Pulmonology
Abstract
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.
Authors
Ye Xiong, Qiong Zhong, Tsvi Palmer, Alison Benner, Lan Wang, Karthik Suresh, Rachel Damico, Franco R. D'Alessio
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
David S. Phelps, Vernon M. Chinchilli, Judith Weisz, Lili Yang, Debra Shearer, Xuesheng Zhang, Joanna Floros
Abstract
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.
Authors
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
Abstract
Ischemia-reperfusion-induced edema (IRE) one of the most significant causes of mortality after lung transplantation can be mimicked ex-vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel studied in endothelium, while its role in the lung epithelium remains elusive. Here we show enhanced IRE in TRPV4-deficient (TRPV4–/–) IPL compared to wild-type (WT) controls, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4–/– ATI cells was reduced and cell barrier function was impaired. Analysis of isolated primary TRPV4-deficient ATII cells revealed a reduced expression of surfactant protein C (SP-C) and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4–/– lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared to WT lungs. Therefore, our data discover essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation.
Authors
Jonas Weber, Suhasini Rajan, Christian Schremmer, Yu-Kai Chao, Gabriela Krasteva-Christ, Martina Kannler, Ali Önder Yildirim, Monika Brosien, Johann Schredelseker, Norbert Weissmann, Christian Grimm, Thomas Gudermann, Alexander Dietrich
Abstract
Based on its clinical benefits, Trikafta, the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) was FDA-approved for treatment of cystic fibrosis (CF) patients carrying deletion of phenylalanine 508 (F508del) of the CF Transmembrane Conductance Regulator (CFTR) on at least one allele. Neither the mechanism of action of VX-445, nor the susceptibility of rare CF folding mutants to Trikafta are known. Here we show that in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1)-membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661+VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to ~62% of wild-type CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K and N1303K), confined to MSD1, MSD2, NBD1 and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.
Authors
Guido Veit, Ariel Roldan, Mark A. Hancock, Dillon F. Da Fonte, Haijin Xu, Maytham Hussein, Saul Frenkiel, Elias Matouk, Tony Velkov, Gergely L. Lukacs
Abstract
Increased metabolism distinguishes myofibroblasts or fibrotic lung fibroblasts (fLfs) from the normal lung fibroblasts (nLfs). The mechanism of metabolic activation in fLfs has not been fully elucidated. Further, the anti-fibrogenic effects of caveolin-1 scaffolding domain peptide CSP/CSP7 involve metabolic reprogramming in fLfs is unclear. We therefore analyzed lactate and succinate levels, and the expression of glycolytic enzymes, and hypoxia inducible factor-1alpha (HIF-1α). Lactate and succinate levels as well as the basal expression of glycolytic enzymes and HIF-1α αwere increased in fLfs. These changes were reversed following restoration of p53 or its transcriptional target microRNA-34a (miR-34a) expression in fLfs. Conversely, inhibition of basal p53 or miR-34a increased glucose metabolism, glycolytic enzymes and HIF-1α in nLfs. Treatment of fLfs or mice having bleomycin- or TGF-beta1-induced lung fibrosis with CSP/CSP7, reduced the expression of glycolytic enzymes and HIF-1α. Further, inhibition of p53 or miR-34a abrogated CSP/CSP7-mediated restoration of glycolytic flux in fLfs in vitro and in mice with pulmonary fibrosis and lacking p53 or miR-34a expression in fibroblasts in vivo. Our data indicate that dysregulation of glucose metabolism in fLfs is causally linked to loss of basal expression of p53 and miR-34a. Treatment with CSP/CSP7 constrains aberrant glucose metabolism through restoration of p53 and miR-34a.
Authors
Venkadesaperumal Gopu, Liang Fan, Rashmi Shetty, MR Nagaraja, Sreerama Shetty
Abstract
Airway mucociliary clearance (MCC) is the main mechanism of lung defense keeping airways free of infection and mucus obstruction. Airway surface liquid volume, ciliary beating, and mucus are central for proper MCC and critically regulated by sodium absorption and anion secretion. Impaired MCC is a key feature of muco-obstructive diseases. The calcium-activated potassium channel KCa.3.1, encoded by Kcnn4, participates in ion secretion, and studies showed that its activation increases Na+ absorption in airway epithelia, suggesting that KCa3.1-induced hyperpolarization was sufficient to drive Na+ absorption. However, its role in airway epithelium is not fully understood. We aimed to elucidate the role of KCa3.1 in MCC using a genetically engineered mouse. KCa3.1 inhibition reduced Na+ absorption in mouse and human airway epithelium. Furthermore, the genetic deletion of Kcnn4 enhanced cilia beating frequency and MCC ex vivo and in vivo. Kcnn4 silencing in the Scnn1b-transgenic mouse (Scnn1btg/+), a model of muco-obstructive lung disease triggered by increased epithelial Na+ absorption, improved MCC, reduced Na+ absorption, and did not change the amount of mucus but did reduce mucus adhesion, neutrophil infiltration, and emphysema. Our data support that KCa3.1 inhibition attenuated muco-obstructive disease in the Scnn1btg/+ mice. K+ channel modulation may be a therapeutic strategy to treat muco-obstructive lung diseases.
Authors
Génesis Vega, Anita Guequén, Amber R. Philp, Ambra Gianotti, Llilian Arzola, Manuel Villalón, Olga Zegarra-Moran, Luis J.V. Galietta, Marcus A. Mall, Carlos A. Flores
Abstract
Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell–mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease–inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.
Authors
Meredith L. Sosulski, Katie M. Stiles, Esther Z. Frenk, Fiona M. Hart, Yuki Matsumura, Bishnu P. De, Stephen M. Kaminsky, Ronald G. Crystal
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