Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BC) express functional taste receptors. Here we report that bitter taste signaling in murine BC induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and Substance P that mediate plasma extravasation, neutrophil recruitment and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BC. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among the most abundant members of the complement system, and is needed to combat Pseudomonas aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.
Monika I. Hollenhorst, Rajender Nandigama, Saskia B. Evers, Igor Gamayun, Noran Abdel Wadood, Alaa Salah, Mario Pieper, Amanda Wyatt, Alexey Stukalov, Anna Gebhardt, Wiebke Nadolni, Wera Burow, Christian Herr, Christoph Beisswenger, Soumya Kusumakshi, Fabien Ectors, Tatjana I. Kichko, Lisa Hübner, Peter Reeh, Antje Munder, Sandra-Maria Wienhold, Martin Witzenrath, Robert Bals, Veit Flockerzi, Thomas Gudermann, Markus Bischoff, Peter Lipp, Susanna Zierler, Vladimir Chubanov, Andreas Pichlmair, Peter König, Ulrich Boehm, Gabriela Krasteva-Christ
Highly effective modulator therapies dramatically improve the prognosis for those with cystic fibrosis (CF). The triple combination of elexacaftor, tezacaftor, and ivacaftor (ETI) benefits many, but not all, of those with the most common F508del mutation in the CF transmembrane conductance regulator (CFTR). Here, we showed that poor sweat chloride concentration responses and lung function improvements upon initiation of ETI were associated with elevated levels of active transforming growth factor β1 (TGF-β1) in the upper airway. Furthermore, TGF-β1 impaired the function of ETI-corrected F508del-CFTR, thereby increasing airway surface liquid (ASL) absorption rates and inducing mucus hyperconcentration in primary CF bronchial epithelial cells in vitro. TGF-β1 not only decreased CFTR mRNA but was also associated with increases in the mRNA expression of tumor necrosis factor alpha (TNFA) and cyclooxygenase-2 (COX2) and TNF-⍺ protein. Losartan improved TGF-β1-mediated inhibition of ETI-corrected F508del-CFTR function and reduced TNFA and COX2 mRNA and TNF⍺ protein expression. This occurred likely by improving correction of mutant CFTR rather than increasing its mRNA (without an effect on potentiation), thereby reversing the negative effects of TGF-β1 and improving ASL hydration in the CF airway epithelium in vitro. Importantly, these effects were independent of type 1 angiotensin II receptor inhibition.
Michael D. Kim, Charles D. Bengtson, Makoto Yoshida, Asef J. Niloy, John S. Dennis, Nathalie Baumlin, Matthias Salathe
AbstractType 2 alveolar epithelial cells (AEC2s) function as progenitor cells in the lung. We have shown previously that failure of AEC2 regeneration results in progressive lung fibrosis in mice and is a cardinal feature of idiopathic pulmonary fibrosis (IPF). In this study, we identified a deficiency of a specific zinc transporter SLC39A8 (ZIP8) in AEC2s from both IPF lungs and lungs of old mice. Loss of ZIP8 expression was associated with impaired renewal capacity of AEC2s and enhanced lung fibrosis. ZIP8 regulation of AEC2 progenitor function was dependent on SIRT1. Replenishment with exogenous zinc and SIRT1 activation promoted self-renewal and differentiation of AEC2s from lung tissues of IPF patients and old mice. Deletion of Zip8 in AEC2s in mice impaired AEC2 renewal, increased susceptibility of the mice to bleomycin injury, and the mice developed spontaneous lung fibrosis. Therapeutic strategies to restore zinc metabolism and appropriate SIRT1 signaling could improve AEC2 progenitor function and mitigate ongoing fibrogenesis.
