Cluster analysis of blood biomarkers to identify molecular patterns in pulmonary fibrosis: assessment of a multicentre, prospective, observational cohort with independent validation.

BACKGROUND: Pulmonary fibrosis results from alveolar injury, leading to extracellular matrix remodelling and impaired lung function. This study aimed to classify patients with pulmonary fibrosis according to blood biomarkers to differentiate distinct disease patterns, known as endotypes. METHODS: In this cluster analysis, we first classified patients from the PROFILE study, a multicentre, prospective, observational cohort of individuals with incident idiopathic pulmonary fibrosis or non-specific interstitial pneumonia in the UK (Nottingham University Hospitals, Nottingham; and Royal Brompton Hospital, London). 13 blood biomarkers representing extracellular matrix remodelling, epithelial stress, and thrombosis were measured by ELISA in the PROFILE study. We classified patients by unsupervised consensus clustering. To evaluate generalisability, a machine learning classifier trained on biomarker signatures derived from consensus clustering was applied to a replication dataset from the Australian Idiopathic Pulmonary Fibrosis Registry (AIPFR). Biomarker associations with mortality and change in percentage of predicted forced vital capacity (FVC%) were assessed, adjusting for age, gender, baseline FVC%, and antifibrotic treatment and steroid treatment before and after baseline. Mortality risk associated with the clusters in the PROFILE cohort was evaluated with Cox proportional hazards models, and mixed-effects models were used to analyse how clustering was associated with longitudinal FVC% in the PROFILE and AIPFR cohorts. FINDINGS: 455 of 580 participants from the PROFILE study (348 [76%] men and 107 [24%] women; mean age 72·4 years [SD 8·3]) were included in the analysis. Within this group, three clusters were identified based on blood biomarkers. A basement membrane collagen (BM) cluster (n=248 [55%]) showed high concentrations of PRO-C4, PRO-C28, C3M, and C6M, whereas an epithelial injury (EI) cluster (n=109 [24%]) showed high concentrations of MMP-7, SP-D, CYFRA211, CA19-9, and CA-125. The third cluster (crosslinked fibrin [XF] cluster; n=98 [22%]) had high concentrations of X-FIB. In the replication dataset (117 of 833 patients from AIPFR; 87 [74%] men and 30 [26%] women; mean age 72·9 years [SD 7·9]), we identified the same three clusters (BM cluster, n=93 [79%]; EI cluster, n=8 [7%]; XF cluster, n=16 [14%]). These clusters showed similarities with clusters in the PROFILE dataset regarding blood biomarkers and phenotypic signatures. In the PROFILE dataset, the EI and XF clusters were associated with increased mortality risk compared with the BM cluster (EI vs BM: adjusted hazard ratio [HR] 1·88 [95% CI 1·42-2·49], p<0·0001; XF vs BM: adjusted HR 1·53 [1·13-2·06], p=0·0058). The EI cluster showed the greatest annual FVC% decline, followed by the BM and XF clusters. A similar FVC% decline pattern was observed in these clusters in the AIPFR replication dataset. INTERPRETATION: Blood biomarker clustering in pulmonary fibrosis identified three distinct blood biomarker signatures associated with lung function and prognosis, suggesting unique pulmonary fibrosis biomarker patterns. These findings support the presence of pulmonary fibrosis endotypes with the potential to guide targeted therapy development. FUNDING: None.

The fibroblast hormone Endotrophin is a biomarker of mortality in chronic diseases.

Fibrosis, driven by fibroblast activities, is an important contributor to morbidity and mortality in most chronic diseases. Endotrophin, a signaling molecule derived from processing of type VI collagen by highly activated fibroblasts, is involved in fibrotic tissue remodeling. Circulating levels of endotrophin have been associated with an increased risk of mortality in multiple chronic diseases. We conducted a systematic literature review collecting evidence from original papers published between 2012 and January 2023 that reported associations between circulating endotrophin (PROC6) and mortality. Cohorts with data available to the study authors were included in an Individual Patient Data (IPD) meta-analysis that evaluated the association of PROC6 with mortality (PROSPERO registration number: CRD42023340215) after adjustment for age, sex and BMI, where available. In the IPD meta-analysis including sixteen cohorts of patients with different non-communicable chronic diseases (NCCDs) (N = 15,205) the estimated summary hazard ratio for 3-years all-cause mortality was 2.10 (95 % CI 1.75-2.52) for a 2-fold increase in PROC6, with some heterogeneity observed between the studies (I2=70 %). This meta-analysis is the first study documenting that fibroblast activities, as quantified by circulating endotrophin, are independently associated with mortality across a broad range of NCCDs. This indicates that, irrespective of disease, interstitial tissue remodeling, and consequently fibroblast activities, has a central role in adverse clinical outcomes, and should be considered with urgency from drug developers as a target to treat.

