European Respiratory Journal

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with limited treatment options. LTI-03 promotes alveolar epithelial cell survival and reduces profibrotic protein expression in experimental models of IPF. In this Phase 1b, placebo-controlled, dose-escalation study, 24 participants with IPF were randomized 3:1 into 2 sequential dose cohorts to LTI-03 (5 or 10 mg/day) or placebo for 14 days (ClinicalTrials.gov: NCT05954988). The primary endpoint was the incidence of treatment-emergent adverse events (TEAEs). Exploratory analyses included pharmacokinetics and changes from baseline in expression of biomarkers related to fibrotic processes and epithelial integrity. Inhaled LTI-03 was well-tolerated, with no treatment-related discontinuations and only mild or moderate TEAEs; cough was the most common treatment-related TEAE. There was no evidence of airway obstruction by symptoms or spirometry. In bronchoscopy-derived deep bronchial brushing samples, both doses of LTI-03 significantly reduced interleukin-11 and thymic stromal lymphopoietin compared to placebo. Additionally, LTI-03 10 mg/day significantly reduced the expression of collagen type 1 alpha chain 1, CXC chemokine ligand 7 and galectin-7 compared to placebo. A trend in the reduction of plasma surfactant protein D was also observed in the LTI-03 10 mg/day group compared to placebo. The favorable safety and tolerability profile in addition to a reduction of disease-related biomarkers supports further evaluation of inhaled LTI-03 for IPF in a Phase 2 study (RENEW; ClinicalTrials.gov: NCT06968845).

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive, fatal disorder with a variable disease trajectory. The aim of this study was to assess the potential of neutrophil-to-lymphocyte ratio (NLR) to predict outcomes for people with IPF. MethodWe adopted a two-stage discovery and validation design using patients from the UCL partners (UCLp) cohort. For the discovery analysis, we included 71 patients from UCLH. In the validation analysis, we included 928 people with IPF, using real-life data from UCLH and 5 other UK centres. Data were collected from patients presenting over a 13-year period with a mean follow up time of 3.7 years. FindingsIn the discovery analysis, we showed that values of NLR (<2.9 vs >/=2.9) were associated with increased risk of mortality (HR 2.04, 95% CI 1.09-3.81; p=0.025). In the validation cohort we confirmed this association of high NLR with mortality (HR 1.65, 95% CI 1.39-1.95; p<0{middle dot}0001) and showed incorporation of baseline NLR in a modified GAP-stage/index (GAP/index)-plus improved predictive ability InterpretationWe have identified NLR as a widely available test that significantly correlates with lung function, can predict outcomes in IPF and refines clinical GAP-staging. NLR may help ILD specialist centres prioritise at risk patients in a timely way, even in the absence of lung function.

