Immune mechanisms in fibrotic interstitial lung disease.

Fibrotic interstitial lung diseases (fILDs) have poor survival rates and lack effective therapies. Despite evidence for immune mechanisms in lung fibrosis, immunotherapies have been unsuccessful for major types of fILD. Here, we review immunological mechanisms in lung fibrosis that have the potential to impact clinical practice. We first examine innate immunity, which is broadly involved across fILD subtypes. We illustrate how innate immunity in fILD involves a complex interplay of multiple cell subpopulations and molecular pathways. We then review the growing evidence for adaptive immunity in lung fibrosis to provoke a re-examination of its role in clinical fILD. We close with future directions to address key knowledge gaps in fILD pathobiology: (1) longitudinal studies emphasizing early-stage clinical disease, (2) immune mechanisms of acute exacerbations, and (3) next-generation immunophenotyping integrating spatial, genetic, and single-cell approaches. Advances in these areas are essential for the future of precision medicine and immunotherapy in fILD.

Dysregulated Lung Commensal Bacteria Drive Interleukin-17B Production to Promote Pulmonary Fibrosis through Their Outer Membrane Vesicles.

Idiopathic pulmonary fibrosis (IPF) is a severe form of lung fibrosis with a high mortality rate. However, the etiology of IPF remains unknown. Here, we report that alterations in lung microbiota critically promote pulmonary fibrosis pathogenesis. We found that lung microbiota was dysregulated, and the dysregulated microbiota in turn induced production of interleukin-17B (IL-17B) during bleomycin-induced mouse lung fibrosis. Either lung-microbiota depletion or IL-17B deficiency ameliorated the disease progression. IL-17B cooperated with tumor necrosis factor-α to induce expression of neutrophil-recruiting genes and T helper 17 (Th17)-cell-promoting genes. Three pulmonary commensal microbes, which belong to the genera Bacteroides and Prevotella, were identified to promote fibrotic pathogenesis through IL-17R signaling. We further defined that the outer membrane vesicles (OMVs) that were derived from the identified commensal microbes induced IL-17B production through Toll-like receptor-Myd88 adaptor signaling. Together our data demonstrate that specific pulmonary symbiotic commensals can promote lung fibrosis by regulating a profibrotic inflammatory cytokine network.

Standigm ASK™: knowledge graph and artificial intelligence platform applied to target discovery in idiopathic pulmonary fibrosis.

Standigm ASK™ revolutionizes healthcare by addressing the critical challenge of identifying pivotal target genes in disease mechanisms-a fundamental aspect of drug development success. Standigm ASK™ integrates a unique combination of a heterogeneous knowledge graph (KG) database and an attention-based neural network model, providing interpretable subgraph evidence. Empowering users through an interactive interface, Standigm ASK™ facilitates the exploration of predicted results. Applying Standigm ASK™ to idiopathic pulmonary fibrosis (IPF), a complex lung disease, we focused on genes (AMFR, MDFIC and NR5A2) identified through KG evidence. In vitro experiments demonstrated their relevance, as TGFß treatment induced gene expression changes associated with epithelial-mesenchymal transition characteristics. Gene knockdown reversed these changes, identifying AMFR, MDFIC and NR5A2 as potential therapeutic targets for IPF. In summary, Standigm ASK™ emerges as an innovative KG and artificial intelligence platform driving insights in drug target discovery, exemplified by the identification and validation of therapeutic targets for IPF.

Homeostatic chemokines as putative therapeutic targets in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic interstitial lung disease (ILD) that affects lung mechanical functions and gas exchange. IPF is caused by increased fibroblast activity and collagen deposition that compromise the alveolar-capillary barrier. Identifying an effective therapy for IPF remains a clinical challenge. Chemokines are key proteins in cell communication that have functions in immunity as well as in tissue homeostasis, damage, and repair. Chemokine receptor signaling induces the activation and proliferation of lung-resident cells, including alveolar macrophages (AMs) and fibroblasts. AMs are an important source of chemokines and cytokines during IPF. We highlight the complexity of this system and, based on insights from genetic and transcriptomic studies, propose a new role for homeostatic chemokine imbalance in IPF, with implications for putative therapeutic targets.

