Extracellular vesicles (EVs) are lipid-membrane bound vesicles that can be beneficial or detrimental depending on the content they carry. As epithelial cells are the first line of defense against harmful particles, this work explored the role of bronchial epithelial cell-derived EVs (CepEVs) in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). RNA sequencing of macrophages stimulated with CepEVs revealed the upregulation of various inflammasome-related genes, alongside significant IL-1b and IL-18 release, which could be attenuated with caspase-1 or NLRP3 inhibition. The proteome of CepEVs was also assessed, which highlighted a significant reduction in antibacterial proteins compared to healthy EVs (HepEVs). When functionally assessed in NTHi infection of THP-1 cells, pre-incubation with HepEVs stimulated NTHi clearance and reduced pro-inflammatory cytokine release by macrophages, which was reduced in CepEV-stimulated cells. This study shows for the first time that CepEVs are able to both prime and activate the inflammasome in healthy macrophages, and highlights EV-induced inflammasome inhibition as a potential therapeutic target for the dysregulated inflammation seen in COPD. Alongside the inflammasome, we were also able to show that CepEVs are deficient for multiple antibacterial proteins, and that one or more of these proteins are essential in mounting an immune response against NTHi in macrophages. This finding contributes to a potential therapeutic pipeline through the supplementation of the depleted antibacterial proteins in CepEVs, allowing for efficient bacterial clearance and reduced consequential inflammatory burden. CepEV co-incubation resulted in a persistent state of inflammation and infection. Both sets of findings contribute to the overall knowledge of COPD pathogenesis, and highlight epithelial EVs as key players in the propagation of inflammation and susceptibility to infection.

In 2024-2025, the Cystic Fibrosis Foundation (US) and Cystic Fibrosis Trust (UK) hosted an International CFRD Consortium round-table webinar series for basic science, translational, and clinical researchers with the goal of sharpening mechanistic understanding of CFRD pathogenesis and prioritizing therapeutic development. This review summarizes the research priorities identified in the International CFRD Consortium, including (i) further investigation into the role of pancreatic fibrosis, vascular abnormalities, and α-cell dysfunction in the development of CFRD; (ii) the creation and refinement of novel animal and human cell- and tissue-based models to understand the complex interplay of exocrine and endocrine cells in the CF pancreas; (iii) development and validation of circulating and imaging biomarkers, together with dynamic glucose testing to explore β-cell function and kinetics in people with CF across the dysglycemia spectrum; and (iv) prospective clinical studies to guide CFRD treatment options and investigate the changing landscape of aging, increasing prevalence of obesity and diabetes and their complications in the era of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. Collectively, these priorities aim to accelerate transition from mechanism to intervention and expand evidence-based care for people with CF at risk of, or living with, CFRD.

