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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.

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.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in the progression and resolution of fibrosis. Identifying the unique fibrogenic macrophages that are found in human fibrotic tissues could lead to new and more effective treatments for fibrosis. Here we used human liver and lung single cell RNA sequencing datasets to identify a unique subset of CD9+ TREM2+ macrophages expressing SPP1, GPNMB, FABP5, and CD63 with strong pro-fibrotic activity. This population was validated across orthogonal techniques, species and tissues. These macrophages were enriched at the outside edges of scarring adjacent to activated mesenchymal cells, and in the fibrotic niche across species and organs. Neutrophils producing the type 3 cytokines GM-CSF and IL-17A, and expressing MMP9, which participates in the activation of TGF-β1, clustered with these scar-associated macrophages. Using in vitro primary human cell assays, we determined that GM-CSF, IL-17A and TGF-β1 drive the differentiation of these scar-associated macrophages, and that co-culture of monocyte-derived macrophages with hepatic stellate cells and TGF-β1 augmented type 1 collagen deposition. In vivo blockade of GM-CSF, IL-17A or TGF-β1 with small or large molecules reduced scar-associated macrophage expansion and fibrosis in multiple models of hepatic and pulmonary fibrosis. Our work demonstrates that a specific scar-associated macrophage population is linked with fibrosis across species and tissues. It further provides a strategy for unbiased discovery, triage and preclinical validation of therapeutic targets within this fibrogenic macrophage population.

Aims Stiffness is a common complication after total knee arthroplasty (TKA). Pathogenesis is not understood, treatment options are limited, and diagnosis is challenging. The aim of this study was to investigate if MRI can be used to visualize intra-articular scarring in patients with stiff, painful knee arthroplasties. Methods Well-functioning primary TKAs (n = 11), failed non-fibrotic TKAs (n = 5), and patients with a clinical diagnosis of fibrosis 1 (n = 8) underwent an MRI scan with advanced metal suppression (Slice Encoding for Metal Artefact Correction, SEMAC) with gadolinium contrast. Fibrotic tissue (low intensity on T1 and T2, low-moderate post-contrast enhancement) was quantified (presence and tissue thickness) in six compartments: supra/infrapatella, medial/lateral gutters, and posterior medial/lateral. Results Fibrotic tissue was identified in all patients studied. However, tissue was significantly thicker in fibrotic patients (4.4 mm ± 0.2 mm) versus non-fibrotic (2.5 mm ± 0.4 mm) and normal TKAs (1.9 mm ± 0.2 mm, p = < 0.05). Significant (> 4 mm thick) tissue was seen in 26/48 (54%) of compartments examined in the fibrotic group, compared with 17/30 (57%) non-fibrotic, and 10/66 (15%) normal TKAs. Although revision surgery did improve range of movement (ROM) in all fibrotic patients, clinically significant restriction remained post-surgery. Conclusion Stiff TKAs contain intra-articular fibrotic tissue that is identifiable by MRI. Studies should evaluate whether MRI is useful for surgical planning of debridement, and as a non-invasive measurement tool following interventions for stiffness caused by fibrosis. Revision for stiffness can improve ROM, but outcomes are sub-optimal and new treatments are required. Cite this article: Bone Joint J 2020;102-B(10):1331–1340.

The αvβ6 integrin plays a key role in the activation of transforming growth factor-β (TGFβ), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvβ6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvβ6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFβ signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvβ6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvβ6, induces prolonged inhibition of TGFβ signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy., 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.

Funder: NIHR Blood and Transplant Unit, Funder: NIHR Newcastle Biomedical Research Centre; Id: http://dx.doi.org/10.13039/501100012295, Funder: NIHR Cambridge Biomedical Research Centre; Id: http://dx.doi.org/10.13039/501100018956, BACKGROUND: Transplantation is an effective treatment for end-stage lung disease, but the donor organ shortage is a major problem. Ex-vivo lung perfusion (EVLP) of extended criteria organs enables functional assessment to facilitate clinical decision-making around utilization, but the molecular processes occurring during EVLP, and how they differ between more or less viable lungs, remain to be determined. METHODS: We used RNA sequencing of lung tissue to delineate changes in gene expression occurring in 10 donor lungs undergoing EVLP and compare lungs that were deemed non-transplantable (n = 4) to those deemed transplantable (n = 6) following perfusion. RESULTS: We found that lungs deemed unsuitable for transplantation had increased induction of innate immune pathways and lower expression of oxidative phosphorylation related genes. Furthermore, the expression of SCGB1A1, a gene encoding an anti-inflammatory secretoglobin CC10, and other club cell genes was significantly decreased in non-transplantable lungs, while CHIT-1 was increased. Using a larger validation cohort (n = 17), we confirmed that the ratio of CHIT1 and SCGB1A1 protein levels in lung perfusate have potential utility to distinguish transplantable from non-transplantable lungs (AUC .81). CONCLUSIONS: Together, our data identify novel biomarkers that may assist with pre-transplant lung assessment, as well as pathways that may be amenable to therapeutic intervention during EVLPAQ6.

