Background Idiopathic pulmonary fibrosis (IPF) is characterised by damage to the epithelial layer, closely associated with the alveolar basement membrane (BM). We aimed to investigate how type IV collagen (COL4) in the BM changes with the progression of IPF. Methods COL4 synthesis (PRO-C4) was detected in blood by the nordicPRO-C4 biomarker in patients with IPF from the two prospective, multicentre, observational, longitudinal cohorts, pulmonary fibrosis biomarker (PFBIO) and prospective observation of fibrosis in the lung clinical endpoints (PROFILE). PRO-C4 trajectories over 12 months were compared between progressors and non-progressors by linear mixed effects regression models. Rate of change in PRO-C4 and lung function were compared by Bayesian bivariate longitudinal models. Cox proportional hazards models analysed baseline PRO-C4 and 3 years mortality. COL4 staining in IPF and non-IPF lungs was evaluated by immunohistochemistry. Results In PFBIO and PROFILE, 51/220 (23.2%) and 221/459 (48.1%) patients, respectively, had progressive disease at 12 months. Longitudinal PRO-C4 levels were higher in progressors versus non-progressors (average differences: PFBIO 21.5% (95% CI 3.4% to 42.9%, p=0.0184); PROFILE 10.9% (95% CI 0.8% to 22.1%; p=0.0340). Monthly rate of change in PRO-C4 was steeper in non-survivors versus survivors (mean difference up to 3.12% (95% CI 0.35% to 5.91%)) and was inversely correlated with the change in lung function. High baseline PRO-C4 was associated with increased mortality risk in PFBIO (HR 2.55 (95% CI 1.27 to 5.12), p=0.0083). COL4 staining was higher in IPF versus non-IPF lung but was less obvious in end-stage tissue. Conclusions High and increasing serological PRO-C4 levels were prognostic for progression in two independent IPF cohorts. This study suggests that COL4 synthesis assessed by PRO-C4 is a pathologically relevant biomarker of alveolar BM repair in IPF.

