Emmet E. McGrath

TNF-related apoptosis-inducing ligand (TRAIL) regulates inflammatory neutrophil apoptosis and enhances resolution of inflammation

Novel therapeutics targeting neutrophilic inflammation are a major unmet clinical need in acute and chronic inflammation. The timely induction of neutrophil apoptosis is critical for inflammation resolution, and it is thought that acceleration of apoptosis may facilitate resolution at inflammatory sites. We previously demonstrated that a death receptor ligand, TRAIL, accelerates neutrophil apoptosis in vitro. We examined the role of TRAIL in neutrophil‐dominant inflammation in WT and TRAIL‐deficient mice. TRAIL deficiency did not alter constitutive neutrophil apoptosis, whereas exogenous TRAIL accelerated apoptosis of murine peripheral blood neutrophils. We compared TRAIL‐deficient and WT mice in two independent models of neutrophilic inflammation: bacterial LPS‐induced acute lung injury and zymosan‐induced peritonitis. In both models, TRAIL‐deficient mice had an enhanced inflammatory response with increased neutrophil numbers and reduced neutrophil apoptosis. Correction of TRAIL deficiency and supraphysiological TRAIL signaling using exogenous protein enhanced neutrophil apoptosis and reduced neutrophil numbers in both inflammatory models with no evidence of effects on other cell types. These data indicate the potential therapeutic benefit of TRAIL in neutrophilic inflammation.

The Toll-like Receptor 3 L412F Polymorphism and Disease Progression in Idiopathic Pulmonary Fibrosis

RATIONALEnIdiopathic pulmonary fibrosis (IPF) is a fatal progressive interstitial pneumonia. The innate immune system provides a crucial function in the recognition of tissue injury and infection. Toll-like receptor 3 (TLR3) is an innate immune system receptor. We investigated the role of a functional TLR3 single-nucleotide polymorphism in IPF.nnnOBJECTIVESnTo characterize the effects of the TLR3 Leu412Phe polymorphism in primary pulmonary fibroblasts from patients with IPF and disease progression in two independent IPF patient cohorts. To investigate the role of TLR3 in a murine model of pulmonary fibrosis.nnnMETHODSnTLR3-mediated cytokine, type 1 IFN, and fibroproliferative responses were examined in TLR3 wild-type (Leu/Leu), heterozygote (Leu/Phe), and homozygote (Phe/Phe) primary IPF pulmonary fibroblasts by ELISA, real-time polymerase chain reaction, and proliferation assays. A murine model of bleomycin-induced pulmonary fibrosis was used in TLR3 wild-type (tlr3(+/+)) and TLR3 knockout mice (tlr3(-/-)). A genotyping approach was used to investigate the role of the TLR3 L412F polymorphism in disease progression in IPF using survival analysis and longitudinal decline in FVC.nnnMEASUREMENTS AND MAIN RESULTSnActivation of TLR3 in primary lung fibroblasts from TLR3 L412F-variant patients with IPF resulted in defective cytokine, type I IFN, and fibroproliferative responses. We demonstrate increased collagen and profibrotic cytokines in TLR3 knockout mice (tlr3(-/-)) compared with wild-type mice (tlr3(+/+)). TLR3 L412F was also associated with a significantly greater risk of mortality and an accelerated decline in FVC in patients with IPF.nnnCONCLUSIONSnThis study reveals the crucial role of defective TLR3 function in promoting progressive IPF.

Hypoxia-inducible factor 2α regulates key neutrophil functions in humans, mice, and zebrafish

Neutrophil lifespan and function are regulated by hypoxia via components of the hypoxia inducible factor (HIF)/von Hippel Lindau/hydroxylase pathway, including specific roles for HIF-1α and prolyl hydroxylase-3. HIF-2α has both distinct and overlapping biological roles with HIF-1α and has not previously been studied in the context of neutrophil biology. We investigated the role of HIF-2α in regulating key neutrophil functions. Human and murine peripheral blood neutrophils expressed HIF-2α, with expression up-regulated by acute and chronic inflammatory stimuli and in disease-associated inflammatory neutrophil. HIF2A gain-of-function mutations resulted in a reduction in neutrophil apoptosis both ex vivo, through the study of patient cells, and in vivo in a zebrafish tail injury model. In contrast, HIF-2α-deficient murine inflammatory neutrophils displayed increased sensitivity to nitrosative stress induced apoptosis ex vivo and increased neutrophil apoptosis in vivo, resulting in a reduction in neutrophilic inflammation and reduced tissue injury. Expression of HIF-2α was temporally dissociated from HIF-1α in vivo and predominated in the resolution phase of inflammation. These data support a critical and selective role for HIF-2α in persistence of neutrophilic inflammation and provide a platform to dissect the therapeutic utility of targeting HIF-2α in chronic inflammatory diseases.

Hot off the breath: triple therapy for idiopathic pulmonary fibrosis—hear the PANTHER roar

Idiopathic pulmonary fibrosis (IPF) is a disease characterised by alveolar epithelial damage followed by an aberrant repair mechanism characterised by fibroblast foci and activated myofibroblasts.1 Despite an incidence of 7.4/100u2008000 person years which is increasing year on year and a median survival of only 2–3u2005years, there is paucity of evidence for effective therapy.2 The current British Thoracic Society guidelines weakly recommend N -acetylcysteine (NAC), prednisolone and azathioprine (based on the IFIGENIA—Idiopathic Pulmonary Fibrosis International Group Exploring N -Acetylcysteine I Annual—trial) whereas the more recent guidelines of the American Thoracic Society/European Respiratory Society recommend lung transplantation or participation in a clinical trial as treatment options.3–5nnu2003Increasing recognition of the clinical need for effective IPF therapy has finally led to a number of clinical trials evaluating potential anti-inflammatory and anti-fibrotic agents. IFIGENIA demonstrated that triple therapy with NAC, azathioprine and prednisolone was better than azathioprine and prednisolone in combination in preserving lung function in IPF patients, suggesting that NAC inclusion was strongly contributing to the benefits …