BackgroundThe clinical course of idiopathic pulmonary fibrosis (IPF) is highly variable and unpredictable, with multiple genetic variants influencing IPF outcomes. Notably, rare pathogenic variants in telomere-related genes are associated with poorer clinical outcomes in these patients. Here we assessed whether rare qualifying variants (QVs) in monogenic adult-onset pulmonary fibrosis (PF) genes are associated with IPF survival. Using polygenic risk scores (PRS), we also evaluated the influence of common IPF risk variants in individuals carrying these QVs. MethodsWe identified QVs in telomere and non-telomere genes linked to monogenic PF forms using whole-genome sequences (WGS) from 888 Pulmonary Fibrosis Foundation Patient Registry (PFFPR) individuals. We also derived a PRS for IPF (PRS-IPF) from 19 previously published common sentinel IPF variants. Using regression models, we then examined the mutual relationships of QVs and PRS-IPF and their association with survival. Validation of results was sought in WGS from an independent IPF study (PROFILE, n=472), and results from the two cohorts were meta-analyzed. ResultsCarriers of QVs in monogenic adult-onset PF genes, representing nearly 1 out of 6 IPF patients, were associated with lower PRS-IPF (Odds Ratio [OR]: 1.79; 95% Confidence Interval [CI]: 1.15-2.81; p=0.010) and shorter survival (Hazard Ratio [HR]: 1.53; 95% CI: 1.12-2.10; p=7.3x10-3). Notably, carriers of pathogenic variants at telomere genes showed the strongest association with survival (HR: 1.76; 95% CI: 1.13-2.76; p=0.013). The meta-analysis of the results showed a consistent direction of effect across both cohorts. ConclusionsWe revealed the opposite effects of QVs and PRS-IPF on IPF survival. Thus, a distinct IPF molecular subtype might be defined by QVs in monogenic adult-onset PF genes. Assessing the carrier status for QVs and modelling PRS-IPF promises to further contribute to predicting disease progression among IPF patients.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive lung disease that primarily afflicts people over the age of 65. IPF is characterized by lung scarring, an elevated senescence burden, and interstitial pneumonia, resulting in disability and mortality. The growing aging population worldwide, limited effectiveness of current treatments, and high economic burden underscore the need for robust models to investigate the underlying mechanisms and test novel interventions. Despite broad preclinical use, the bleomycin-induced murine model has notable limitations. Animals subjected to a single dose of bleomycin administration undergo recovery in weight and behavior between 21 and 28 days after administration, which is contrary to the progressive nature of IPF. Previous reports have shown that repetitive instillation of bleomycin phenocopies this aspect of the human disease. However, these methods are time-consuming and complex. Although IPF is typically associated with advanced age, most research is conducted in 6 to 8-week-old mice, which lack the age-related structural and metabolic deficits seen in humans. In this study, we report an improved model of IPF using 17-month-old UM-HET3 mice subjected to a single oropharyngeal bleomycin dosing that better mimics the persistent nature of the disease. Lung histology and immunohistochemistry (IHC) confirm persistence of fibrosis and senescence in mice 10 weeks after bleomycin administration. Furthermore, bulk RNA sequencing (RNA-Seq) analysis revealed a distinct set of gene expression signatures that is more consistent with chronic human fibrosis. This model offers greater insight into IPF pathogenesis, and we anticipate that it will enhance confidence in the human translatability of candidate therapeutic interventions.

RationaleThe diagnosis of idiopathic pulmonary fibrosis (IPF) requires exclusion of known underlying autoimmunity, as present in interstitial lung diseases associated with connective tissue diseases (CTD-ILD). However, autoantibodies of unknown significance have been repeatedly detected in IPF patients. ObjectivesWe aimed to characterize autoreactivities in IPF patients beyond clinically established autoimmune panels by establishing an unbiased assay for de novo discovery of autoantigens in different forms of ILD and healthy controls. MethodsWe developed the proteomic Differential Antigen Capture (DAC) assay, capturing patient antibodies from plasma, followed by affinity purification of lung proteins coupled to mass spectrometry. Plasma antibodies from patients with IPF (n=35), CTD-ILD (n=24) and age-matched controls (n=32) were analyzed and validated in an independent cohort (IPF: n=40; CTD-ILD: n=20). Plasma antibody binding profiles were associated with clinical meta-data including diagnosis, lung function and transplant free survival. Measurements and Main ResultsWe identified 586 putative autoantigens in both study cohorts with a broad heterogeneity among disease entities and cohorts. The prevalence of autoantibodies was higher in IPF compared to CTD-ILD. We identified a predictive autoimmune signature that was significantly associated with reduced transplant free survival in IPF. In particular, presence of autoantibodies to Thrombospondin 1 (THBS1) was associated with a significantly reduced survival in patients with IPF (p=0.002), independent of the study cohort, suggesting clinical relevance as predictive biomarker. ConclusionsUnbiased proteomic profiling reveals that the overall prevalence of autoantibodies is similar in IPF and CTD-ILD patients and identifies novel IPF specific autoantigens associated with patient survival.