Background Pulmonary fibrosis (PF) is a shared characteristic of chronic interstitial lung diseases of mixed aetiology. Previous studies on PF highlight a pathogenic role for common and rare genetic variants. This study aimed to identify rare pathogenic variants that are enriched in distinct biological pathways and dysregulated gene expression. Methods Rare variants were identified using whole genome sequencing (WGS) from two independent PF cohorts, the PROFILE study and the Genomics England 100K (GE100KGP) cohort, with the gnomAD database as a reference. Four pathogenic variant categories were defined: loss of function variants, missense variants, protein altering variants, and protein truncating variants. Gene burden testing was performed for rare variants defined as having a minor allele frequency <0.1%. Overrepresentation analysis of gene ontology terms and gene concept network analysis were used to interpret functional pathways. Integration of publicly available transcriptomic datasets was performed using weighted gene co-expression network analysis of idiopathic pulmonary fibrosis (IPF) lung tissue compared with healthy controls. Results Burden testing was performed on 507 patients from the PROFILE study and 451 PF patients from GE100KGP cohort, compared with 76,156 control participants from the gnomAD database. Ninety genes containing significantly more pathogenic rare variants in cases than in controls were observed in both cohorts. Fifty-six genes included missense variants and 87 genes included protein altering variants. For missense variants, HMCN1 , encoding hemicentin-1, and RGPD1 , encoding a protein with a RanBD1 domain, were highly associated with PF in both PROFILE (p=5.70E-22 and p=4.48E-51, respectively) and GE100KGP cohorts (p=2.27E-24 and p=1.59E-36, respectively). 56 of 90 genes with significant burden were observed within modules correlated with disease in transcriptomic analysis, including HMCN1 and RGPD1 . Enriched functional categories from genetic and transcriptomic analyses included pathways involving extracellular matrix constituents, cell adhesion properties and microtubule organisation. Conclusions Rare pathogenic variant burden testing and weighted gene co-expression network analysis of transcriptomic data provided complementary evidence for pathways regulating cytoskeletal dynamics in PF pathogenesis. Functional validation of candidates could provide novel targets for intervention strategies. ### Competing Interest Statement LVW receives research funding from Orion Pharma, GSK, and Genentech, consulting fees from Galapagos, Boehringer Ingelheim, and GSK, and travel support from Genentech, has research collaboration with AstraZeneca, Nordic Bioscience, and Sysmex (OGT), and serves on advisory board for Galapagos. TMM, via his institution, has received industry-academic funding from Astra Zeneca and GlaxoSmithKline R and D; and consultancy or speaker fees from Abbvie, Amgen, Astra Zeneca, Bayer, Boehringer Ingelheim, BMS, CSL Behring, Endeavor, Fibrogen, Galapagos, Galecto, GlaxoSmithKline, IQVIA, Merck, Pliant, Pfizer, Qureight, Roche, Sanofi-Aventis, Structure Therapeutics, Trevi and Vicore. He is supported by an NIHR Clinician Scientist Fellowship (NIHR Ref: CS-2013-13-017) British Lung Foundation Chair in Respiratory Research (C17-3). RGJ received grant funding to institution from AstraZeneca, Biogen, Galecto, GlaxoSmithKline, Nordic Bioscience, RedX, and Pliant Therapeutics, consulting fees from AstraZeneca, Brainomix, Bristol Myers Squibb, Chiesi, Cohbar, Daewoong, GlaxoSmithKline, Veracyte, Resolution Therapeutics, and Pliant Therapeutics, payments or honoraria from Boehringer Ingelheim, Chiesi, Roche, PatientMPower, and AstraZeneca, payment for expert testimony from Pinsent Masons LLP, is the president of Action for Pulmonary Fibrosis and in the leadership of NuMedii.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal disease characterized by excessive extracellular matrix deposition within the lung. Recent advances in single-cell RNA sequencing have identified unique populations of epithelial cells lacking KRT5 while expressing KRT17; fibroblasts characterized by high collagen production, and the expression of CTHRC1; as well as a distinct subset of SPP1 positive macrophages. However, the precise interactions among these and potentially other cells that lead to the formation of fibrotic niche remain unclear. Methods: Using spatial transcriptomics and Hyperion imaging mass cytometry we compared the cellular populations in formalin fixed paraffin embedded fibrotic lesions (n=9 patients) with control lung (n=9), and using CellChat we investigated the cellular interactions. Results: Spatial transcriptomic analysis identified 180,067 cells which demonstrated three unique fibrotic niches enriched for KRT5-/KRT17+ epithelial cells, SPP1+ macrophages and collagen producing fibroblasts as well as a unique DCN+ expressing plasma B cell. Ligand receptor analysis inferred that most fibrotic cells interacted with ATII cells, with the exception of KRT5-/KRT17+ epithelial cells which interacted primarily with macrophages. Hyperion Mass Cytometry identified 55,979 cells, which identified similar cell populations clustered into two unique fibrotic niches: a stromal fibrotic niche characterized by CTHRC1+ fibroblasts interacting with fibrotic associated plasma cells, and a second niche containing KRT5-/KRT17+ cells and SPP1+ macrophages. This latter niche appeared likely to originate from the control bronchial niche with progressive loss of KRT5. Conclusion: These findings describe the characteristics of the fibrotic niche, support the hypothesis that basal cells can also contribute to the origin of the fibrotic epithelium and describe a novel fibrotic plasma cell population, shedding light on the complex cellular dynamics within fibrotic human lung tissues. ### Competing Interest Statement R.G.J. reports honoraria from Boehringer Ingelheim, Chiesi, Roche, PatientMPower, AstraZeneca, GSK, and consulting fees from AbbVie, AdALta, Apollo Therapeutics, Brainomix, Bristol Myers Squibb, Chiesi, Cohbar, GlaxoSmithKline Pliant, RedX. A.E.J is founder and shareholder of Alevin Therapeutics. R.H. reports consulting and speaker fees from Boehringer Ingelheim. S.R.J declares personal travel awards from Ferrer. L.V.W. claims consultancy fees from Galapagos, Boehringer Ingelheim and GSK. B.L., I.S., J.M., M.C.Z., E.L.-J., A.L.T., R.L.C., N.L., N.M., A.K.R., E.A.R., P.M.G., and A.U.W. declare no competing interests. This study was funded by a Medical Research Council Programme Grant (MR/V00235X/1) to RGJ. RGJ was funded by an NIHR Research Professorship (RP-2017-08-ST2-014). B.L. is a research fellow funded by Action for pulmonary fibrosis.