RATIONALE:Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease driven by nodules containing TSC2 -/- "LAM cells" and recruited LAM-associated fibroblasts. Although rapamycin reduces lung function loss, some patients continue to decline, meaning additional therapies are needed. OBJECTIVES:To investigate how the LAM nodule environment affects LAM cell proliferation and the response to rapamycin. METHODS:Proteins altered in advanced LAM were identified using shotgun proteomics and immunohistochemistry in tissue from closely phenotyped patients. Genes associated with rapamycin insensitivity on LAM derived extracellular matrix were identified by RNA sequencing and validated using pharmacological inhibitors. RESULTS:More advanced disease was associated a greater decline in forced expiratory volume in 1 s when treated with rapamycin (p=0.005). In advanced LAM, using proteomics analysis, an upregulation of protein clusters comprising extracellular matrix, glucose metabolism and the actin cytoskeleton was identified. RNA sequencing and immunohistochemistry confirmed expression of collagens I and VI in LAM-associated fibroblasts and LAM nodules, and increased markers of collagen turnover in patient serum (p=0.0048). Growth of LAM patient-derived cells in vitro was faster on LAM associated fibroblast-derived extracellular matrix (p<0.0001) and incompletely suppressed by rapamycin. RNA sequencing identified upregulation of pathways driving cell cycle control, transcription and metabolism by extracellular matrix. Tractable, pro-proliferative, upregulated genes included CDK7, GAS6, PLAUR and PLAU. Inhibitors of these pathways reduced LAM cell proliferation and enhanced the antiproliferative effect of rapamycin. CONCLUSIONS:Extracellular matrix deposition upregulates the expression of genes which may blunt the response to rapamycin, but offer additional therapeutic opportunities for patients with established LAM.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is an irreversible lung disease characterized by chronic inflammation, emphysema and airways obstruction caused by airway abnormalities and mucus accumulation. Tanimilast is a selective inhaled phosphodiesterase-4 inhibitor (PDE4i) in advanced clinical phase for COPD. Tanimilast has shown an anti-inflammatory activity in preclinical animal models and in human clinical trials, but no data are available on its effect on mucus overproduction in preclinical assays. Here, we tested the efficacy of tanimilast in modulating mucus hyperproduction, inflammation and fibrosis in COPD patient-derived Precision Cut Lung Slices (PCLS). METHODS: PCLS were prepared from 4 explanted COPD lungs collected from patients undergoing lung transplantation and cultured in a 96 well format at 37 °C with 5% CO2. Eight PCLS per group were rested for 48hrs and after, they were cultured in the presence of ALK5i (10 uM) or budesonide (10 uM) as experimental controls, and tanimilast (0.1uM). PCLS culture media, including all inhibitors, were refreshed and harvested at 24hrs intervals from 48hrs and harvested at 144hrs to assess metabolic viability. Secretion of markers of fibrosis (Col1a1), inflammation (MCP1) and mucus hyperproduction (Muc5AC and Muc5B) were analysed at 144h by ELISA quantification. RESULTS: The tested concentration of tanimilast showed no signs of toxicity according to Resazurin analysis. In the PCLS treated with tanimilast 0.1 µM, a significant reduction in MUC5AC (-50.6%) and MUC5B (-31%) secretion was detected relative to untreated slices. Moreover, an inhibition of collagen (Col1a1) (-45.8%) and MCP-1 (-44%) secretion were also detected. CONCLUSION: The obtained results confirm the anti-inflammatory effect of tanimilast in the context of COPD and underline its ability to affect mucus hyperproduction in PCLS derived from COPD patients. This data further consolidates the potential therapeutic role of tanimilast in the treatment of COPD.

Chronic liver injury characterized by unresolved hepatitis leads to fibrosis, potentially progressing to cirrhosis and hepatocellular carcinoma. Effective treatments for halting or reversing liver fibrosis are currently lacking. This study investigates the potential of HDAC6 as a therapeutic target in liver fibrosis. We synthesized two selective HDAC6 inhibitors, DR‐3 and FDR2, and assessed their effects on hepatic stellate cell (HSC) activation and liver fibrosis using human precision cut liver slices (hPCLS). Molecular docking, deacetylation inhibition assays, and various cellular assays were employed to evaluate the specificity and anti‐fibrotic efficacy of these inhibitors. DR‐3 and FDR2 demonstrated high selectivity for HDAC6 over HDAC1, significantly inhibiting HSC activation markers and fibrogenic gene expression. Both inhibitors increased acetylation of α‐tubulin and suppressed TGF‐β1‐induced SMAD signaling in HSCs. In human precision cut liver slices (hPCLS), DR‐3 and FDR2 reduced fibrogenic protein levels and collagen deposition. The selective inhibition of HDAC6 by DR‐3 and FDR2 effectively reduces HSC activation and fibrogenesis in liver models, supporting further investigation of HDAC6 inhibitors as potential anti‐fibrotic therapies.