BackgroundOlder age is the main risk factor for chronic lung diseases including idiopathic pulmonary fibrosis (IPF). Halting or reversing progression of IPF remains an unmet clinical need due to limited knowledge of underlying mechanisms. The lung circulatory system, composed of blood (pulmonary and bronchial) and lymphatic vessels networks, has been implicated in IPF pathophysiology in elderly people, based solely on reports of altered density and increased permeability of vessels. AimWe aimed to define heterogeneity and IPF-associated changes of lung endothelial cells (EC or endothelium) by comparing gene expression in tissues from elderly people - transplant donors and recipients with IPF. MethodsSingle-cell RNA sequencing (scRNAseq) datasets of "ageing lung" tissues were selected only from those publicly available sources that contain age-matching samples for both groups (49- 77 years old donors and IPF patients; nine pairs in total), integrated and compared. Findings were validated by immunohistochemistry using EC-specific markers. ResultsThe generation of integrated single-cell maps of ageing lung tissues revealed 17 subpopulations of endothelium (12 for blood and 5 for lymphatic vessels, including 9 novel), with distinct transcriptional profiles. In IPF lung, the heterogeneity of ageing lung endothelium was significantly altered - both in terms of cell numbers (linked to disease- related changes in tissue composition) and differentially expressed genes (associated with fibrosis, inflammation, differentiation and vasodilation) in individual pulmonary, bronchial and lymphatic EC subpopulations. ConclusionsThese findings reveal underappreciated extent of heterogeneity and IPF-associated changes of ageing lung endothelium. Our data suggest direct involvement of specific subpopulations of ageing lung endothelium in IPF pathophysiology, uncovering cellular and molecular targets which may have potential diagnostic, prognostic and therapeutic relevance. This study creates a conceptual framework for appreciating the disease-specific heterogeneity of ageing lung endothelium as a hallmark of IPF.

BackgroundInterstitial lung disease (ILD) is a known complication of rheumatoid arthritis (RA) with a lifetime risk in any individual of 7.7%. The TRAIL1 trial was a randomized, double-blinded, placebo-controlled, phase 2 study of safety, tolerability, and efficacy of pirfenidone for the treatment of patients with RA-ILD. MethodsThe TRAIL1 was a phase 2 trial intended to enroll 270 adult patients (18 to 85 years) with established RA-ILD at 33 sites in 4 countries. Patients were randomly assigned (1:1) to 2,403 mg oral pirfenidone or placebo daily. The primary endpoint was the incidence of the composite endpoint of decline from baseline in percent predicted forced vital capacity (FVC%) of 10% or greater or death during the 52-week treatment period. Key secondary endpoints included change in absolute and FVC% over 52 weeks. FindingsThe trial was stopped early due to slow recruitment and soon after the shutdown of clinical trials as a consequence of the coronavirus disease 2019 (COVID-19) pandemic. Data from 123 patients enrolled were analyzed. The primary endpoint was met by 11.1% on pirfenidone vs. 15% on placebo [OR=0.67 (0.22, 2.03), p=0.48]. Subjects receiving pirfenidone had a slower rate of decline in lung function as measured by estimated annual change in FVC(ml) (-66 vs. -146, p=0.0082) and FVC(%) (-1.02 vs. -3.21, p=0.0028). This effect on decline was also seen when analyzed within participants with baseline usual interstitial pneumonia (UIP) pattern on HRCT (FVC(ml) (-43 vs. -169, p=0.0014) and FVC% (-0.2 vs. -3.81, p=0.0002)). There was no significant difference in the rate of treatment-emergent serious adverse events. InterpretationDue to early termination of the study, results should be interpreted with caution. Despite being underpowered to evaluate the primary endpoint, pirfenidone slowed the rate of decline of FVC over time in subjects with RA-ILD. Safety in patients with RA-ILD was similar to that seen in other pirfenidone trials. FundingFunding for this investigator initiated trial was provided by Genentech, Inc. to Ivan O. Rosas, MD, on behalf of the TRAIL1 Investigators.

