Idiopathic pulmonary fibrosis (IPF) is a devastating pulmonary disease with no curative treatment other than lung transplantation that results from maladaptive responses to lung epithelial injury; however, the underlying mechanisms remain unclear, and treatment options are limited. Here, we showed that deficiency in the innate immune receptor toll-like receptor 5 (TLR5) is associated with IPF in humans and with increased susceptibility to bleomycin-induced pulmonary fibrosis in mice and that activation of lung epithelial TLR5 through a synthetic flagellin analog protected mice from experimental fibrosis. Mechanistically, epithelial TLR5 activation induced antimicrobial gene expression and ameliorated lung dysbiosis after injury. In contrast, TLR5 deficiency in mice and patients with IPF was associated with lung dysbiosis. Elimination of the microbiome in mice through administration of antibiotics abolished the protective effect of TLR5, and reconstitution of the microbiome by fecal microbiota transplantation rescued the observed phenotype. In conclusion, these studies revealed that TLR5 protects against pulmonary fibrosis through effects on the lung microbiota, providing insight into therapeutic approaches that may ultimately benefit patients with IPF.

Rationale:Idiopathic pulmonary fibrosis (IPF) is a rare, chronic, progressive lung disease with high mortality and few treatment options. Using an additive genetic model, genome-wide association studies (GWAS) have identified multiple risk loci highlighting new genes and pathways of interest. Since IPF risk could also be influenced by non-additive effects, we hypothesised that association analyses using alternative genetic models may provide additional mechanistic insight. Objectives:To perform GWAS of IPF susceptibility to detect associations where the underlying effects are consistent with recessive or dominant genetic models. Methods:We performed GWAS of IPF susceptibility, with logistic regression assuming dominant or recessive genetic models, including 5,159 IPF cases, from clinically-curated sources, and 27,459 controls. We functionally annotated independent signals and performed variant-to-gene mapping, applying fine-mapping to define potentially causal variants and genes. We assessed differential expression levels of genes of interest in publicly available single cell RNAseq data and in primary cells derived from IPF donors and controls. Main Results:We identified five genome-wide significant signals, under a recessive model, that had not been reported previously. These included exonic variants in the cell-cycle gene Polyamine-Modulated Factor 1 (PMF1) and in Epsin 3 (EPN3) genes. We also observed evidence of increased PMF1 expression in airway basal cells of IPF patients compared to controls. Conclusions:Using alternative genetic models in IPF susceptibility GWAS identified new signals and genes, providing new insights into IPF pathogenesis and potential future therapies.

RATIONALE:Restrictive allograft syndrome (RAS) is a major cause of mortality following lung transplantation due to progressive fibrosis of the lung allograft with no therapeutic options. Knowledge of the cellular and molecular mechanisms driving fibrosis in RAS remains limited. OBJECTIVE:To characterize the cellular and molecular changes in human RAS lungs through single-cell transcriptomic profiling. METHODS:Single-nucleus RNA-sequencing (snRNA-seq) was performed in peripheral lung tissues from 15 RAS patients undergoing lung re-transplantation, and from 9 healthy control lungs. Findings were validated and extended using histologic techniques including immunofluorescence, RNA in situ hybridization, Elastica-van-Gieson immunohistochemistry, quantitative histological analyses, and micro-CT scans. MEASUREMENTS AND MAIN RESULTS:snRNA-seq analysis of RAS lungs revealed previously undescribed aberrant basaloid cells, ectopic COL15A1+ peribronchial vascular endothelial cells (pVECs), and CTHRC1+ fibrotic fibroblasts. Histologic stains disclosed distinctive distribution patterns: aberrant basaloid cells, primarily localized at the fibrotic edge, together with juxtaposed CTHRC1+ fibrotic fibroblasts and ectopic COL15A1+ pVECs form the fibrotic niche of alveolar fibroelastosis (AFE). PRX+ alveolar microvasculature is partially lost in AFE areas. Micro-CT scans revealed changes from pulmonary to systemic perfusion, facilitated by COL15A1+ pVECs. Last, our data reveals potential therapeutic targets in RAS, including integrin αvβ6, activator of TGFβ. CONCLUSION:Considering the multifaceted differences of RAS and idiopathic pulmonary fibrosis, we revealed a surprising general principle of an entity-spanning composition of the fibrotic niche by aberrant basaloid cells localized at the fibrotic edge, ectopic COL15A1+ pVECs and CTHRC1+ fibrotic fibroblasts. This suggests a flexible but cellular pathogenesis-guided transferability of potential therapeutic approaches between progressive fibrotic lung diseases.