BACKGROUND:Disease modeling is vital for our understanding of disease mechanisms and for developing new therapeutic strategies. Accurately modeling the intact tumor microenvironment (TME) is increasingly recognized as essential for gaining insights into cancer biology and therapeutic response. Preclinical mouse models have provided utility for studying the evolving TME, but these models are costly and can lead to animal suffering and the discontinuation of drug investigations. To address these limitations, particularly in hepatocellular carcinoma (HCC), we have developed an ex vivo model using tumor precision-cut slices (TPCS) derived from orthotopic liver tumors. METHODS:Murine HCC tumors were generated via intrahepatic injection of Hep-53.4 cells, providing a source of tumor tissue for TPCS generation. Subsequent scaling to a 96-well format and modification to include a secreted luciferase enabled longitudinal ex vivo screening of 26 drugs applied at 2 doses over an 8-day period, using just 5 tumors. One drug identified in the screen, salinomycin, was then validated in vivo via intraperitoneal injection of mice with orthotopic liver tumors. RESULTS:Histological characterization determined that TPCS maintain the architecture, cellular complexity, and drug responsiveness of the original HCC-TME under simplified culture conditions that preserve viability and metabolic activity. In addition to typical HCC therapies, sorafenib and anti-PD1 immunotherapy, the screen identified 2 drugs as potent anticancer agents capable of impacting the viability of TPCS: salinomycin and rottlerin. Salinomycin was further validated in vivo, significantly reducing tumor burden without evidence of toxicity. CONCLUSIONS:We present a 3Rs (Reduction, Refinement, Replacement) approach for studying HCC biology and performing 96-well-scale drug screening within an intact, metabolically active TME, offering a more ethical and effective platform for drug discovery.

Cellular senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The mechanisms of senescence in the bronchial epithelium, however, remain largely unknown. In this study, we aimed to elucidate whether cellular senescence in COPD epithelial cells contributes to the pathogenesis of the disease and investigated the potential molecular mechanisms involved. Single-cell RNA sequencing was performed on well-differentiated primary bronchial epithelial cells from patients with COPD and healthy subjects. We evaluated the abundance and distribution of senescence markers in key epithelial differentiated subtypes and senescence-associated secretory phenotype involved in airway epithelial dysfunction. The effects of IFN-pathway inhibitors on cellular senescence were also investigated. There was increased expression of cellular senescence genes in the COPD cohort, which was predominantly in basal and club cells. Enhanced expression of cellular senescence markers, p16 and p21, was observed in COPD cultures, which was histologically confirmed in the lung tissue of patients with COPD. There was also a notable increase in IFN-β and IFN-γ. Senescence-associated secretory phenotype productions were increased in COPD and were attenuated by JAK-STAT or cGAS-STING pathway inhibitors (baricitinib or C-176). These inhibitors also effectively suppressed expression of senescence markers. COPD bronchial epithelium displays a senescence-driven phenotype which is mediated by Type I/II IFNs. Inhibition of JAK-STAT or STING-cGAS IFN pathways may represent targets to alleviate cellular senescence and chronic inflammation in COPD.

Disease modelling is vital for improving knowledge of disease mechanisms and for development of new therapeutic molecules and strategies. Modelling the intact living tumour microenvironment (TME) is increasingly considered to be vital not only for gaining a better understanding of the biology of cancer but for examining the efficacy of novel oncology drugs. To date, pre-clinical mouse models of cancer have represented the mainstay methodology for studying the evolving TME and for determining the effects of potential therapeutic molecules on tumour evolution and growth. Regarding drug screening, in vivo mouse models are expensive, require the use of large cohorts of mice and involve the administration of drugs with unknown toxicities to animals which often result in adverse effects that can cause animal suffering and the discontinuation of drug investigations. Hepatocellular carcinoma (HCC) is a primary cancer of the liver for which there is an urgent need for improved systemic treatments due to the disease usually being diagnosed at an advanced stage and current treatments having limited efficacy. To provide a practical solution to the screening of drugs for their likely efficacy in HCC we have developed an ex-vivo model in which orthotopic tumours are excised from the liver and subsequently processed to generate precision-cut tumour slices (PCTS) which provide an intact culture model of the HCC-TME. We describe simplified culture conditions that maintain the viability and metabolic activity of live PCTS which maintain the architecture, cellular complexity, drug sensitivity and responsiveness to immunotherapy of the original tumour. Importantly, we show that HCC derived PCTS can be miniaturised to 96-well scale and modified to express soluble luciferase, which in combination enabled non-destructive screening of a library of 26 drugs at two doses using just 5 tumours as the source for PCTS. This screen identified two small molecules, salinomycin and rottlerin, that have potent anti-tumour activities in HCC-PCTS and subsequently validated salinomycin as effective in vivo . In summary, we report a 3Rs (reduction, refinement and replacement) solution for study of HCC biology and for 96-well-scale screening of potential therapeutic agents in the context of an intact, metabolically active TME. ### Competing Interest Statement Competing interests: L.A.B, F.O, D.A.M are directors of Fibrofind limited. L.A.B, H.P, J.L, F.O and D.A.M are shareholders in Fibrofind limited. * ALD : Alcohol-related liver disease AST : Aspartate aminotransferase H&E : haematoxylin and eosin HCC : hepatocellular carcinoma IMC : Imaging Mass Cytometry LDH : lactate dehydrogenase MASLD : metabolic dysfunction-associated steatotic liver disease MASH : metabolic associated steatohepatitis PCLS : Precision cut liver slices PCTS : precision cut tumour slices RLTD : relative level of transcriptional difference TUNEL : Terminal deoxynucleotidyl transferase-mediated dUTP nick end TME : tumour microenvironment TKIs : tyrosine kinase inhibitors WT : wild type

