessels in lung fibrosis, the contribution of lymphatics to fibrosis is unknown. We examined the mechanism and consequences of lymphatic remodeling in mice with lung fibrosis after bleomycin injury or telomere dysfunction. Widespread lymphangiogenesis was observed after bleomycin treatment and in fibrotic lungs of prospero homeobox 1-enhanced green fluorescent protein (Prox1-EGFP) transgenic mice with telomere dysfunction. In loss-of-function studies, blocking antibodies revealed that lymphangiogenesis 14 days after bleomycin treatment was dependent on vascular endothelial growth factor (Vegf) receptor 3 signaling, but not on Vegf receptor 2. Vegfc gene and protein expression increased specifically. Extensive extravasated plasma, platelets, and macrophages at sites of lymphatic growth were potential sources of Vegfc. Lymphangiogenesis peaked at 14 to 28 days after bleomycin challenge, was accompanied by doubling of chemokine (C-C motif) ligand 21 in lung lymphatics and tertiary lymphoid organ formation, and then decreased as lung injury resolved by 56 days. In gain-of-function studies, expansion of the lung lymphatic network by transgenic overexpression of Vegfc in club cell secretory protein (CCSP)/VEGF-C mice reduced macrophage accumulation and fibrosis and accelerated recovery after bleomycin treatment. These findings suggest that lymphatics have an overall protective effect in lung injury and fibrosis and fit with a mechanism whereby lung lymphatic network expansion reduces lymph stasis and increases clearance of fluid and cells, including profibrotic macrophages.

Progressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by variable histopathological and clinical features. A subset of subjects with CHP have usual interstitial pneumonia and appear to be clinically similar to subjects with idiopathic pulmonary fibrosis (IPF).Objectives: To determine the common and unique molecular features of CHP and IPF.Methods: Transcriptome analysis of lung samples from CHP (n = 82), IPF (n = 103), and unaffected controls (n = 103) was conducted. Differential gene expression was determined adjusting for sex, race, age, and smoking history and using false discovery rate to control for multiple comparisons.Measurements and Main Results: When compared with controls, we identified 413 upregulated and 317 downregulated genes in CHP and 861 upregulated and 322 downregulated genes in IPF. Concordantly upregulated or downregulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP (differentially expressed in CHP when compared with control and not differentially expressed in IPF) were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene coexpression network analysis, we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function, respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing.Conclusions: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP.

Telomere dysfunction is associated with multiple fibrotic lung processes, including chronic lung allograft dysfunction (CLAD)-the major limitation to long-term survival following lung transplantation. Although shorter donor telomere lengths are associated with an increased risk of CLAD, it is unknown whether short telomeres are a cause or consequence of CLAD pathology. Our objective was to test whether telomere dysfunction contributes to the pathologic changes observed in CLAD. Histopathologic and molecular analysis of human CLAD lungs demonstrated shortened telomeres in lung epithelial cells quantified by teloFISH, increased numbers of surfactant protein C immunoreactive type II alveolar epithelial cells, and increased expression of senescence markers (β-galactosidase, p16, p53, and p21) in lung epithelial cells. TRF1F/F (telomere repeat binding factor 1 flox/flox) mice were crossed with tamoxifen-inducible SCGB1a1-cre mice to generate SCGB1a1-creTRF1F/F mice. Following 9 months of tamoxifen-induced deletion of TRF1 in club cells, mice developed mixed obstructive and restrictive lung physiology, small airway obliteration on microcomputed tomography, a fourfold decrease in telomere length in airway epithelial cells, collagen deposition around bronchioles and adjacent lung parenchyma, increased type II aveolar epithelial cell numbers, expression of senescence-associated β-galactosidase in epithelial cells, and decreased SCGB1a1 expression in airway epithelial cells. These findings demonstrate that telomere dysfunction isolated to airway epithelial cells leads to airway-centric lung remodeling and fibrosis similar to that observed in patients with CLAD and suggest that lung epithelial cell telomere dysfunction may be a molecular driver of CLAD.

BackgroundLung transplantation and related medications are associated with pathobiological changes that can induce frailty, a state of decreased physiological reserve. Causes of persistent or emergent frailty after lung transplantation, and whether such transplant-related frailty is associated with key outcomes, are unknown.MethodsFrailty and health-related quality of life (HRQL) were prospectively measured repeatedly for up to 3 years after lung transplantation. Frailty, quantified by the Short Physical Performance Battery (SPPB), was tested as a time-dependent binary and continuous predictor. The association of transplant-related frailty with HRQL and mortality was evaluated using mixed effects and Cox regression models, respectively, adjusting for age, sex, ethnicity, diagnosis, and for body mass index and lung function as time-dependent covariates. We tested the association between measures of body composition, malnutrition, renal dysfunction and immunosuppressants on the development of frailty using mixed effects models with time-dependent predictors and lagged frailty outcomes.ResultsAmong 259 adults (56% male; mean age 55.9±12.3 years), transplant-related frailty was associated with lower HRQL. Frailty was also associated with a 2.5-fold higher mortality risk (HR 2.51; 95% CI 1.21 to 5.23). Further, each 1-point worsening in SPPB was associated, on average, with a 13% higher mortality risk (HR 1.13; 95% CI 1.04 to 1.23). Secondarily, we found that sarcopenia, underweight and obesity, malnutrition, and renal dysfunction were associated with the development of frailty after transplant.ConclusionsTransplant-related frailty is associated with lower HRQL and higher mortality in lung recipients. Abnormal body composition, malnutrition and renal dysfunction may contribute to the development of frailty after transplant. Confirming the role of these potential contributors and developing interventions to mitigate frailty may improve lung transplant success.

Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered a Wnt5a-Ror2-Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This includes PDGF secretion from alveolar type I and type II cells, cell shape changes of type I cells and migration of myofibroblasts. All these cellular properties are conferred by changes in the cytoskeleton and represent a new facet of PCP function. These results extend our current model of PCP signaling from polarizing a field of epithelial cells to conferring new properties at subcellular levels to regulate collective cell behavior.

Collagen-producing cells maintain the complex architecture of the lung and drive pathologic scarring in pulmonary fibrosis. Here we perform single-cell RNA-sequencing to identify all collagen-producing cells in normal and fibrotic lungs. We characterize multiple collagen-producing subpopulations with distinct anatomical localizations in different compartments of murine lungs. One subpopulation, characterized by expression of Cthrc1 (collagen triple helix repeat containing 1), emerges in fibrotic lungs and expresses the highest levels of collagens. Single-cell RNA-sequencing of human lungs, including those from idiopathic pulmonary fibrosis and scleroderma patients, demonstrate similar heterogeneity and CTHRC1-expressing fibroblasts present uniquely in fibrotic lungs. Immunostaining and in situ hybridization show that these cells are concentrated within fibroblastic foci. We purify collagen-producing subpopulations and find disease-relevant phenotypes of Cthrc1-expressing fibroblasts in in vitro and adoptive transfer experiments. Our atlas of collagen-producing cells provides a roadmap for studying the roles of these unique populations in homeostasis and pathologic fibrosis.

Shorter peripheral blood leukocyte (PBL) telomere length (TL) has been associated with poor outcomes in various chronic lung diseases. Whether PBL-TL is associated with survival from critical illness was tested in this study.We analysed data from a prospective observational cohort study of 937 critically ill patients at Vanderbilt University Medical Center (VUMC). PBL-TL was measured using quantitative PCR of DNA isolated from PBLs. Findings were validated in an independent cohort of 394 critically ill patients with sepsis admitted to the University of California San Francisco (UCSF).In the VUMC cohort, shorter PBL-TL was associated with worse 90-day survival (adjusted hazard ratio (aHR) 1.3, 95% CI 1.1-1.6 per 1 kb TL decrease; p=0.004); in subgroup analyses, shorter PBL-TL was associated with worse 90-day survival for patients with sepsis (aHR 1.5, 95% CI 1.2-2.0 per 1 kb TL decrease; p=0.001), but not trauma. Although not associated with development of acute respiratory distress syndrome (ARDS), among ARDS subjects, shorter PBL-TL was associated with more severe ARDS (OR 1.7, 95% CI 1.2-2.5 per 1 kb TL decrease; p=0.006). The associations of PBL-TL with survival (adjusted HR 1.6, 95% CI 1.2-2.1 per 1 kb TL decrease; p=0.003) and risk for developing severe ARDS (OR 2.5, 95% CI 1.1-6.3 per 1 kb TL decrease; p=0.044) were validated in the UCSF cohort.Short PBL-TL is strongly associated with worse survival and more severe ARDS in critically ill patients, especially patients with sepsis. These findings suggest that telomere dysfunction may contribute to outcomes from critical illness.

Rationale: Rare genetic variants in telomere-related genes have been identified in familial, idiopathic, and rheumatoid arthritis-associated pulmonary fibrosis. Short peripheral blood leukocyte (PBL) telomere length predicts poor outcomes in chronic hypersensitivity pneumonitis (CHP).Objectives: Determine the prevalence and clinical relevance of rare protein-altering variants in telomere-related genes in patients with CHP.Methods: Next-generation sequences from two CHP cohorts were analyzed to identify variants in TERT (telomerase reverse transcriptase), TERC (telomerase RNA component), DKC1 (dyskerin pseudouridine synthase 1), RTEL1 (regulator of telomere elongation helicase 1), PARN (poly[A]-specific RNase), and TINF2 (TERF1-interacting nuclear factor 2). To qualify, variants were required to have a minor allele frequency less than 0.005 and be predicted to be damaging to protein function. Variant status (binary variable) was used in statistical association tests, including Cox proportional hazard models for transplant-free survival. PBL telomere length was measured using quantitative PCR.Measurements and Main Results: Qualifying variants were identified in 16 of 144 patients (11.1%; 95% confidence interval [CI], 6.5-17.4) in the discovery cohort and 17 of 209 patients (8.1%; 95% CI, 4.8-12.7) in the replication cohort. Age- and ancestry-adjusted PBL telomere length was significantly shorter in the presence of a variant in both cohorts (discovery: -561 bp; 95% CI, -933 to -190; P = 0.003; replication: -612 bp; 95% CI, -870 to -354; P = 5.30 × 10-6). Variant status was significantly associated with transplant-free survival in both cohorts (discovery: age-, sex-, and ancestry-adjusted hazard ratio, 3.73; 95% CI, 1.92-7.28; P = 0.0001; replication: hazard ratio, 2.72; 95% CI, 1.26-5.88; P = 0.011).Conclusions: A substantial proportion of patients diagnosed with CHP have rare, protein-altering variants in telomere-related genes, which are associated with short peripheral blood telomere length and significantly reduced transplant-free survival.

