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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The IL-1 cytokine family comprises 11 members (7 ligands with agonist activity, 3 receptor antagonists and 1 anti-inflammatory cytokine) and is recognised as a key mediator of inflammation and fibrosis in multiple tissues including the lung. IL-1 targeted therapies have been successfully employed to treat a range of inflammatory conditions such as rheumatoid arthritis and gouty arthritis. This review will introduce the members of the IL-1 cytokine family, briefly discuss the cellular origins and cellular targets and provide an overview of the role of these molecules in inflammation and fibrosis in the lung.

Arthrofibrosis is a fibroproliferative disease characterised by excessive deposition of extracellular matrix components intra-articularly leading to pain and restricted range of movement. Although frequently observed following total knee arthroplasty (TKA) no therapeutic options exist. A pilot study demonstrated that intra-articular injection of Anakinra, an IL-1R antagonist, improved range of movement and pain in patients with arthrofibrosis however the mechanism of action is unknown. We hypothesise that IL-1α/β will drive an inflammatory phenotype in fibroblasts isolated from the knee, therefore identifying a potential mechanism of action for Anakinra in arthrofibrosis following TKA. Fibroblasts isolated from synovial membranes and infra-patellar fat pad of patients undergoing TKA express high levels of IL-1R1. Stimulation with IL-1α/β induced a pro-inflammatory phenotype characterised by increased secretion of GMCSF, IL-6 and IL-8. No significant difference in the inflammatory response was observed between fibroblasts isolated from synovial membrane or infra-patellar fat pad. IL-1α/β treatments induced a pro-inflammatory phenotype in fibroblasts from both synovial membrane and infra-patellar fat pad and therefore Anakinra can likely have an inhibitory effect on fibroblasts present in both tissues in vivo. It is also likely that fibroblast responses in the tissues are controlled by IL-1α/β availability and not their ability to respond to it.

Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.

Bacterial infections after lung transplantation cause airway epithelial injury and are associated with an increased risk of developing bronchiolitis obliterans syndrome. The damaged epithelium is a source of alarmins that activate the innate immune system, yet their ability to activate fibroblasts in the development of bronchiolitis obliterans syndrome has not been evaluated. Two epithelial alarmins were measured longitudinally in bronchoalveolar lavages from lung transplant recipients who developed bronchiolitis obliterans syndrome and were compared to stable controls. In addition, conditioned media from human airway epithelial cells infected with Pseudomonas aeruginosa was applied to lung fibroblasts and inflammatory responses were determined. Interleukin‐1 alpha (IL‐1α) was increased in bronchoalveolar lavage of lung transplant recipients growing P. aeruginosa (11.5 [5.4–21.8] vs. 2.8 [0.9–9.4] pg/mL, p, This study demonstrates that a Pseudomonas aeruginosa challenge of lung epithelial cells causes the release of IL‐1α and the induction of an inflammatory phenotype in lung fibroblasts, and provides evidence of an association between infection with P. aeruginosa, IL‐1α levels in bronchoalveolar lavage fluid, and the development of bronchiolitis obliterans syndrome following lung transplantation.

The roles of macrophages in type 2-driven inflammation and fibrosis remain unclear. Here, using CD11b-diphtheria toxin receptor (DTR) transgenic mice and three models of interleukin 13 (IL-13)-dependent inflammation, fibrosis, and immunity, we show that CD11b(+) F4/80(+) Ly6C(+) macrophages are required for the maintenance of type 2 immunity within affected tissues but not secondary lymphoid organs. Direct depletion of macrophages during the maintenance or resolution phases of secondary Schistosoma mansoni egg-induced granuloma formation caused a profound decrease in inflammation, fibrosis, and type 2 gene expression. Additional studies with CD11c-DTR and CD11b/CD11c-DTR double-transgenic mice suggested that macrophages but not dendritic cells were critical. Mechanistically, macrophage depletion impaired effector CD4(+) T helper type 2 (Th2) cell homing and activation within the inflamed lung. Depletion of CD11b(+) F4/80(+) Ly6C(+) macrophages similarly reduced house dust mite-induced allergic lung inflammation and suppressed IL-13-dependent immunity to the nematode parasite Nippostrongylus brasiliensis. Consequently, therapeutic strategies targeting macrophages offer a novel approach to ameliorate established type 2 inflammatory diseases.