[Background and Aims] NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD., [Approach and Results] We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian target of rapamycin complex (mTORC) 1 and the LD coating protein perilipin (Plin) 3 in these processes. We show that the autophagy machinery is recruited to the LD on hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins focal adhesion interaction protein 200 KDa and autophagy-related 16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation., [Conclusions] These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid-mediated toxicity, thus offering a therapeutic strategy in NAFLD., Supported by C0120R3166, C0245R4032, and BH182173 from Newcastle University. M. G.-M. is a Sara Borrell Postdoctoral fellow (CD18/00203) from the Ministerio de Ciencia, Innovación y Universidades (Spain). J. P. B. is funded by the Agencia Estatal de Investigación, grants PID2019-105699RB-I00/AEI/10.13039/501100011033 and RED2018-102576-T, Instituto de Salud Carlos III (CB16/10/00282), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA, and Fundación Ramón Areces. V. I. K. acknowledges support from Biotechnology and Biological Sciences Research Council (BB/M023389/1, BB/R008167/1, BB

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-β (TGFβ) via the alpha-V beta-6 (αvβ6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvβ6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvβ6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103 induced concentration- and time-dependent internalisation of αvβ6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvβ6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvβ6 integrin and inhibit the activation of TGFβ in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvβ6 engagement, TGFβ signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvβ6 inhibitor that attenuates TGFβ signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

Airway epithelium plays a pivotal role in maintaining immune homeostasis and orchestrates the innate and adaptive responses of the lung against inhaled insults. However, the aetiology of pathological changes on airway epithelium during COPD pathogenesis is not completely understood, in particular factors linked to the susceptibility to severe disease. To address this, we characterized well-differentiated primary bronchial epithelial cells (WD-PBECs) derived from severe COPD patients undergoing transplantation and age-matched healthy controls employing an air-liquid interface (ALI) 3D in vitro/ex-vivo culture model. WD-PBECs derived from severe COPD patients showed an indistinguishable morphology compared with controls. However, aberrant epithelium differentiation in terms of a decrease in the number of ciliated cells and an increase in the number of Club cells and goblet cells in was evidenced in COPD cultures compared with healthy controls. Tight junction integrity was also compromised in the former. Moreover, there was enhanced basolateral secretion of IL-6, IL-8, GM-CSF, IL-1alpha, IP-10, CCL1 as well as altered secretion of Th1 and Th2 cytokines by COPD cells. Our data demonstrate that primary bronchial epithelial cells from patients with severe COPD show alterations in individual cell types during differentiation and enhanced secretion of inflammatory mediators including an alteration in Th1 and Th2 cytokines. Such changes in COPD bronchial epithelium during severe disease may represent an underlying perturbation in cellular function in these cells which may contribute to inflammation and infection in the COPD airways.

Significance The circadian clock plays an essential role in energy metabolism and inflammation. In contrast, the importance of the clock in the pathogenesis of fibrosis remains poorly explored. This study describes a striking alteration in circadian biology during pulmonary fibrosis where the relatively arrhythmic alveolar structures gain circadian but asynchronous rhythmicity due to infiltration by fibroblasts. Disruption of the clock in these cells, which are not widely implicated in circadian pathophysiology, results in a profibrotic phenotype. Translation of these findings in humans revealed previously unrecognized important circadian risk factors for pulmonary fibrosis (sleep length, chronotype, and shift work). In addition, targeting REVERBα repressed collagen secretion from human fibrotic lung tissue, making this protein a promising therapeutic target., Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

Chronic pain and restricted knee motion is a significant problem following the total knee arthroplasty (TKA). The molecular pathogenesis of pain post‐TKA is not known and no targeted therapeutic intervention is available. The aim of this study was to investigate whether pro‐inflammatory mediators are elevated in revision knee patients, indicating an active, ongoing inflammatory process that may contribute to pain. Twelve key markers (pro‐inflammatory cytokines granulocyte‐macrophage colony‐stimulating factor [GM‐CSF], interleukin 5 [IL‐5], IL‐8 and IL‐10, chemokines CCL2, CCL3, CCL4, and CCL13, mediators of angiogenesis Flt‐1, vascular endothelial growth factor, and cell migration vascular cell adhesion molecule 1 and intercellular adhesion molecule 1) were measured in knee tissue and synovial fluid (SF) from primary TKA (n = 29) and revision patients (n = 32). Indications for surgery were osteoarthritis (OA) for primary TKA, and component loosening (n = 11), stiffness (n = 11), laxity pattern (n = 8), or progression of OA in patella resurfacing (n = 3) for revision surgery. Pain levels (WOMAC score) were higher in revision than primary patients (p ≤ 0.05). Time from primary to revision ranged from 8 months to 30 years (median 10 years). All markers were elevated in revision TKA; there was no trend toward decreasing levels with greater time from primary surgery for any marker studied in SF. Similar results were seen in knee tissue. We found no differences comparing indications for revision surgery (p ≥ 0.05). The elevation of inflammatory mediators in painful post‐TKA knees requiring revision suggests active, chronic inflammation. Characterization of upregulated markers provides rationale for targeted therapy, even many years from the primary surgery. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2316–2324, 2019

Precision cut liver slices (PCLSs) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono- or co-cultures. The aim of this study was to develop a bioreactor system to increase the healthy life span of PCLSs and model fibrogenesis. PCLSs were generated from normal rat or human liver, or fibrotic rat liver, and cultured in our bioreactor. PCLS function was quantified by albumin enzyme-linked immunosorbent assay (ELISA). Fibrosis was induced in PCLSs by transforming growth factor beta 1 (TGFβ1) and platelet-derived growth factor (PDGFββ) stimulation ± therapy. Fibrosis was assessed by gene expression, picrosirius red, and α-smooth muscle actin staining, hydroxyproline assay, and soluble ELISAs. Bioreactor-cultured PCLSs are viable, maintaining tissue structure, metabolic activity, and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell-cultured PCLSs rapidly deteriorate, and albumin secretion is significantly impaired by 48 hours. TGFβ1/PDGFββ stimulation of rat or human PCLSs induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts, and histological fibrosis. Fibrogenesis slowly progresses over 6 days in cultured fibrotic rat PCLSs without exogenous challenge. Activin receptor-like kinase 5 (Alk5) inhibitor (Alk5i), nintedanib, and obeticholic acid therapy limited fibrogenesis in TGFβ1/PDGFββ-stimulated PCLSs, and Alk5i blunted progression of fibrosis in fibrotic PCLS. Conclusion: We describe a bioreactor technology that maintains functional PCLS cultures for 6 days. Bioreactor-cultured PCLSs can be successfully used to model fibrogenesis and demonstrate efficacy of antifibrotic therapies. ispartof: HEPATOLOGY vol:70 issue:4 pages:1377-1391 ispartof: location:United States status: published

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca2+ imaging studies. We found that both CS, and CSE, rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but excluded store-operated and L-type Ca2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke.