Lactate accumulation induced by Akt2-PDK1 signaling promotes pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with an abnormal accumulation of fibrotic tissue in the lung parenchyma and elevated glycolysis level in associated cells without effective therapy options. Lactate accumulation in pulmonary fibrotic tissue is a significant factor aggravating IPF development, but the main mechanism regulating glycolysis needs further investigation. In this study, lung fibrosis model was induced by bleomycin (BLM) intratracheally in female C57BL/6 mice. The changes of lactate level and fibrotic markers were detected. For in vitro studies, cell lines of alveolar epithelial cell and lung fibroblast cell were stimulated with TGF-ß1 and BLM respectively, to detect changes in their fibrotic properties. The function of lactate accumulation on facilitating fibrosis was verified. We demonstrated that BLM-induced pulmonary fibrosis is accompanied by lactate accumulation owing to glycolysis upregulation. Significantly high PDK1 expression in lung fibrotic tissue promotes glycolysis. Moreover, PDK1 stimulated trans-differentiation of lung fibroblasts and epithelial-mesenchymal transition (EMT) of alveolar epithelial cells. Furthermore, phosphorylated Akt2 activated PDK1 to cause pulmonary fibrosis and inhibitors of Akt2 and PDK1 could suppress fibrotic process. This study is the first to consider PDK1 facilitated lactate accumulation through glycolysis as a vital factor in pulmonary fibrosis and could be initiated by Akt2. We concluded that the pro-fibrotic properties of PDK1 are associated with Akt2 phosphorylation and thus provide new potential therapeutic targets for pulmonary fibrosis.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Tetrahedral DNA loaded siCCR2 restrains M1 macrophage polarization to ameliorate pulmonary fibrosis in chemoradiation-induced murine model.

Idiopathic pulmonary fibrosis (IPF) is a chronic lethal disease in the absence of demonstrated efficacy for preventing progression. Although macrophage-mediated alveolitis is determined to participate in myofibrotic transition during disease development, the paradigm of continuous macrophage polarization is still under-explored due to lack of proper animal models. Here, by integrating 2.5 U/kg intratracheal Bleomycin administration and 10 Gy thorax irradiation at day 7, we generated a murine model with continuous alveolitis-mediated fibrosis, which mimics most of the clinical features of our involved IPF patients. In combination with data from scRNA-seq of patients and a murine IPF model, a decisive role of CCL2/CCR2 axis in driving M1 macrophage polarization was revealed, and M1 macrophage was further confirmed to boost alveolitis in leading myofibroblast activation. Multiple sticky-end tetrahedral framework nucleic acids conjunct with quadruple ccr2-siRNA (FNA-siCCR2) was synthesized in targeting M1 macrophages. FNA-siCCR2 successfully blocked macrophage accumulation in pulmonary parenchyma of the IPF murine model, thus preventing myofibroblast activation and leading to the disease remitting. Overall, our studies lay the groundwork to develop a novel IPF murine model, reveal M1 macrophages as potential therapeutic targets, and establish new treatment strategy by using FNA-siCCR2, which are highly relevant to clinical scenarios and translational research in the field of IPF.

Role of transient receptor potential ankyrin 1 in idiopathic pulmonary fibrosis: modulation of M2 macrophage polarization.