Distinct phenotypes of chronic lung allograft dysfunction (CLAD) after lung transplantation are emerging with lymphocytic bronchiolitis (LB)/azithromycin reversible allograft dysfunction (ARAD), classical or fibrotic bronchiolitis obliterans syndrome (BOS), and restrictive allograft syndrome (RAS) proposed as separate entities. We have additionally identified lung transplant recipients with prior LB, demonstrating persistent airway neutrophilia (PAN) despite azithromycin treatment. The aim of this study was to evaluate differences in the airway microenvironment in different phenotypes of CLAD. Bronchoalveolar lavage (BAL) from recipients identified as stable (control), LB/ARAD, PAN, BOS, and RAS were evaluated for differential cell counts and concentrations of IL-1α, IL-1β, IL-6, IL-8, and TNF-α. Primary human bronchial epithelial cells were exposed to BAL supernatants from different phenotypes and their viability measured. BOS and RAS showed increased BAL neutrophilia but no change in cytokine concentrations compared with prediagnosis. In both LB/ARAD and PAN, significant increases in IL-1α, IL-1β, and IL-8 were present. BAL IL-6 and TNF-α concentrations were increased in PAN and only this phenotype demonstrated decreased epithelial cell viability after exposure to BAL fluid. This study demonstrates clear differences in the airway microenvironment between different CLAD phenotypes. Systematic phenotyping of CLAD may help the development of more personalized approaches to treatment. ispartof: TRANSPLANT INTERNATIONAL vol:27 issue:8 pages:857-867 ispartof: location:Switzerland status: published

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL-1β and IL-8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL-1β than alternatively activated macrophages and dramatically accentuate TGF-β1-driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL-1β, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti-TNFα treatment improved forced expiratory volume in 1 second and 6-min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.

Physiological lung epithelial wound repair is a complex, highly orchestrated process presenting numerous points where dysregulation may occur, leading to the development of several pulmonary disorders. Current studies are limited by a lack of relevant lung injury models, with much work relying on other organ models such as the skin or in vitro cultures. However, much promising investigative work is being undertaken, some of which is described in this article. This article attempts to describe the processes required to heal a severe wound to the airway epithelium, characteristic of several chronic pulmonary disorders, highlighting areas where dysregulation may occur, which in turn leads to the development or continuation of a disease state.

Introduction Survival after lung transplantation is limited to a median of 5-year due to development of bronchiolitis obliterans syndrome (BOS). BOS is the clinical manifestation of chronic allograft dysfunction, characterised by inflammation and fibrosis of small/medium-sized airways leading to airflow obstruction. Numerous insults to the transplanted lungs have been associated with BOS development. Alarmins are cell derived danger signals released from damaged tissue which activate innate and adaptive immune responses. We hypothesised that the release of alarmins into the airway compartment after lung transplantation may contribute to BOS pathogenesis. Methods A retrospective longitudinal study of 52 lung transplant recipients from 2005 to present was performed (26 recipients developed BOS; 26 remained free from BOS). All recipients had lung function and bronchoalveolar lavage (BAL) performed at 1, 3, 6 and 12 months post transplant. Further samples were taken if the diagnostic criteria for BOS were fulfilled. A total of 214 BAL samples were analysed by ELISA for the alarmins Interleukin-1a (IL-1a) and High Motility Group-Box1 (HMG-B1). Data were analysed using Mann–Whitney tests. Results Both BOS and non-BOS recipients with culture positive BAL samples had significantly higher concentrations of IL-1a and HMG-B1 than culture negative samples. Due to the confounding effect of infection, our analysis excluded 90 BAL samples with positive cultures. Concentrations of IL-1a were significantly higher in culture negative BAL from BOS patients (median 2.411, range [AJF1] 0.073–19.078 pg/ml) than from Non-BOS patients (median 1.424, range [AJF2] 1.159–17.41 pg/ml; p=0.001). No significant difference in HMG-B1 concentrations between the two groups was observed (BOS median 58.906, range 0–197.5; Non-BOS median 76.25, range 0–211.563 ng/ml; p=0.2378). Longitudinal measurements of IL-1a in BOS patients showed significantly higher levels 3 months before or after BOS diagnosis (median 3.935, range 1.122–13.544 pg/ml), compared to >3 months before BOS diagnosis (median 2.015, range 0.073–14.669 pg/ml; p=0.0153). There was no such difference in HMG-B1 concentrations (p=0.9164). Conclusions An increase in the alarmin IL-1a, but not HMG-B1, is associated with BOS development. The cellular source of IL-1a requires further evaluation but may be a marker of airway epithelial injury and/or play a mechanistic role in BOS development via its secretion by other cell types.