Jiurong Liang, Guanling Huang, Xue Liu, Forough Taghavifar, Ningshan Liu, Yizhou Wang, Nan Deng, Changfu Yao, Ting Xie, Vrishika Kulur, Kristy Dai, Ankita Burman, Simon C. Rowan, S. Samuel Weigt, John Belperio, Barry Stripp, William C. Parks, Dianhua Jiang, Paul W. Noble
The respiratory tract surface is protected from inhaled pathogens by a secreted layer of mucus rich in mucin glycoproteins. Abnormal mucus accumulation is a cardinal feature of chronic respiratory diseases but the relationship between mucus and pathogens during exacerbations is poorly understood. We identified elevations in airway MUC5AC and MUC5B concentrations during spontaneous and experimentally-induced chronic obstructive pulmonary disease (COPD) exacerbations. MUC5AC was more sensitive to changes in expression during exacerbation and was therefore more predictably associated with virus load, inflammation, symptom severity, decrements in lung function, and secondary bacterial infections. MUC5AC was functionally related to inflammation as Muc5ac-deficient (Muc5ac-/-) mice had attenuated rhinovirus (RV)–induced airway inflammation and exogenous MUC5AC glycoprotein administration augmented inflammatory responses and increased release of extracellular adenosine triphosphate (ATP) in mice and human airway epithelial cell cultures. Hydrolysis of ATP suppressed MUC5AC augmentation of rhinovirus-induced inflammation in mice. Therapeutic suppression of mucin production using an epidermal growth factor receptor (EGFR) antagonist ameliorated immunopathology in a mouse COPD exacerbation model. The coordinated virus induction of MUC5AC and MUC5B suggests that non-Th2 mechanisms trigger mucin hypersecretion during exacerbations. Our data identifies a pro-inflammatory role for MUC5AC during viral infection and suggest that MUC5AC inhibition may ameliorate COPD exacerbations.
Aran Singanayagam, Joseph Footitt, Matthias Marczynski, Giorgia Radicioni, Michael T. Cross, Lydia J. Finney, Maria-Belen Trujillo-Torralbo, Maria Adelaide Calderazzo, Jie Zhu, Julia Aniscenko, Thomas B. Clarke, Philip L. Molyneaux, Nathan W. Bartlett, Miriam F. Moffatt, William O. Cookson, Jadwiga A. Wedzicha, Christopher M. Evans, Richard C. Boucher, Mehmet Kesimer, Oliver Lieleg, Patrick Mallia, Sebastian L. Johnston
Women have higher prevalence of asthma compared to men. In asthma, allergic airway inflammation is initiated by IL-33 signaling through ST2, leading to increased IL-4, IL-5, and IL-13 production and eosinophil infiltration. Foxp3+ Tregs suppress and ST2+ Tregs promote allergic airway inflammation. Clinical studies showed the androgen, dehydroepiandrosterone (DHEA), reduced asthma symptoms in patients, and mouse studies showed androgen receptor (AR) signaling decreased allergic airway inflammation. Yet, the role of AR signaling on lung Tregs remains unclear. Using AR deficient and Foxp3 fate-mapping mice, we determined that AR signaling increased Treg suppression during Alternaria extract (Alt Ext, allergen) challenge by stabilizing Foxp3+ Tregs and limiting the number of ST2+ ex-Tregs and IL-13+ Th2 and ex-Tregs. AR signaling also decreased Alt Ext-induced ST2+ Tregs in mice by limiting Gata2 expression, a transcription factor for ST2, and by decreasing Alt Ext-induced IL-33 production from murine airway epithelial cells. We confirmed our findings in human cells where 5α-dihydrotestosterone (DHT), an androgen, decreased IL-33-induced ST2 expression in lung Tregs and decreased Alt Ext induced IL-33 secretion in human bronchial epithelial cells. Our findings showed that AR signaling stabilized Treg suppressive function, providing a mechanism for the sex difference in asthma.
Vivek D. Gandhi, Jacqueline-Yvonne Cephus, Allison E. Norlander, Nowrin U. Chowdhury, Jian Zhang, Zachary J. Ceneviva, Elie Tannous, Vasiliy V. Polosukhin, Nathan D. Putz, Nancy Wickersham, Amrit Singh, Lorraine B. Ware, Julie A Bastarache, Ciara M. Shaver, Hong Wei Chu, Ray S. Peebles Jr, Dawn C. Newcomb
Altered redox biology challenges all cells, with compensatory responses often determining a cell’s fate. When 15 lipoxygenase-1 (15LO1), a lipid peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine binding protein-1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase-4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter, SLC7A11, critically restores/maintains intracellular GSH. We hypothesized high 15LO1-PEBP1-GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG), to enhance Type-2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11 and T2 biomarkers (western blot and RNAseq) were measured in asthmatic and healthy control (HC) cells/fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared to HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, T2 cytokine (IL-13) induced 15LO1 generated hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers and worsened clinical outcomes. Thus, 15LO1 pathway-induced redox biology perturbations worsen T2 inflammation and asthma control, supporting15LO1 as a therapeutic target.