Healthy repair of the alveoli requires alveolar stem cells to differentiate into cells designed for gas exchange. In chronic lung fibrotic disease like idiopathic pulmonary fibrosis (IPF), alveolar epithelial cells regenerate abnormally. The cause of this is unknown but its highly cellular, inflamed and structurally altered regenerating niche is likely to be relevant. Here, in unique sets of human lung tissues capturing advancing fibrosis, and with a 33-plex single cell imaging mass cytometry (IMC), we provide a high resolution and comprehensive temporo-spatial cell atlas of the regenerating alveolar niches. Using a suite of mathematical tools, we expose an organized immune network and identify CD206hi alveolar macrophages as a central immune cell in the immune-alveolar epithelial interactome. A spatially-directed receptor-ligand analysis offers an in-silico mechanism by which these macrophages influenced alveolar regeneration. Our study unravels a complex cellular environment and identifies key interactions that influence alveolar regeneration in a fibrotic lung. ### Competing Interest Statement Andrew Fisher declares the following: Grant Award to Newcastle University from Mallinckrodt Pharmaceuticals, Chiesi and Eurofins/TGI Consultancy via Newcastle University with Fibrofind, Mallinckrodt and Sanofi James Shaw declares the following: Scientific Advisory Board for Mogrify (consultancy fee paid to Newcastle University). ### Funding Statement The study is funded by combination of grants from MRC UKRMP Grant MR/S020918/1; NIHR Oxford Biomedical Research Centre grant; University of Oxford Medical Science Division and Newcastle University Flow Core Facility. LPH is supported by MRC (grant CFR01480) and the NIHR Oxford Biomedical Research Centre. AJF is supported by the National Institute for Health and Care Research (NIHR) Blood and Transplant Research Unit in Organ Donation and Transplantation (NIHR203332), a partnership between NHS Blood and Transplant, University of Cambridge and Newcastle University. The views expressed are those of the author(s) and not necessarily those of the NIHR, NHS Blood and Transplant or the Department of Health and Social Care. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All patients provided informed consent for use of their tissue via the Cellular and Molecular Mechanisms in Chronic Lung Diseases (EXPLANT) study which was approved by the NHS Research Ethics Service (11/NE/0291) and was sponsored by Newcastle Upon Tyne Hospitals NHS Foundation Trust ( R&D ref 5885). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes

Cellular senescence is not only associated with ageing but also impacts physiological and pathological processes, such as embryonic development and wound healing. Factors secreted by senescent cells affect their microenvironment and can induce spreading of senescence locally. Acute severe liver disease is associated with hepatocyte senescence and frequently progresses to multi-organ failure. Why the latter occurs is poorly understood. Here we demonstrate senescence development in extrahepatic organs and associated organ dysfunction in response to liver senescence using liver injury models and genetic models of hepatocyte-specific senescence. In patients with severe acute liver failure, we show that the extent of hepatocellular senescence predicts disease outcome, the need for liver transplantation and the occurrence of extrahepatic organ failure. We identify the TGFβ pathway as a critical mediator of systemic spread of senescence and demonstrate that TGFβ inhibition in vivo blocks senescence transmission to other organs, preventing liver senescence induced renal dysfunction. Our results highlight the systemic consequences of organ-specific senescence, which, independent of ageing, contributes to multi-organ dysfunction.