To the Editor: IL-13 is a pleiotropic cytokine that provokes diverse pathophysiological outcomes. While its effect during gastrointestinal (GI) helminth infection is prototypic of a protective Th2 response (increased peristalsis, goblet cell hyperplasia and mucus secretion, eosinophil recruitment, fibroblast activation and wound repair), temporal or spatial dysregulation of this response is thought to underlie diseases such as asthma, allergic hyperreactivity and organ fibrosis (1). IL-13 signals via the IL-13Rα1/IL-4Rα heterodimer to induce several genes specific to Th2 inflammation including CCL26, CCL11, POSTN, and MUC5AC (2). IL-13Rα2 binds IL-13 with significantly higher affinity, and the secreted form found in mice acts as a decoy receptor, protecting mice from IL-13 induced immunopathology. However humans do not alternatively splice IL13RA2 transcript and therefore IL-13Rα2 is only expressed as a cell surface protein (3). The factors that regulate the expression of IL-13Rα2 are unclear and the biological function of the endogenously expressed IL-13Rα2 remains controversial (4, 5). In this study, we show that the inflammatory cytokines TNFα and IL-17, often associated with severe asthma, synergize to induce IL-13Rα2 in primary human lung fibroblasts and mouse lungs, and evaluate its biological role using a novel IL-13Rα2 blocking antibody. Primary human neonatal lung fibroblasts (NLF) were grown to confluence and incubated with TNFα, IL-4 and IL-17 for 72 hours, at which time cells lysates were analyzed by qPCR with primers specific for IL13RA2 (Methods in Online Repository). While TNFα and IL-4 synergistically induced IL13RA2 as described (6), a combination of TNFα and IL-17 induced higher expression (Fig 1A). In contrast, IL13RA1 decreased in the presence of TNFα (Fig 1B). Fig 1 TNFα and IL-17 synergistically induce IL-13Rα2. Primary NLF (A, B) were incubated with specified cytokines for 72 hr. Kinetics of IL13RA2 expression in NLF, assayed at 24, 48 or 72 hr post stimulation (C) and decay post-cytokine withdrawal ... IL-13Rα2 mRNA progressively increased over time, peaking at 72 hours (Fig 1C) and longer incubations did not consistently result in any further increase (data not shown); therefore, 72 hrs was chosen as the ideal endpoint for subsequent experiments. To determine whether IL-13Rα2 expression was sustainable, NLF were incubated with a combination of TNF/IL-17 for 72 hours, after which the supernatants were removed and the wells rinsed and replenished with fresh media without added cytokines. Upon removal of TNFα and IL-17, IL-13Rα2 expression declined between 24 hours and 72 hours post-wash, though remained elevated (Fig 1D). We confirmed that primary lung fibroblasts derived from healthy adult donors (ALF) also manifested similar synergy to TNFα and IL-17 (Fig E1). Using flow cytometry, we verified that the transcriptional induction of IL-13Rα2 was associated with augmented surface expression of protein (Fig 1E-F). In order to ascertain if this phenomenon can be recapitulated in vivo, we administered multiple doses of the cytokines into mouse airways. IL-13Rα2 transcripts were strikingly higher in the lungs of mice that received both TNF and IL-17, while IL-13Rα1 message changed negligibly (Fig 1G). Next, we examined whether TNF/IL-17-induced expression of IL-13Rα2 was capable of altering the biological effects of IL-13. NLFs were incubated with TNF/IL-17 for 72 hours to upregulate IL-13Rα2. Subsequently, the wells were washed with media, and treated with IL-13 in increasing concentrations for 24 hours. Cellular RNA was assayed by real time quantitative PCR for CCL26 (eotaxin-3) expression, as a marker for the canonical IL-13 signaling through IL-13Rα1. Whereas the expression of CCL26 increased in a dose-dependent fashion upon IL-13 stimulation in control wells where IL-13Rα2 was at baseline levels, CCL26 expression was abrogated in conditions where IL-13Rα2 expression was augmented (Fig 2A-B). Similar data were obtained with ALF (Fig E2). Fig 2 NLF were treated with TNFα and IL-17 for 72 hr, washed and then treated with IL-13 in increasing concentrations (0.1 to 2.5 ng/ml) for 24 hours. Increased IL13RA2 induction is associated with loss of IL-13 induced CCL26 production (A-B). Recovery ... While inhibition of IL-13 activity in the setting of increased IL-13Rα2 was likely due to the IL-13Rα2 receptor functioning as a decoy, a number of other alternative explanations, such as concomitant down-regulation of IL-13Rα1 or dampening of intracellular signaling networks could have also led to the attenuation of IL-13 bioactivity following TNF/IL-17 treatment. To investigate these possibilities, NLF were first incubated with TNF/IL-17 to induce high IL-13Rα2 expression and prior to the addition of IL-13, some wells were treated with a highly selective neutralizing anti-human IL-13Rα2 monoclonal antibody. Subsequently, wells were treated with IL-13 in increasing concentrations for 24 hours and CCL26 expression was assayed by qPCR. As before, in wells expressing basal levels of IL-13Rα2, CCL26 was induced in a dose-dependent fashion following IL-13 stimulation. In wells were IL-13Rα2 was induced with TNF/IL-17, CCL26 expression was suppressed. However, in wells treated with anti-IL-13Rα2 antibody, addition of IL-13 resulted in restoration of CCL26 expression in a dose-dependent fashion, confirming that the diminution of IL-13 activity by TNF/IL-17 was due to increased expression of IL-13Rα2 (Fig 2C). While the above results confirm potent decoy activity for IL-13Rα2 (7), they fail to explore its signaling activity, which has been proposed in some studies (5). To uncover possible signaling potential for this ligand-receptor pair in fibroblasts, we performed a transcriptome-wide microarray analysis, in which the effect of IL-13 stimulation in NLF with or without TNF/IL17 pretreatment, in the presence or absence of the anti-IL-13Rα2 mAb were compared. With this approach, the effects of IL-13 signaling through IL-13Rα1 and IL-13Rα2 could be distinguished. CCL26 was the most abundant IL-13 induced transcript in this genome-wide screen, upregulated 12.3-fold (i.e. 23.6, q-value=6×10−8) over baseline (Fig 2D and E3). Upon TNF/IL17 pretreatment, this diminished to 3.2-fold (21.7), in accord with the above-described IL-13Rα2 mediated inhibition. Surprisingly however, while IL-13 binding to IL-13Rα2 reduced the transcript abundance of CCL26, it did not result in any additional statistically significant changes in the transcriptome profile of fibroblasts (Fig 2D and E3) that were distinguishable from the pretreatment controls . Blocking IL-13Rα2 with the Ab restored IL-13-induced CCL26 expression (Fig 2D and E3). These data indicate that in NLF, IL-13 binding to the high affinity IL-13Rα2 serves to dampen canonical IL-13 signaling, as opposed to inducing other signaling pathways. Asthma syndrome is a heterogeneous mixture of distinct phenotypic subtypes and only cohorts that have a ‘IL-13 high’ signature, are indeed likely to benefit from therapeutic modalities that block IL-13/IL-13Rα1 interactions (8). Steroid-resistant and severe asthma subtypes are often associated with neutrophilia, TNFα and IL-17 activity (9) and therefore may not benefit from blockade of IL-13, and perhaps the mechanisms we describe above already operate in these patients to quench endogenous IL-13 activity via IL-13Rα2. Indeed, due caution must be exercised, as blocking IL-13/IL-13Rα1 interactions in these patients might actually lead to disease aggravation, as IL-13 has been shown to down modulate inflammation induced by TNFα and IL-17 (10).