Diseases where fibrosis plays a major role accounts for enormous morbidity and mortality and yet we have very little in our therapeutic arsenal despite decades of research and clinical trials. Our understanding of fibrosis biology is primarily built on data generated in conventional mono-culture or co-culture systems and in vivo model systems. While these approaches have undoubtedly enhanced our understanding of basic mechanisms, they have repeatedly failed to translate to clinical benefit. Recently, there had been a push to generate more physiologically relevant platforms to study fibrosis and identify new therapeutic targets. Here we review the state-of-the-art regarding the development and application of 3D complex cultures, bio-printing and precision cut slices to study pulmonary, hepatic and renal fibrosis.

Correction to: Nature Metabolism https://doi.org/10.1038/s42255-020-00306-2, published online 9 November 2020. In the version of this article initially published, in the ×40 diseased human kidney images in Supplementary Fig. 1, the FSGS image duplicated the DN image. The error has been corrected in the HTML version of the article.

Neutrophils are the most abundant inflammatory cells at the earliest stages of wound healing and play important roles in wound repair and fibrosis. Formyl peptide receptor 1 (FPR-1) is abundantly expressed on neutrophils and has been shown to regulate their function, yet the importance of FPR-1 in fibrosis remains ill defined. FPR-1–deficient (fpr1–/–) mice were protected from bleomycin-induced pulmonary fibrosis but developed renal and hepatic fibrosis normally. Mechanistically, we observed a failure to effectively recruit neutrophils to the lungs of fpr1–/– mice, whereas neutrophil recruitment was unaffected in the liver and kidney. Using an adoptive transfer model we demonstrated that the defect in neutrophil recruitment to the lung was intrinsic to the fpr1–/– neutrophils, as C57BL/6 neutrophils were recruited normally to the damaged lung in fpr1–/– mice. Finally, C57BL/6 mice in which neutrophils had been depleted were protected from pulmonary fibrosis. In conclusion, FPR-1 and FPR-1 ligands are required for effective neutrophil recruitment to the damaged lung. Failure to recruit neutrophils or depletion of neutrophils protects from pulmonary fibrosis., FPR-1 is the primary receptor driving neutrophil recruitment to the injured lung, and absence of FPR-1 or depletion of neutrophils protects from pulmonary fibrosis.

ObjectivesNF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc.MethodsFibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel−/− murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls.ResultscRel expression was elevated in murine lung and skin fibrosis models. Rel−/− mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts.ConclusioncRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease.

The ionic liquid 1-octyl-3-methylimidazolium (M8OI) has been found in the environment and identified as a hazard for triggering the liver disease primary biliary cholangitis (PBC). Given limited toxicity data for M8OI and other structurally-related ionic liquids, target organs for M8OI toxicity were examined. Adult male C57Bl6 mice were acutely exposed to 0–10 mg/kg body weight M8OI via 2 intraperitoneal injections (time zero and 18 h) and effects examined at 24 h. At termination, tissue histopathology, serum and urinary endpoints were examined. No overt pathological changes were observed in the heart and brain. In contrast, focal and mild to multifocal and moderate degeneration with a general trend for an increase in severity with increased dose was observed in the kidney. These changes were accompanied by a dose-dependent increased expression of Kim1 in kidney tissue, marked elevations in urinary Kim1 protein and a dose-dependent increase in serum creatinine. Hepatic changes were limited to a significant dose-dependent loss of hepatic glycogen and a mild but significant increase in portal tract inflammatory recruitment and/or fibroblastic proliferation accompanied by a focal fibrotic change. Cultured mouse tissue slices reflected these in vivo effects in that dose-dependent injury was observed in kidney slices but not in the liver. Kidney slices accumulated higher levels of M8OI than liver slices (e.g. at 10 μM, greater than 4 fold) and liver slices where markedly more active in the metabolism of M8OI. These data indicate that the kidney is a target organ for the toxic effects of M8OI accompanied by mild cholangiopathic changes in the liver after intraperitoneal administration., Highlights • The ionic liquid M8OI [C8mim] has been found in the environment. • Mice were exposed acutely over 24 h to M8OI via i.p. injection. • The kidney was the target organ for toxicity, heart and brain appeared normal. • Hepatic changes were limited to glycogen depletion and mild portal tract changes. • Kidney tissue slices accumulated higher levels of M8OI than liver tissue slices.

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-β1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease. ispartof: NATURE METABOLISM vol:2 issue:11 ispartof: location:Germany status: published