BackgroundGuideline-based therapy (GBT) drugs for Mycobacterium avium-complex (MAC) lung disease (LD) were chosen in part because they have low MICs. Despite these low MICs, GBT achieves six-month sustained sputum culture conversion in only 43% of patients. MethodsFirst, we co-incubated tigecycline with MAC for seven days in time-kill studies and calculated the exposure mediating 50% of maximal effect (Emax) or EC50. Next, we performed tigecycline exposure-effect studies in the hollow fiber system of MAC (HFS-MAC) inoculated with the reference ATCC#700898 isolate. Third, we performed an exposure-effect study in the HFS-MAC inoculated with 5 different isolates. Finally, the target exposure (EC80) was used to identify a clinical dose of inhaled tigecycline for MAC-LD in 10,000 subject Monte Carlo experiments (MCE). ResultsIn time-kill studies the EC50 was 0-24h area under the concentration-time curve-to-MIC (AUC0-24/MIC) of 62.24 for extracellular and 0.14 for intracellular MAC (p<0.001). In the HFS-MAC inoculated with ATCC#700898, the EC50 statistically differed between sampling days by 2,370.7%. However, studies with five different isolates demonstrated a stable and robust day-to-day EC50 (%CV=18.18%), with an EC80 AUC0-24/MIC of 33.65. The Emax was 4.84 log10 CFU/mL. In MCE, tigecycline inhalational doses of 35-40 mg/day achieved the EC80 target in >90% of virtual patients, with and an MIC breakpoint of 256 mg/L. ConclusionTime-kill studies do not inform on PK/PD target exposures or extent of kill. Inclusion of multiple MAC isolates in HFS-MAC studies improves precision of pharmacokinetic/pharmacodynamic parameter estimates. Tigecycline via the inhalational route could contribute to treatment of MAC-LD.

IntroductionAccurate pre-lung transplant biomarkers of post-lung transplant survival are lacking in Idiopathic Pulmonary Fibrosis (IPF). MethodsThis was a retrospective, observational study including consecutive patients diagnosed with IPF at the University of South Florida/ Tampa General Hospital. First, we compared survival differences in patients with IPF that received lung transplant versus non- recipients, then we investigated whether pre-transplant monocyte counts could predict post- lung transplant survival, Primary Graft Dysfunction (PGD), Acute Cellular Rejection (ACR), Antibody-Mediated Rejection (AMR) and Chronic Lung Allograft Dysfunction (CLAD) using Cox Proportional Hazards (CoxPH) models adjusted to Gender, Age and Physiology index (GAP). ResultsA total of 201 patients with IPF were included in the analysis [lung transplant recipients: n=103, non-recipients of lung transplant: n=98]. Patients with IPF that did not undergo lung transplantation had significantly worse survival compared to patients with IPF that underwent lung transplantation [3.13 years (95% CI: 2.30 to 3.72) vs 7.05 years (95% CI: 5.41 to 8.48), HR: 2.95 (95% CI: 2.18 to 4.00), p<0.0001]. Patients with IPF and pre-lung transplant monocyte counts>700 K/L had increased risk of post-lung transplant mortality [HR: 1.71 (95%CI: 1.10 to 2.65), p=0.016] or adverse outcomes defined as either PGD, ACR, AMR or CLAD, [HR: 2.05 (95% CI: 1.11 to 3.78), p=0.02] compared to patients with monocyte counts[&le;]700 K/L. ConclusionLung transplantation substantially prolongs survival of patients with IPF. Incorporation of pre-lung transplant monocyte counts in the pre-transplant evaluation of patients with IPF could optimize the selection of ideal lung transplant candidates with increased probability of survival.

