Explore the vast range of titles available.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating condition leading to respiratory failure and >3000 deaths/year in the UK. Therapeutic approaches are limited and there is an urgent need to better understand mechanisms driving pulmonary fibrosis to support development of new anti-fibrotics. There is spatial and temporal heterogeneity of pathological changes within IPF tissue, which may correlate to changes in pathophysiological mediators of disease and clinical progression. Here, we utilise an intrapatient approach for target identification and validation by comparing histologically distinct regions of tissue from within the same IPF lung.MethodsMacroscopically ‘normal’, ‘intermediate’ and end-stage ‘fibrotic’ tissue was sampled under pathology guidance from the upper left lobe of explant IPF lungs (n=8) collected from patients undergoing lung transplantation. Histological assessment of the regions confirmed distinct pathology before samples were subject to unbiased proteomics assessment alongside aged-matched non-diseased unused donor (UD) lungs (n=10). Ingenuity Pathway Analysis (IPA) was performed to identify novel upstream regulators of fibrosis, from which inhibitory compounds targeting these regulators were selected and anti-fibrotic efficacy was assessed in IPF-derived precision cut slices (PCS).ResultsPrincipal component analysis showed IPF samples clustered based on region of tissue and became less similar to UD controls in correlation with disease severity. We identified markers/pathways significantly modulated in the intermediate region compared to other regions of the IPF lung. The intermediate region is the site of active tissue remodelling and therefore the region that needs to be targeted therapeutically to limit disease progression. Validation of PCS from these distinct regions showed that only intermediate-derived PCS increased collagen-1α1 secretion spontaneously throughout culture, suggesting enhanced disease progression in these PCS. A total of n=18 candidate compounds targeting upregulated markers/pathways modulated in the IPF intermediate region were assessed, of which n=10 exhibited robust anti-fibrotic effects in IPF-derived PCS.ConclusionWe have identified distinct patterns of protein expression that are modulated in line with changes in disease severity. Interrogation of protein heterogeneity identified novel targets that have been validated in the PCS system via inhibitors, confirming involvement in disease pathogenesis.Please refer to page A286 for declarations of interest related to this abstract.

IntroductionMucosal-associated invariant T (MAIT) cells are the most abundant unconventional T cells in the lung. We and others have recently described a potential role in promoting tissue repair. It is unknown what role MAIT cells play during sterile tissue damage, or the functional consequences of their tissue repair capabilities in the lung.MethodsWe assessed MAIT cell activation and function using a bleomycin-induced murine lung injury model. We used flow cytometry, spectral analysis (Cytek Aurora), histology, qPCR, immunoassays, bulk and single cell RNA sequencing and adoptive transfer in wild-type and MAIT cell-deficient Mr1-/- mice.Results In vivo bleomycin challenge caused recruitment and potent activation of pulmonary MAIT cells and induced their tissue repair program. Mr1-/- mice exhibited more severe weight loss and a more robust immune response following bleomycin challenge compared with their wild-type counterparts. MAIT-cell dependent early accumulation of pulmonary CD103+ type 1 dendritic cell (cDC1) was impaired in Mr1-/- mice, and MAIT cells were required for the early differentiation of CCR2+ Ly6Chi monocytes into cDC1. The early weight loss of Mr1-/- mice was rescued by adoptive transfer of bone marrow-derived dendritic cells (BMDCs) but this effect was lost when DNGR-1 was blocked in the mice. scSeq data revealed MAIT cells drive marked baseline differences in macrophage, monocyte and neutrophil expression of Pbx1, a major physiological and selective transcriptional mediator of apoptotic-cell-induced macrophage IL10 gene expression.ConclusionOverall, our data suggested that MAIT cells played a protective role against bleomycin-induced lung tissue damage by promoting monocyte differentiation into cDC1, which limited tissue damage via the DNGR-1 signal. These findings establish a novel mechanism by which MAIT cells function to reduce damage during sterile tissue injury and provide insight into the potential use of MAIT cells in pulmonary tissue repair. Furthermore they imply a homeostatic role for MAIT cells in promoting an anti-inflammatory which markedly alters the lungs response to subsequent tissue injury.Please refer to page A286 for declarations of interest related to this abstract.