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-4Rαflox/deltaLysMCre 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 F4/80hiCD11bhi macrophages, in contrast to resident tissue macrophages, express lower levels of Lyz2 explaining why this population resists LysMCre-mediated deletion. We show that in response to IL-4 and IL-13, Lyz2loIL-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 Lyz2hiIL-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., Author Summary Chronic injury and inflammation lead to irreversible fibrosis in a range of diseases and infections. Macrophages alternatively activated by the immune system are capable of regulating inflammation and fibrosis, but our understanding of the source and function of these cells is incomplete. Mice genetically engineered to specifically prevent macrophages from becoming alternatively activated have been used to study the cells' role following infection with the parasite, Schistosoma mansoni. To our surprise, we found these mice prevent alternative activation only in macrophages that have had time to mature and some, perhaps more nascent, macrophages can become alternatively activated following exposure to S. mansoni eggs. We detected lower expression of Lyz2 gene in these cells, leading to less expression of the enzyme excising the receptor gene necessary for alternative activation. Following S. mansoni infection, the livers of these mice have similar levels of fibrosis but significantly more inflammation compared to controls. We conclude that during schistosomiasis, distinct populations of alternatively activated macrophages control inflammation and fibrosis: macrophages expressing low levels of Lyz2 express Arg1 and thus are sufficient to control fibrosis, while more mature Lyz2-expressing macrophages are required for downmodulation of egg-induced inflammation in chronic schistosomiasis.