During postprandial state, the liver is exposed to high levels of dietary fatty acids (FAs) and carbohydrates. FAs are re-esterified into triglycerides, which can be stored in lipid droplets (LDs) in the liver. Aberrant accumulation of LDs can lead to diseases such as alcoholic liver disease and non-alcoholic fatty liver disease, the latter being the most common liver pathology in western countries. Improved understanding of LD biology has potential to unlock new treatments for these liver diseases. The Perilipin (Plin) family is the group of proteins that coat LDs, controlling their biogenesis, stabilization, and preventing their degradation. Recent studies have revealed that autophagy is involved in LD degradation and, therefore, may be crucial to avoid lipid accumulation. Here, we show that a phosphorylated form of Plin3 is required for selective degradation of LDs in fibroblasts, primary hepatocytes and human liver slices. We demonstrate that oleic acid treatment induces the recruitment of the autophagy machinery to the surface of LDs. When Plin3 is silenced, this recruitment is suppressed resulting in accumulation of lipid. Plin3 pulldowns revealed interactions with the autophagy initiator proteins Fip200 and Atg16L indicating that Plin3 may function as a docking protein involved in lipophagy activation. Of particular importance, we define Plin3 as a substrate for mTORC1-dependent phosphorylation and show that this event is decisive for lipophagy. Our study therefore reveals that Plin3, and its phosphorylation by mTORC1, is crucial for degradation of LDs by autophagy. We propose that stimulating this pathway to enhance LD autophagy in hepatocytes will help protect the liver from lipid-mediated toxicity thus offering new therapeutic opportunities in human steatotic liver 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 summaryTranscriptional changes in lungs undergoing ex vivo normothermic perfusion identify chitinase1 and club cell genes as potential biomarkers to guide utilisation

OBJECTIVES: Availability of donor lungs suitable for transplant falls short of current demand and contributes to waiting list mortality.\ud Ex vivo lung perfusion (EVLP) offers the opportunity to objectively assess and recondition organs unsuitable for immediate transplant.\ud Identifying robust biomarkers that can stratify donor lungs during EVLP to use or non-use or for specific interventions could further improve its clinical impact.\ud METHODS: In this pilot study, 16 consecutive donor lungs unsuitable for immediate transplant were assessed by EVLP. Key inflammatory mediators and tissue injury markers were measured in serial perfusate samples collected hourly and in bronchoalveolar lavage fluid (BALF) collected before and after EVLP. Levels were compared between donor lungs that met criteria for transplant and those that did not.\ud RESULTS: Seven of the 16 donor lungs (44%) improved during EVLP and were transplanted with uniformly good outcomes. Tissue and vascular injury markers lactate dehydrogenase, HMGB-1 and Syndecan-1 were significantly lower in perfusate from transplanted lungs. A model combining IL-1b and IL-8 concentrations in perfusate could predict final EVLP outcome after 2 h assessment. In addition, perfusate IL-1b concentrations showed an inverse correlation to recipient oxygenation 24 h post-transplant.\ud CONCLUSIONS: This study confirms the feasibility of using inflammation and tissue injury markers in perfusate and BALF to identify donor lungs most likely to improve for successful transplant during clinical EVLP. These results support examining this issue in a larger study.

AimsThe aim of this consensus was to develop a definition of post-operative fibrosis of the knee.Patients and MethodsAn international panel of experts took part in a formal consensus process composed of a discussion phase and three Delphi rounds.ResultsPost-operative fibrosis of the knee was defined as a limited range of movement (ROM) in flexion and/or extension, that is not attributable to an osseous or prosthetic block to movement from malaligned, malpositioned or incorrectly sized components, metal hardware, ligament reconstruction, infection (septic arthritis), pain, chronic regional pain syndrome (CRPS) or other specific causes, but due to soft-tissue fibrosis that was not present pre-operatively. Limitation of movement was graded as mild, moderate or severe according to the range of flexion (90° to 100°, 70° to 89°, < 70°) or extension deficit (5° to 10°, 11° to 20°, > 20°). Recommended investigations to support the diagnosis and a strategy for its management were also agreed.ConclusionThe development of standardised, accepted criteria for the diagnosis, classification and grading of the severity of post-operative fibrosis of the knee will facilitate the identification of patients for inclusion in clinical trials, the development of clinical guidelines, and eventually help to inform the management of this difficult condition. Cite this article: Bone Joint J 2016;98-B:1479–88.

Acidic mammalian chitinase (AMCase) is known to be induced by allergens and helminths, yet its role in immunity is unclear. Using AMCase-deficient mice, we show that AMCase deficiency reduced the number of group 2 innate lymphoid cells during allergen challenge but was not required for establishment of type 2 inflammation in the lung in response to allergens or helminths. In contrast, AMCase-deficient mice showed a profound defect in type 2 immunity following infection with the chitin-containing gastrointestinal nematodes Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. The impaired immunity was associated with reduced mucus production and decreased intestinal expression of the signature type 2 response genes Il13, Chil3, Retnlb, and Clca1. CD103(+) dendritic cells, which regulate T cell homing, were also reduced in mesenteric lymph nodes of infected AMCase-deficient mice. Thus, AMCase functions as a critical initiator of protective type 2 responses to intestinal nematodes but is largely dispensable for allergic responses in the lung.

Epithelial injury has been recognised as a key event in the pathogenesis of lung fibrosis.1 Damaged epithelial cells are a major source of danger signals referred to as alarmins or damage associated molecular patterns (DAMPs). Previously published work from our group has shown that IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts via an IL-1R1 dependant pathway.2 3 Critically mice that are globally deficient in IL-1α or IL-1R1 are protected from pulmonary fibrosis. In this study we specifically investigate the importance of IL-1R1 on fibroblasts using both ex-vivo and in-vivo models. IL-1R1 floxed mice were crossed with LysM Cre mice and PDGFRββ Cre mice to generate mice deficient in IL-1R1 expression in myeloid (IL-1R1ΔMono) and mesenchymal (IL-1R1ΔFib) cells. Initially, Precision Cut Lung Slices (PCLS) were prepared from IL-1R1ΔMono, IL-1R1ΔFib as well as IL-1R1 floxed mice, LysM Cre and PDGFRββ Cre control mice. Exogenous challenge with IL-1β induced a dose-dependent inflammatory response in PCLS from control mice as characterised by elevated secretion of IL-6, KC and CCL2. Critically, inflammation in PCLS from IL-1R1ΔFib mice was attenuates ˜70% whereas no effect was observed in PCLS from IL-1R1ΔMono mice. To determine the importance of IL-1R1 on fibroblasts in vivo we challenged IL-1R1ΔMono and IL-1R1ΔFib mice intratracheally with bleomycin. Critically IL-1R1ΔFib mice were protected from bleomycin induced pulmonary inflammation and fibrosis whereas IL-1R1ΔMono mice developed pulmonary fibrosis normally. In summary, the fibroblasts is the primary cell responding to epithelial damage in intact lung tissue and selective depletion of IL-1R1 on fibroblasts in vivo attenuates pulmonary inflammation and fibrosis. These data suggest that the fibroblasts directly drives pulmonary inflammation and fibrosis in response to epithelial damage. References Chambers CR, Mercer FP. Mechanisms of alveolar epithelial injury, repair, and fibrosis. ATS Journal 2015;12:S16–S20. Suwara MI, et al. IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunology2014;7:684–93. Borthwick AL. Pseudomonas aeruginosa Induced Airway Epithelial Injury Drives Fibroblast Activation: A Mechanism in Chronic Lung Allograft Dysfunction. American Journal of Transplantation2015;16:1751–65.