Introduction and ObjectivesPulmonary Fibrosis (PF) is a fatal lung disease that accounts for 1% of UK deaths. Based on the literature1 and local clinical cohort observations, PF is overtly heritable in approximately 20% of cases. Low polygenic risk can counteract causal variant penetrance implied by positive family history across a range of diseases.2 Interactions between sex hormones, telomere length and pulmonary fibrosis are also reported.3 We hypothesised that penetrance of ‘probably damaging’ variants in genes associated with pulmonary fibrosis is moderated by both polygenic risk and sex hormone levels.MethodsWe derived 3,424 carriers of ‘probably damaging’ rare variants from exome sequencing in 8 genes for which carrier status was associated with PF prevalence amongst 82,795 male and 101,682 female UK Biobank participants. We created polygenic risk scores for telomere length in males and females separately. We generated percentage of bioavailable testosterone (bioT%) in males and free androgen index (FAI) in females from biomarkers measured at registration. We used median values to group rare variant carriers into four quadrants of risk. Within each of these quadrants we examined both PF prevalence and PF death within ten years of biomarker measurements.ResultsThere were 45 PF cases amongst our 3,424 ‘probably damaging’ rare variant carriers. Dichotomising our cohort by median telomere length polygenic risk score split this group by 18:25 for low:high risk (chi2 P=0.142). Dichotomising by bioT% for males and FAI for females split this group by 5:38 for low:high sex hormone levels (chi2 P<0.001). Combining these two categories into quadrants showed a marked difference in penetrance of PF for rare variant carriers. Mean age of death with PF also varied considerably by quadrant.Abstract S97 Figure 1Quadrants with differing percentage penetrance of pulmonary fibrosis for carriers of rare probably damaging variants, categorised by above or below median values of polygenic risk score (PRS) for short telomere and sex hormone levels in males and female combinedConclusionsPenetrance of ‘probably damaging’ rare variants in genes associated with PF prevalence appears to be affected by both polygenic risk for short telomeres and blood sex hormone levels. These results carry important implications for PF screening, prognostication and preventive treatment with sex hormones.ReferencesLiu Q, et al. Am J Respir Crit Care Med 2023.Mars N, et al. Am J Hum Genet 2022.Duckworth A, et al. medRxiv 2022.

BackgroundHuman Epididymis Protein 4 (HE4), a biomarker in ovarian cancer, has been found to be associated with fibrotic lung diseases. However, the effects of HE4 on lung fibrosis and inflammation remain unclear. The inflammation regulator A20 and its transcriptional suppressor DREAM have also been found to be dysregulated in lung diseases, including connective tissue disease-related interstitial lung disease.AimsWe aim to elucidate the effect of HE4 on inflammation-driven lung fibrosis and investigate its potential effect on the A20/DREAM-pathway.MethodsHE4 induction by hypoxia (6h 1% O2, 18h 21% O2) was assessed using ELISA and confirmed with Western Blot in bronchial epithelial (16HBE14o-), endothelial (HUVEC), and alveolar (A549) cell lines. The fibrogenic and inflammation-modulating effects of recombinant human HE4 (rHE4) (7.5 nM, 24h) on pulmonary fibroblasts (CCD-Lu11) was compared to those of TGF-beta1 (10 ng/mL, 24h). Type 1 collagen (COL1A1), alpha-SMA, vimentin, S100A4, IL1-beta, IL-6, IL-8 and TNF-alpha expression was assessed at mRNA level using qRT-PCR. Collagen deposition was further investigated using Sirius RED staining and inflammatory marker secretion was confirmed with ELISA. The effects of rHE4 on DREAM and A20 transcription were assessed by qRT-PCR and A20 expression was confirmed by Western Blot.ResultsHuman bronchial epithelial cells showed significantly higher HE4 levels at baseline compared to endothelial and alveolar cells. Exposure to hypoxia induced HE4 secretion in 16HBE14o-, HUVEC and A549 cells. rHE4 significantly increased mRNA levels of COL1A1 and vimentin, but not alpha-SMA in pulmonary fibroblasts. Similar to TGF-beta1 exposure, collagen deposition was significantly increased at 24h following rHE4 exposure. Expression and secretion of inflammatory mediators (IL-6, IL-8) were significantly induced in rHE4-exposed lung fibroblasts. TGF-beta1 increased A20 mRNA expression, but in response to rHE4 A20 remained at control levels and A20 protein levels were markedly decreased following rHE4 exposure. DREAM mRNA was significantly increased in response to TGF-beta1 and rHE4.Abstract S99 Figure 1Pro-fibrotic and pro-inflammatory effects of HE4. (A) HE4 is induced in pulmonary cells by hypoxia (H:6h 1% O2, 18h 21% O2) compared to normoxia (N). (B) Exposure of CCD-Lu11 pulmonary fibroblasts to rHE4 (7.5 nM) increases collagen deposition and (C) expression of pro-inflammatory cytokines. All data n=3, mean±SEM, One-Way ANOVA , ***p<0.001, **p<0.01, *p<0.05 vs Control/24hConclusionHE4 is induced by hypoxia and secreted by respiratory cells to exert a fibrogenic and inflammation-modulating effect on fibroblasts, possibly through DREAM-dependent suppression of A20. HE4 and its downstream targets could therefore prove promising therapeutic targets in fibrotic lung diseases.

