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BackgroundCystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation.MethodsBlood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR.ResultsCF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF.ConclusionsCF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

Background Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. Methods Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography–tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. Results Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1−/− mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. Conclusions We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.

Background: Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. Methods: A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age-and sex-matched volunteers. Growth of pathogens at >10(4) colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from "non-VAP". Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. Results: Seventy-two patients had recoverable lavaged-24% had VAP. BALF interleukin-1 beta (IL-1 beta), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1 alpha were significantly higher in the VAP group (all p Conclusions: BALF IL-1 beta and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.

Introduction The immediate detection of pathogens in the lungs of patients with unexplained pulmonary opacities in the intensive care unit would represent a significant advance in their management. An optical imaging strategy, including the endobronchial administration of bacterial specific Smartprobes, would confer a number of advantages over conventional techniques such as bronchoalveolar lavage, principally real-time detection to immediately inform antimicrobial therapy. The aims of this study were to fluorescently label and iteratively develop anti-microbial peptides to image bacteria in situ in the lung using fibered confocal fluorescence microscopy (FCFM). Methods Antimicrobial peptides (AMP) have been synthesised on a dendrimeric scaffold (AMP-1) and conjugated to an environmentally sensitive fluorophore called NBD, following the continuous development a linear counterpart. A further construct consists of an AMP with gram-selectivity conjugated to the NBD fluorophore (AMP-2). These are combined with FCFM to allow distal alveolar imaging at micron resolution in an ex vivo ovine model of bacterial infection. Results AMP-1 demonstrates bacterial binding affinity in a concentration dependent manner and labels a diverse panel of bacteria, including a panel consisting of >70% of ventilator-associated pneumonia causing organisms and the pathogenic fungi Aspergillus fumigatus. AMP-1 demonstrates significantly higher fluorescence over isomolar linear equivalents for E. coli, K. pneumoniae, P. aeruginosa , MSSA, A. baumannii and S. pneumoniae (all p E. coli, K. pneumoniae, P. aeruginosa and MSSA in situ in an ex vivo ovine model when instilled endobronchially and imaged with FCFM (pin vitro and remains selective for gram-negative bacteria over mammalian cells. In the ex-vivo model AMP-2 selectively labels the gram-negative bacterial segments ( P. aeruginosa, K. pneumonia and E. coli ) over the gram-positive (MSSA, MRSA and S. pneumoniae ) or control pulmonary segments (all p Conclusions A Smartprobe/FCFM strategy to immediately detect bacteria with gram selectivity in size relevant pre-clinical models is described, and are undergoing first-in-man translation.

Background—The success of haematopoietic (bone marrow or peripheral blood) stem cell transplantation (SCT) is compromised by pulmonary complications. We hypothesised that a proinflammatory alveolar microenvironment, reflected in alveolar macrophage (AM) cytokine production, would predispose to such complications. Methods—AM were isolated from adult SCT recipients by bronchoalveolar lavage before SCT (n=32) and during posttransplant pancytopenia (n=23). Concentrations of tumour necrosis factor (TNF)AE, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-1‚, IL-6, and IL-8 in 24 hour AM culture medium were measured by enzyme linked immunosorbent assay and compared with both the occurrence of postSCT lung disease and with subjects’ previous respiratory histories. Results—Eleven subjects developed lung disease within 6 months of SCT. These subjects had higher median pretransplant AM TNFAE (8 (IQR 1‐8) v 2 (1‐5) ng/10 6 AM,p=0.01,median diVerence (D) = 3, 95% CI 0.1 to 7), GM-CSF (5 (0.7‐8) v 0.2 (0.1‐0.8), p=0.006, D = 4, 95% CI 0.5 to 7), and IL-6 (0.5 (0.1‐1) v 0.1 (0.02‐0.3), p=0.049, D = 0.3, 95% CI 0.0002 to 1) production than remaining subjects; IL-1‚ and IL-8 did not diVer. During pancytopenia high AM GM-CSF production again predicted later lung disease (1 (0.7‐9) v 0.1 (0.06‐0.3), p=0.01, D = 1, 95% CI 0.1 to 6). A history of recent chest disease was associated with high AM TNFAE and GM-CSF production and with postSCT lung disease. Pre-SCT lung function was unrelated to post-SCT lung disease. Conclusions—Recent respiratory disease and persistent proinflammatory AM behaviour detectable before transplantation are associated with lung disease following SCT. These associations may prove useful in pre-transplant risk assessment. (Thorax 2001;56:941‐946)