Background:Idiopathic pulmonary fibrosis (IPF) is characterised by progressive loss of pulmonary function and poor survival. Although biomarkers for disease progression and mortality exist, their reliability in large studies remains unproven. This study investigates prognostic biomarkers from the ISABELA trials, the largest IPF cohort to date, to identify those predicting worse clinical outcomes. Methods:Plasma from 1280 IPF patients in ISABELA 1 and 2 (NCT03711162, NCT03733444) was analysed for 17 circulating soluble disease-related biomarkers at multiple time-points and for the MUC5B (rs35705950_T) genotype. Statistical learning algorithms investigated biomarker levels/status with disease progression (≥10% decline in forced vital capacity (FVC) or mortality within 1 year) and pharmacotherapy. Results:Patients with ≥10% annual decline in FVC had higher median baseline of matrix metalloproteinase-7 (MMP-7) versus those with <10% decline (5.5 versus 4.2 µg·L-1; p<0.005). Patients with baseline MMP-7 ≥5.2 μg·L-1 and/or C-C motif chemokine ligand 18 (CCL18) ≥75.2 μg·L-1 had increased risk of mortality (p<0.0001); with patients having both elevated biomarkers at an even greater risk. Machine learning identified CCL18 changes by week 26 as a predictor of disease progression. The rs35705950_T genotype predicted neither mortality nor disease progression. Conclusions:We provide new insights into the prognostic value of MMP-7 and CCL18 in identifying high-risk IPF patients in the largest cohort to date. The combination of high baseline MMP-7 and CCL18 levels, along with longitudinal changes in CCL18, has the potential to enhance risk stratification and support efficacy assessment and monitoring in clinical trials.

The geroscience hypothesis suggests that understanding underlying ageing mechanisms will enable us to delay aging and lessen age-related disability and diseases. While hallmarks of ageing list multiple contributing factors, role of mechanics has only been recently recognized and increasingly appreciated. Here, we use mouse models of ageing to investigate changes in mechanics of the proximal pulmonary artery, lung and right ventricle function in ageing. We found an age-related decline in the capacity to store energy and increased circumferential stiffness of the proximal pulmonary artery with age that associated with a reorientation of collagen towards the circumferential direction, decreased exercise ability, and decreased function of the lung and right ventricle. The observed compromised mechanics in proximal pulmonary artery is consistent across multiple mouse models of accelerated ageing. Further, transcriptional changes in proximal pulmonary artery indicate that aging is associated with senescence of perivascular macrophages, adventitial fibroblasts, and medial smooth muscle cells. Older pulmonary arteries increase expression of genes associated with ECM turnover (including genes in the TGFβ pathway) and increased intercellular signaling amongst perivascular macrophages, fibroblasts and smooth muscle cells. Our results provide promising biomarkers of ageing for diagnosis and potential pathways and molecular targets for targeting anti-ageing therapies.

