Respiratory complicˆations are the major cause of morbidity and mortality among preterm infants, which is partially prevented by the administration of antenatal corticosteroids (ACS). Most very preterm infants are exposed to chorioamnionitis, but short- and long-term effects of ACS treatment in this setting are not well defined. In low-resource settings, ACS increased neonatal mortality by perhaps increasing infection. We report that treatment with low-dose ACS in the setting of inflammation induced by intraamniotic lipopolysaccharide (LPS) in rhesus macaques improves lung compliance and increases surfactant production relative to either exposure alone. RNA sequencing shows that these changes are mediated by suppression of proliferation and induction of mesenchymal cellular death via TP53. The combined exposure results in a mature-like transcriptomic profile with inhibition of extracellular matrix development by suppression of collagen genes COL1A1, COL1A2, and COL3A1 and regulators of lung development FGF9 and FGF10. ACS and inflammation also suppressed signature genes associated with proliferative mesenchymal progenitors similar to the term gestation lung. Treatment with ACS in the setting of inflammation may result in early respiratory advantage to preterm infants, but this advantage may come at a risk of abnormal extracellular matrix development, which may be associated with increased risk of chronic lung disease.
Augusto F. Schmidt, Paranthaman S. Kannan, James Bridges, Pietro Presicce, Courtney M. Jackson, Lisa A. Miller, Suhas G. Kallapur, Claire A. Chougnet, Alan H. Jobe
The G/T transversion, rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and non-expressing lineages. In aggregate, our results indicate that the MUC5B-associated variant, rs35705950, resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.
Fabienne Gally, Sarah K. Sasse, Jonathan Kurche, Margaret A. Gruca, Jonathan H. Cardwell, Tsukasa Okamoto, Hong Wei Chu, Xiaomeng Hou, Olivier Poirion, Justin Buchanan, Sebastian Preissl, Bing Ren, Sean P. Colgan, Robin D. Dowell, Ivana V. Yang, David A. Schwartz, Anthony N. Gerber
COPD is a chronic respiratory disease characterized by small airway remodeling and alveolar emphysema due to environmental stresses such as cigarette smoking (CS). Oxidative stress is commonly implicated in COPD pathology, but recent findings suggest that one oxidant-producing NADPH oxidase homolog, dual oxidase 1 (DUOX1), is downregulated in the airways of COPD patients. We evaluated lung tissue sections from COPD patients for small airway epithelial DUOX1 protein expression, in association with measures of lung function and small airway and alveolar remodeling. We also addressed the impact of DUOX1 for lung tissue remodeling in mouse models of COPD. Small airway DUOX1 levels were decreased in advanced COPD, and correlated with loss of lung function and markers of emphysema and remodeling. Similarly, DUOX1 downregulation in correlation with extracellular matrix remodeling was observed in a genetic model of COPD, transgenic SPC-TNF-α mice. Finally, development of subepithelial airway fibrosis in mice due to exposure to the CS-component acrolein, or alveolar emphysema induced by administration of elastase, were in both cases exacerbated in Duox1-deficient mice. Collectively, our studies highlight that downregulation of DUOX1 may be a contributing feature of COPD pathogenesis, likely related to impaired DUOX1-mediated innate injury responses involved in epithelial homeostasis.
Caspar Schiffers, Cheryl van de Wetering, Robert A. Bauer, Aida Habibovic, Milena Hristova, Christopher M. Dustin, Sara Lambrichts, Pamela M. Vacek, Emiel F.M. Wouters, Niki L. Reynaert, Albert van der Vliet
The pathogenesis of Chronic Obstructive Pulmonary Disease (COPD) involves aberrant responses to cellular stress caused by chronic cigarette smoke (CS) exposure. However, not all smokers develop COPD and the critical mechanisms that regulate cellular stress responses to increase COPD susceptibility are not understood. Because microRNAs are well-known regulators of cellular stress responses, we evaluated microRNA expression arrays performed on distal parenchymal lung tissue samples from 172 subjects with and without COPD. We identified miR-24-3p as the microRNA that best correlated with radiographic emphysema (ρ=-0.353, P=1.3e-04), and validated this finding in multiple cohorts. In a CS-exposure mouse model, inhibition of miR-24-3p increased susceptibility to apoptosis, including alveolar type II epithelial cell (AECII) apoptosis, and emphysema severity. In lung epithelial cells, miR-24-3p suppressed apoptosis through the BH3-only protein BIM and suppressed homology-directed DNA repair and the DNA repair protein BRCA1. Finally, we found BIM and BRCA1 are increased in COPD lung tissue, and BIM and BRCA1 expression inversely correlate with miR-24-3p. We concluded that miR-24-3p, a regulator of the cellular response to DNA damage, is decreased in COPD, and decreased miR-24-3p increases susceptibility to emphysema through increased BIM and apoptosis.