Defining the mechanism of galectin-3-mediated TGF-ß1 activation and its role in lung fibrosis.

Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.

Rheumatoid arthritis and idiopathic pulmonary fibrosis: a bidirectional Mendelian randomisation study.

BACKGROUND: A usual interstitial pneumonia (UIP) pattern of lung injury is a key feature of idiopathic pulmonary fibrosis (IPF) and is also observed in up to 40% of individuals with rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD). The RA-UIP phenotype could result from either a causal relationship of RA on UIP or vice versa, or from a simple co-occurrence of RA and IPF due to shared demographic, genetic or environmental risk factors. METHODS: We used two-sample bidirectional Mendelian randomisation (MR) to test the hypothesis of a causal effect of RA on UIP and of UIP on RA, using variants from genome-wide association studies (GWAS) of RA (separately for seropositive (18 019 cases and 991 604 controls) and seronegative (8515 cases and 1 015 471 controls) RA) and of IPF (4125 cases and 20 464 controls) as genetic instruments. Sensitivity analyses were conducted to assess the robustness of the results to violations of the MR assumptions. FINDINGS: IPF showed a significant causal effect on seropositive RA, with developing IPF increasing the risk of seropositive RA (OR=1.06, 95% CI: 1.04 to 1.08, p<0.001) which was robust under all models. For the MR in the other direction, seropositive RA showed a significant protective effect on IPF (OR=0.93; 95% CI: 0.87 to 0.99; p=0.032), but the effect was not significant when sensitivity analyses were applied. This was likely because of bias due to exclusion of patients with RA from among the cases in the IPF GWAS, or possibly because our genetic instruments did not fully capture the effect of the complex human leucocyte antigen region, the strongest RA genetic risk factor. INTERPRETATION: Our findings support the hypothesis that RA-UIP may be due to a cause-effect relationship between UIP and RA, rather than due to a coincidental occurrence of IPF in patients with RA. The significant causal effect of IPF on seropositive RA suggests that pathomechanisms involved in the development of UIP may promote RA, and this may help inform future guidelines on screening for ILD in patients with RA.

The Burden and Impact of Cough in Patients with Idiopathic Pulmonary Fibrosis: An Analysis of the Prospective Observational PROFILE Study.

Rationale: Cough is a commonly reported symptom in idiopathic pulmonary fibrosis (IPF) that negatively impacts patient-reported quality of life (QoL). However, both the burden of cough at diagnosis and the behavior of cough over time have not been systematically described in patients with IPF. Objectives: By utilizing data prospectively collected as part of the PROFILE study, we sought to assess cough burden and the impact that this has on QoL within a cohort of patients with newly diagnosed IPF. We also reexamined the previously described relationship between cough and mortality and the association of cough with the MUC5B promoter polymorphism. Methods: The PROFILE study is a multicenter, prospective, observational, longitudinal cohort study of incident IPF. Scores on the Leicester Cough Questionnaire (LCQ) were recorded at baseline in 632 subjects and then repeated 6 monthly in a subset (n = 216) of the cohort. Results: The median LCQ score at diagnosis was 16.1 (interquartile range, 6.5). LCQ scores remained stable over the subsequent year in the majority of patients. There was a weak association between LCQ score and baseline lung function, with worse cough-related QoL associated with more severe physiological impairment. Cough scores were not associated with subsequent mortality after correcting for baseline lung function. Furthermore, there was no relationship between LCQ score and MUC5B promoter polymorphism status. Conclusions: The burden of cough in IPF is high. Although cough is weakly associated with disease severity at baseline, cough-specific QoL, as measured by the LCQ, confers no prognostic value. Cough-specific QoL burden remains relatively stable over time and does not associate with MUC5B promoter polymorphism.

Effects of sleep disturbance on dyspnoea and impaired lung function following hospital admission due to COVID-19 in the UK: a prospective multicentre cohort study.