Background Chronic lung allograft dysfunction (CLAD) is the leading cause of late mortality after lung transplantation. Bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) are the main underlying clinical entities. Their molecular and cellular signatures are unclear and, therefore, we aimed to identify molecular programmes associated with morphological disease severity in CLAD. Methods We performed high-resolution imaging-based gene expression profiling of 128 lung samples from explanted CLAD and donor lungs, using weighted gene co-expression network analysis, cellular and pathway enrichment, and hub gene identification. Findings were validated across four datasets, including a murine transplant model, human BOS lungs, transbronchial biopsies, and bronchoalveolar lavage fluid of lung transplant recipients. Results Unsupervised clustering revealed two transcriptomic CLAD endotypes aligning with mild-fibrotic (BOS, mild RAS) and advanced-fibrotic disease (moderate/severe RAS). Samples from the same patient often diverged molecularly, underscoring intra-patient heterogeneity and limitations of current phenotypical classification. Five molecular programmes emerged: (1) epithelial stress and innate immunity in early-fibrotic CLAD, (2) progressive adaptive immunity and cytotoxicity in advanced CLAD, (3) transient extracellular matrix remodelling, (4) progressive endothelial loss/dysfunction, and (5) progressive loss of homeostasis, wherein multiple potential druggable targets were detected. Finally, we identified a 26 CLAD hub gene-panel, that showed robust diagnostic performance to discriminate CLAD. Conclusion CLAD is a spatially heterogeneous, yet molecularly continuous disease process, wherein BOS and RAS represent variable stages of a shared immunopathological continuum. Our findings support the development of lung-specific molecular classifiers to guide diagnostics and reveal novel targets for personalised therapies in transplantation.

Fibrosis, marked by excess extracellular matrix (ECM) deposition, is the end stage of many diseases. Single-cell studies have highlighted the emergence of disease-specific fibroblast populations, including a high collagen-synthesizing CTHRC1+ subpopulation. The profibrotic cytokine TGF-β1 promotes fibrogenesis via cooperation between Smad and mTORC1/4E-BP1 signaling axes. Using CRISPR-Cas9 gene editing, we report that more than one-third of TGF-β1-regulated matrisome genes are under mTORC1 control. Mapping the transcriptome of TGF-β1-stimulated fibroblasts revealed similarity to CTHRC1+ fibroblasts identified in idiopathic pulmonary fibrosis (IPF). This overlap is lost when mTORC1 is disabled. Using the selective mTORC1 inhibitor RMC-5552, we confirm a causal role for mTORC1 in promoting the acquisition of the collagen-high, CTHRC1+ phenotype in response to TGF-β1 stimulation in fibroblasts derived from patients with either IPF or lung adenocarcinoma. We conclude that mTORC1 plays a key role in shaping the transcriptional identity of these fibroblasts, with implications for therapeutic inhibition of mTORC1 in fibrosis and cancer.

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with limited treatment options. Our group previously identified the antifibrotic potential of thyroid hormone, triiodothyronine (T3); however, clinical translation of thyroid hormone therapy is limited by its systemic adverse effects. In this study, we investigate whether sobetirome, a selective and well-tolerated thyroid hormone receptor beta (THRB) agonist, offers antifibrotic benefits of thyroid hormone while minimizing systemic toxicity. Our study reveals that sobetirome, administered via intraperitoneal or inhalational routes, effectively mitigates bleomycin-induced pulmonary fibrosis in mice, with no evidence of toxicity. We identified that sobetirome restores mitochondrial homeostasis via activating the THRB–PPARGC1α axis. This protects alveolar type II epithelial cells from injury-induced apoptosis while selectively inducing apoptosis and metabolic reprogramming in apoptosis-resistant IPF fibroblasts. Cell-specific deletion of Ppargc1α in either alveolar epithelial cells or fibroblasts abolishes sobetirome-mediated protection, establishing PPARGC1α as an essential mediator of therapeutic response. Importantly, sobetirome reverses fibrosis-associated transcriptional programs in human IPF lung tissue, reducing expression of key fibrosis-associated genes, including collagen I alpha 1 ( COL1A1 ), collagen III alpha 1 ( COL3A1 ), periostin ( POSTN), cathepsin K ( CTSK ), and Chitinase 3 Like 1 ( CHI3L1 ), while promoting extracellular matrix remodeling, epithelial restoration, and tissue homeostasis. Collectively, our findings identify THRB activation as a novel metabolic strategy for reversing pulmonary fibrosis. Across complementary in vitro , in vivo , and human ex vivo models, sobetirome restores mitochondrial function, modulates apoptotic pathways in pathogenic cells, and promotes fibrosis resolution, highlighting its potential as a lung-targeted therapeutic approach for IPF and other fibrotic lung diseases. One sentence summary Sobetirome, a thyroid hormone receptor beta agonist, exerts potent antifibrotic effects in preclinical models of pulmonary fibrosis across in vitro , in vivo , and ex vivo settings by acting on both lung epithelial and fibroblast cells.