IntroductionFibrosis is involved in up to one-third of deaths globally and can affect many organ systems. Individuals with fibrosis in one organ may be more likely to develop fibrosis in another. Genome-wide association studies (GWAS) have shown that genetics plays a key role in fibrotic diseases, including pulmonary fibrosis. Identifying genetic variants associated with a disease increases our understanding and aids in the development of novel treatments. We hypothesised that there could be genetic variants associated with fibrosis across multiple organs.ObjectiveTo identify fibrosis associated genetic variants across different organ systems, and investigate the correlating genetic architecture between respiratory fibrosis and fibrosis in other organs.MethodsWe used unrelated individuals of European ancestry in UK Biobank. Fibrotic diseases, as defined by a published consensus list, were combined across 13 organs or systems (Respiratory, Liver, Bile, Cardiomyopathy, Intestinal-Pancreatic, Integumentary, Skeletal, Systemic, Reproductive, Urinary, Blood-vessel, Atherosclerosis and Diabetes). Individuals with at least one fibrotic condition recorded in their hospital or mortality records were defined as cases (all others defined as controls). We then performed a GWAS to test the association between each genetic variant across the genome with each organ fibrosis separately. We selected genetic variants that reached genome-wide significance (p<5x10-8) as associated with respiratory fibrosis. We calculated genome-wide genetic correlation between fibrotic diseases using LD score regression.ResultsThere were 2,600 cases and 415,455 controls included in our respiratory fibrosis GWAS. We identified three genetic signals associated with respiratory fibrosis. These included signals located near the MUC5B and TERT genes that have been previously reported as associated with idiopathic pulmonary fibrosis and a potential novel genetic association near SYNPO2 (rs6844137, OR=1.18, p=4.89x10-8). None of these variants reached genome-wide significance in the GWAS of fibrosis in other organs. However, there was genetic correlation observed between respiratory fibrosis and fibrosis in other organs. Of the 12 organs investigated, six had a genetic correlation (r2>0.4) with respiratory fibrosis, with skeletal (r2=0.79) and urinary (r2=0.63) fibrosis showing the strongest correlations.ConclusionThese results suggest there may be biological mechanisms involved in developing respiratory fibrosis that are shared with fibrotic diseases in other organs.Please refer to page A286 for declarations of interest related to this abstract.

