Integrins, the extracellular matrix receptors that facilitate cell adhesion and migration, are necessary for organ morphogenesis; however, their role in maintaining adult tissue homeostasis is poorly understood. To define the functional importance of β1 integrin in adult mouse lung, we deleted it post-development in type 2 alveolar epithelial cells (AECs). Aged β1 integrin-deficient mice exhibited chronic obstructive pulmonary disease (COPD)-like pathology characterized by emphysema, lymphoid aggregates, and increased macrophage infiltration. These histopathological abnormalities were preceded by β1 integrin-deficient AEC dysfunction such as excessive reactive oxygen species production and up-regulation of NF-κB-dependent chemokines, including CCL2. Genetic deletion of the CCL2 receptor, Ccr2, in mice with β1 integrin-deficient type 2 AECs impaired recruitment of monocyte-derived macrophages and resulted in accelerated inflammation and severe premature emphysematous destruction. These lungs exhibited reduced AEC efferocytosis and excessive numbers of inflamed type 2 AECs, demonstrating the requirement for recruited monocyte-macrophages in limiting lung injury and remodeling in the setting of a chronically inflamed epithelium. These studies support a critical role for β1 integrin in alveolar homeostasis in the adult lung.
Erin J. Plosa, John T. Benjamin, Jennifer M. Sucre, Peter M. Gulleman, Linda A. Gleaves, Wei Han, Seunghyi Kook, Vasiliy V. Polosukhin, Scott M. Haake, Susan H. Guttentag, Lisa R. Young, Ambra Pozzi, Timothy S. Blackwell, Roy Zent
Background. We hypothesized that dynamic perfluorinated gas magnetic resonance imaging (19F MRI) would sensitively detect mild cystic fibrosis (CF) lung. Methods. This prospective study enrolled 20 healthy volunteers and 24 stable subjects with CF, including a subgroup of subjects with normal FEV1 (>80% predicted, n = 9). Dynamic 19F MRI images were acquired during sequential breath holds while breathing perfluoropropane (PFP) and during gas wash-out. Outcomes included the fraction of lung without significant ventilation (ventilation defect percent, VDP) and time constants that described PFP wash-in and wash-out kinetics. Results. VDP values (mean ± SD) of healthy controls (3.87% ± 2.7%) were statistically different from moderate CF subjects (19.5% ± 15.5%, P = 0.001) but not from mild CF subjects (10.4% ± 9.9%, P = 0.24) . The fractional lung volume with slow gas wash-out was elevated both in subjects with mild (9.61% ± 4.87%; P = 0.0066) and moderate CF (16.01% ± 5.01%; P = 0.0002) when compared to healthy controls (3.84% ± 2.16%). Conclusion. 19F MRI detected significant ventilation abnormalities in subjects with cystic fibrosis. Assessment of gas wash-out kinetics was more sensitive to mild CF lung disease than quantitation of steady state ventilation defects making 19F MRI a potentially valuable method for the characterization of early lung disease in CF.
Jennifer L. Goralski, Sang Hun Chung, Tyler M. Glass, Agathe S. Ceppe, Esther O. Akinnagbe-Zusterzeel, Aaron T. Trimble, Richard C. Boucher, Brian J. Soher, H. Cecil Charles, Scott H. Donaldson, Yueh Z. Lee
The MEK1/2–ERK1/2 pathway has been implicated in regulating the inflammatory response to lung injury and infection, and pharmacologic MEK1/2 inhibitor compounds are reported to reduce detrimental inflammation in multiple animal models of disease, in part through modulation of leukocyte responses. However, the specific contribution of myeloid MEK1 in regulating acute lung injury (ALI) and its resolution remain unknown. Here, the role of myeloid Mek1 was investigated in a murine model of LPS-induced ALI (LPS-ALI) by genetic deletion using the Cre-floxed system (LysMCre × Mekfl), and human alveolar macrophages from healthy volunteers and patients with acute respiratory distress syndrome (ARDS) were obtained to assess activation of the MEK1/2–ERK1/2 pathway. Myeloid Mek1 deletion results in a failure to resolve LPS-ALI, and alveolar macrophages lacking MEK1 had increased activation of MEK2 and the downstream target ERK1/2 on day 4 of LPS-ALI. The clinical significance of these findings is supported by increased activation of the MEK1/2–ERK1/2 pathway in alveolar macrophages from patients with ARDS compared with alveolar macrophages from healthy volunteers. This study reveals a critical role for myeloid MEK1 in promoting resolution of LPS-ALI and controlling the duration of macrophage proinflammatory responses.