Rationale:Preclinical familial pulmonary fibrosis (FPF) represents an early stage of fibrotic lung disease, yet the compartment- and cell-specific molecular programs preceding fibrosis remain poorly understood. Objective:To define spatially organized molecular signatures associated with preclinical FPF and identify tissue-informed circulating biomarkers linked to early fibrotic remodeling. Methods:We performed integrated multi-omic profiling of histologically preserved and remodeled lung regions from subjects with preclinical FPF, Idiopathic Pulmonary Fibrosis (IPF), and controls using spatial transcriptomics, single-nucleus RNA sequencing (snRNAseq), and blood proteomics. Differential expression and pathway enrichment analyses were performed across spatial compartments and epithelial cell states. Results:Histologically preserved lung regions in preclinical FPF demonstrated transcriptional abnormalities including stress-response, ciliary, and extracellular matrix-associated programs despite minimal architectural distortion. Spatial analyses identified alterations in alveolar niche molecular programs accompanied by increasing profibrotic signaling across preserved and tissue remodeled lung compartments. Compared with advanced IPF, preclinical FPF retained epithelial repair and surfactant-associated signatures. Integration with snRNAseq demonstrated enrichment of alveolar and airway epithelial cell dysregulated states associated with transitional phenotypes previously implicated in IPF. Compartment- and epithelial-associated transcriptional signatures identified in lung tissue were partially represented in the peripheral blood. Conclusion:Preclinical FPF is characterized by compartment- and cell-specific molecular programs that precede established fibrosis. We identified distinct alveolar, airway, and vascular molecular signatures and epithelial remodeling states represented in the peripheral blood. These findings provide an initial framework for molecular classification of early stages of pulmonary fibrosis and support future studies evaluating minimally invasive approaches for disease stratification and precision therapeutics. At a Glance Commentary:Scientific Knowledge on the Subject: The molecular events preceding a diagnosis of pulmonary fibrosis remain poorly understood. Most mechanistic studies in Idiopathic Pulmonary Fibrosis (IPF) have relied on end-stage explanted lungs, limiting insight into the compartment- and cell-specific molecular programs associated with early stages of pulmonary fibrosis.What this study adds to the field: Using integrated spatial transcriptomics, single-cell sequencing, and peripheral blood proteomic profiling, we demonstrate that preclinical familial pulmonary fibrosis (FPF) is characterized by compartment- and cell-specific molecular programs that precede clinically detectable fibrosis. Spatial analyses identified distinct alveolar, airway, and vascular molecular signatures, while single cell analysis confirmed the presence of epithelial dysregulated states. These signatures are partially represented in the peripheral blood. Our findings provide an initial framework for biologically informed classification of early stages of pulmonary fibrosis and future minimally invasive approaches for disease stratification.