Introduction and objectives Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). We have shown that Pellino-1, an E3 ubiquitin ligase, is involved in viral-induced TLR3 signalling; thus we sought to describe the role of Pellino-1 in the host response to viral stimulation of the airway. Methods Pellino-1 expression was examined in bronchial sections from patients with GOLD stage 2 COPD (n=7) and healthy controls (n=8). Primary bronchial epithelial cells (PBECs, n=5) in which Pellino-1 expression had been knocked down using siRNA, were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1-/- mice and wild type littermates were subjected to intranasal infection with the clinically-relevant respiratory viruses rhinovirus (RV1B) and influenza A, and responses monitored up to 8 days later. Results We show that Pellino-1 is expressed in the airways of normal subjects and those with COPD. In the absence of Pellino-1, PBECs showed significant reduction in proinflammatory cytokines, including CXCL8 and IL-6, upon TLR3 activation. Surprisingly however, knockout of Peli1 in the murine lung resulted in increased production of the proinflammatory cytokines IL-6 and TNFα with viral infection. This was accompanied by an enhanced recruitment of immune cells to the airways in these animals, including a population of innate B cells, without loss of viral replication. Conclusions We conclude that Pellino-1 functions as a positive regulator for antiviral responses in isolated bronchial epithelial cells and in systemic responses to TLR3 activation, but it has the opposite effect in the lung. Here, Pellino-1 has a distinct function in the down-regulation of antiviral inflammation; a role dissociated from viral replication. Our data therefore suggest that Pellino-1 may offer therapeutic potential to limit viral inflammation in COPD and asthma.

Burkholderia cepacia complex (BCC) bacteria are a group of opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult, due to the inherent and acquired multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. Here, we demonstrate that the novel compound Q22, which is related to the bacterial cytoskeleton destabilising compound A22, can reduce the growth rate and inhibit growth of BCC bacteria. We further analysed the phenotypic effects of Q22 treatment on BCC virulence traits, to assess its feasibility as an antimicrobial. BCC bacteria were grown in the presence of Q22 with a broad phenotypic analysis, including resistance to H2O2-induced oxidative stress, changes in the inflammatory potential of cell surface components, and in-vivo drug toxicity studies. The influence of the Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide, and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H2O2-induced oxidative stress, with some strains showing increased resistance after treatment. There was strain-to-strain variation in the pro-inflammatory ability of bacterial lysates to elicit TNF&alpha, and IL-1&beta, from human myeloid cells. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in-vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. In summary, destabilisation of the bacterial cytoskeleton in BCC, using compounds such as Q22, led to increased virulence-related traits in vitro. These changes appear to vary depending on strain and BCC species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects translate into the in-vivo environment of the CF infection.

Summary boxWhat is already known about this subject?Currently there are no effective anti-fibrotic drugs to treat liver fibrosis and there is an urgent unmet need to increase our knowledge of the disease process and develop better tools for anti-fibrotic drug discovery.Preclinical in vitro cell cultures and animal models are widely used to study liver fibrosis and test anti-fibrotic drugs, but have shortfalls; cell culture models lack the relevant complex cell-cell interactions of the liver and animal models only reproduce some features of human disease.Precision Cut Liver Slices (PCLS) are structurally representative of the liver and can be used to model liver fibrosis and test anti-fibrotic drugs. However, PCLS are typically cultured in elevated, non-physiological oxygen levels and only have a healthy lifespan of 48h.What are the new findings?We have developed a novel bioreactor culture system that increases the longevity of functional PCLS to up to 6 days under normoxic conditions.Bioreactor cultured PCLS can be used to model fibrogenesis in both normal and fibrotic PCLS using a combination of biochemical and histological outputs.Administration of an Alk5 inhibitor effectively limits fibrogenesis in normal rodent and human PCLS and in rodent PCLS with established fibrosis.How might it impact on clinical practice in the foreseeable future?The extended longevity of bioreactor cultured PCLS represent a novel pre-clinical tool to investigate the cellular and molecular mechanisms of liver fibrosis.Bioreactor cultured human PCLS offer a clinically relevant system to test efficacy of anti-fibrotic drugs.AbstractObjectivePrecision cut liver slices (PCLS) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono or co-cultures. The objective of this study was to develop a bioreactor system to increase the healthy lifespan of PCLS and model fibrogenesis.DesignPCLS were generated from normal rat or human liver, or 4-week carbon tetrachloride-fibrotic rat liver and cultured in our patented bioreactor. PCLS function was quantified by albumin ELISA. Fibrosis was induced in PCLS by TGFβ1 and PDGFββ stimulation. Alk5 inhibitor therapy was used. Fibrosis was assessed by fibrogenic gene expression, Picrosirius Red and αSmooth Muscle Actin staining, hydroxyproline assay and collagen 1a1, fibronectin and hyaluronic acid ELISA.ResultsBioreactor cultured PCLS are viable, maintaining tissue structure and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell cultured PCLS rapidly deteriorate and albumin secretion is significantly impaired by 48 hours. TGFβ1 and PDGFββ stimulation of rat or human PCLS induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts and histological fibrosis. Fibrogenesis slowly progresses over 6-days in cultured fibrotic rat PCLS without exogenous challenge. Alk5 inhibitor limited fibrogenesis in both TGFβ1 and PDGFββ stimulated PCLS and fibrotic PCLS.ConclusionWe describe a new bioreactor technology which maintains functional PCLS cultures for 6 days. Bioreactor cultured PCLS can be successfully used to model fibrogenesis and demonstrate efficacy of an anti-fibrotic therapy.