Tadao Nagasaki, Alexander J. Schuyler, Jinming Zhao, Svetlana N. Samovich, Kazuhiro Yamada, Yanhan Deng, Scott P. Ginebaugh, Stephanie A. Christenson, Prescott G. Woodruff, John V. Fahy, John B. Trudeau, Detcho Stoyanovsky, Anuradha Ray, Yulia Y. Tyurina, Valerian E. Kagan, Sally E. Wenzel
Air pollution is a well-known contributor to asthma. Air toxics are hazardous air pollutants that cause or may cause serious health effects. While individual air toxics have been associated with asthma, only a limited number of studies have specifically examined combinations of air toxics associated with the disease. We geocoded air toxic levels from the US National Air Toxics Assessment (NATA) to residential locations for participants of our AiRway in Asthma (ARIA) study. We then applied Data-driven ExposurE Profile extraction (DEEP), a novel machine learning-based method, to discover combinations of early-life air toxics associated with current use of daily asthma controller medication, lifetime emergency department visit for asthma, and lifetime overnight hospitalization for asthma. We discovered 20 multi-air toxic combinations and 18 single air toxics associated with at least one outcome. The multi-air toxic combinations included those containing acrylic acid, ethylidene dichloride, and hydroquinone, and they were significantly associated with asthma outcomes with odds ratios of 1.60 to 3.19. Several air toxic members of the combinations would not have been identified by single air toxic analyses, supporting the use of machine learning-based methods designed to detect combinatorial effects. Our findings provide knowledge about air toxic combinations associated with childhood asthma.
Yan-Chak Li, Hsiao-Hsien Leon Hsu, Yoojin Chun, Po-Hsiang Chiu, Zoe Arditi, Luz Claudio, Gaurav Pandey, Supinda Bunyavanich
In this study, we demonstrate that Forkhead Box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, is retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as a key player in lung allograft remodeling and fibrosis. Utilizing Foxf1_tdTomato BAC (Foxf1_tdTomato) and Foxf1_tdTomato;Col1a1_GFP mice, we show that Lin-Foxf1+ cells encompass the Sca1+CD34+ subset of collagen I-expressing mesenchymal cells (MCs) with capacity to generate colony forming units and lung epithelial organoids. Histologically, Foxf1-expressing MCs formed a three-dimensional network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA sequencing confirmed distinct transcriptional signatures of Foxf1pos/neg MCs, with Foxf1-expressing cells delineated by their high Gli1 and low Integrin α8 expression, from other collagen-expressing MCs. Foxf1+Gli1+ MCs demonstrated proximity to Sonic hedgehog (Shh) expressing bronchial epithelium, and mesenchymal Foxf1/Gli1 expression was found to be dependent on the paracrine Shh signaling in epithelial organoids. Utilizing a murine lung transplant model, we show dysregulation of the epithelial mesenchymal Shh/Gli1/Foxf1 crosstalk and expansion of this specific peri-bronchial MC population in chronically rejecting fibrotic lung allografts.
Russell R. Braeuer, Natalie M. Walker, Keizo Misumi, Serina Mazzoni-Putman, Yoshiro Aoki, Ruohan Liao, Ragini Vittal, Gabriel G. Kleer, David S. Wheeler, Jonathan Z. Sexton, Carol F. Farver, Joshua D. Welch, Vibha N. Lama
Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here we identify a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaces normal airspaces in a mouse model of progressive post-viral lung disease due to Sendai virus. Single-cell and lineage-tracing technologies identify a distinct subset of basal epithelial stem cells (basal-ESCs) that extend into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset is selectively expanded by crossing a cell growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that is independent of IL-33-receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupts homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuates post-viral disease in the lung based on down-regulation of remodeling and inflammation. We thereby identify a basal-ESC strategy to deploy innate-immune machinery that appears to overshoot the primordial goal of self-defense. The findings reveal new targets to stratify and correct chronic and often deadly post-viral disease.
Kangyun Wu, Kenji Kamimoto, Yong Zhang, Kuangying Yang, Shamus P. Keeler, Benjamin J. Gerovac, Eugene V. Agapov, Stephen P. Austin, Jennifer Yantis, Kelly A. Gissy, Derek E. Byers, Jennifer Alexander-Brett, Christy M. Hoffmann, Matthew Wallace, Michael E. Hughes, Erika C. Crouch, Samantha A. Morris, Michael J. Holtzman
Without CFTR-mediated HCO3- secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time-course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNFα and IL-17, two inflammatory cytokines that are elevated in CF airways. TNFα+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl-/HCO3- exchanger. Moreover, when CF epithelia were exposed to TNFα+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator-induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO3- secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.
Tayyab Rehman, Philip H. Karp, Ping Tan, Brian J. Goodell, Alejandro A. Pezzulo, Andrew L. Thurman, Ian M. Thornell, Samantha L. Durfey, Michael E. Duffey, David A. Stoltz, Edward F. McKone, Pradeep K. Singh, Michael J. Welsh