In a phase 2 trial (NCT04308681), treatment with admilparant (BMS-986278), an oral lysophosphatidic acid receptor 1 (LPA1) antagonist, reduced lung function decline in patients with idiopathic pulmonary fibrosis (IPF) or progressive pulmonary fibrosis (PPF). In this exploratory analysis, we evaluated post-treatment changes in circulating biomarkers of lung fibrosis to elucidate mechanisms of admilparant action. Patients with IPF or PPF were randomized 1:1:1 to receive twice-daily admilparant (30 or 60 mg) or placebo for 26 weeks; background antifibrotics were allowed. The IPF and PPF cohorts were analyzed separately. Changes from baseline (CfB) in serum proteins associated with epithelial injury, inflammation, and fibrosis were measured by quantitative immunoassays at 4, 12, and 26 weeks, and compared between patients who received admilparant versus placebo. Pharmacodynamic biomarker changes were evaluated by clinical response status at week 26. Plasma samples from patients with IPF were assessed post hoc by SomaScan v4.1 proteomics assay. Statistical evaluations used linear mixed-effects models. In the IPF cohort (n=276), nine serum proteins showed significant CfB (p<0.05) at week 26 in patients treated with 60-mg admilparant versus placebo, including increased adiponectin, MMIF, CD163, CEA, and ENRAGE, and decreased markers of epithelial injury and fibrosis (CA-125/MUC16, MMP-7, TN-C, PRO-FIB). In the PPF cohort (n=116), significant differences at week 26 (p<0.05) were observed for 11 serum proteins, including increased CEA and decreased periostin, IL6Rβ, CD163, KIM-1, multiple inflammatory markers (YKL-40, VCAM-1, PECAM-1, ferritin), and collagen degradation markers (C3M and C4M). Two serum proteins, CA-125 and TN-C, showed significantly greater CfB (p<0.05) in responders compared with non-responders in the IPF cohort. Plasma proteomic analysis identified differential expression (adjusted p<0.1) of adiponectin, CKMT1A, ANGPTL3, PDCD1LG2, and IGFBP6 at week 26 in patients with IPF treated with 60-mg admilparant versus placebo. Treatment with 60-mg admilparant improved circulating biomarkers associated with epithelial injury and fibrosis in IPF, and with inflammation, fibrosis, and collagen degradation in PPF. Proteomic analysis identified additional biomarkers associated with mitochondrial and metabolic pathways in IPF. These findings expand our understanding of potential mechanisms of LPA1 antagonism with admilparant and identify biomarkers that may help evaluate treatment response and disease activity.

Rationale:Fibrotic hypersensitivity pneumonitis (fHP) is an antigen-driven, life-threatening interstitial lung disease characterized by heterogeneous radiologic features, clinical outcomes, and treatment responses. Objectives:To identify blood-based fHP endotypes that inform mechanism, prognosis and therapeutic response. Methods:We performed integrative analyses of multi-compartment transcriptomic data derived from whole blood, peripheral blood mononuclear cells, bronchoalveolar lavage, and surgical lung biopsies, alongside circulating plasma proteomics. Multiple clustering algorithms were cross-compared to ensure robustness and reproducibility of endotypes identification. Immune cell composition was inferred using bulk RNA-seq deconvolution and annotated with BAL single-cell RNA-seq. Pathway activities were characterized using Gene Set Enrichment Analysis. Transplant-free survival (TFS) was evaluated for endotype and corticosteroid exposure by Kaplan-Meier methods, with hazard ratios analyzed using multivariable Cox proportional hazards models. Results:Two molecular endotypes, lymphocytic-associated (L-fHP) and non-lymphocytic-associated (N-fHP), were identified and validated. L-fHP showed enrichment of adaptive immune signaling and lymphocyte predominance, whereas N-fHP demonstrated myeloid-cell activation with neutrophil and macrophage predominance. Corticosteroid exposure was associated with worse TFS in L-fHP but not in N-fHP after adjusting for age, sex, and baseline pulmonary function. Compared to L-fHP, N-fHP had poorer baseline pulmonary function, faster 12-month FVC decline, and shorter TFS. N-fHP also exhibited elevated neutrophil-associated markers, including matrix metalloproteinase-9, across paired transcriptomic and proteomic datasets, supporting a neutrophil-driven, cross-compartment disease process. Conclusion:Multi-omic, multi-compartment analysis identifies two reproducible fHP endotypes with distinct clinical outcomes and corticosteroid responses, supporting a precision medicine approach beyond current clinical and radiologic classification.