Idiopathic pulmonary fibrosis (IPF) poses significant challenges due to limited treatment options despite its complex pathogenesis involving cellular and molecular mechanisms. This study investigated the role of transient receptor potential ankyrin 1 (TRPA1) channels in regulating M2 macrophage polarization in IPF progression, potentially offering novel therapeutic targets. Using a bleomycin-induced pulmonary fibrosis model in C57BL/6J mice, we assessed the therapeutic potential of the TRPA1 inhibitor HC-030031. TRPA1 upregulation was observed in fibrotic lungs, correlating with worsened lung function and reduced survival. TRPA1 inhibition mitigated fibrosis severity, evidenced by decreased collagen deposition and restored lung tissue stiffness. Furthermore, TRPA1 blockade reversed aberrant M2 macrophage polarization induced by bleomycin, associated with reduced Smad2 phosphorylation in the TGF-ß1-Smad2 pathway. In vitro studies with THP-1 cells treated with bleomycin and HC-030031 corroborated these findings, highlighting TRPA1's involvement in fibrotic modulation and macrophage polarization control. Overall, targeting TRPA1 channels presents promising therapeutic potential in managing pulmonary fibrosis by reducing pro-fibrotic marker expression, inhibiting M2 macrophage polarization, and diminishing collagen deposition. This study sheds light on a novel avenue for therapeutic intervention in IPF, addressing a critical need in the management of this challenging disease.

Biological Age, Chronological Age and Survival in Pulmonary Fibrosis: A Causal Mediation Analysis.

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

Molecular Endotypes of Idiopathic Pulmonary Fibrosis: A Latent Class Analysis of Two Multicenter Observational Cohorts.

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

Epidemiology and Prognostic Significance of Cough in Fibrotic Interstitial Lung Disease.

Rationale: Cough is a key symptom in patients with fibrotic interstitial lung disease (ILD). Objective: This study evaluated the prevalence, longitudinal change, associations, and prognostic significance of cough severity in patients with fibrotic ILD. Methods: We included consecutive patients with idiopathic pulmonary fibrosis (IPF) and non-IPF fibrotic ILD who completed the 100mm-Cough Severity Visual Analog Scale (VAS) from the prospective multi-center Canadian Registry for Pulmonary Fibrosis. Baseline cough severity and associations with patient demographics and clinical factors were determined. Relationships between baseline cough severity and health outcomes were evaluated. Measurements and Main Results: Patients with IPF (n=1061) had higher median baseline cough severity than those with non-IPF fibrotic ILD (n=2825) [24 vs 20mm, p<0.001], with worse cough associated with gastroesophageal reflux disease for both cohorts. Worse cough severity was independently associated with worse health-related quality of life at baseline, larger annualized decline in DLCO, development of disease progression, and reduced transplant-free survival in both IPF and non-IPF fibrotic ILD cohorts. The IPF cohort (2.2mm, 95% CI 1.6-2.9mm) had larger annualized increments in cough severity compared to the non-IPF fibrotic ILD cohort (1.1mm, 95% CI 0.8-1.4mm; p=0.004). There was no difference in worsening cough over time comparing those receiving and not receiving ILD-targeted therapy or with and without lung function decline. Conclusion: Cough is common in patients with IPF and non-IPF fibrotic ILD, with increasing cough severity over time irrespective of ILD-targeted therapy. Patient-reported cough severity has prognostic implications on health-related quality of life, disease progression, and survival in fibrotic ILD.

Single-Cell Profiling Reveals Immune Aberrations in Progressive Idiopathic Pulmonary Fibrosis.

RATIONALE: Changes in peripheral blood cell populations have been observed but not detailed at single-cell resolution in idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. METHODS: Peripheral blood mononuclear cells (PBMCs) from IPF patients and controls were profiled using 10x Chromium 5' single-cell RNA sequencing (scRNA-seq). Flow cytometry was used for validation. Protein concentrations of Regulatory T-cells (Tregs) and Monocytes chemoattractants were measured in plasma and lung homogenates from patients and controls. MEASUREMENTS AND MAIN RESULTS: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched controls yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in progressive and stable IPF compared to controls (32.1%, 25.2%, 17.9%, respectively, p<0.05). Total lymphocytes were decreased in IPF vs controls, and in progressive vs stable IPF (52.6% vs 62.6%, p=0.035). Tregs were increased in progressive vs stable IPF (1.8% vs 1.1% of all PBMC, p=0.007), although not different than controls, and may be associated with decreased survival (P=0.009 in Kaplan-Meier analysis; P=0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of IPF patients. Fraction of Tregs out of all T cells was also increased in two cohorts of lung scRNA-seq. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. CONCLUSIONS: The single-cell atlas of the peripheral immune system in IPF, reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

Hospitalization Rates in Interstitial Lung Disease: An Analysis of the Pulmonary Fibrosis Foundation Registry.