Background Chronic obstructive pulmonary disease (COPD) is characterised by repeated viral or bacterial exacerbations which increase morbidity, mortality and accelerate lung function decline. Chronic bacterial colonisation, frequently recognised in stable COPD patients, may contribute to airway inflammation and promote disease progression. Cigarette smoke has previously been shown to alter responses to LPS via the TLR-4 receptor in cell lines. We hypothesised that cigarette smoke would suppress the innate immune responses of airway epithelial cells and macrophages to Haemophilus influenzae (HI) favouring persistence. Methods Primary bronchial epithelial cells (PBEC) and macrophage-like cells (THP-1) were incubated with varying concentrations of cigarette smoke extract (CSE) before stimulation with whole cell lysates of HI prepared from clinical isolates. Cell viability was assessed by flow cytometry and PBEC proliferation was analysed using an XTT assay. Pro-inflammatory cytokine release (IL-8, TNFα, IL-1β) was assessed in PBEC and PMA-differentiated THP-1 cells by ELISA. Results CSE concentrations ≤10% had no significant effect on PBEC or THP-1 cell viability, however CSE concentrations >5% inhibited PBEC proliferation (p 0.05). Co-treatment of THP-1 cells with HI + CSE increased IL-8 release (61%), but reduced TNF-α (40%) and IL-1β (24%) release compared to treatment with HI alone (n=3, p Conclusions CSE alters epithelial cell and macrophage responses to bacterial pathogens by promoting release of the neutrophil chemokine IL-8 whilst at the same time suppressing TNFα and IL-1 β release. This dysregulation may promote continued neutrophil inflammation in the airway whilst insufficiently clearing pathogens and could be an important mechanism in COPD.

