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Introduction: Heparin sulfate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signaling, 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 localisation of SULF2 was 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 cells 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 versus 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 release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In Human PBMCs, SULF2 preferentially induced migration of macrophage precursors (monocytes), inducing a phenotypic chance 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.

The αvβ6 integrin is key in activating the pro-fibrotic cytokine TGFβ in idiopathic pulmonary fibrosis. Here, the authors show an inhaled small molecule αvβ6 inhibitor GSK3008348 induces prolonged inhibition of TGFβ signaling pathways in human and murine models of lung fibrosis via αvβ6 degradation.

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.

Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Pellino-1 is an E3 ubiquitin ligase known to regulate virally-induced inflammation. We wished to determine the role of Pellino-1 in the host response to respiratory viruses in health and disease. Pellino-1 expression was examined in bronchial sections from patients with GOLD stage two COPD and healthy controls. Primary bronchial epithelial cells (PBECs) in which Pellino-1 expression had been knocked down were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1−/− mice and wild type littermates were subjected to intranasal infection with clinically-relevant respiratory viruses: rhinovirus (RV1B) and influenza A. We found that Pellino-1 is expressed in the airways of normal subjects and those with COPD, and that Pellino-1 regulates TLR3 signaling and responses to airways viruses. In particular we observed that knockout of Pellino-1 in the murine lung resulted in increased production of proinflammatory cytokines IL-6 and TNFα upon viral infection, accompanied by enhanced recruitment of immune cells to the airways, without any change in viral replication. Pellino-1 therefore regulates inflammatory airway responses without altering replication of respiratory viruses.

Pulmonary epithelial cells are widely considered to be the first line of defence in the lung and are responsible for coordinating the innate immune response to injury and subsequent repair. Consequently, epithelial cells communicate with multiple cell types including immune cells and fibroblasts to promote acute inflammation and normal wound healing in response to damage. However, aberrant epithelial cell death and damage are hallmarks of pulmonary disease, with necrotic cell death and cellular senescence contributing to disease pathogenesis in numerous respiratory diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and coronavirus disease (COVID)-19. In this review, we summarise the literature that demonstrates that epithelial damage plays a pivotal role in the dysregulation of the immune response leading to tissue destruction and abnormal remodelling in several chronic diseases. Specifically, we highlight the role of epithelial-derived damage-associated molecular patterns (DAMPs) and senescence in shaping the immune response and assess their contribution to inflammatory and fibrotic signalling pathways in the lung.

Joint fibrosis affects many synovial joints (including hip, knee and shoulder) causing stiffness and pain. The mechanism of joint fibrosis remains unknown, although genetic factors may contribute. Defects in maintenance of telomere length resulting from impaired telomere repair have been shown to cause lung and liver fibrotic disease. Here we tested the hypothesis that joint fibrosis and other soft tissue fibrotic conditions are also associated with telomere length.5,200 participants in the TwinsUK registry had data on telomere length (measured by qPCR) and the traits of interest (hip and knee stiffness, total joint replacement (TJR, hip or knee) and fibrotic conditions (Dupuytren's disease, frozen shoulder).Multivariable logistic regression analyses showed a significant association between telomere length and fibrotic conditions (hip stiffness, knee stiffness and frozen shoulder, p = ≤0.002) even after taking age into account. No association was found between TJR and telomere length.These findings suggest that defects in telomere repair contribute to joint fibrosis, and that fibrosis shares a common mechanistic pathway in different organs. Therapeutic strategies to combat telomere shortening may offer novel treatments for fibrotic joint disease.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

Mice expressing a Cre recombinase from the lysozyme M-encoding locus (Lyz2) have been widely used to dissect gene function in macrophages and neutrophils. Here, we show that while naïve resident tissue macrophages from IL-4Rαf(lox/delta)LysM(Cre) mice almost completely lose IL-4Rα function, a large fraction of macrophages elicited by sterile inflammatory stimuli, Schistosoma mansoni eggs, or S. mansoni infection, fail to excise Il4rα. These F4/80(hi)CD11b(hi) macrophages, in contrast to resident tissue macrophages, express lower levels of Lyz2 explaining why this population resists LysM(Cre)-mediated deletion. We show that in response to IL-4 and IL-13, Lyz2(lo)IL-4Rα(+) macrophages differentiate into an arginase 1-expressing alternatively-activated macrophage (AAM) population, which slows the development of lethal fibrosis in schistosomiasis. In contrast, we identified Lyz2(hi)IL-4Rα(+) macrophages as the key subset of AAMs mediating the downmodulation of granulomatous inflammation in chronic schistosomiasis. Our observations reveal a limitation on using a LysMCre mouse model to study gene function in inflammatory settings, but we utilize this limitation as a means to demonstrate that distinct populations of alternatively activated macrophages control inflammation and fibrosis in chronic schistosomiasis.

Th2-driven lung inflammation increases Arginase 1 (Arg1) expression in alternatively-activated macrophages (AAMs). AAMs modulate T cell and wound healing responses and Arg1 might contribute to asthma pathogenesis by inhibiting nitric oxide production, regulating fibrosis, modulating arginine metabolism and restricting T cell proliferation. We used mice lacking Arg1 in myeloid cells to investigate the contribution of Arg1 to lung inflammation and pathophysiology. In six model systems encompassing acute and chronic Th2-mediated lung inflammation we observed neither a pathogenic nor protective role for myeloid-expressed Arg1. The number and composition of inflammatory cells in the airways and lungs, mucus secretion, collagen deposition, airway hyper-responsiveness, and T cell cytokine production were not altered if AAMs were deficient in Arg1 or simultaneously in both Arg1 and NOS2. Our results argue that Arg1 is a general feature of alternative activation but only selectively regulates Th2 responses. Therefore, attempts to experimentally or therapeutically inhibit arginase activity in the lung should be examined with caution.

Understanding where mutant CFTR is localised in airway epithelia is essential in guiding the best therapeutic approach to correct the dysfunction of the CFTR protein. The widely held paradigm is that CF patients harbouring the commonest mutation, CFTR-delF508, trap CFTR within the endoplasmic reticulum and target it for degradation. However there are conflicting reports concerning expression and localisation of CFTR-delF508 in lung tissue. To attempt to resolve this fundamental issue we developed a novel approach to measure CFTR-delF508 in the lower airways of patients who have undergone lung transplantation for advanced CF. By sampling CF and non-CF epithelium simultaneously from the same individual, confounding factors of different airway microenvironments which may have influenced previous observations can be overcome.Epithelia sampled by bronchial brushing above (CF) and below (non-CF) the bronchial anastomosis were stained for CFTR and the localisation and level of expression assessed (n = 12).There was no significant difference in the proportion of tall columnar cells showing CFTR immunostaining as a discrete band at the apical membrane in cells harbouring the CFTR-delF508 mutation compared to non-CF cells (p = 0.21, n = 12). However, the amount of CFTR expressed at the apical surface was reduced by ∼50% in CF cells compared to non-CF cells (p = 0.04, n = 5).Our novel observation challenges the prevailing paradigm that CFTR is essentially absent from the apical membrane of respiratory cells harbouring the CFTR-delF508 mutation. Moreover, it raises the possibility that the new generation of CFTR potentiators may offer a realistic therapeutic option for CF patients.