RATIONALE: Little is known about hospitalization in other types of interstitial lung disease (ILD) besides idiopathic pulmonary fibrosis (IPF). OBJECTIVES: We sought to determine the frequency of hospitalizations in various types of ILD and elucidate the association of hospitalization with outcomes. METHODS: An analysis of the Pulmonary Fibrosis Foundation Patient Registry data was performed. Inpatient hospitalization rates and survival following hospitalization were compared for various types of ILD. RESULTS: Hospitalization rates were similar across ILD types (40.6% of IPF participants, 42.8% of connective tissue disease related ILD (CTD-ILD), 44.9% of non-IPF idiopathic interstitial pneumonia (IIPs), 46.5% of chronic hypersensitivity pneumonitis (CHP) participants, and 53.3% of "other" ILD participants). All-cause hospitalization was not associated with decreased transplant-free survival (adjusted hazard ratio (AHR) 1.20, 95% CI: 0.98, 1.46, p=0.0759) after adjusting for co-morbidities and severity of illness; however respiratory-related hospitalization was (AHR 1.53, 95% CI: 1.23, 1.90, p=0.0001). CTD-ILD (HR 0.43, 95% CI: 0.25, 0.75, p=0.0031) and non-IPF IIP (HR 0.3, 95% CI: 0.15, 0.58, p=0.005) had a lower risk of death following hospitalization compared to IPF while CHP (HR 0.67, 95% CI: 0.37, 1.20, p=0.1747) and "other-ILD" (HR 0.54, 95% CI: 0.19, 1.54, p=0.25) had a comparable risk to IPF. CONCLUSION: Rates of hospitalization are similar across ILD subtypes. The risk of death or transplant following hospitalization is lower in CTD-ILD, CHP and non-IPF IIP compared to IPF participants. In a mixed population of ILD participants, all-cause hospitalizations were not associated with decreased transplant-free survival; however respiratory-related hospitalizations were.

Single Cell Analysis Reveals Novel Immune Perturbations in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3hi/CCL4hi and S100Ahi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZMhi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFß and TNFα/NFκB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions: Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZMhi cytotoxic CD4+ and CD8+ T cells - reflecting this disease's unique inflammatory T-cell driven nature - as well as increased S100Ahi and CCL3hi/CCL4hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

Machine Learning of Plasma Proteomics Classifies Diagnosis of Interstitial Lung Disease.

RATIONALE: Distinguishing connective tissue disease associated interstitial lung disease (CTD-ILD) from idiopathic pulmonary fibrosis (IPF) can be clinically challenging. OBJECTIVES: Identify proteins that separate and classify CTD-ILD from IPF patients. METHODS: Four registries with 1247 IPF and 352 CTD-ILD patients were included in analyses. Plasma samples were subjected to high-throughput proteomics assays. Protein features were prioritized using Recursive Feature Elimination (RFE) to construct a proteomic classifier. Multiple machine learning models, including Support Vector Machine, LASSO regression, Random Forest (RF), and imbalanced-RF, were trained and tested in independent cohorts. The validated models were used to classify each case iteratively in external datasets. MEASUREMENT AND MAIN RESULTS: A classifier with 37 proteins (PC37) was enriched in biological process of bronchiole development and smooth muscle proliferation, and immune responses. Four machine learning models used PC37 with sex and age score to generate continuous classification values. Receiver-operating-characteristic curve analyses of these scores demonstrated consistent Area-Under-Curve 0.85-0.90 in test cohort, and 0.94-0.96 in the single-sample dataset. Binary classification demonstrated 78.6%-80.4% sensitivity and 76%-84.4% specificity in test cohort, 93.5%-96.1% sensitivity and 69.5%-77.6% specificity in single-sample classification dataset. Composite analysis of all machine learning models confirmed 78.2% (194/248) accuracy in test cohort and 82.9% (208/251) in single-sample classification dataset. CONCLUSIONS: Multiple machine learning models trained with large cohort proteomic datasets consistently distinguished CTD-ILD from IPF. Identified proteins involved in immune pathways. We further developed a novel approach for single sample classification, which could facilitate honing the differential diagnosis of ILD in challenging cases and improve clinical decision-making.