Jessica Nouws, Feng Wan, Eric Finnemore, Willy Roque, So-Jin Kim, Isabel S. Bazan, Chuan-Xing Li, C. Magnus Sköld, Qile Dai, Xiting Yan, Maurizio Chioccioli, Veronique Neumeister, Clemente J. Britto, Joann Sweasy, Ranjit S. Bindra, Åsa M. Wheelock, Jose L. Gomez, Naftali Kaminski, Patty J. Lee, Maor Sauler
Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible fibrotic disease of the distal lung alveoli that culminates in respiratory failure and reduced lifespan. Unlike normal lung repair in response to injury, IPF is associated with the accumulation and persistence of fibroblasts and myofibroblasts and continued production of collagen and other extracellular matrix (ECM) components. Prior in vitro studies have led to the hypothesis that the development of resistance to Fas-induced apoptosis by lung fibroblasts and myofibroblasts contibributes to their accumulation in the distal lung tissues of IPF patients. Here, we test this hypothesis in vivo in the resolving model of bleomycin-induced pulmonary fibrosis in mice. Using genetic loss-of-function approaches to inhibit Fas signaling in fibroblasts, novel flow cytometry strategies to quantify lung fibroblast subsets and transcriptional profiling of lung fibroblasts by bulk and single cell RNA-sequencing, we show that Fas is necessary for lung fibroblast apoptosis during homeostatic resolution of bleomycin-induced pulmonary fibrosis in vivo. Furthermore, we show that loss of Fas signaling leads to the persistence and continued pro-fibrotic functions of lung fibroblasts. Our studies provide novel insights into the mechanisms that contribute to fibroblast survival, persistence and continued ECM deposition in the context of IPF and how failure to undergo Fas-induced apoptosis prevents fibrosis resolution.
Elizabeth F. Redente, Sangeeta Chakraborty, Satria P. Sajuthi, Bart P. Black, Ben L. Edelman, Max A. Seibold, David W.H. Riches
Current treatments for pneumonia (PNA) are focused on the pathogens. Mortality from PNA-induced acute lung injury (PNA-ALI) remains high, underscoring the need for additional therapeutic targets. Clinical and experimental evidence exists for potential sex differences in PNA survival, with males having higher mortality. In a model of severe pneumococcal PNA, when compared to males, age-matched female mice exhibited enhanced resolution characterized with decreased alveolar and lung inflammation and increased numbers of Regulatory T cells (Tregs). Recognizing the critical role of Tregs in lung injury resolution, we evaluated if improved outcomes in females were due to estradiol (E2) effects on Treg biology. E2 promoted Treg suppressive phenotype in vitro and resolution of PNA in vivo. Systemic rescue administration of E2 promoted resolution of PNA in males independent of lung bacterial clearance. E2 augmented Treg expression of Foxp3, CD25 and GATA3, an effect that required ERb, and not ERa signaling. Importantly, the in vivo therapeutic effects of E2 were lost in Treg depleted mice (Foxp3DTR). Adoptive transfer of ex vivo E2-treated Tregs rescued S. pneumoniae-induce PNA-ALI, a salutary effect that required Treg ERβ expression. E2-ERβ was required for Tregs to control macrophage pro-inflammatory responses. Our findings support the therapeutic role for E2 in promoting resolution of lung inflammation after PNA via ERβ Tregs.
Ye Xiong, Qiong Zhong, Tsvi Palmer, Alison Benner, Lan Wang, Karthik Suresh, Rachel Damico, Franco R. D'Alessio
Understanding the distinct pathogenic mechanisms that culminate in allograft fibrosis and chronic graft failure is key in improving outcomes after solid organ transplantation. Here, we describe an F1 → parent orthotopic lung transplant model of restrictive allograft syndrome (RAS), a particularly fulminant form of chronic lung allograft dysfunction (CLAD), and identify a requisite pathogenic role for humoral immune responses in development of RAS. B6D2F1/J (H2-b/d) donor lungs transplanted into the parent C57BL/6J (H2-b) recipients demonstrated a spectrum of histopathologic changes, ranging from lymphocytic infiltration, fibrinous exudates, and endothelialitis to peribronchial and pleuroparenchymal fibrosis, similar to those noted in the human RAS lungs. Gene expression profiling revealed differential humoral immune cell activation as a key feature of the RAS murine model, with significant B cell and plasma cell infiltration noted in the RAS lung allografts. B6D2F1/J lung allografts transplanted into μMt–/– (mature B cell deficient) or activation-induced cytidine deaminase (AID)/secretory μ-chain (μs) double-KO (AID−/−μs−/−) C57BL/6J mice demonstrated significantly decreased allograft fibrosis, indicating a key role for antibody secretion by B cells in mediating RAS pathology. Our study suggests that skewing of immune responses determines the diverse allograft remodeling patterns and highlights the need to develop targeted therapies for specific CLAD phenotypes.
