Pulmonology
Abstract
Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing, and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.
Authors
Bryce Piper, Srimathi Bogamuwa, Tanvir Hossain, Daniela Farkas, Lorena Rosas, Adam C. Green, Geoffrey Newcomb, Nuo Sun, Jose A. Ovando-Ricardez, Jeffrey C. Horowitz, Aneel R. Bhagwani, Hu Yang, Tatiana V. Kudryashova, Mauricio Rojas, Ana L. Mora, Pearlly Yan, Rama K. Mallampalli, Elena A. Goncharova, David M. Eckmann, Laszlo Farkas
Abstract
Idiopathic Pulmonary Fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins, and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin (KRT) 8 genetic variants are associated with IPF. Krt8-/- mice are protected from fibrosis and accumulation of the transitional state. Keratin (K) 8 regulates expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promote accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs are highly senescent, basaloid, and do not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induce and are maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolve, whereas in human IPF, transitional AECs evolve into an aberrant basaloid state which persists with progressive fibrosis.
Authors
Fa Wang, Christopher Ting, Kent A. Riemondy, Michael T. Douglas, Kendall M. Foster, Nisha Patel, Norihito Kaku, Alexander E. Linsalata, Jean Nemzek, Brian M. Varisco, Erez Cohen, Jasmine A. Wilson, David W.H. Riches, Elizabeth F. Redente, Diana M. Toivola, Xiaofeng Zhou, Bethany B. Moore, Pierre A. Coulombe, M. Bishir Omary, Rachel L. Zemans
Abstract
Secondary lung infection by inhaled Staphylococcus aureus (SA) is a common and lethal event in individuals infected with influenza A virus (IAV). How IAV disrupts host defense to promote SA infection in lung alveoli, where fatal lung injury occurs, is not known. We addressed this issue using the first real-time determinations of alveolar responses to IAV in live, intact, perfused lungs. Our findings show IAV infection blocked defensive alveolar wall liquid (AWL) secretion and induced airspace liquid absorption, thereby reversing normal alveolar liquid dynamics and inhibiting alveolar clearance of inhaled SA. Loss of AWL secretion resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in the alveolar epithelium, and airspace liquid absorption was caused by stimulation of the alveolar epithelial Na+ channel (ENaC). Loss of AWL secretion promoted alveolar stabilization of inhaled SA, but rescue of AWL secretion protected against alveolar SA stabilization and fatal SA-induced lung injury in IAV-infected mice. These findings reveal a central role for AWL secretion in alveolar defense against inhaled SA and identify AWL inhibition as a critical mechanism of IAV lung pathogenesis. AWL rescue may represent a new therapeutic approach for IAV-SA coinfection.
Authors
Stephanie Tang, Ana Cassandra De Jesus, Deebly Chavez, Sayahi Suthakaran, Sarah K.L. Moore, Keshon Suthakaran, Sonya Homami, Raveen Rathnasinghe, Alison J. May, Michael Schotsaert, Clemente J. Britto, Jahar Bhattacharya, Jaime L. Hook
Abstract
The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl– into the airway surface liquid through CFTR (cystic fibrosis transmembrane conductance regulator) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl– channels in their basolateral membrane. Disrupting barttin/Cl– channel function impaired liquid absorption, and overexpressing barttin/Cl– channels increased absorption. Together, apical CFTR and basolateral barttin/Cl– channels provide an electrically conductive pathway for Cl– flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.