Matthew E. Long, Ke-Qin Gong, William E. Eddy, Joseph S. Volk, Eric D. Morrell, Carmen Mikacenic, T. Eoin West, Shawn J. Skerrett, Jean Charron, W. Conrad Liles, Anne M. Manicone
Introduction: The airways of obese asthmatics have been shown to be nitric oxide (NO) deficient, which contributes to airway dysfunction and reduced response to inhaled corticosteroids (ICS). In cultured airway epithelial cells, L-citrulline, a precursor of L-arginine recycling and NO formation, has been shown to prevent asymmetric di-methyl arginine (ADMA)-mediated NO synthase (NOS2) uncoupling, restoring NO and reducing oxidative stress. Methods: In a proof of concept, pre – post open label pilot study, we hypothesized that 15g/day of L-citrulline for two weeks would: a) increase the fractional excretion of NO (FeNO); b) improve asthma control and c) improve lung function. To do this, we recruited obese (body mass index [BMI] >30) asthmatics on controller therapy with a baseline fractional exhaled nitric oxide (FeNO) ≤ 30 ppb from the University of Colorado Medical Center and Duke University Health System. Results: A total of 41 subjects with an average FeNO of 17 ppb (95% 19 - 20) and poorly controlled asthma (average asthma control questionnaire [ACQ] 1.5 [95% 1.2 – 1.8) completed the study. Compared to baseline, L-citrulline increased (values represent the mean delta and 95%CI): plasma L-citrulline (190uM, 84 – 297), plasma L-arginine (67uM, 38 – 95), plasma L-arginine/ADMA (117, 67 - 167), but not ADMA or arginase concentration. FeNO increased by 4.2ppb (1.7 – 6.7); ACQ decreased by -0.46 (-0.67 – -0.27); the forced vital capacity (FVC) and forced exhalation volume in one second (FEV1) respectively changed by 86 ml (10 – 161), and 52 ml (-11 – 132). In a secondary analysis, the greatest FEV1 increments occurred in those subjects with late onset asthma (>12 years) (63 ml [95%CI 1 – 137]), in females (80 ml [95%CI 5 – 154]), with a greater change seen in late onset females (100ml, [95%CI 2 – 177]). The changes in lung function or asthma control were not significantly associated with the pre-post changes in L-arginine/ADMA or FeNO. Conclusion: Short-term L-citrulline treatment improved asthma control and FeNO levels in obese asthmatics with low or normal FeNO. Larger FEV1 increments were observed in those with late onset asthma and in females.
Fernando Holguin, Hartmut Grasemann, Sunita Sharma, Daniel Winnica, Karen Wasil, Vong Smithphone, Margaret H. Cruse, Nancy Perez, Erika Coleman, Timothy J. Scialla, Loretta Que
Accumulation of senescent cells is associated with the progression of pulmonary fibrosis but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM) lipid extracts and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by anti-fibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR90) induced pro-fibrotic signaling in naïve fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl leukotriene in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cells removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time that senescent cells secrete functional LTs, significantly contributing to the LTs pool known to cause or exacerbate IPF.