BACKGROUND: Sleep disturbance is common following hospital admission both for COVID-19 and other causes. The clinical associations of this for recovery after hospital admission are poorly understood despite sleep disturbance contributing to morbidity in other scenarios. We aimed to investigate the prevalence and nature of sleep disturbance after discharge following hospital admission for COVID-19 and to assess whether this was associated with dyspnoea. METHODS: CircCOVID was a prospective multicentre cohort substudy designed to investigate the effects of circadian disruption and sleep disturbance on recovery after COVID-19 in a cohort of participants aged 18 years or older, admitted to hospital for COVID-19 in the UK, and discharged between March, 2020, and October, 2021. Participants were recruited from the Post-hospitalisation COVID-19 study (PHOSP-COVID). Follow-up data were collected at two timepoints: an early time point 2-7 months after hospital discharge and a later time point 10-14 months after hospital discharge. Sleep quality was assessed subjectively using the Pittsburgh Sleep Quality Index questionnaire and a numerical rating scale. Sleep quality was also assessed with an accelerometer worn on the wrist (actigraphy) for 14 days. Participants were also clinically phenotyped, including assessment of symptoms (ie, anxiety [Generalised Anxiety Disorder 7-item scale questionnaire], muscle function [SARC-F questionnaire], dyspnoea [Dyspnoea-12 questionnaire] and measurement of lung function), at the early timepoint after discharge. Actigraphy results were also compared to a matched UK Biobank cohort (non-hospitalised individuals and recently hospitalised individuals). Multivariable linear regression was used to define associations of sleep disturbance with the primary outcome of breathlessness and the other clinical symptoms. PHOSP-COVID is registered on the ISRCTN Registry (ISRCTN10980107). FINDINGS: 2320 of 2468 participants in the PHOSP-COVID study attended an early timepoint research visit a median of 5 months (IQR 4-6) following discharge from 83 hospitals in the UK. Data for sleep quality were assessed by subjective measures (the Pittsburgh Sleep Quality Index questionnaire and the numerical rating scale) for 638 participants at the early time point. Sleep quality was also assessed using device-based measures (actigraphy) a median of 7 months (IQR 5-8 months) after discharge from hospital for 729 participants. After discharge from hospital, the majority (396 [62%] of 638) of participants who had been admitted to hospital for COVID-19 reported poor sleep quality in response to the Pittsburgh Sleep Quality Index questionnaire. A comparable proportion (338 [53%] of 638) of participants felt their sleep quality had deteriorated following discharge after COVID-19 admission, as assessed by the numerical rating scale. Device-based measurements were compared to an age-matched, sex-matched, BMI-matched, and time from discharge-matched UK Biobank cohort who had recently been admitted to hospital. Compared to the recently hospitalised matched UK Biobank cohort, participants in our study slept on average 65 min (95% CI 59 to 71) longer, had a lower sleep regularity index (-19%; 95% CI -20 to -16), and a lower sleep efficiency (3·83 percentage points; 95% CI 3·40 to 4·26). Similar results were obtained when comparisons were made with the non-hospitalised UK Biobank cohort. Overall sleep quality (unadjusted effect estimate 3·94; 95% CI 2·78 to 5·10), deterioration in sleep quality following hospital admission (3·00; 1·82 to 4·28), and sleep regularity (4·38; 2·10 to 6·65) were associated with higher dyspnoea scores. Poor sleep quality, deterioration in sleep quality, and sleep regularity were also associated with impaired lung function, as assessed by forced vital capacity. Depending on the sleep metric, anxiety mediated 18-39% of the effect of sleep disturbance on dyspnoea, while muscle weakness mediated 27-41% of this effect. INTERPRETATION: Sleep disturbance following hospital admission for COVID-19 is associated with dyspnoea, anxiety, and muscle weakness. Due to the association with multiple symptoms, targeting sleep disturbance might be beneficial in treating the post-COVID-19 condition. FUNDING: UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council.

Mediators of systemic sclerosis-associated interstitial lung disease (SSc-ILD): systematic review and meta-analyses.