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.

Background Idiopathic 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. Methods Macroscopically '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). Results Principal 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. Conclusion We 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.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9 + TREM2 + macrophages that express SPP1 , GPNMB , FABP5 , and CD63 . In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-β1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-β1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-β1–induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-β1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

BACKGROUND:The association between interleukin-1β (IL-1β) concentrations during ex vivo lung perfusion (EVLP) with donor organ quality and post-lung transplant outcome has been demonstrated in several studies. The mechanism underlying IL-1β-mediated donor lung injury was investigated using a paired single-lung EVLP model. METHODS:Human lung pairs were dissected into individual lungs and perfused on identical separate EVLP circuits, with one lung from each pair receiving a bolus of IL-1β. Fluorescently labeled human neutrophils isolated from a healthy volunteer were infused into both circuits and quantified in perfusate at regular timepoints. Perfusates and tissues were subsequently analyzed, with perfusates also used in functional assays. RESULTS:Neutrophil numbers were significantly lower in perfusate samples collected from the IL-1β-stimulated lungs consistent with increased neutrophil adhesion ( P = 0.042). Stimulated lungs gained significantly more weight than controls ( P = 0.046), which correlated with soluble intercellular adhesion molecule-1 (R 2 = 0.71, P = 0.0043) and von-Willebrand factor (R 2 = 0.39, P = 0.040) in perfusate. RNA expression patterns for inflammatory genes were differentially regulated via IL-1β. Blockade of IL-1β significantly reduced neutrophil adhesion in vitro ( P = 0.025). CONCLUSION:These data illustrate the proinflammatory functions of IL-1β in the context of EVLP, suggesting this pathway may be susceptible to therapeutic modulation before transplantation.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

miRNAs are 22 nucleotides long and belong to a class of noncoding RNAs that plays an important role in regulating gene expression at a post-transcriptional level. Studies show aberrant levels of miRNAs to be associated with profibrotic processes in idiopathic pulmonary fibrosis (IPF). However, most of these studies used whole IPF tissue or in vitro monocultures in which fibrosis was artificially induced. The current study used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, isolated miRNAs, and compared their expression levels with those found in whole IPF lung tissue and/or in vitro cultured fibroblast from IPF or normal lungs. Sequencing libraries were generated, and data generated were bioinformatically analyzed. A total of 18 miRNAs were significantly overexpressed in FF tissue when compared with whole IPF tissue. Of those, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impacted several signaling pathways. The miRNA composition of FF is both overlapping and distinct from that of whole IPF tissue or cultured IPF fibroblasts and highlights the importance of characterizing FF biology as a phenotyp-ically and functionally discrete tissue microenvironment. (Am J Pathol 2023, 193: 417-429; https:// doi.org/10.1016/j.ajpath.2022.12.015)

Introduction: Heparin sulphate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signalling, modulated by sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME. Methods: In liver biopsies from 60 patients with HCC, expression and localization of SULF2 were analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids, and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, β-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68, and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cell phenotype using fluorescent activated cell sorting. Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type 2 diabetes, SULF2 was more commonly expressed in cancer-associated fibroblasts (CAFs) (52%) and independently associated with shorter survival (7.2 vs. 29.2 months, p = 0.003). Stromal SULF2 modulated glypican-3/β-catenin signalling in vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFβ1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFβ1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRβ/STAT3 signalling was evident in stromal cells, with the release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In human PBMCs, SULF2 preferentially induced the migration of macrophage precursors (monocytes), inducing a phenotypic change consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2-induced paracrine activation of the IKKβ/NF-κB pathway, tumour cell proliferation, invasion, and sorafenib resistance. Conclusion: SULF2 derived from CAFs modulates glypican-3/β-catenin signalling but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKβ/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC.