BackgroundNormal lung repair involves tightly regulated epithelial-mesenchymal crosstalk and extracellular matrix (ECM) generation, and dysregulation of repair results in conditions such as pulmonary fibrosis and emphysema. Mesenchymal Gαq/11 knockout causes emphysema with altered ECM composition,1 however the role of the ECM in driving this phenotype is unknown.AimUnderstand how Gαq/11 -/- fibroblast-deposited ECM influences epithelial repair processes.MethodsWild-type (WT) and Gnaq-/-;Gna11-/- (Gαq/11 -/-) murine embryonic fibroblasts (MEFs), and E10 mouse lung epithelial cells were cultured on ECM generated by WT or Gαq/11 -/- MEFs. Wound healing was assessed using a scratch wound assay (injury) and by seeding cells around ‘fences’ (non-injury model). Cell viability was quantified using MTT assays. Transforming growth factor-β (TGFβ) signalling was assessed using transformed mink lung reporter cells (TMLC) and Pai-1 mRNA expression.ECM was isolated in soluble and insoluble fractions in RIPA and urea buffers, respectively.ResultsEpithelial cell injury-related healing was attenuated (13.8% vs 26.3% 8-hour wound healing, p=0.05), and noninjury defect closure was slower (5.6% vs 15.7%, p=0.02) on Gαq/11 -/- MEF-generated ECM compared with WT ECM. This was not due to cell death, as E10 and MEF viability was similar on both ECM types.TGFβ signalling was lower when TMLCs were cultured on Gαq/11 -/--generated ECM compared with WT ECM (0.44 relative TMLC luciferase activity (RLA), p<0.05). While total protein concentrations from Gαq/11 -/--generated and WT ECM were comparable, these data indicate reduced TGFβ content of ECM deposited by Gαq/11 -/- MEFs. This was supported by reduced Pai-1 mRNA expression in epithelial cells cultured on Gαq/11 -/--generated ECM compared with WT ECM.WT MEFs released less active TGFβ into the culture media when grown on Gαq/11 -/- ECM compared with WT ECM (0.53 RLA, p<0.05). Conversely, Gαq/11 -/- MEFs increased active TGFβ release on WT ECM compared with Gαq/11 -/-ECM (1.8 RLA, p<0.05). Therefore, the ECM may also influence lung repair via mesenchymal TGFβ signalling.ConclusionFibroblast Gαq/11 signalling controls epithelial repair via modulation of ECM properties, a key aspect of which is TGFβ content. Further evaluation of these mechanisms may identify therapeutic methods of manipulating lung repair.ReferenceGoodwin, et al. Development 2023:150(9):dev201046.Please refer to page A286 for declarations of interest related to this abstract.

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation.

Computational modeling is a tool for exploring both normal electrical propagation in healthy hearts and cardiac arrhythmias in patients. While numerous human ventricular cell models exist, the ten Tusscher (TT2) model is one of the most used for simulating ventricular arrhythmia. Recently, the Tomek model has been proposed, offering improved accuracy by better reproducing key depolarization, repolarization, and calcium dynamics in healthy ventricular cardiomyocytes. However, a quantitatively comprehensive comparison of these models at the single‐cell, tissue, and organ levels has not been conducted. This study systematically compared the TT2 and Tomek models by evaluating electrophysiological parameters and reentrant properties in 0‐dimensional(0D), 1‐dimensional(1D), 2‐dimensional (2D), and 3‐dimensional (3D) simulations. Additionally, the effects of ion currents modifications to simulate ischemic scar‐related tissue were analyzed. These results reveal that although the TT2 and Tomek models demonstrate distinct 0D, 1D, and 2D characteristics, their 3D reentrant properties—specifically in terms of reentry locations and critical conduction channels—are highly comparable. Therefore, both models are suitable for simulating post‐infarct ventricular tachycardia (VT), as their shared 3D features effectively capture the essential mechanisms underlying this arrhythmia.

It has been demonstrated that scar tissue and fibrosis may increase the likelihood of developing malignancies. Specifically, scar tissue has been linked to the occurrence and progression of lung cancer (LC), though the precise mechanisms necessitate further research for explanation. Lung scarring can stem from various causes, with carcinogenesis on scarring lesions in pulmonary tuberculosis (PTB) being the most frequent (accounting for approximately 75% of cases). Notably, having previously cured, PTB is the second most common risk factor for LC after smoking, with approximately 3% of PTB patients experiencing LC as a secondary condition. This essay will delve into the mechanisms, treatment, and prognosis of tuberculosis scar carcinoma (TSC).

The formation of severe scars still represents the result of the closure process of extended and deep skin wounds. To address this issue, different bioengineered skin substitutes have been developed but a general consensus regarding their effectiveness has not been achieved yet. It will be shown that bioengineered skin substitutes, although representing a valid alternative to autografting, induce skin cells in repairing the wound rather than guiding a regeneration process. Repaired skin differs from regenerated skin, showing high contracture, loss of sensitivity, impaired pigmentation and absence of cutaneous adnexa (i.e., hair follicles and sweat glands). This leads to significant mobility and aesthetic concerns, making the development of more effective bioengineered skin models a current need. The objective of this review is to determine the limitations of either commercially available or investigational bioengineered skin substitutes and how advanced skin tissue engineering strategies can be improved in order to completely restore skin functions after severe wounds.