Normal wound repair is a complex and highly orchestrated process that allows the restoration of tissue homeostasis after injury. However, when the injury is sustained, the wound-healing response becomes dysregulated leading to fibrosis. Fibrosis can affect any organ, and it is estimated that 45 % of deaths in the western world are attributed to diseases where fibrosis plays a major pathogenic role. Fibrosis is an extremely complex process that requires numerous cell types and a range of cellular processes including proliferation, differentiation, apoptosis/necrosis, ingress/egress, to name but a few. These key cellular events are controlled by a plethora of cytokines, chemokines, and growth factors including TGF-β1 and IL-13, which have been identified as critical pro-fibrotic mediators in many studies. This review will focus on the roles of TGF-β1 and IL-13 in liver fibrosis and discuss the current state of the art for therapeutic targets for these proteins and their receptors.

Liver fibrosis, the accumulation of extracellular matrix proteins caused by chronic liver damage, results from the activation of hepatic stellate cells. In a new study, Arriazu et al. explore in detail the mechanisms by which the multifunctional protein osteopontin drives hepatic fibrosis, and show that interaction with HMGB1 mediates the fibrogenic response.

Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countries and is the second leading indication for liver transplantation due to the extensive fibrosis it causes. NAFLD progression is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity; however, the specific immunological mechanisms regulating the progression of NAFLD-associated fibrosis in the liver are unclear. To investigate the immunopathogenesis of NAFLD more completely, we investigated adipose dysfunction, nonalcoholic steatohepatitis (NASH), and fibrosis in mice that develop polarized type 1 or type 2 immune responses. Unexpectedly, obese interleukin-10 (IL-10)/IL-4-deficient mice (type 1-polarized) were highly resistant to NASH. This protection was associated with an increased hepatic interferon-γ (IFN-γ) signature. Conversely, IFN-γ-deficient mice progressed rapidly to NASH with evidence of fibrosis dependent on transforming growth factor-β (TGF-β) and IL-13 signaling. Unlike increasing type 1 inflammation and the marked loss of eosinophils seen in expanding adipose tissue, progression of NASH was associated with increasing eosinophilic type 2 liver inflammation in mice and human patient biopsies. Finally, simultaneous inhibition of TGF-β and IL-13 signaling attenuated the fibrotic machinery more completely than TGF-β alone in NAFLD-associated fibrosis. Thus, although type 2 immunity maintains healthy metabolic signaling in adipose tissues, it exacerbates the progression of NAFLD collaboratively with TGF-β in the liver.

Fibroblasts persist within fibrotic scar tissue and exhibit considerable phenotypic and functional plasticity. Herein, we hypothesized that scar-associated fibroblasts may be a source of stress-induced inflammatory exacerbations and pain. To test this idea, we used a human model of surgery-induced fibrosis, total knee arthroplasty (TKA). Using a combination of tissue protein expression profiling and bioinformatics, we discovered that many months after TKA, the fibrotic joint exists in a state of unresolved chronic inflammation. Moreover, the infrapatellar fat pad, a soft tissue that becomes highly fibrotic in the post-TKA joint, expresses multiple inflammatory mediators, including the monocyte chemoattractant, chemokine (C-C motif) ligand (CCL) 2, and the innate immune trigger, IL-1α. Fibroblasts isolated from the post-TKA fibrotic infrapatellar fat pad express the IL-1 receptor and on exposure to IL-1α polarize to a highly inflammatory state that enables them to stimulate the recruitment of monocytes. Blockade of fibroblast CCL2 or its transcriptional regulator NF-κB prevented IL-1α–induced monocyte recruitment. Clinical investigations discovered that levels of patient-reported pain in the post-TKA joint correlated with concentrations of CCL2 in the joint tissue, such that the chemokine is effectively a pain biomarker in the TKA patient. We propose that an IL-1α–NF-κB–CCL2 signaling pathway, operating within scar-associated fibroblasts, may be therapeutically manipulated for alleviating inflammation and pain in fibrotic joints and other tissues.