Keizo Misumi, David S. Wheeler, Yoshiro Aoki, Michael P. Combs, Russell R. Braeuer, Ryuji Higashikubo, Wenjun Li, Daniel Kreisel, Ragini Vittal, Jeffrey Myers, Amir Lagstein, Natalie M. Walker, Carol F. Farver, Vibha N. Lama
Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short life spans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of ~ 40 days and died of gastrointestinal disease, but therapeutic regimens aimed at restoring gastrointestinal transit extended median survival to ~ 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression pattern in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the upper and lower airways. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1 year-old CF rabbits as compared to WT littermates, which were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.
Jie Xu, Alessandra Livraghi-Butrico, Xia Hou, Carthic Rajagopalan, Jifeng Zhang, Jun Song, Hong Jiang, Hong-guang Wei, Hui Wang, Mohamad Bouhamdan, Jinxue Ruan, Dongshan Yang, Yining Qiu, Xie Youming, Ronald P. Barrett, Sharon A. McClellan, Hongmei Mou, Qingtian Wu, Xuequn Chen, Troy D. Rogers, Kristen J. Wilkinson, Rodney C. Gilmore, Charles R. Esther Jr., Khalequz Zaman, Xiubin Liang, Michael Sobolic, Linda Hazlett, Kezhong Zhang, Raymond A. Frizzell, Martina Gentzsch, Wanda K. O'Neal, Barbara R. Grubb, Y Eugene Chen, Richard C. Boucher, Fei Sun
Alveolar macrophages (AM) are differentially regulated by human surfactant protein-A (SP-A)1 or SP-A2. However, AM are very heterogeneous and differences are difficult to characterize in intact cells. Using the Toponome Imaging System (TIS), an imaging technique that uses sequential immunostaining to identity patterns of biomarker expression or combinatorial molecular phenotypes (CMP), we studied individual single cells and identified subgroups of AM (n=168) from SP-A knockout (KO) mice and mice expressing either SP-A1 or SP-A2. The effects, as shown by CMPs, of SP-A1 and SP-A2 on AM were significant and differed. SP-A1 AM were the most diverse and shared the fewest CMPs with KO and SP-A2. Clustering analysis of each group showed three clusters where the CMP-based phenotype was distinct in each cluster. Moreover, a clustering analysis of all 168 AM revealed ten clusters, many dominated by one group. Some CMP, overlap among groups was observed with SP-A2 AM sharing the most CMPs and SP-A1 AM the fewest. The CMP-based patterns identified here provide a basis for not only understanding AM diversity, but, most importantly, the molecular basis for the diversity of functional differences in mouse models where the impact of genetics of innate immune molecules on AM has been studied.
David S. Phelps, Vernon M. Chinchilli, Judith Weisz, Lili Yang, Debra Shearer, Xuesheng Zhang, Joanna Floros
Primary varicella-zoster virus (VZV) infection in adults is often complicated by severe pneumonia, which is difficult to treat and associated with high morbidity and mortality. Here, the simian varicella virus (SVV) nonhuman primate (NHP) model was used to investigate the pathogenesis of varicella pneumonia. SVV infection resulted in transient fever, viremia and robust virus replication in alveolar pneumocytes and bronchus-associated lymphoid tissue. Clearance of infectious virus from lungs coincided with robust innate immune responses, leading to recruitment of inflammatory cells, mainly neutrophils and lymphocytes, and finally severe acute lung injury. SVV infection caused neutrophil activation and formation of neutrophil extracellular traps (NETs) in vitro and in vivo. Notably, NETs were also detected in lung and blood specimens of varicella pneumonia patients. Lung pathology in the SVV NHP model was associated with dysregulated expression of alveolar epithelial cell tight junction proteins (claudin-2, claudin-10 and claudin-18) and alveolar endothelial adherens junction protein VE-cadherin. Importantly, factors released by activated neutrophils, including NETs, were sufficient to reduce claudin-18 and VE-cadherin expression in NHP lung slice cultures. Collectively, the data indicate that local inflammatory responses involving activated neutrophils contribute to impaired alveolar epithelial/endothelial barrier integrity in varicella pneumonia and possibly other virus-induced acute lung injuries.
Werner J.D. Ouwendijk, Henk Jan van den Ham, Mark W. Delany, Jeroen J.A. van Kampen, Gijsbert P. van Nierop, Tamana Mehraban, Fatiha Zaaraoui-Boutahar, Wilfred F.J. van IJcken, Judith M.A. van den Brand, Rory D. De Vries, Arno C. Andeweg, Georges M.G.M. Verjans
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