Systemic lupus erythematosus (SLE or lupus), an autoimmune disease characterized by autoantibody production and inflammation, exhibits clinicopathologic heterogeneity. Here we investigated the heterogeneity and functions of mononuclear phagocytes (MPs), including monocytes (Mo) and macrophages (MΦ), driven by lupus immune complex (IC) stimulation. Our single cell RNA sequencing (scRNA-seq) analysis of human Mo incubated with U1-snRNP lupus IC revealed an expansion of pro-inflammatory Mo with increased expression of inflammatory genes including cytokines, chemokines, and transcription factors. These transcriptomic changes were reflected at the protein level, with a strong correlation between gene and protein expression, as determined by proteomic analysis. Mo developed similar pro-inflammatory transcriptomic changes in response to other IC containing dsDNA and Ro60. Interrogation of scRNA-seq datasets from the skin, kidneys, and peripheral blood of lupus patients revealed the presence and expansion of pro-inflammatory Mo and MΦ populations, with transcriptomic signatures similar to those seen in lupus IC-stimulated Mo. Some of these cells with increased expression of the snRNP IC gene signature exhibited low expression of the type I IFN signature, suggesting that lupus IC and type I IFN stimulation may independently affect Mo subsets. In lupus nephritis tissue, infiltration of CD68+ cells expressing NLRP3, a molecule upregulated in Mo stimulated with snRNP IC, was associated with treatment outcomes, supporting an important role for MPs in lupus nephritis. Collectively, our findings provide novel insights into the critical role of pro-inflammatory MPs arising from lupus IC stimulation in the pathogenesis of SLE.

Background:Idiopathic pulmonary fibrosis (IPF) is a rare, incurable lung disease with a median survival of 3-5 years after diagnosis. Treatment options are limited. Genetic association studies can identify new genes involved in disease that might represent potential new drug targets, and it has been shown that drug targets with support from genetic studies are more likely to be successful in clinical development. Previous genome-wide association studies (GWAS) of IPF susceptibility have identified more than 20 signals implicating genes involved in multiple mechanisms, including telomere dysfunction, cell-cell adhesion, host defence immunity, various signalling pathways and, more recently, mitotic spindle assembly complex. Aim:To leverage new datasets and genotype imputation to discover further genes involved in development of IPF that could yield new pathobiological avenues for exploration and to guide future drug target discovery. Methods:We conducted a GWAS of IPF susceptibility including seven IPF case-control studies comprising 5,159 IPF cases and 27,459 controls of European ancestry, where IPF diagnosis was made by a respiratory clinician according to international guidelines. Genotypes were obtained from Whole Genome Sequencing (WGS) or from array-based imputation to the TOPMed WGS reference panel. New signals were replicated in independent biobanks with IPF defined using Electronic Healthcare Records. Bayesian fine-mapping was performed to identify the most likely causal variant(s) and bioinformatic investigation undertaken to map associated variants to putative causal genes. Results:We identified three novel genetic signals of association with IPF susceptibility. Genes prioritised by functional evidence at these signals included MUC1, which encodes a large transmembrane glycoprotein and known biomarker of lung fibrosis, and NTN4 encoding Netrin-4 whose known roles include angiogenesis. The third signal may map to SLC6A6, a taurine and beta-alanine transporter gene, previously implicated in retinal, cardiac and kidney dysfunction. Conclusion:Our study has identified new associations not previously identified by previous large biobank-based studies thereby highlighting the value of utilising clinically-curated IPF case-control studies, and new genotype imputation. We present new evidence for disease-driving roles of MUC1 and of endothelial cell and vascular changes in IPF.

Acute exacerbations (AEs) occur both in patients with idiopathic pulmonary fibrosis (IPF) and non-IPF fibrotic interstitial lung disease (fILD). These events confer high morbidity and mortality, with a lack of proven effective therapeutic interventions. The objective of this state-of-the-art document is to summarize latest evidence since the 2016 international working group report on AE-IPF, expanding it across the spectrum of all fILDs. A comprehensive literature review on the epidemiology, associated and risk factors, prognosis, and management of AE-fILD is summarized. In addition to revising the AE definition and diagnostic criteria for broad application across different fILDs, a conceptual framework for acute respiratory worsening (ARW) has been proposed to encompass a variety of acute respiratory deteriorations, both related and unrelated to AE. This allows structured evaluation in both clinical and research settings. The proposed revised definition for AE-fILD is an acute respiratory event characterized by increased respiratory symptoms or signs and associated with radiologic or histologic features consistent with diffuse alveolar damage (with or without superimposed organizing pneumonia) in a patient with known or newly diagnosed fILD. On the other hand, ARW refers to a heterogeneous group of clinical events with acute symptom worsening not attributable to DAD in patients with fILD, such as pulmonary edema, bronchitis, and pneumonia, although severe pneumonia can trigger AE-fILD. Additionally, we discuss considerations for inclusion of AE as a clinical trial endpoint, as well as research priorities for advancing knowledge on the pathogenic mechanisms, event prediction, risk stratification, and development of drugs and supportive treatments.