Acute inflammation is a recognised part of normal wound healing. However, when inflammation fails to resolve and a chronic inflammatory response is established this process can become dysregulated resulting in pathological wound repair, accumulation of permanent fibrotic scar tissue at the site of injury and the failure to return the tissue to normal function. Fibrosis can affect any organ including the lung, skin, heart, kidney and liver and it is estimated that 45% of deaths in the western world can now be attributed to diseases where fibrosis plays a major aetiological role. In this review we examine the evidence that cytokines play a vital role in the acute and chronic inflammatory responses that drive fibrosis in injured tissues. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Liver fibrosis is characterized by excessive deposition of collagenous scar tissue after persistent liver damage. The primary liver-scar–forming cell is the hepatic myofibroblast (HM), which is derived from quiescent hepatic stellate cells (qHSCs).1 Liver injury instructs qHSCs to undergo a transdifferentiation program from retinoid-storing cells into HMs, which produce collagen I and enzymes, which prevent collagen degradation, the tissue inhibitors of matrix metalloproteinase.1 Continued damage causes perpetuation of the HM phenotype and an imbalance in the deposition and breakdown of fibrotic matrix and, ultimately, the progression of liver fibrosis. Fibrosis in both rodents and humans is a dynamic process that can reverse, as well as progress,2 and removal of the injury stimulus or promoting HM apoptosis is associated with fibrolysis.3,4 Nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) is a transcription factor that contains five subunits, RelA (p65), p50, c-Rel, RelB, and p52, which form either homo-or heterodimers to bind DNA. NF-κB is a master regulator of essential cellular functions, including cell cycle, survival, and immunity.5,6 The pathway is activated by canonical (RelA, p50, and c-Rel subunits) or noncanonical (RelB and p52 subunit) signaling.5 In unstimulated cells, canonical NF-κB is predominantly bound to its inhibitor (IκBα) and retained in the cytoplasm. Stimulation by an appropriate signal, for example, tumor necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS), promotes IκB kinase (IKK)-dependent degradation of IκBα, nuclear translocation of NF-κB, and subsequent DNA binding. In the noncanonical pathway, RelB is bound to p100, the precursor of p52. Stimulation by CD40 or lymphotoxin activates NF-κB-inducing kinase, which then activates IKKα. This promotes the phosphorylation and polyubiquitination of p100, which is then degraded by the proteasome, releasing active p52/RelB. Canonical NF-κB signaling influences liver fibrosis and promotes HM survival. In models of liver damage, mice lacking nfkb1 (p105/p50) develop severe inflammation and fibrosis,7 whereas crel knockout mice develop less fibrosis, but have impaired liver regeneration, compared to wild-type controls.8 Pharmacological blockade of NF-κB in HM promotes their apoptosis and enhanced reversal of liver fibrosis.9,10 However, long-term global NF-κB blockade may alter immune responses or cause cancer.11,12 In HM, NF-κB is constitutively active. Deregulation of healthy NF-κB activity is controlled by at least two reprogramming events that occur during HSC transdifferentiation. The first is persistent down-regulation in IκBα levels, which is mediated by epigenetic changes,13 and the second is phosphorylation of RelA at serine 536 (RelA-P-Ser536).9 This post-translational modification promotes RelA nuclear translocation and increases the ability of RelA-containing dimers to interact with coactivators as well as the transcriptional machinery to enhance the transactivation potential of RelA in multiple cells.14–16 RelA-P-Ser536 is a feature of HM in culture and diseased human liver and can be controlled by autocrine renin-angiotensin system (RAS) signaling.9 However, angiotensin blockade does not completely inhibit RelA-P-Ser536 in cultured HM, suggesting that other stimuli or signaling pathways regulate RelA-P-Ser536. Here, we report that in human HM, as with other cells, this modification can be induced by TNF-α stimulation.15,17 We show that a cell-permeable RelA-P-Ser536 competing peptide (P6) inhibits RelA-P-Ser536 in HM, both in vitro and in vivo, and has antifibrotic, but not anti-inflammatory, effects in multiple models of liver injury.

Thymic stromal lymphopoietin (TSLP), interleukin-25 (IL-25), and IL-33 are important initiators of type 2–associated mucosal inflammation and immunity. However, their role in the maintenance of progressive type 2 inflammation and fibrosis is much less clear. Using chronic models of helminth infection and allergic lung inflammation, we show that collective disruption of TSLP, IL-25, and IL-33 signaling suppresses chronic and progressive type 2 cytokine–driven inflammation and fibrosis. In a schistosome lung granuloma model or during chronic Schistosoma mansoni infection in the liver, individual ablation of TSLP, IL-25, or IL-33/ST2 had no impact on the development of IL-4/IL-13–dependent inflammation or fibrosis. However, significant reductions in granuloma-associated eosinophils, hepatic fibrosis, and IL-13–producing type 2 innate lymphoid cells (ILC2s) were observed when signaling of all three mediators was simultaneously disrupted. Combined blockade through monoclonal antibody (mAb) treatment also reduced IL-5 and IL-13 expression during primary and secondary granuloma formation in the lungs. In a model of chronic house dust mite–induced allergic lung inflammation, combined mAb treatment did not decrease established inflammation or fibrosis. TSLP/IL-33 double-knockout mice treated with anti–IL-25 mAb during priming, however, displayed decreased inflammation, mucus production, and lung remodeling in the chronic phase. Together, these studies reveal partially redundant roles for TSLP, IL-25, and IL-33 in the maintenance of type 2 pathology and suggest that in some settings, early combined targeting of these mediators is necessary to ameliorate progressive type 2–driven disease.

Fibrotic remodelling of lung parenchymal and airway compartments is the major contributor to life-threatening organ dysfunction in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD). Since transforming growth factor-β1 (TGF-β1) is believed to play a key role in disease pathogenesis and markers of oxidative stress are also commonly detected in bronchoalveolar lavage (BAL) from such patients we sought to investigate whether both factors might be interrelated. Here we investigated the hypothesis that oxidative stress to the lung epithelium promotes fibrotic repair by driving epithelial-to-mesenchymal transition (EMT) via the augmentation of TGF-β1. We show that in response to 400μM hydrogen peroxide (H(2)O(2)) A549 cells, used a model for alveolar epithelium, and human primary bronchial epithelial cells (PBECs) undergo EMT displaying morphology changes, decreased expression of epithelial markers (E-cadherin and ZO-1), increased expression of mesenchymal markers (vimentin and α-smooth muscle actin) as well as increased secretion of extracelluar matrix components. The same oxidative stress also promotes expression of TGF-β1. Inhibition of TGF-β1 signalling as well as treatment with antioxidants such as phenyl tert-butylnitrone (PBN) and superoxide dismutase 3 (SOD3) prevent the oxidative stress driven EMT-like changes described above. Interventions also inhibited EMT-like changes. This study identifies a link between oxidative stress, TGF-β1 and EMT in lung epithelium and highlights the potential for antioxidant therapies to limit EMT and its potential contribution to chronic lung disease.