Deficiency of tumour necrosis factor-related apoptosis-inducing ligand exacerbates lung injury and fibrosis

Background The death receptor ligand tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) shows considerable clinical promise as a therapeutic agent. TRAIL induces leukocyte apoptosis, reducing acute inflammatory responses in the lung. It is not known whether TRAIL modifies chronic lung injury or whether TRAIL has a role in human idiopathic pulmonary fibrosis (IPF). We therefore explored the capacity of TRAIL to modify chronic inflammatory lung injury and studied TRAIL expression in patients with IPF. Methods TRAIL−/− and wild-type mice were instilled with bleomycin and inflammation assessed at various time points by bronchoalveolar lavage and histology. Collagen deposition was measured by tissue hydroxyproline content. TRAIL expression in human IPF lung samples was assessed by immunohistochemistry and peripheral blood TRAIL measured by ELISA. Results TRAIL−/− mice had an exaggerated delayed inflammatory response to bleomycin, with increased neutrophil numbers (mean 3.19±0.8 wild type vs 11.5±5.4×104 TRAIL−/−, p<0.0001), reduced neutrophil apoptosis (5.42±1.6% wild type vs 2.47±0.5% TRAIL−/−, p=0.0003) and increased collagen (3.45±0.2 wild type vs 5.8±1.3u2005mg TRAIL−/−, p=0.005). Immunohistochemical analysis showed induction of TRAIL in bleomycin-treated wild-type mice. Patients with IPF demonstrated lower levels of TRAIL expression than in control lung biopsies and their serum levels of TRAIL were significantly lower compared with matched controls (38.1±9.6 controls vs 32.3±7.2u2005pg/ml patients with IPF, p=0.002). Conclusion These data suggest TRAIL may exert beneficial, anti-inflammatory actions in chronic pulmonary inflammation in murine models and that these mechanisms may be compromised in human IPF.

Black hairy tongue: What is your call?

A 73-year-old male smoker attended a follow-up outpatient clinic 18 months after starting antibiotic therapy (rifampicin, isoniazid and clarithromycin) for a nontuberculous mycobacterial lung infection. His infection was clinically improving, but the patient had noticed that his tongue had become

S111 The role of TNF-related apoptosis inducing ligand (TRAIL) in pulmonary fibrosis

Introduction The concept of driving cellular apoptosis as a potential therapy for diseases characterised by inappropriate cellular persistence or proliferation is of widespread interest. We previously showed a death receptor ligand, TRAIL, accelerates neutrophil apoptosis without associated cell activation (J Immunol 170:1027–33) and other work revealed TRAIL-induced apoptosis of human lung fibroblasts. The aims of this project were to study the role of TRAIL in a bleomycin lung injury model in wild-type and TRAIL−/− mice and in patients with idiopathic pulmonary fibrosis (IPF). Methods Mice received intratracheal bleomycin or saline control. Bronchoalveolar lavage (BAL) at 3, 7, 16 and 23u2005days was analysed by cytospin morphology and haemocytometer count for % neutrophils, % neutrophil apoptosis, total number of neutrophils and total number of apoptotic cells. Flow cytometry was also used to analyse apoptosis. Collagen deposition in whole lung samples was analysed using a hydroxyproline assay. TRAIL expression and TUNEL positive events were also analysed. Serum and lung tissue from IPF patients/controls were examined for TRAIL expression and concentration. Lung function and survival data were retrieved from patient charts. Results BAL analysis revealed statistically significant differences between TRAIL−/− and wild-type mice, with TRAIL−/− mice showing increased neutrophil numbers and reduced neutrophil apoptosis as absolute count or as % total cell count. Collagen deposition was statistically greater in TRAIL−/− mice at 16u2005days. At dayu200523, TRAIL−/− mice had decreased TUNEL positive events compared to wild-type mice. Histological analysis of murine lung sections revealed specific TRAIL expression in bronchus associated lymphoid tissue and alveolar macrophages. IPF patient lung section analysis revealed an absence of TRAIL expression compared to controls. IPF patients had significantly lower serum levels of TRAIL than controls which inversely correlated with TLCO (% predicted) and positively correlated with survival from diagnosis. Conclusions We demonstrated that the neutrophilic inflammatory response to bleomycin is increased in TRAIL−/− compared with wild-type mice and that this finding is associated with increased collagen deposition. We also demonstrated reduced pulmonary and systemic expression of TRAIL in IPF, which correlates with worse pulmonary function and clinical outcome. This data suggests TRAIL may have biomarker potential and therapeutic benefit in pulmonary fibrosis.

Mycobacterium malmoense and underlying lung disease.

A 71 year old male ex-miner presented with a 3 month history of dyspnoea, lethargy and a cough which was productive of brown sputum. He had a past medical history of pneumoconiosis and rheumatoid arthritis. His chest x-ray demonstrated extensive fibrotic changes in both lungs consistent with pneumoconiosis. CT thorax also demonstrated a right upper lobe cavity (Fig. 1). Sputum culture for acid-fast bacilli (AFB) and tuberculin skin test were negative. Bronchoscopy was unremarkable and the bronchoalveolar lavage (BAL) grew no pathogens. His second sputum was reported positive for AFB and he was commenced on standard anti-tuberculous treatment of rifampicin, isoniazid, ethambutol and pyrazinamide. One month later we received a report that this sputum, along with several others, had actually grown a