Background Alarmins are molecular ‘danger signals’ released by injured cells that can contribute to the innate immune response by activating immune cells via multiple receptors including Toll-like receptors (TLR), Nod-like receptors (NLR) and the receptor for advanced glycationend products (RAGE). Pulmonary fibrosis is associated with the upregulation of alarmins such as Interleukin 1 α (IL-1α) and High mobility group box 1 (HMGB1) in bronchoalveolar lavage fluid (BAL), however it remains unclear whether alarmins can contribute directly to the fibrogenic process by interacting with fibroblasts. We hypothesised that alarmins released from damaged epithelial cells act as damage associated molecular patterns (DAMPs) which are recognised by fibroblasts and lead to their activation. Methods The 16HBE14o- human bronchial epithelial cell line was damaged by hydrogen peroxide (H 2 O 2 ) induced oxidative stress and alarmin release (Heat Shock Protein 60 (HSP-60), HMGB1, IL-1α) measured via ELISA. MRC5 human lung fibroblasts were treated with media from damaged lung epithelia and cell proliferation (XTT proliferation assay), phosphorylation of downstream TLR signalling molecules (interleukin 1 receptor associated kinase 1 (IRAK1), TGFβ associated kinase 1 (TAK1)—western blotting) and gene expression of proinflammatory cytokines (interleukin 6 (IL-6) and Interferon β (IFNβ)—qRT-PCR) assessed. Results Conditioned media from 16HBE14o- cells damaged with 200 μM H 2 O 2 contained elevated concentrations of HSP-60 (16.7 vs 0.64 ng/ml; p Conclusions The results suggest that alarmins such as the intrerleukin-1 family, released by damaged human lung epithelia may be implicated in activation of fibroblasts and could contribute to fibrogenic responses in lung disease. However, further studies are required to confirm relative importance within the family and reveal mechanisms of action.

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.

Background IL33, a member of the IL-1 superfamily has been implicated in the pathogenesis of asthma and postulated to play an aetiological role in several non-pulmonary fibrotic diseases. IL-33 expression has predominantly been reported in mucosal surfaces. We hypothesised lung epithelium might act as a source of IL33 release into the microenvironment as a damage associated molecular pattern (DAMP); propagating pro-inflammatory/fibrotic pathways. We evaluated tissue expression of IL-33 from a range of chronic lung diseases and assessed release in response to airway epithelial damage in vitro . Finally, we determined if IL-33 was detectable in BAL of lung transplant patients developing Bronchiolitis Obliterans Syndrome (BOS). Methods Expression of IL-33 in chronic lung disease was evaluated by immunohistochemistry from patients with IPF(n=3), COPD(n=3), Bronchiectasis(n=3) and CF(n=3). Epithelial damage was induced in Primary Bronchial Epithelial Cells and 16HBE14o- cells by oxidative stress or freeze/thaw and release of IL33 evaluated by ELISA and Western Blot. BAL was prospectively collected(n=207) from post lung transplant patients(n=26) and IL-33 concentration measured by ELISA. BAL samples were classified as Non –BOS(n=116) or BOS(n=91) on the basis of histological and clinical data. Co-existing presence of infection was identified by standard microbiological culture. Results IL33 was strongly expressed in airway epithelia with a predominant nuclear location. This was more marked in chronic lung diseases with an infective aetiology (CF and bronchiectasis). IL33 was not detectable in response to airway epithelial cell injury in vitro . However, IL-33 levels were elevated in BAL of individuals with BOS(p=0.011). Longitudinal analysis of 26 individuals spanning the time frame of initial BOS diagnosis demonstrated a trend towards increased concentration of IL-33 in BAL in the immediate period following BOS diagnosis. There was a strong association between elevated IL-33 levels and the presence of BOS with concomitant infection(p Conclusion IL33 is strongly expressed in the airway epithelium in chronic respiratory diseases but does not appear to be passively released as a DAMP in airway epithelial cell damage. Elevated IL33 with infection in the post transplant population suggests sources other than epithelium may be important but further work is required to evaluate the relevance and significance of these observations.