Introduction and Objectives Terminally-differentiated neutrophils are short-lived but key effector cells of the innate immune response and play a prominent role in the pathogenesis and propagation of many inflammatory diseases including interstitial lung disease. BAL neutrophilia has been correlated with poor outcome in idiopathic pulmonary fibrosis (IPF) and neutrophils are the dominant cell type in acute exacerbations of IPF as well as in diffuse alveolar damage. Non-specific cyclin-dependent kinase inhibitor drugs have previously been shown to resolve neutrophil-dominant inflammation in the murine bleomycin lung injury model an established model of IPF. Here we elucidate the mechanism by which CDK inhibitor drugs mediate neutrophil apoptosis and hence promote resolution of inflammation. Methods We isolated peripheral blood neutrophils from healthy human donors and performed a variety of experiments including microarray, confocal microscopy and apoptosis assays (flow cytometry, fluorometric assay, DNA ladders and mitochondrial integrity assay and western blotting) to show that specific effects on neutrophil transcriptional capacity are responsible for CDK-inhibitor driven neutrophil apoptosis. Finally we show that specific CDK inhibitors drive resolution of neutrophil-dominant inflammation in the murine bleomycin lung injury model (Histology, BAL analysis). Results CDK inhibitors drive neutrophil apoptosis. Neutrophil apoptosis is dependent on a critical balance of pro- and anti-apoptotic proteins. Functional transcriptional machinery is present in human peripheral blood neutrophils. CDKs are present in neutrophils and their activity is both demonstrated and modulated with the use of specific CDK inhibitors. Specific CDK inhibition is a successful strategy for the resolution of murine bleomycin lung injury. Conclusion We highlight a novel mechanism that controls both neutrophil transcription and apoptosis that could be targeted by selective CDK inhibitor drugs to resolve established neutrophil-dominant inflammation.

Introduction and objectives Idiopathic pulmonary fibrosis (IPF) remains one of the few respiratory conditions for which there are no effective therapies. The role of monocytes and macrophages in IPF has been disputed as anti-inflammatory therapies produce questionable benefit. Corticosteroids, however, actually induce an alternatively activated, pro-fibrotic, macrophage phenotype. We sought to determine whether monocytes and macrophages play a role in disease pathogenesis in an attempt to explain why current hypotheses and anti-inflammatory therapies have produced limited clinical benefit despite years of research. Methods Using multiple in vivo depletional strategies, backed up by an adoptive transfer technique, we extensively investigated the role of monocytes and macrophages during lung fibrogenesis. We performed studies on samples from patients with IPF in an attempt to determine the translational importance of our findings. Results Depletion of lung macrophages during fibrogenesis reduced pulmonary fibrosis as measured by lung collagen (p=0.0079), fibrosis score (p=0.0051), and qPCR for surrogate markers of fibrosis Col1 (p=0.0083) and a-smooth muscle actin (p=0.0349). There was an associated reduction in expression of markers of alternative macrophage activation, Ym1 (p=0.0179), and Arginase1 . This reduction was confirmed by immunohistochemistry (IHC) for Ym1 (p=0.0233). IHC on lung macrophages from patients with IPF demonstrated the novel finding of expression of the human alternative macrophage marker CD163. Depletion of Ly6C hi circulating monocytes reduced pulmonary fibrosis (p=0.0052). Adoptive transfer of Ly6C hi BMDMS during fibrogenesis exacerbated pulmonary fibrosis (p=0.0304). Furthermore, depletion of circulating Ly6C hi monocytes lead to a subsequent reduction in the number of Ym1-positive alternatively activated lung macrophages (p=0.0310) with a concomitant reduction in the expression of Ym1 and Arginase1 . Conclusions We have demonstrated that monocytes and macrophages do modulate pulmonary fibrosis and suggest that Ly6C hi monocytes (possible fibrocyte precursors) are precursors of alternatively activated, pro-fibrotic, lung macrophages. These findings could link the ‘inflammatory’ and aberrant wound healing hypotheses and explain the lack of effectiveness of corticosteroids in treating IPF. By enhancing our understanding of the pathogenesis of this dreadful disease, our results may enable new therapeutic targets to be developed, facilitate targeted cell-based therapy, and bring hope to one of the longstanding enigmas of respiratory medicine.