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is rare, poorly understood, with heterogeneous characteristics resulting in difficult diagnosis. We aimed to systematically review evidence of soluble markers in peripheral blood or bronchoalveolar lavage fluid (BALF) as biomarkers in SSc-ILD. METHOD: Five databases were screened for observational or interventional, peer-reviewed studies in adults published between January 2000 and September 2021 that assessed levels of biomarkers in peripheral blood or BALF of SSc-ILD patients compared with healthy controls. Qualitative assessment was performed using Critical Appraisal Skills Programme (CASP) checklists. Standardised mean difference (SMD) in biomarkers were combined in random-effects meta-analyses where multiple independent studies reported quantitative data. RESULTS: 768 published studies were identified; 38 articles were included in the qualitative synthesis. Thirteen studies were included in the meta-analyses representing three biomarkers: KL6, SP-D and IL-8. Greater IL-8 levels were associated with SSc-ILD in both peripheral blood and BALF, overall SMD 0.88 (95% CI 0.61 to 1.15; I2=1%). Greater levels of SP-D and KL-6 were both estimated in SSc-ILD peripheral blood compared with healthy controls, at an SMD of 1.78 (95% CI 1.50 to 2.17; I2=8%) and 1.66 (95% CI 1.17 to 2.14; I2=76%), respectively. CONCLUSION: We provide robust evidence that KL-6, SP-D and IL-8 have the potential to serve as reliable biomarkers in blood/BALF for supporting the diagnosis of SSc-ILD. However, while several other biomarkers have been proposed, the evidence of their independent value in diagnosis and prognosis is currently lacking and needs further investigation. PROSPERO REGISTRATION NUMBER: CRD42021282452.

Forced vital capacity trajectories in patients with idiopathic pulmonary fibrosis: a secondary analysis of a multicentre, prospective, observational cohort.

BACKGROUND: Idiopathic pulmonary fibrosis is a progressive fibrotic lung disease with a variable clinical trajectory. Decline in forced vital capacity (FVC) is the main indicator of progression; however, missingness prevents long-term analysis of patterns in lung function. We aimed to identify distinct clusters of lung function trajectory among patients with idiopathic pulmonary fibrosis using machine learning techniques. METHODS: We did a secondary analysis of longitudinal data on FVC collected from a cohort of patients with idiopathic pulmonary fibrosis from the PROFILE study; a multicentre, prospective, observational cohort study. We evaluated the imputation performance of conventional and machine learning techniques to impute missing data and then analysed the fully imputed dataset by unsupervised clustering using self-organising maps. We compared anthropometric features, genomic associations, serum biomarkers, and clinical outcomes between clusters. We also performed a replication of the analysis on data from a cohort of patients with idiopathic pulmonary fibrosis from an independent dataset, obtained from the Chicago Consortium. FINDINGS: 415 (71%) of 581 participants recruited into the PROFILE study were eligible for further analysis. An unsupervised machine learning algorithm had the lowest imputation error among tested methods, and self-organising maps identified four distinct clusters (1-4), which was confirmed by sensitivity analysis. Cluster 1 comprised 140 (34%) participants and was associated with a disease trajectory showing a linear decline in FVC over 3 years. Cluster 2 comprised 100 (24%) participants and was associated with a trajectory showing an initial improvement in FVC before subsequently decreasing. Cluster 3 comprised 113 (27%) participants and was associated with a trajectory showing an initial decline in FVC before subsequent stabilisation. Cluster 4 comprised 62 (15%) participants and was associated with a trajectory showing stable lung function. Median survival was shortest in cluster 1 (2·87 years [IQR 2·29-3·40]) and cluster 3 (2·23 years [1·75-3·84]), followed by cluster 2 (4·74 years [3·96-5·73]), and was longest in cluster 4 (5·56 years [5·18-6·62]). Baseline FEV1 to FVC ratio and concentrations of the biomarker SP-D were significantly higher in clusters 1 and 3. Similar lung function clusters with some shared anthropometric features were identified in the replication cohort. INTERPRETATION: Using a data-driven unsupervised approach, we identified four clusters of lung function trajectory with distinct clinical and biochemical features. Enriching or stratifying longitudinal spirometric data into clusters might optimise evaluation of intervention efficacy during clinical trials and patient management. FUNDING: National Institute for Health and Care Research, Medical Research Council, and GlaxoSmithKline.

Lysyl oxidase like 2 is increased in asthma and contributes to asthmatic airway remodelling.