Hermansky-Pudlak syndrome (HPS) is a group of rare genetic disorders, with several subtypes leading to fatal adult-onset pulmonary fibrosis (PF) and no effective treatment. Circulating biomarkers detecting early PF have not been identified. We investigated whether endocannabinoids could serve as blood biomarkers of PF in HPS. We measured endocannabinoids in the serum of HPS, IPF, and healthy human subjects and in a mouse model of HPSPF. Pulmonary function tests (PFT) were correlated with endocannabinoid measurements. In a pale ear mouse model of bleomycin-induced HPSPF, serum endocannabinoid levels were measured with and without treatment with zevaquenabant (MRI-1867), a peripheral CB1R and iNOS antagonist. In three separate cohorts, circulating anandamide levels were increased in HPS-1 patients with or without PF, compared to healthy volunteers. This increase was not observed in IPF patients or in HPS-3 patients, who do not have PF. Circulating anandamide (AEA) levels were negatively correlated with PFT. Furthermore, a longitudinal study over the course of 5-14 years with HPS-1 patients indicated that circulating AEA levels begin to increase with the fibrotic lung process even at the subclinical stages of HPSPF. In pale ear mice with bleomycin-induced HpsPF, serum AEA levels were significantly increased in the earliest stages of PF and remained elevated at a later fibrotic stage. Zevaquenabant treatment reduced the increased AEA levels and attenuated progression in bleomycin-induced HpsPF. Circulating AEA may be a prognostic blood biomarker for PF in HPS-1 patients. Further studies are indicated to evaluate endocannabinoids as potential surrogate biomarkers in progressive fibrotic lung diseases.

ObjectiveCirculating fibrocytes are elevated in idiopathic pulmonary fibrosis, but the relationship between fibrocyte level with lung function decline and outcomes is lacking replication in prospective clinical study. We aim to validate the utility of circulating fibrocyte levels as a prognostic biomarker in idiopathic pulmonary fibrosis. MethodsWe tested associations between circulating fibrocyte levels, mortality, disease progression and longitudinal lung function in a well-defined prospective observational study of pulmonary fibrosis (PROFILE; NCT01134822). A subset of recruited participants had blood samples processed for fibrocyte measurement, with flow cytometry based on CD45 and collagen-I gating. Associations were tested using univariable and multivariable generalised linear models. Mortality data were subsequently combined with an independent cohort in a mixed-effect multilevel analysis. ResultsIn 102 participants with idiopathic pulmonary fibrosis, an empirically defined cutpoint of 2.22% was associated with a greater risk of overall mortality in adjusted analysis (Hazard Ratio 2.24 95% CI 1.06-4.72). A 2.5 fold greater risk of mortality was supported in a pooled analysis with a historic cohort for a larger sample of 162 participants of idiopathic pulmonary fibrosis, specifically (Hazard Ratio 2.49 95% CI 2.41-2.56). A previously defined mortality risk threshold of 5% circulating fibrocytes was not reproducible in this cohort, circulating fibrocytes were not significantly elevated above non-specific interstitial pneumonia or healthy controls.. We found no association of fibrocytes with lung function or disease progression. ConclusionsIn a prospective clinical cohort of idiopathic pulmonary fibrosis, circulating fibrocytes of 2.22% or above are associated with greater mortality, but do not associate with disease related decline in lung function. What is the key question?Can circulating fibrocytes provide reproducible prognostic biomarker value in fibrotic lung disease? What is the bottom line?Greater proportions of fibrocytes isolated from circulating leukocyte populations are associated with an increase risk of mortality in idiopathic pulmonary fibrosis, but no association was observed with disease related decline in lung function. Why read on?We present associations and limitations of circulating fibrocytes in the largest sample of individuals with pulmonary fibrosis, recruited into a prospective observational study, and replicate prognositic insights from a historic cohort.