BACKGROUND:Disorders in pulmonary vascular integrity are a prominent feature in many lung diseases. Paracrine signaling is highly enriched in the lung and plays a crucial role in regulating vascular homeostasis. However, the specific local cell-cell crosstalk signals that maintain pulmonary microvascular stability in adult animals and humans remain largely unexplored. METHODS:In this study, we used single-cell RNA-sequencing-based computational pipelines to systematically profile ligand-receptor interactions within the lung microvascular niche and identified VEGF-D (vascular endothelial growth factor-D) as a key local factor with previously unrecognized barrier-protective properties in models of acute lung injury. RESULTS:Our single-cell RNA-sequencing data revealed that, under physiological conditions, soluble ligand-receptor interactions between mesenchymal cells, in particular alveolar fibroblasts, and microvascular endothelial cells are predominantly associated with pathways involved in maintaining vascular integrity as compared with all other cells. On treatment with top identified ligands, we found that VEGF-D significantly enhanced endothelial barrier function and conferred protection against inflammatory challenges induced by TNF-α (tumor necrosis factor-α), IL (interleukin)-1β, and thrombin. This barrier-protective effect of VEGF-D was significantly attenuated by inhibition of VEGFR2 (vascular endothelial growth factor receptor 2), either through small interfering RNA (siRNA) knockdown or pharmacological blockade using specific VEGFR2 inhibitors. Intravenous administration of recombinant VEGF-D in lipopolysaccharide-induced acute lung injury models significantly reduced vascular permeability (7339±2510 arbitrary unit [a.u.] [lipopolysaccharides] versus 5350±1821 a.u. [lipopolysaccharides+VEGF-D]; P<0.05), immune cell infiltration (0.791±0.199×106 whole blood cells/mL [lipopolysaccharides] versus 0.540±0.190×106 whole blood cells/mL [lipopolysaccharide+VEGF-D]; P<0.01), and the expression of proinflammatory markers TNF-α, IL-6, and keratinocyte chemoattractant in the lung tissue. This effect was abolished in VEGFR2iECKO (VEGFR2 inducible endothelial cells knockout) mice, confirming that VEGF-D mediates its effects via VEGFR2-dependent signaling. CONCLUSIONS:This study demonstrates an unexpected protective role for VEGF-D in promoting lung endothelial barrier integrity and suggests that paracrine signaling from the alveolar fibroblast niche contributes critically to lung capillary homeostasis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease in which the earliest cellular events driving fibrosis remain poorly defined. Here, we analyzed lung samples from three independent and unique cohorts of patients with early disease and preserved lung function (Florence, NIH, Forli), applying an integrated multi-modal approach combining single-nucleus RNA sequencing, bulk transcriptomics, immunostaining, and spatial transcriptomics. Single nuclear RNA sequencing of samples obtained by diagnostic bronchoscopic cryobiopsy (Florence, n= 22) revealed that early IPF is characterized by a marked shift in alveolar epithelial composition, with loss of AT1 and AT2 cells and the emergence of aberrant basaloid cells and alveolar epithelial intermediate cells. These populations exhibited transcriptional programs associated with epithelial plasticity and profibrotic signaling and closely resembled those observed in end-stage IPF. Higher proportions of aberrant basaloid and alveolar epithelial intermediate cells were associated with subsequent disease progression, whereas AT2 cell abundance correlated with preserved lung function. Fibrotic CTHRC1+ fibroblasts are largely restricted to advanced disease, while endothelial remodeling and inflammatory fibroblast states are already evident in early IPF. Spatial transcriptomic analyses confirmed early disruption of the alveolar niche, with replacement of normal epithelial-capillary interactions by aberrant epithelial and venous endothelial cells (Forli, n= 24); the findings were replicated through single cell RNA sequencing of samples obtained by video assisted thoracoscopy two decades earlier (NIH n=9). Together, these findings identify that alveolar niche remodeling with loss of its normal components, and emergence of aberrant basaloid cells are features of early IPF, highlighting epithelial dysfunction as a key potential target for therapeutic interventions in early disease.