BACKGROUND: Airway smooth muscle (ASM) cells are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyperresponsiveness and airway remodelling. The extracellular matrix (ECM) can influence tissue remodelling pathways; however, to date no study has investigated the effect of ASM ECM stiffness and cross-linking on the development of asthmatic airway remodelling. We hypothesised that transforming growth factor-ß (TGF-ß) activation by ASM cells is influenced by ECM in asthma and sought to investigate the mechanisms involved. METHODS: This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF-ß activation and expression of ECM cross-linking enzymes. Human bronchial biopsies from asthmatic and nonasthmatic donors were used to confirm lysyl oxidase like 2 (LOXL2) expression in ASM. A chronic ovalbumin (OVA) model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling. RESULTS: We found that asthmatic ASM cells activated more TGF-ß basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-ß activated. Our data demonstrate that the ECM cross-linking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGF-ß activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an OVA mouse model of asthma. CONCLUSION: These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma.

Evolution of lung pathology in lymphangioleiomyomatosis: associations with disease course and treatment response.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease with a variable clinical course. The lungs are infiltrated by nodules of LAM cells, stromal cells and inflammatory cells, causing lung cysts and respiratory failure. We used immunohistochemical markers in lung biopsy and transplant samples from a national cohort of women with LAM with linked clinical data to understand how LAM nodule cell populations changed with disease progression. Marker distribution was examined qualitatively by dual immunohistochemistry, and markers for LAM cells, fibroblasts, lymphatics, mast cells, proliferation, cathepsin K and mTOR pathway activity were quantitated in LAM nodules and compared with clinical features and prospective lung function loss. The LAM cell marker PNL2 was more extensively expressed in those with higher forced expiratory volume in one second (FEV1 ), higher diffusion in the lung for carbon monoxide (DLCO ) and less extensive disease involvement whilst the converse was true for the protease cathepsin K. Each percentage increase in cathepsin K reactivity was associated with a 0.65% decrease in FEV1 (95% CI -1.11 to -0.18) and a 0.50% decrease in DLCO (95% CI -0.96 to -0.05). Higher reactivity to the mTOR complex 1 activation marker, phospho-ribosomal protein S6, was associated with a better lung function response to rapamycin (p = 0.0001). We conclude that LAM nodules evolve with disease progression, with LAM cells becoming outnumbered by fibroblasts. Increasing cathepsin K expression is associated with more severe disease and lung function loss. Markers of mTOR activation predict the response to rapamycin, suggesting that more advanced LAM may be less mTOR responsive and treatments specifically targeted towards LAM associated fibroblasts may have roles as adjuncts to mTOR inhibition.

Loss of ELK1 has differential effects on age-dependent organ fibrosis.

ETS domain-containing protein-1 (ELK1) is a transcription factor important in regulating αvß6 integrin expression. αvß6 integrins activate the profibrotic cytokine Transforming Growth Factor ß1 (TGFß1) and are increased in the alveolar epithelium in idiopathic pulmonary fibrosis (IPF). IPF is a disease associated with aging and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where αvß6 mediated TGFß activation has been implicated. Here we identify that Elk1-knockout (Elk1-/0) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1-/0 aged mice demonstrated increased collagen deposition, in particular collagen 3α1, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1-/0 animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1 in human disease we exposed human lung epithelial cells and murine lung slices to cigarette smoke extract, which lead to reduced ELK1 expression andmay explain the loss of ELK1 in human disease. These data support a fundamental role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke.

Metabolic heat production by human and animal populations in cities.

Anthropogenic heating from building energy use, vehicle fuel consumption, and human metabolism is a key term in the urban energy budget equation. Heating from human metabolism, however, is often excluded from urban energy budgets because it is widely observed to be negligible. Few reports for low-latitude cities are available to support this observation, and no reports exist on the contribution of domestic animals to urban heat budgets. To provide a more comprehensive view of metabolic heating in cities, we quantified all terms of the anthropogenic heat budget at metropolitan scale for the world's 26 largest cities, using a top-down statistical approach. Results show that metabolic heat release from human populations in mid-latitude cities (e.g. London, Tokyo, New York) accounts for 4-8% of annual anthropogenic heating, compared to 10-45% in high-density tropical cities (e.g. Cairo, Dhaka, Kolkata). Heat release from animal populations amounts to <1% of anthropogenic heating in all cities. Heat flux density from human and animal metabolism combined is highest in Mumbai-the world's most densely populated megacity-at 6.5 W m-2, surpassing heat production by electricity use in buildings (5.8 W m-2) and fuel combustion in vehicles (3.9 W m-2). These findings, along with recent output from global climate models, suggest that in the world's largest and most crowded cities, heat emissions from human metabolism alone can force measurable change in mean annual temperature at regional scale.