RationaleData on the prognostic value of peripheral blood mononuclear cell (PBMC) expression profiles, when used in fibrotic hypersensitivity pneumonitis (fHP) patients as an adjunct to traditional clinical assessment in predicting disease progression, remains limited. ObjectivesTo determine whether a baseline and time-course transcriptomic signature in PBMC from patients with fHP can enhance progression-free survival (PFS) prediction and complement clinical risk stratification. MethodsThe prospective multicenter study cohort included 133 participants with fHP. Lasso regression was employed to create a baseline and time-course (baseline to 12-month change) gene signature. We developed multivariable models incorporating clinical variables (age, sex, smoking status, exposure history, FVC% and quantitative measurements of lung fibrosis--derived from data-driven textural analysis, DTA) both with and without the baseline and time-course gene expression, and evaluated these models using receiver operating characteristic curves. Kaplan-Meier curves displaying high and low gene expression risk scores are presented. ResultsThe addition of a baseline gene expression profile to the logistic regression model of 24-month PFS using baseline clinical parameters markedly improved the predictive accuracy, increasing the area under the curve (AUC) from 0.77 to 0.93. Similarly, a logistic regression model of 12-month PFS using age, sex, smoking status, exposure history, and changes in FVC% and DTA over 12 months had an AUC of 0.81, which improved to 0.95 with the inclusion of changes in gene expression over the same period. A difference in PFS was noted when the cohort was divided based on whether their gene principal component analysis (PCA) scores were above or below the median. At baseline, patients with higher gene PCA scores had lower median survival than those with lower scores (267 days vs. not available; P = 0.008). Similarly, when assessing the change in gene expression from baseline to 12 months, patients with higher gene PCA scores had lower median survival than those with lower scores (203 days vs. 449 days; P = <0.001). ConclusionsIn fHP, a risk-indicative gene expression signature in peripheral blood at baseline, along with its changes over 12 months, predicts disease progression at 24 months and during the subsequent 12 months, respectively. Prognostic models based on gene expression and clinical factors strongly outperform models based solely on clinical factors.

AimsPulmonary hypertension (PH) is associated with significant morbidity and mortality and leads to progressive right heart failure. In patients with PAH, haemodynamic parameters measured at catheterisation relate to clinical worsening events, in patients with heart failure proactive pulmonary artery pressure based therapeutic intervention reduces hospitalisation. We therefore investigated use of a novel implanted pulmonary artery (PA) pressure monitor to detect clinically relevant changes in pressure in large animal models of pulmonary hypertension (PH). Methods and ResultsPrototype pulmonary artery pressure sensors (Endotronix) were implanted using standard interventional techniques. Acute PH was induced by infusion of thromboxane A2 in domestic swine. Over a physiological range pressure monitors remained concordant to reference catheter (bias -0.43, 95%CI-5.3-4.4). Chronic PH was induced by i.p. injection of monocrotaline. Implanted pressure sensors demonstrated a gradual rise in PA pressure over 30 days (baseline: 20.7+/-0.4 vrs day-30: 31.74+/-1.4, p<0.01). Pressure sensor derived readings matched reference catheter at baseline and day-30. Pressure sensors remained stable and no adverse events were identified by clinical and histological examination. ConclusionsThe development of PA pressure monitors provide long-term haemodynamic data that identified clinically meaningful changes in pulmonary artery pressure. In addition to proactive heart failure management, such devices may be used to optimise or personalize patient therapy, investigate aspects of physiology and pathology essential to the understanding of disease and provide the opportunity to assess therapeutic interventions in clinical studies.

RationaleIdiopathic 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. ObjectivesTo perform GWAS of IPF susceptibility to detect associations where the underlying effects are consistent with recessive or dominant genetic models. MethodsWe 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 ResultsWe 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. ConclusionsUsing alternative genetic models in IPF susceptibility GWAS identified new signals and genes, providing new insights into IPF pathogenesis and potential future therapies.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial pneumonia of unknown etiology often leading to respiratory failure. Over half of IPF patients present with discordant features of usual interstitial pneumonia on high-resolution computed tomography at diagnosis which warrants surgical lung biopsy to exclude the possibility of other interstitial lung diseases (ILDs). Therefore, there is a need for non-invasive biomarkers for expediting the differential diagnosis of IPF. MethodsUsing mass spectrometry, we performed proteomic analysis of plasma extracellular vesicles (EVs) in a cohort of subjects with IPF, chronic hypersensitivity pneumonitis, nonspecific interstitial pneumonitis, and healthy subjects (HS). A five-protein signature was identified by lasso regression and was validated in an independent cohort using ELISA. We evaluated the concordance between plasma EV proteome and the lung transcriptome data. Lastly, we compared the molecular pathways overrepresented in IPF by differentially expressed proteins and transcripts from EVs and lung tissues, respectively. ResultsThe five-protein signature derived from mass spectrometry data showed area under the receiver operating characteristic curve of 0.915 (95%CI: 0.819-1.011) and 0.958 (95%CI: 0.882-1.034) for differentiating IPF from other ILDs and from HS, respectively. We also found that the EV protein expression profiles mirrored their corresponding mRNA expressions in IPF lungs. Further, we observed an overlap in the EV proteome- and lung mRNA-associated molecular pathways. ConclusionsWe discovered a plasma EV-based protein signature for differential diagnosis of IPF and validated this signature in an independent cohort. The signature needs to be tested in large prospective cohorts to establish its clinical utility.