Background Hypoxia-inducible factor (HIF-1) is a master regulator of the cellular hypoxic response and has been implicated in the pathogenesis of inflammatory and fibrotic disease including IPF. Aims To study the role of hypoxia and HIF-1 activation in macrophages in the i.t. bleomycin-induced lung fibrosis model. Methods The i.t. bleomycin model was used to study the effect of HIF-1 manipulation in mice. The primary end-point was lung collagen content at day 24 post i.t. bleomycin instillation. The HIF-1α inducer dimethyloxallyl glycine (DMOG) was administered i.p. on days 14, 17 and 21. The role of myeloid-HIF-1 activity in lung fibrosis was determined using mice in which either HIF-1α or vHL (the dominant negative-regulator of HIF-1α) was selectively knocked out of lysosyme M expressing cells (LysM-Cre-Hif-1 and Cre-LysM-vHL). Lung tissue hypoxia was determined using Hypoxyprobe-1TM administered on day 24. Alternative activation status of HIF-1 null and vHL null macrophages was studied in bone-marrow derived cells from LysM-Cre-Hif-1 and Cre-LysM-vHL mice. Results Pharmacological induction of HIF-1 in the late period of the bleomycin model with i.p. dimethyloxallyl glycine (DMOG) resulted in significantly enhanced lung collagen (mean±s.e.mμg/lung) on day 24 compared to controls (193±15 vs 152±8, p 7 per gp). Hypoxyprobe-1 staining in the bleomycin-injured lung revealed hypoxic alveolar macrophages even in areas of lung distant to patches if severe fibrosis, implying a role for hypoxic/HIF-1 expressing alveolar macrophages in lung fibrosis. However, lung collagen content was identical in myeloid-cell Hif-1 null mice and wild-type litter-mate controls (276±23 vs 277±22, n=8 per gp). In contrast, myeloid-cell vHL-null mice exhibited significantly enhanced lung collagen deposition versus controls (373±36 vs 282±54, p 9 per gp). Isolated vHL-null macrophages exhibited enhanced expression of the alternative activation markers YM-1, mannose receptor, arginase-1 and FIZZ-1. Conclusions vHL deletion in macrophages enhances alternative activation and promotes lung fibrosis independent of HIF-1.

In a double-blind placebo-controlled trial in 24 patients fulfilling the MRC criteria for chronic bronchitis, oral salbutamol 4 mg and slow-release aminophylline (Phyllocontin) 450 mg produced similar and significant (p less than 0.05) mean increases in forced expiratory volume in one second (FEV1). A significantly greater increase in mean FEV1 and forced vital capacity (FVC) was seen when both drugs were given although there was no statistical evidence of synergistic interaction. Salbutamol significantly increased the mean distance walked in 12 minutes (12MD) (p less than 0.02) by 56 metres and a similar increase of 54 metres (p less than 0.001) was seen after Phyllocontin. With both drugs in combination mean 12MD increased by 51 metres (p less than 0.02 cf placebo), a change not significantly different from that observed with either drug alone. Oral salbutamol and Phyllocontin improve exercise tolerance in chronic bronchitis. The significantly greater changes in FEV1 and FVC resulting from simultaneous administration of the two drugs are not associated with further improvement in exercise tolerance.