Michael Gibbons

Regulation of Transforming Growth Factor-beta 1-driven Lung Fibrosis by Galectin-3

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. OBJECTIVES To examine the role of galectin-3 in pulmonary fibrosis. METHODS We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1-induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β-induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF. CONCLUSIONS This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.

Monocytes Control Second-Phase Neutrophil Emigration in Established Lipopolysaccharide-induced Murine Lung Injury

RATIONALE Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood. OBJECTIVES To determine the influence of peripheral blood monocytes (PBMs) in established ALI. METHODS In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2(hi) monocytes. MEASUREMENTS AND MAIN RESULTS PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1(hi) and Gr1(lo) PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes. CONCLUSIONS These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.

Endobronchial ultrasound-guided fine-needle aspiration and liquid-based thin-layer cytology

Background: Optimal management of patients with lung cancer requires accurate cell typing of tumours and staging at the time of diagnosis. Endobronchial ultrasound-guided lymph node aspiration as a method of diagnosing and staging lung cancer is a relatively new technique. Aim: To report the use of liquid-based-thin-layer cytology for the processing and reporting of these specimens. Methods: The specimens obtained from 80 patients were processed using the ThinPrep system, with the remainder of the samples being processed as a cell block. Results: 40 of the 81 procedures yielded malignant cells (30 non-small cell carcinoma, 8 small-cell carcinoma and 2 combined small-cell carcinoma/non-small-cell carcinoma). The cell blocks were found to contain sufficient material to allow the immunohistochemical characterisation of tumour cells with a range of antibodies. Conclusion: The use of liquid-based-thin-layer cytological techniques provides high-quality specimens for diagnostic purposes. When used in conjunction with cell blocks, sufficient material may be obtained to allow immunohistochemical studies to confirm the tumour cell type. Given the current move towards centralisation of pathology services, this approach gives the pathologist high-quality specimens without the need for direct onsite support at the time of the procedure.

British Thoracic Society quality standards for home oxygen use in adults

Introduction The purpose of the quality standards document is to provide healthcare professionals, commissioners, service providers and patients with a guide to standards of care that should be met for home oxygen provision in the UK, together with measurable markers of good practice. Quality statements are based on the British Thoracic Society (BTS) Guideline for Home Oxygen Use in Adults. Methods Development of BTS Quality Standards follows the BTS process of quality standard production based on the National Institute for Health and Care Excellence process manual for the development of quality standards. Results 10 quality statements have been developed, each describing a key marker of high-quality, cost-effective care for home oxygen use, and each statement is supported by quality measures that aim to improve the structure, process and outcomes of healthcare. Discussion BTS Quality Standards for home oxygen use in adults form a key part of the range of supporting materials that the society produces to assist in the dissemination and implementation of a guideline’s recommendations.

Delving Deep into the Proteome of Lung Fibrosis Brings Plasma Cells to the Surface

This is the author accepted manuscript. The final version is available from American Thoracic Society via the DOI in this record

S125 Ly6Chi circulating monocytes direct alternatively activated, pro-fibrotic, lung macrophage regulation of pulmonary fibrosis

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 Ly6Chi circulating monocytes reduced pulmonary fibrosis (p=0.0052). Adoptive transfer of Ly6Chi BMDMS during fibrogenesis exacerbated pulmonary fibrosis (p=0.0304). Furthermore, depletion of circulating Ly6Chi 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 Ly6Chi 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.

S124 Macrophage deletion of vHL results in alternative activation and enhanced lung fibrosis independent of HIF-1

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<0.05, n>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<0.05, n>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.