BackgroundAntineutrophil cytoplasmic antibodies (ANCAs) have been reported to occur in 7% to 10% of patients with idiopathic pulmonary fibrosis (IPF), but their clinical relevance remains unclear. The aim of this study was to estimate the prevalence of ANCAs in a North American population with IPF and evaluate their clinical significance.MethodsThis was a retrospective study of two independent cohorts of patients diagnosed with IPF at the University of California San Francisco (discovery cohort) and the University of Chicago (replication cohort). Myeloperoxidase (MPO) and proteinase 3 (PR3) ANCAs were measured in all patients. Prevalence and associations of ANCAs with clinical characteristics and transplant-free survival were evaluated.ResultsA total of 14 of 353 (4.0%; 95% CI, 2.2-6.5) and 20 of 392 (5.1%; 95% CI, 3.1-7.8) patients with IPF were positive for ANCAs at the time of diagnosis in the discovery and replication cohorts, respectively. Among those positive for MPO antibodies, two of six (33%) in the discovery cohort and three of 12 (25%) in the replication cohort developed vasculitis. None of the patients who were PR3-positive developed vasculitis. Patients who were ANCA-positive were more likely to be women than patients who were ANCA-negative, and were more likely to have some ground-glass opacities on CT scan. In the combined cohort of 745 patients, median transplant-free survival was not significantly different in patients who were ANCA-positive vs ANCA-negative (P = .57).ConclusionsANCA positivity is uncommon in North American patients with IPF and not associated with baseline disease severity or transplant-free survival; however, a significant proportion of patients who are MPO-positive with IPF develop clinical vasculitis.

At the center of fibrosing diseases is the aberrant activation of tissue fibroblasts. The cellular and molecular mechanisms of how the immune system augments fibroblast activation have been described; however, little is known about how the immune system controls fibroblast function in tissues. Here, we identify regulatory T cells (Tregs) as important regulators of fibroblast activation in skin. Bulk cell and single-cell analysis of Tregs in murine skin and lungs revealed that Tregs in skin are transcriptionally distinct and skewed toward T helper 2 (TH2) differentiation. When compared with Tregs in lung, skin Tregs preferentially expressed high levels of GATA3, the master TH2 transcription factor. Genes regulated by GATA3 were highly enriched in skin "TH2 Treg" subsets. In functional experiments, Treg depletion resulted in a preferential increase in TH2 cytokine production in skin. Both acute depletion and chronic reduction of Tregs resulted in spontaneous skin fibroblast activation, profibrotic gene expression, and dermal fibrosis, all of which were exacerbated in a bleomycin-induced murine model of skin sclerosis. Lineage-specific deletion of Gata3 in Tregs resulted in an exacerbation of TH2 cytokine secretion that was preferential to skin, resulting in enhanced fibroblast activation and dermal fibrosis. Together, we demonstrate that Tregs play a critical role in regulating fibroblast activation in skin and do so by expressing a unique tissue-restricted transcriptional program that is mediated, at least in part, by GATA3.

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.

AimsTo evaluate the clinical significance of bronchiolocentric fibrosis (BCF) in patients with a histopathological pattern of usual interstitial pneumonia (UIP).Methods and resultsTwo hundred and fifty-two patients with pathological UIP pattern were identified. Two hundred and fifteen of these patients (215 of 252) had the multidisciplinary diagnosis of idiopathic pulmonary fibrosis (IPF). Prospectively defined clinical, radiological and pathological features (including BCF) were recorded, and peripheral blood MUC5B genotype and telomere length were measured. BCF was observed in 38% (96 of 252) of all patients and 33% (72 of 215) of IPF patients; its presence was associated with a non-IPF diagnosis on multivariate analysis (odds ratio = 3.71, 95% confidence interval = 1.68-8.19). BCF was not significantly associated with environmental exposures, gastroesophageal reflux, cigarette smoking or radiological patterns. There was no significant association of BCF with MUC5B genotype or telomere length. BCF has no significant impact on survival time.ConclusionsMost patients with BCF and a histopathological pattern of UIP have IPF. However, this combined fibrotic pattern is associated with a non-IPF multidisciplinary diagnosis, with approximately one-quarter of these patients being diagnosed as chronic hypersensitivity pneumonia or unclassifiable interstitial fibrosis. The presence of BCF in these patients is not significantly associated with presumed clinical risk factors for bronchiolocentric involvement, radiological findings, MUC5B genotype, telomere length or survival time.