Background Activation of the innate immune system plays a key role in exacerbations of chronic lung disease. Myeloid cells are classically considered to drive innate immune responses yet the potential of fibroblasts to act as immune cells has been postulated. We hypothesized that alarmins released from lung epithelium during environmental insults such as oxidant injury and viral infection might induce innate immune responses in lung fibroblasts. Methods Human bronchial epithelial cells (BECs) and human lung fibroblasts (HLFs) were cultured from brushings taken from lung transplant recipients and resected lung tissue respectively. Cytokine concentrations were measured by ELISA or multiplex platform (MSD). Gene expression was assessed by qRT-PCR. Wild-type and Il1a-/- mice were infected with Influenza (PR8). Data were analysed using t-Student or Mann-Whitney U Test. Correlations were assessed using Spearman rank correlation coefficient. Results Conditioned media from PBECs subjected to oxidant injury contained elevated levels of alarmins. Treatment of HLFs with conditioned media significantly upregulated proinflammatory cytokine expression. Anti-IL-1α or IL-1Ra significantly reduced induction of IL-8 (93% and 95%), IL-6 (90% and 91%), MCP-1 (92% and 93%) and GM-CSF (95% and 94%). Anti-IL-1β had no effect. Co-stimulation with Poly I:C significantly accentuated the IL-1 α induced inflammatory phenotype in HLFs. Bronchoalveolar lavage (BAL) form Influenza infected wild-type mice contains elevated levels of IL-1α and activates innate immune signalling in wild-type murine lung fibroblasts (MLFs) but not Il1r1-/- MLFs. BAL from Il1a-/- mice had no effect on MLFs and demonstrated a blunted neutrophilic response to Influenza. Clinically we show that IL-1α is increased in BAL of lung transplant recipients with infections and within 3 months of developing bronchiolitis obliterans syndrome (BOS) (p Conclusions We propose a new paradigm of innate immune signalling in exacerbations of lung disease, where epithelial damage triggers a potent inflammatory phenotype in resident fibroblasts. The pivotal role of IL-1α in this process is accentuated in the presence of viral infection. This novel pathway warrants further evaluation of its therapeutic potential to limit the repeated cycles of injury and exacerbation in chronic lung diseases.

Introduction Obliterative Bronchiolitis (OB) is characterised by fibrotic obliteration of small airways which adversely affecting graft function and survival after lung transplantation. It has been shown in vitro and in vivo that primary bronchial epithelial cells (PBEC) from the transplanted lung can undergo epithelial to mesenchymal transition (EMT) and this process may contribute to the development of OB. We have shown that activated macrophages can disrupt epithelial wound repair by accentuating TGF-β1-driven EMT. We hypothesised that this effect might be limited to macrophages with an M1 phenotype and that their secretory products might be a target for limiting the inflammatory accentuation of EMT. Methods and materials The THP-1 monocytic cell line was stimulated with clinical isolates of Pseudomonas aeruginosa (PA) and the effect of the activated cells or conditioned media on TGF-β1-driven EMT assessed in PBEC (Western blotting, confocal microscopy). In addition, THP-1 cells were differentiated to an M1 phenotype by treatment with IFNγ and an M2 phenotype with IL-4/IL-13 and cytokine release (ELISA) and their effect on TGF-β1 driven EMT assessed. The effect of blocking TNFα secreted from activated THP-1 cells on EMT was assessed using an anti-TNFα antibody. Results Treatment with TGF-β1+activated THP-1 cells had no effect on EMT marker expression (p>0.05 n=6). However, co-treatment with TGF-β1+conditioned media from activated THP-1 cells dramatically accentuated TGF-β1-driven EMT (p Conclusion The secretory products of M1, but not M2, macrophages significantly accentuate TGF-β1 driven EMT. TNFα appears to be a major constituent of this accentuating action. This raises the possibility that either TNFα targeted therapies or modulation of macrophage phenotype may inhibit the inflammatory accentuation of EMT in airway epithelium.