Christopher D. Wiley, Alexis N. Brumwell, Sonnet S. Davis, Julia R. Jackson, Alexis Valdovinos, Cheresa Calhoun, Fatouma Alimirah, Carlos A. Castellanos, Richard Ruan, Ying Wei, Harold A. Chapman, Arvind Ramanathan, Judith Campisi, Claude Jourdan Le Saux
Loss of Thy-1 expression in fibroblasts correlates with lung fibrogenesis; however, the clinical relevance of therapeutic targeting of myofibroblasts via Thy-1–associated pathways remains to be explored. Using single (self-resolving) or repetitive (nonresolving) intratracheal administration of bleomycin in type 1 collagen-GFP reporter mice, we report that Thy-1 surface expression, but not mRNA, is reversibly diminished in activated fibroblasts and myofibroblasts in self-resolving fibrosis. However, Thy-1 mRNA expression is silenced in lung with nonresolving fibrosis following repetitive bleomycin administration, associated with persistent activation of αv integrin. Thy1-null mice showed progressive αv integrin activation and myofibroblast accumulation after a single dose of bleomycin. In vitro, targeting of αv integrin by soluble Thy-1-Fc (sThy-1), but not RLE-mutated Thy-1 or IgG, reversed TGF-β1–induced myofibroblast differentiation in a dose-dependent manner, suggesting that Thy-1’s integrin-binding RGD motif is required for the reversibility of myofibroblast differentiation. In vivo, treatment of established fibrosis induced either by single-dose bleomycin in WT mice or by induction of active TGF-β1 by doxycycline in Cc10-rtTA-tTS-Tgfb1 mice with sThy-1 (1000 ng/kg, i.v.) promoted resolution of fibrosis. Collectively, these findings demonstrate that sThy-1 therapeutically inhibits the αv integrin–driven feedback loop that amplifies and sustains fibrosis.
Chunting Tan, Min Jiang, Simon S. Wong, Celia R. Espinoza, Ceonne Kim, Xiaoping Li, Edward Connors, James S. Hagood
Pulmonary drug delivery presents a unique opportunity to target lower airway inflammation, which is often characterized by the massive recruitment of neutrophils from blood. However specific therapies are lacking that can modulate airway neutrophil function, and difficult challenges must be overcome to achieve therapeutic efficacy against pulmonary inflammation, notably drug hydrophobicity, mucociliary and macrophage-dependent clearance, and high extracellular protease burden. Here, we present a multi-stage, aerodynamically favorable delivery platform that uses extracellular proteolysis to its advantage in order to deliver nanoparticle-embedded hydrophobic drugs to neutrophils within the lower airways. Our design consists in a self-regulated nanoparticle-in-microgel system, in which microgel activation is triggered by extracellular elastase (degranulated by inflammatory neutrophils), and nanoparticles are loaded with Nexinhib20, a potent neutrophil degranulation inhibitor. Successful in vivo delivery of Nexinhib20 to the airways and into neutrophils promoted resolution of the inflammatory response by dampening neutrophil recruitment and degranulation, pro-inflammatory cytokine production in both airway and systemic compartments, as well as the presence of neutrophil-derived pathological extracellular vesicles in the lung fluid. Our findings showcase a new platform that overcomes challenges in pulmonary drug delivery and allows customization to match the proteolytic footprint of given diseases.
Joscelyn C Mejías, Osric A Forrest, Camilla Margaroli, David A. Frey Rubio, Liliana Viera, Jindong Li, Xin Xu, Amit Gaggar, Rabindra Tirouvanziam, Krishnendu Roy
To develop a systems biology model of fibrosis progression within the human lung we performed RNAseq and microRNA analysis on 95 samples obtained from 10 idiopathic pulmonary fibrosis (IPF) and 6 control lungs. Extent of fibrosis in each sample was assessed by microCT measured alveolar surface density (ASD) and confirmed by histology. Regulatory gene expression networks were identified using linear mixed-effect models and dynamic regulatory events miner (DREM). Differential gene expression analysis identified a core set of genes increased or decreased before fibrosis was histologically evident that continued to change with advanced fibrosis. DREM generated a systems biology model of fibrosis progression (available at http: www.sb.cs.cmu.edu/IPFReg) that identified progressively divergent gene expression tracks with microRNAs and transcription factors that specifically regulate early or advanced fibrosis. We confirmed model predictions by demonstrating that expression of POU2AF1, previously unassociated with lung disease but proposed by the model as regulator, is increased in B-lymphocytes in IPF lungs and that POU2AF1 knockout mice were protected from bleomycin induced lung fibrosis. Our results reveal distinct regulation of gene expression changes in IPF tissue that remained structurally normal compared with moderate or advanced fibrosis and suggest distinct regulatory mechanisms for each stage.