BACKGROUND:The association between immune-cell-specific transcriptomic profiles and mortality in idiopathic pulmonary fibrosis (IPF) is unknown. METHODS:We profiled peripheral blood mononuclear cells by single-cell RNA sequencing (scRNA-seq) and investigated which immune-cell-specific transcriptomic profile predicted IPF outcomes consistently. Prognostic accuracy was investigated in peripheral blood mononuclear cells (PBMCs), bronchoalveolar lavage (BAL) and lung tissue. Findings were validated by flow cytometry, analysis of independent scRNA-seq datasets and cellular deconvolution. We investigated the function of this transcriptomic profile and its cellular source in lung tissue (overall sample size, n=1054; IPF, n=555; other, n=499). Connectivity map analysis and LASSO regression were used to identify drug candidates and a subset of genes with prognostic potential, respectively. RESULTS:A 230-gene up-score (Pittsburgh PBMC cohort) from CD14+CD163-HLA-DRlow monocytes predicted mortality in the Chicago PBMC cohort (HR 6.58, 95% CI 2.15-20.13; p=0.001), in BAL pooled analysis (HR 2.20, 95% CI 1.44-3.37; p=0.0003), and negatively correlated with forced vital capacity in lung tissues (ρ= -0.2, p=0.02). Proportions of CD14+CD163-HLA-DRlow monocytes were higher in progressive versus stable IPF (12.59%, 95% CI 9.66-16.23%, versus 7.61%, 95% CI 6.68-10.21%; p=0.014). High-risk patients with IPF had decreased expression of T-cell co-stimulatory genes (Pittsburgh and Chicago, p<0.01). CD14+HLA-DRlow monocytes had higher expression of profibrotic, proangiogenic and chemotactic factors compared to CD14+HLA-DRhi monocytes (p<0.05). The 230-gene up-score correlated with the secreted phosphoprotein 1 (SPP1)+ fibrosis-associated macrophages gene-score in lung tissues (ρ=0.19, p<2.2e-16). Connectivity map analysis identified drug categories to reverse the 230-gene signature. A subset of six genes retained predictive performance (pooled PBMC cohorts HR 4.79, 95% CI 2.58-8.92; p<0.0001). CONCLUSIONS:The transcriptome of CD14+CD163-HLA-DRlow monocytes is associated with increased mortality in patients with IPF. Its reversal should be investigated as a precision-based therapy in IPF.

Pleuroparenchymal fibroelastosis (PPFE) is a progressive interstitial lung disease (ILD) with defining histology of intra-alveolar fibrosis with septal elastosis (AFE), suggesting unique cellular disease processes. Here, we present a binational single-nucleus RNA sequencing atlas of PPFE, based on explanted lungs from 40 patients. Immunofluorescence microscopy, RNA in situ hybridization, micro-computed tomography (CT), and hierarchical phase-contrast (HiP) synchrotron CT provided spatial context. We identify PPFE-associated adventitial and elastofibrotic fibroblasts as key drivers of elastotic remodeling within an inflammatory microenvironment, maintained by immune cells forming tertiary lymphoid structures. Spatial mapping reveals an intriguing zonation of AFE, maintained by intercellular circuits between PPFE-associated cell types. Comparative analysis with idiopathic pulmonary fibrosis highlights CTHRC1+ fibrotic fibroblasts and aberrant basaloid cells as conserved profibrotic cellular machinery mediating collagen deposition across ILDs. This integrative atlas defines the cellular landscape of PPFE and dissects elastotic from fibrotic remodeling, providing a molecular rationale for niche-specific therapeutic strategies.