Microscopic Small Airway Abnormalities Identified in Early Idiopathic Pulmonary Fibrosis In Vivo Using Endobronchial Optical Coherence Tomography.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) affects subpleural lung, but is considered to spare small airways. Micro-CT studies demonstrated small airway reduction in end-stage IPF explanted lungs, raising questions about small airway involvement in early-stage disease. Endobronchial optical coherence tomography (EB-OCT) is a volumetric imaging modality that detects microscopic features from subpleural to proximal airways. We use EB-OCT to evaluate small airways in early IPF and control subjects in vivo. METHODS: EB-OCT was performed in 12 IPF and 5 control subjects (matched by age, sex, smoking-history, height, BMI). IPF subjects had early disease with mild restriction (FVC: 83.5% predicted), diagnosed per current guidelines and confirmed by surgical biopsy. EB-OCT volumetric imaging was acquired bronchoscopically in multiple, distinct, bilateral lung locations (total: 97 sites). IPF imaging sites were classified by severity into affected (all criteria for UIP present) and less affected (some but not all criteria for UIP present) sites. Bronchiole count and small airway stereology metrics were measured for each EB-OCT imaging site. RESULTS: Compared to control subjects (mean: 11.2 bronchioles/cm3; SD: 6.2), there was significant bronchiole reduction in IPF subjects (42% loss; mean: 6.5/cm3; SD: 3.4; p=0.0039), including in IPF affected (48% loss; mean: 5.8/cm3; SD: 2.8; p<0.00001) and IPF less affected (33% loss; mean: 7.5/cm3; SD: 4.1; p=0.024) sites. Stereology metrics showed IPF affected small airways were significantly larger and more distorted/irregular than in IPF less affected sites and control subjects. IPF less affected and control airways were statistically indistinguishable for all stereology parameters (p=0.36-1.0). CONCLUSION: EB-OCT demonstrated marked bronchiolar loss in early IPF (between 30 and 50%), even in areas minimally affected by disease, compared to matched controls. These findings support small airway disease as a feature of early IPF, providing novel insight into pathogenesis and potential therapeutic targets.

Meaningful Endpoints for Idiopathic Pulmonary Fibrosis (IPF) Clinical Trials: Emphasis on 'Feels, Functions, Survives'. Report of a Collaborative Discussion in a Symposium with Direct Engagement from Representatives of Patients, Investigators, the National Institutes of Health, a Patient Advocacy Organization, and a Regulatory Agency.

Background: Idiopathic pulmonary fibrosis (IPF) carries significant mortality and unpredictable progression, with limited therapeutic options. Designing trials with patient-meaningful endpoints, enhancing the reliability and interpretability of results, and streamlining the regulatory approval process are of critical importance to advancing clinical care in IPF. Methods: A landmark in-person symposium in June 2023 assembled 43 participants from the US and internationally, including patients with IPF, investigators, and regulatory representatives, to discuss the immediate future of IPF clinical trial endpoints. Patient advocates were central to discussions, which evaluated endpoints according to regulatory standards and the FDA's 'feels, functions, survives' criteria. Results: Three themes emerged: 1) consensus on endpoints mirroring the lived experiences of patients with IPF; 2) consideration of replacing forced vital capacity (FVC) as the primary endpoint, potentially by composite endpoints that include 'feels, functions, survives' measures or FVC as components; 3) support for simplified, user-friendly patient-reported outcomes (PROs) as either components of primary composite endpoints or key secondary endpoints, supplemented by functional tests as secondary endpoints and novel biomarkers as supportive measures (FDA Guidance for Industry (Multiple Endpoints in Clinical Trials) available at: https://www.fda.gov/media/162416/download). Conclusions: This report, detailing the proceedings of this pivotal symposium, suggests a potential turning point in designing future IPF clinical trials more attuned to outcomes meaningful to patients, and documents the collective agreement across multidisciplinary stakeholders on the importance of anchoring IPF trial endpoints on real patient experiences-namely, how they feel, function, and survive. There is considerable optimism that clinical care in IPF will progress through trials focused on patient-centric insights, ultimately guiding transformative treatment strategies to enhance patients' quality of life and survival.