Integrin-mediated activation of the pro-fibrotic mediator transforming growth factor-β1 (TGF-β1), 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 hypothesised that galectin-3 potentiates TGF-β1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-β1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt (LPA)-induced integrin-mediated TGF-β1 activation. Surface plasmon resonance (SPR) analysis confirmed that galectin-3 binds to αv integrins, αvβ1, αvβ5 and αvβ6 and to the TGFβRII 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 β1 integrin was validated in vitro by co-immunoprecipitation in HLFs. Proximity ligation assays indicated galectin-3 and β1 integrin colocalize closely (≤40 nm) on the cell surface, that colocalization is increased by TGF-β1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-β1 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 HA secretion to a similar degree as TGF-β type I receptor inhibitor. These data suggest galectin-3 promotes TGF-β1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-β1 signaling cascade.

COVID-19 is a viral respiratory tract infection caused by SARS-CoV-2. Initial infection with the first wave strain and subsequent variants, up to and including the Delta variant were associated with considerable acute lung injury (ALI) and fibrotic sequelae. However, the Omicron variant has been associated with less ALI, although whether this is due to immunity or intrinsic differences in the biology of the virus is not known. It is important to understand the mechanisms of alveolar damage during the SARS-CoV-2 infection to prevent the development of ALI and any subsequent complications. The parent SARS-CoV-2 spike protein contains a cryptic RGD motif that is exposed upon binding to the ACE2 receptor, which is lost in Omicron/BA.2 and subsequent variants. We therefore wanted to understand the role of the RGD motif in viral cell entry and cellular pathology. Using a solid phase binding assay we show that the parent SARS-CoV-2 spike protein can bind the αvβ6 integrin in a concentration and EDTA dependent manner (Calver J, et al. Thorax 2021; 76:A22-A23). Using a pseudoviral assay we demonstrate that expression of the αvβ6 integrin can augment ACE2 mediated viral cell entry and using immunocytochemistry we demonstrate co-localisation of the spike protein, ACE2 and αvβ6. BA.2 Omicron spike protein doesn’t contain the RGD motif and we are studying whether this mutation has an effect on the cell internalisation. Our prior data have suggested people with conditions associated with high levels of the αvβ6 integrin were more likely to develop severe complications of COVID-19 supporting a role for RGD binding in the pathogenesis of COVID-19 ALI. These data might indicate that the reduction in ALI following the omicron variant is due in part to reduced ability to engage alveolar integrins.