John E. McDonough, Farida Ahangari, Qin Li, Siddhartha Jain, Stijn E. Verleden, Jose Herazo-Maya, Milica Vukmirovic, Giuseppe DeIuliis, Argyrios Tzouvelekis, Naoya Tanabe, Fanny Chu, Xiting Yan, Johny Verschakelen, Robert J. Homer, Dimitris V. Manatakis, Junke Zhang, Jun Ding, Karen Maes, Laurens De Sadeleer, Robin Vos, Arne Neyrinck, Panayiotis V. Benos, Ziv Bar-Joseph, Dean Tantin, James C. Hogg, Bart M. Vanaudenaerde, Wim A. Wuyts, Naftali Kaminski
Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF). The aim of this study was to test the therapeutic effects of MSC-extracellular vesicles/exosomes (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate putative mechanisms of action. Exosomes were isolated from media conditioned by human bone marrow MSCs. Adult mice (C57BL/6 strain) were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently or for reversal models, at day 7 or 21. Experimental groups were assessed at day 7 and/or at day 14 or 28. Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented or reversed by a single dose of MEx. Furthermore, MEx therapy modulated whole lung macrophage phenotype and shifted the proportion of lung ‘proinflammatory’ classical monocytes, non-classical monocytes and alveolar macrophages to favor the monocyte/macrophage profiles of untreated-control mice. A parallel immunomodulatory effect was demonstrated in the bone marrow. Notably, transplantation of MEx-preconditioned bone marrow-derived monocytes alleviated core features of pulmonary fibrosis and lung inflammation. Proteomic analysis further revealed a signature enriched in non-inflammatory monocyte genes following MEx therapy supporting the immuno-regulatory, anti-inflammatory effect of MEx.We conclude that a bolus dose of MEx prevents and reverts core features of bleomycin-induced pulmonary fibrosis, and that the beneficial actions of MEx may be mediated via systemic modulation of monocyte phenotypes.
Nahal Mansouri, Gareth R. Willis, Angeles Fernandez-Gonzalez, Monica Reis, Sina Nassiri, Alex Mitsialis, Stella Kourembanas
Oxidative stress is a major contributor to chronic lung diseases. Antioxidants such as N-acetylcysteine (NAC) are broadly viewed as protective molecules that prevent the mutagenic effects of reactive oxygen species. Antioxidants may, however, increase the risk of some forms of cancer and accelerate lung cancer progression in murine models. Here, we investigated chronic NAC treatment in aging mice displaying lung oxidative stress and cell senescence due to inactivation of the transcription factor JunD, which is downregulated in diseased human lungs. NAC treatment decreased lung oxidative damage and cell senescence and protected from lung emphysema but concomitantly induced the development of lung adenocarcinoma in 50% of JunD-deficient mice and 10% of aged control mice. This finding constitutes the first evidence to our knowledge of a carcinogenic effect of antioxidant therapy in the lungs of aged mice with chronic lung oxidative stress and warrants the utmost caution when considering the therapeutic use of antioxidants.
Marielle Breau, Amal Houssaini, Larissa Lipskaia, Shariq Abid, Emmanuelle Born, Elisabeth Marcos, Gabor Czibik, Aya Attwe, Delphine Beaulieu, Alberta Palazzo, Jean-Michel Flaman, Brigitte Bourachot, Guillaume Collin, Jeanne Tran Van Nhieu, David Bernard, Fatima Mechta-Grigoriou, Serge Adnot
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