Novel Small Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline due to scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial to mesenchymal transition and oxidative stress. The RhoA/ROCK signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in IPF patients and is a promising target for IPF therapy. However, due to hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ~80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain (p-MLC) and reduced the expression of fibrosis-related proteins, such as collagen, fibronectin, and alpha-smooth muscle actin (α-SMA) in various in vitro cellular models. GNS-3595 also prevented transforming growth factor beta (TGF-ß)-induced fibroblast-to-myofibroblast transition (FMT). Additionally, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

Lipid Deficiency Contributes to Impaired Alveolar Progenitor Cell Function in Aging and Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated interstitial lung disease resulting from repeated epithelial injury and inadequate epithelial repair. Alveolar type II cells (AEC2) are progenitor cells that maintain epithelial homeostasis and repair the lung after injury. In the current study, we assessed lipid metabolism in AEC2s from human lungs of IPF patients and healthy donors, as well as AEC2s from bleomycin-injured young and old mice. Through single cell RNA sequencing (scRNA-seq), we observed that lipid metabolism-related genes were downregulated in IPF AEC2s and bleomycin-injured mouse AEC2s. Aging aggravated this decrease and hindered recovery of lipid metabolism gene expression in AEC2s after bleomycin injury. Pathway analyses revealed down-regulation of genes related to lipid biosynthesis and fatty acid -oxidation in AEC2s from IPF lungs and bleomycin-injured, aged mouse lungs compared to the respective controls. We confirmed decreased cellular lipid content in AEC2s from IPF lungs and bleomycin-injured, aged mouse lungs using immunofluorescence staining and flow cytometry. We further show that lipid metabolism was associated with AEC2 progenitor function. Lipid supplementation and peroxisome proliferator activated receptor gamma (PPARγ) activation promoted progenitor renewal capacity of both human and mouse AEC2s in 3D organoid cultures. Lipid supplementation also increased AEC2 proliferation and expression of SFTPC in AEC2s. In summary, we identified a lipid metabolism deficiency in AEC2s from lungs of patients with IPF and bleomycin-injured aged mice. Restoration of lipid metabolism homeostasis in AEC2s might promote AEC2 progenitor function and offer new opportunities for therapeutic approaches to IPF. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Extracellular Matrix as a Driver of Chronic Lung Diseases.

The extracellular matrix (ECM) is not just a three-dimensional scaffold that provides stable support for all cells in the lungs, but also an important component of chronic fibrotic airway, vascular, and interstitial diseases. It is a bioactive entity that is dynamically modulated during tissue homeostasis and disease, that controls structural and immune cell functions and drug responses, and that can release fragments that have biological activity and that can be used to monitor disease activity. There is a growing recognition of the importance of considering ECM changes in chronic airway, vascular, and interstitial diseases, including 1) compositional changes, 2) structural and organizational changes, and 3) mechanical changes and how these affect disease pathogenesis. As altered ECM biology is an important component of many lung diseases, disease models must incorporate this factor to fully recapitulate disease-driver pathways and to study potential novel therapeutic interventions. Although novel models are evolving that capture some or all of the elements of the altered ECM microenvironment in lung diseases, opportunities exist to more fully understand cell-ECM interactions that will help devise future therapeutic targets to restore function in chronic lung diseases. In this perspective article, we review evolving knowledge about the ECM's role in homeostasis and disease in the lung.