Authors
Lei Lei, Soumba Traore, Guillermo S. Romano Ibarra, Philip H. Karp, Tayyab Rehman, David K. Meyerholz, Joseph Zabner, David A. Stoltz, Patrick L. Sinn, Michael J. Welsh, Paul B. McCray, Jr., Ian M. Thornell
Abstract
Authors
Nirmal S. Sharma, Kapil Patel, Ezgi Sari, Shruti Shankar, Maria G. Gastanadui, Diego Moncada-Giraldo, Yixel M. Soto-Vázquez, Delores A. Stacks, Louise Hecker, Kevin G. Dsouza, Mudassir Banday, Edward O'Neill, Paul Benson, Gregory A. Payne, Camilla Margaroli, Amit Gaggar
Abstract
BACKGROUND. Despite guidelines promoting the prevention and aggressive treatment of ventilator-associated pneumonia (VAP), the importance of VAP as a driver of outcomes in mechanically ventilated patients, including patients with severe COVID-19, remains unclear. We aimed to determine the contribution of unsuccessful treatment of VAP to mortality in patients with severe pneumonia. METHODS. We performed a single-center prospective cohort study of 585 mechanically ventilated patients with severe pneumonia and respiratory failure, 190 of whom had COVID-19, who underwent at least one bronchoalveolar lavage. A panel of ICU physicians adjudicated pneumonia episodes and endpoints based on clinical and microbiologic data. Given the relatively long ICU length of stay among patients with COVID-19, we developed a machine learning approach called CarpeDiem, which groups similar ICU patient-days into clinical states based on electronic health record data. RESULTS.CarpeDiem revealed that the long ICU length of stay among patients with COVID-19 is attributable to long stays in clinical states characterized primarily by respiratory failure. While VAP was not associated with mortality overall, mortality was higher in patients with one episode of unsuccessfully treated VAP compared with successfully treated VAP (76.4% versus 17.6%, P < 0.001). In all patients, including those with COVID-19, CarpeDiem demonstrated that unresolving VAP was associated with transitions to clinical states associated with higher mortality. CONCLUSIONS. Unsuccessful treatment of VAP is associated with greater mortality. The relatively long length of stay among patients with COVID-19 is primarily due to prolonged respiratory failure, placing them at higher risk of VAP. FUNDING. U19AI135964
Authors
Catherine A. Gao, Nikolay S. Markov, Thomas Stoeger, Anna E. Pawlowski, Mengjia Kang, Prasanth Nannapaneni, Rogan A. Grant, Chiagozie Pickens, James M. Walter, Jacqueline M. Kruser, Luke V. Rasmussen, Daniel Schneider, Justin Starren, Helen K. Donnelly, Alvaro Donayre, Yuan Luo, G.R. Scott Budinger, Richard G. Wunderink, Alexander V. Misharin, Benjamin D. Singer
Abstract
BACKGROUND Lower respiratory tract infection (LRTI) is a leading cause of death in children worldwide. LRTI diagnosis is challenging because noninfectious respiratory illnesses appear clinically similar and because existing microbiologic tests are often falsely negative or detect incidentally carried microbes, resulting in antimicrobial overuse and adverse outcomes. Lower airway metagenomics has the potential to detect host and microbial signatures of LRTI. Whether it can be applied at scale and in a pediatric population to enable improved diagnosis and treatment remains unclear.METHODS We used tracheal aspirate RNA-Seq to profile host gene expression and respiratory microbiota in 261 children with acute respiratory failure. We developed a gene expression classifier for LRTI by training on patients with an established diagnosis of LRTI (n = 117) or of noninfectious respiratory failure (n = 50). We then developed a classifier that integrates the host LRTI probability, abundance of respiratory viruses, and dominance in the lung microbiome of bacteria/fungi considered pathogenic by a rules-based algorithm.RESULTS The host classifier achieved a median AUC of 0.967 by cross-validation, driven by activation markers of T cells, alveolar macrophages, and the interferon response. The integrated classifier achieved a median AUC of 0.986 and increased the confidence of patient classifications. When applied to patients with an uncertain diagnosis (n = 94), the integrated classifier indicated LRTI in 52% of cases and nominated likely causal pathogens in 98% of those.CONCLUSION Lower airway metagenomics enables accurate LRTI diagnosis and pathogen identification in a heterogeneous cohort of critically ill children through integration of host, pathogen, and microbiome features.FUNDING Support for this study was provided by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Heart, Lung, and Blood Institute (UG1HD083171, 1R01HL124103, UG1HD049983, UG01HD049934, UG1HD083170, UG1HD050096, UG1HD63108, UG1HD083116, UG1HD083166, UG1HD049981, K23HL138461, and 5R01HL155418) as well as by the Chan Zuckerberg Biohub.
Authors
Eran Mick, Alexandra Tsitsiklis, Jack Kamm, Katrina L. Kalantar, Saharai Caldera, Amy Lyden, Michelle Tan, Angela M. Detweiler, Norma Neff, Christina M. Osborne, Kayla M. Williamson, Victoria Soesanto, Matthew Leroue, Aline B. Maddux, Eric A.F. Simões, Todd C. Carpenter, Brandie D. Wagner, Joseph L. DeRisi, Lilliam Ambroggio, Peter M. Mourani, Charles R. Langelier
Abstract
BACKGROUND. Lung infections are among the most consequential manifestations of cystic fibrosis (CF) and are associated with reduced lung function and shortened survival. Drugs called CFTR modulators improve activity of dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channels, which is the physiological defect causing CF. However, it is unclear how improved CFTR activity affects CF lung infections. METHODS. We performed a prospective, multicenter, observational study to measure the effect of the newest and most effective CFTR modulator, elexacaftor/tezacaftor/ivacaftor (ETI) on CF lung infections. We studied sputum from 236 people with CF during their first 6 months of ETI using bacterial cultures, PCR and sequencing. RESULTS. Mean sputum densities of Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Achromobacter and Burkholderia spp. decreased by 2-3 log10 CFU/ml after 1 month of ETI. However, most participants remained culture-positive for the pathogens cultured from their sputum before starting ETI. In those becoming culture-negative after ETI, the pathogens present before treatment were often still detectable by PCR months after sputum converted to culture-negative. Sequence-based analyses confirmed large reductions in CF pathogen genera, but other bacteria detected in sputum were largely unchanged. ETI treatment increased average sputum bacterial diversity and produced consistent shifts in sputum bacterial composition. However, these changes were caused by ETI-mediated decreases in CF pathogen abundance rather than changes in other bacteria. CONCLUSIONS. Treatment with the most effective CFTR modulator currently available produced large and rapid reductions in traditional CF pathogens in sputum, but most participants remain infected with the pathogens present before modulator treatment. TRIAL REGISTRATION. The trial registered at www.ClinicalTrials.gov as NCT04038047. FUNDING. This study was funded by the Cystic Fibtosis Foundation (PROMISE-MICRO18K1 and SINGH19R0) and NIH (R01HL148274).