BackgroundIdiopathic 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. AimTo 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. MethodsWe 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. ResultsWe 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. ConclusionOur 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.

Rationale: While rodent lung fibrosis models are routinely used to evaluate novel antifibrotics, these models have largely failed to predict clinical efficacy of novel drug candidates for Idiopathic Pulmonary Fibrosis (IPF). Moreover, single target therapeutic strategies for IPF have failed and current multi-target standard of care drugs are not curative. Caveolin-1 (CAV-1) is an integral membrane protein, which, via its caveolin scaffolding domain (CSD), interacts with caveolin binding domains (CBD). CAV-1 regulates homeostasis, and its expression is decreased in IPF lungs. LTI-03 is a seven amino acid peptide derived from the CSD and formulated for dry powder inhalation; it was well tolerated in normal volunteers (NCT04233814) and a safety trial is underway in IPF patients (NCT05954988). Objectives: Anti-fibrotic efficacy of LTI-03 and other CSD peptides has been observed in IPF lung monocultures, and rodent pulmonary, dermal, and heart fibrosis models. This study aimed to characterize progressive fibrotic activity in IPF PCLS explants and to evaluate the antifibrotic effects of LTI-03 and nintedanib in this model. Methods: First, CBD regions were identified in IPF signaling proteins using in silico analysis. Then, IPF PCLS (n=8) were characterized by COL1A1 immunostaining, multiplex immunoassays, and bulk RNA sequencing following treatment every 12hrs with LTI-03 at 0.5, 3.0, or 10 M; nintedanib at 0.1 M or 1 M; or control peptide (CP) at 10 M. Measurements and Main Results: CBDs were present in proteins implicated in IPF, including VEGFR, FGFR and PDGFR. Increased expression of profibrotic mediators indicated active fibrotic activity in IPF PCLS over five days. LTI-03 dose dependently decreased COL1A1 staining, and like nintedanib, decreased profibrotic proteins and transcripts. Unlike nintedanib, LTI-03 did not induce cellular necrosis signals. Conclusion: IPF PCLS explants demonstrate molecular activity indicative of fibrosis during 5 days in culture and LTI-03 broadly attenuated pro-fibrotic proteins and pathways, further supporting the potential therapeutic effectiveness of LTI-03 for IPF.

BackgroundThe extracellular matrix (ECM) is a dynamic network that provides structural support and maintains tissue homeostasis. Collagens are the main structural components of the ECM, occupying distinct tissue compartments and serving specialized roles. Dysregulated ECM remodeling involves an imbalance between collagen production and degradation, generating neoepitope-specific fragments that can be released into circulation. Serological measurements of these fragments can be used as biomarkers of disease and have been associated with progression and mortality in different fibrotic diseases, including pulmonary fibrosis (PF). This study aimed to investigate whether these systemic biomarkers originate from human lung tissue in patients with PF and non-fibrotic controls. MethodsLung tissue was collected from patients with PF (n = 21) and non-fibrotic controls (n = 21) and processed in parallel as formalin-fixed paraffin-embedded or snap-frozen samples. Serum samples were collected from patients with PF and healthy controls (n = 21). Neoepitope-specific biomarkers reflecting type III, IV, and VI collagen production (PRO-C3, PRO-C4, and PRO-C6) and degradation (C3M, C4M, C4Ma3, and C6M) were quantified in serum and proteolytically degraded lung tissue, and their spatial distribution was assessed by immunohistochemistry in lung tissue sections. ResultsAll collagen remodeling biomarkers were significantly increased in serum of patients with PF compared with healthy controls (PRO-C3: p = 0.0006, all others: p < 0.0001). Collagen degradation fragments (C3M, C4M, and C6M) could be generated and released from both non-fibrotic and fibrotic human lung tissue following proteolytic cleavage with pepsin, collagenase, and/or MMP-9. All biomarkers were detected in lung tissue by immunohistochemical staining, with widespread distribution of type III and IV collagen fragments, whereas type VI collagen (PRO-C6) production showed a more compartment-specific pattern. ConclusionsThese findings demonstrated that neoepitope-specific collagen remodeling biomarkers, usually detected in circulation, are present and can be released from human lung tissue. Their spatial distribution suggests that ECM remodeling is heterogeneous and differs according to collagen type and distinct tissue compartments. Collectively, our findings support the use of collagen remodeling biomarkers as tools to assess ECM remodeling in pulmonary disease.