Alveolar development and repair require tight spatiotemporal regulation of numerous signalling pathways that are influenced by chemical and mechanical stimuli. Mesenchymal cells play key roles in numerous developmental processes. Transforming growth factor-β (TGFβ) is essential for alveologenesis and lung repair, and the G protein α subunits G αq and G α11 (G αq/11 ) transmit mechanical and chemical signals to activate TGFβ in epithelial cells. To understand the role of mesenchymal G αq/11 in lung development, we generated constitutive ( Pdgfrb-Cre +/− ;Gnaq fl/fl ;Gna11 −/− ) and inducible ( Pdgfrb-Cre/ERT2 +/− ;Gnaq fl/fl ;Gna11 −/− ) mesenchymal G αq/11 deleted mice. Mice with constitutive G αq/11 gene deletion exhibited abnormal alveolar development, with suppressed myofibroblast differentiation, altered mesenchymal cell synthetic function, and reduced lung TGFβ2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal G αq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGFβ2 and elastin deposition. Cyclical mechanical stretch-induced TGFβ activation required G αq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGFβ2. These data highlight a previously undescribed mechanism of cyclical stretch-induced G αq/11 -dependent TGFβ2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis. Summary statement Mesenchymal cell G αq/11 signalling regulates myofibroblast function and stretch-mediated TGFβ2 signalling, which are important for alveologenesis and organ homeostasis. These mechanisms are relevant to both developmental and adult lung disease.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) with limited treatment options. Interleukin-33 (IL-33) is proposed to play a role in the development of IPF however the exclusive use of prophylactic dosing regimens means that the therapeutic benefit of targeting this cytokine in IPF is unclear. IL-33 expression was assessed in ILD lung sections and human lung fibroblasts (HLFs) by immunohistochemistry and gene/protein expression and responses of HLFs to IL-33 stimulation measured by qPCR. In vivo, the fibrotic potential of IL-33:ST2 signalling was assessed using a murine model of bleomycin (BLM)-induced pulmonary fibrosis and therapeutic dosing with an ST2-Fc fusion protein. Lung and bronchoalveolar lavage fluid were collected for measurement of inflammatory and fibrotic endpoints. Human precision-cut lung slices (PCLS) were stimulated with transforming growth factor-β (TGFβ) or IL-33 and fibrotic readouts assessed. IL-33 was expressed by fibrotic fibroblasts in situ and was increased by TGFβ treatment in vitro. IL-33 treatment of HLFs did not induce IL6, CXCL8, ACTA2 and COL1A1 mRNA expression with these cells found to lack the IL-33 receptor ST2. Similarly, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by PCLS. Despite having effects on inflammation suggestive of target engagement, therapeutic dosing with the ST2-Fc fusion protein failed to reduce BLM-induced fibrosis measured by hydroxyproline content or Ashcroft score. Together these findings suggest the IL-33:ST2 axis does not play a central fibrogenic role in the lungs with therapeutic blockade of this pathway unlikely to surpass the current standard of care for IPF.

Airway remodelling occurs in chronic asthma leading to increased airway smooth muscle (ASM) mass and extra-cellular matrix (ECM) deposition. Whilst extensively studied in murine airways; studies report only selected larger airways at one time point meaning the spatial distribution and resolution of remodelling are poorly understood. Here we use a new method allowing comprehensive assessment of the spatial and temporal changes in ASM, ECM and epithelium in large numbers of murine airways after allergen challenge. Using image processing to analyse 20-50 airways from a whole lung section revealed increases in ASM and ECM after allergen challenge were greater in small and large rather than intermediate airways. ASM predominantly accumulated adjacent to the basement membrane whereas ECM was distributed across the airway wall. Epithelial hyperplasia was most marked in small and intermediate airways. Post challenge, ASM changes resolved over seven days whereas ECM and epithelial changes persisted. The new method suggests large and small airways remodel differently and the long-term consequences of airway inflammation may depend more on ECM and epithelial changes than ASM. The method reduces the number of animals needed, reveals important spatial differences in remodelling and could set new analysis standards for murine asthma models.

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-β (TGF-β) 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-β 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-β basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-β 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-β 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.

Mental health and QoL are often interdependent. Depression, an increasingly prevalent condition, can have significant impact on QoL. In-order-to understand the extent of this effect, we aim to compare different PRO instruments available to measure QoL among patients with depression.

IPF is a progressive disease occurring in adults, limited to the lungs for which nintedanib and pirfenidone are approved drugs by the FDA. In this review, we aim to evaluate the incidence of hepatic adverse events (AEs) with pirfenidone and nintedanib.