Epithelial to mesenchymal transition (EMT) is a process by which an epithelial cell alters its phenotype to that of a mesenchymal cell and plays a critical role in embryonic development, tumour invasion and metastasis and tissue fibrosis. Transforming growth factor-β1 (TGF-β1) continues to be regarded as the key growth factor involved in driving EMT however recently tumour necrosis factor α (TNFα) has been demonstrated to accentuate TGF-β1 driven EMT. In this study we investigate how various signalling pathways contribute to this accentuated effect. A549 cells were treated with TGF-β1 (10 ng/ml), TNFα (20 ng/ml) or a combination of both for 72 h and EMT assessed. The effect of selective inhibition of the SMAD, MAPK and NF-κB pathways on EMT was assessed. A549 cells treated with TGF-β1 downregulate the expression of epithelial markers, increase the expression of mesenchymal markers, secrete matrix-metalloproteinases and become invasive. Significantly, TGF-β1 driven EMT is accentuated by co-treatment with TNFα. SMAD 3 inhibition attenuated TGF-β1 driven EMT but has no effect on the accentuation effect of TNFα. However, inhibiting IKKβ blocked both TGF-β1 driven EMT and the accentuating action of TNFα. Inhibiting p38 and ERK signalling had no effect on EMT. TNFα accentuates TGF-β1 driven EMT in A549 cells via a SMAD 2/3 independent mechanism involving the NF-κB pathway independent of p38 and ERK 1/2 activation.

Epithelial-to-mesenchymal transition (EMT) has been implicated in the dysregulated epithelial wound repair that contributes to obliterative bronchiolitis (OB) after lung transplantation. Acquisition of Pseudomonas aeruginosa in the transplanted airway has been shown to be a risk factor for the development of OB. We investigated the potential of P. aeruginosa to drive EMT in primary bronchial epithelial cells (PBECs) isolated from lung transplant recipients. Changes in the expression of epithelial and mesenchymal markers was assessed in cells challenged with clinical isolates of P. aeruginosa or co-cultured with P. aeruginosa-activated monocytic cells (THP-1) in the presence or absence of transforming growth factor (TGF)-β1. P. aeruginosa did not drive or accentuate TGF-β1-driven EMT directly. Co-culturing P. aeruginosa-activated THP-1 cells with PBECs did not drive EMT. However, co-culturing P. aeruginosa-activated THP-1 cells with PBECs significantly accentuated TGF-β1-driven EMT. P. aeruginosa, via the activation of monocytic cells, can accentuate TGF-β1-driven EMT. These in vitro observations may help explain the in vivo clinical observation of a link between acquisition of P. aeruginosa and an increased risk of developing OB.

The formation of a heterotetrameric complex between annexin 2 (anx 2) and S100A10 plays an important role in regulating the cellular distribution and biochemical properties of anx 2. A major distinction between the anx 2-S100A10 complex and other annexin-S100 complexes is that S100A10 binding to anx 2 occurs independently of calcium. Here we describe a cyclic 3',5'-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA, EC 2.7.1.37)-dependent mechanism regulating anx 2-S100A10 complex formation and its interaction with the transient receptor potential vanilloid type 6 channel (TRPV6) in airway and gut epithelia. In both 16HBE14o- and Caco-2 cells, forskolin (FSK) stimulated increased anx 2-S100A10 complex formation, which was attenuated by either PKA inhibitors or calcineurin A (CnA) inhibitors. The anx 2-S100A10 complex association with TRPV6 was dependent on FSK-induced CnA-dependent dephosphorylation of anx 2. Analysis of the significance of the cAMP/PKA/CnA pathway on calcium influx showed that both PKA and CnA inhibitors attenuated Ca(45) uptake in Caco-2, but not 16HBE14o-, cells. Thus, the cAMP/PKA/CnA-induced anx 2-S100A10/TRPV6 complex may require additional factors for calcium influx or play a role independent of calcium influx in airway epithelia. In conclusion, our data demonstrates that cAMP/PKA/CnA signalling is important for anx 2-S100A10 complex formation and interaction with target molecules in both absorptive and secretory epithelia.

Chronic lung allograft dysfunction, manifesting as bronchiolitis obliterans syndrome (BOS), is characterized by airway epithelial injury, impaired epithelial regeneration and subsequent airway remodeling. Increased cellular senescence has been reported in renal and liver allografts affected by chronic allograft dysfunction but the significance of cellular senescence in the airway epithelium of the transplanted lung is unknown. Thirty-four lung transplant recipients, 20 with stable graft function and 14 with BOS, underwent transbronchial lung biopsy and histochemical studies for senescence markers in small airways. Compared to nontransplant control lung tissue (n = 9), lung allografts demonstrate significantly increased airway epithelial staining for senescence-associated beta galactosidase (SA beta-gal) (p = 0.0215), p16(ink4a) (p = 0.0002) and p21(waf1/cip) (p = 0.0138) but there was no difference in expression of these markers between stable and BOS affected recipients (p > 0.05). This preliminary cross-sectional study demonstrates that cellular senescence occurs with increased frequency in the airway epithelium of the lung allograft but does not establish any association between airway epithelial senescence and BOS. A prospective longitudinal study is required to better address any potential causal association between airway epithelial senescence in stable allograft recipients and the subsequent development of BOS.