BackgroundRespiratory viral infections are closely associated with COPD exacerbations, hospitalisations, and significant morbidity and mortality. The consequences of the persisting inflammation and differentiation status in virus associated severe disease is not fully understood. The aim of this study was to evaluate barrier function, cellular architecture, the inflammatory response in severe COPD bronchial epithelium to human rhinovirus (HRV) induced pathological changes and innate immune responses.MethodsWell-differentiated primary bronchial epithelial cells (WD-PBECs) derived from severe COPD patients and age-matched healthy controls were cultured in the air-liquid interface (ALI) model. The differentiation phenotype, epithelial barrier integrity, pathological response and cytokine secreting profile of these cultures before and after HRV infection were investigated.ResultsWD-PBECs derived from severe COPD patients showed aberrant epithelium differentiation with a decreased proportion of ciliated cells but increased numbers of club cells and goblet cells compared with healthy controls. Tight junction integrity was compromised in both cultures following HRV infection, with heightened disruptions in COPD cultures. HRV induced increased epithelial cell sloughing, apoptosis and mucus hypersecretion in COPD cultures compared with healthy controls. A Th1/Th2 imbalance and a strong interferon and pro-inflammatory cytokine response was also observed in COPD cultures, characterized by increased levels of IFNγ, IFNβ, IP-10, IL-10 and decreased TSLP and IL-13 cytokine levels prior to HRV infection. Significantly enhanced basolateral secretion of eotaxin 3, IL-6, IL-8, GM-CSF were also observed in both mock and HRV infected COPD cultures compared with corresponding healthy controls. In response to HRV infection, all cultures displayed elevated levels of IFNλ1 (IL-29), IP-10 and TNFα compared with mock infected cultures. Interestingly, HRV infection dramatically reduced IFNλ levels in COPD cultures compared with healthy subjects.ConclusionAn altered differentiation phenotype and cytokine response as seen in severe COPD WD-PBECs may contribute to increased disease susceptibility and an enhanced inflammatory response to HRV infection.

The Interleukin 1 family remains a topic of intense research and clinical investigation. Currently comprising of 11 cytokines and 11 receptors, it is clear that we are only just beginning to elucidate the broad range of physiological and pathological processes wherein IL-1 family members play critical roles. In this chapter we seek to summarize the literature with regard to the established and emerging roles of the IL-1 family in the lung and discuss their potential as therapeutic targets in respiratory diseases.

Transplantation is an effective treatment for end-stage lung disease but donor organ shortage is a major problem. Ex-vivo lung perfusion (EVLP) of marginal organs enables functional assessment under normothermic conditions to facilitate clinical decision-making around utilisation, but the molecular processes occurring during EVLP, and how they differ between more or less viable lungs, remains to be determined. Here we used RNA sequencing to delineate changes in gene expression occurring in n=10 donor lungs undergoing EVLP, comparing lungs that were deemed transplantable (n=6) to those deemed unusable (n=4). We found that lungs deemed suitable for transplantation following EVLP had reduced induction of a number of innate immune pathways during EVLP, but a greater increase in genes involved in oxidative phosphorylation, a critical ATP-degenerating pathway. Furthermore, SCGB1A1 , a gene encoding an anti-inflammatory secretoglobin CC10, and other club cell genes were significantly increased in transplantable lungs following perfusion, whilst CHIT-1 was decreased. Using a larger validation cohort (n=18), we confirmed that the ratio of CHIT1 and SCGB1A1 protein levels in lung perfusate have potential utility to distinguish transplantable and non-transplantable lungs (AUC 0.81). Together, our data identify novel biomarkers that may assist with pre-transplant lung assessment, as well as pathways that may amenable to therapeutic intervention during EVLP. Single sentence summary Transcriptional changes in lungs undergoing ex vivo normothermic perfusion identify chitinase1 and club cell genes as potential biomarkers to guide utilisation