IntroductionInterstitial lung abnormalities (ILAs) are subtle or mild parenchymal abnormalities observed in more than 5 % of the lungs on CT scans in patients in whom interstitial lung disease was not previously clinically suspected and is considered. ILA is considered to be partly undeveloped stages of idiopathic pulmonary fibrosis (IPF) or progressive pulmonary fibrosis (PPF). This study aims to clarify the frequency of subsequent IPF or PPF diagnosis, the natural course from the preclinical status of the diseases, and the course after commencing treatment. Methods and analysisThis is an ongoing, prospective, multicentre observational cohort study of patients with ILA referred from general health screening facilities with more than 70000 annual attendances. Up to 500 participants will be enrolled annually over 3 years, with 5-year assessments every six months. Treatment intervention including anti-fibrotic agents will be introduced in disease progression cases. The primary outcome is the frequency of subsequent IPF or PPF diagnoses. Additionally, secondary and further endpoints are associated with the efficacy of early therapeutic interventions in cases involving disease progression. Ethics and disseminationThis study protocol and informed consent documents have been approved by the Institutional Review Boards of Kumamoto University Hospital (approval number: 2368), Saiseikai Kumamoto Hospital (approval number: 809), and each participating institution. Additionally, written informed consent will be obtained from all participants. Patient recruitment commenced on 20 June 2022. The results will be disseminated through peer-reviewed publications and international conferences. Trial registration numberUMIN000045149 Strengths and limitations of this studyThis is the first prospective, multicentre, observational study to clarify the following points: O_LIthe aetiological data of patients with ILA from a large general health check-up population C_LIO_LIthe natural course of IPF or PPF from the asymptomatic stage C_LIO_LIthe effects and outcomes of early therapeutic intervention including anti-fibrotic agents for progressive cases of ILA. C_LI The limitations of the study: O_LIparticipants missing the regular 6-monthly visits after consenting to participate in the study because of their asymptomatic or the pandemic viral infection. C_LI

Lung function is a key outcome used in the evaluation of disease progression in cystic fibrosis. The variability of individual lung function measurements over time (within-individual variability) has been shown to predict subsequent lung function changes. Nevertheless, the association between within-individual lung function variability and demographic and genetic covariates is not quantified. We performed a longitudinal analysis of data from a cohort of 7099 adults with cystic fibrosis (between 18 and 49 years old) from the UK cystic fibrosis registry, containing annual review data between 1996 and 2020. A mixed-effects location-scale model is used to quantify mean FEV1 (forced expiratory volume in 1 second) trajectories and FEV1 within-individual variability as a function of sex, age at annual review, age at diagnosis, genotype and birth cohort. Mean FEV1 decreased with age and lung function variability showed an approximately quadratic trend by age. Males showed higher FEV1 mean and variability than females across the whole age range. Individuals who died during follow-up showed on average higher lung function variability than those who survived. This work opens new avenues for further research to understand the role of within-individual lung function variability in disease progression and prediction of key outcomes such as mortality.