Authors
David P. Nichols, Sarah J. Morgan, Michelle Skalland, Anh T. Vo, Jill M. Van Dalfsen, Sachinkumar B.P. Singh, Wendy Ni, Lucas R. Hoffman, Kailee McGeer, Sonya L. Heltshe, John P. Clancy, Steven M. Rowe, Peter K. Jorth, Pradeep K. Singh
Abstract
BACKGROUND. The fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). T-helper (Th) cells orchestrate immune responses against fungi, but the types of A. fumigatus-specific Th-cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized. METHODS. We used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls. RESULTS. We show that clonally expanded, high-avidity A. fumigatus-specific effector Th-cells develop in pwCF, which are absent in healthy donors. Individual patients were characterized by distinct Th1, Th2, or Th17 dominated responses that remained stable over years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th-cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2-cells that cross-recognize various filamentous fungi. CONCLUSION. Our data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2-cells as a potential risk factor for ABPA. FUNDING. This research was supported by grants from the German Research Foundation (DFG) under Germany’s Excellence Strategy - EXC 2167-390884018 “Precision Medicine in Chronic Inflammation”, EXC 2051-390713860 “Balance of the Microverse”; the Oskar Helene Heim Stiftung; the Christiane Herzog Stiftung, Stuttgart, Germany; the Mukoviszidose Institut gGmbH, Bonn, the research and development arm of the German Cystic Fibrosis Association Mukoviszidose e.V; the German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath, BMBF 03ZZ0838A+B.
Authors
Carsten Schwarz, Patience Eschenhagen, Henrijette Schmidt, Thordis Hohnstein, Christina Iwert, Claudia Grehn, Jobst Roehmel, Eva Steinke, Mirjam Stahl, Laura Lozza, Ekaterina Tikhonova, Elisa Rosati, Ulrik Stervbo, Nina Babel, Jochen G. Mainz, Hilmar Wisplinghoff, Frank Ebel, Lei-Jie Jia, Matthew G. Blango, Peter Hortschansky, Sascha Brunke, Bernhard Hube, Axel A. Brakhage, Olaf Kniemeyer, Alexander Scheffold, Petra Bacher
Abstract
KRAS is one of the most frequently activated oncogenes in human cancers. While the role of KRAS mutation in tumorigenesis and tumor maintenance has been extensively studied, the relationship between KRAS and the tumor immune microenvironment is not fully understood. Herein, we identified a novel role of KRAS in driving tumor evasion from innate immune surveillance. In lung adenocarcinoma patient samples and Kras-driven genetic mouse models of lung cancer, mutant KRAS activated the expression of cluster of differentiation 47 (CD47), an antiphagocytic signal in cancer cells, leading to decreased phagocytosis of cancer cells by macrophages. Mechanistically, mutant KRAS activated PI3K-STAT3 signaling, which restrained miR-34a expression and relieved the post-transcriptional repression of miR-34a on CD47. In three independent lung cancer patient cohorts, KRAS mutation status positively correlated with CD47 expression. Therapeutically, disruption of the KRAS-CD47 signaling axis with KRAS siRNA, the KRASG12C inhibitor AMG 510 or miR-34a mimic suppressed CD47 expression, enhanced the phagocytic capacity of macrophages and restored innate immune surveillance. Our results revealed a direct mechanistic link between active KRAS and innate immune evasion and identified CD47 as a major effector underlying KRAS-mediated immunosuppressive tumor microenvironment.
Authors
Huanhuan Hu, Rongjie Cheng, Yanbo Wang, Xiaojun Wang, Jianzhuang Wu, Yan Kong, Shoubin Zhan, Zhen Zhou, Hongyu Zhu, Ranran Yu, Gaoli Liang, Qingyan Wang, Xiaoju Zhu, Chen-Yu Zhang, Rong Yin, Chao Yan, Xi Chen
Copyright © 2024 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)