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
Tuberculosis (TB) is a persistent global pandemic and standard treatment has not changed for thirty years. Mycobacterium tuberculosis (Mtb) has undergone prolonged co-evolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesised that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a non-infectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared three primary human cell culture models at the whole transcriptome level, and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified twelve intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.
Michaela T. Reichmann, Liku B. Tezera, Andres F. Vallejo, Milica Vukmirovic, Rui Xiao, James Reynolds, Sanjay Jogai, Susan Wilson, Ben Marshall, Mark G. Jones, Alasdair Leslie, Jeanine M. D'Armiento, Naftali Kaminski, Marta E. Polak, Paul Elkington
Lung-resident memory B cells (BRM cells) are elicited after influenza infections of mice, but connections to other pathogens and hosts — as well as their functional significance — have yet to be determined. We postulate that BRM cells are core components of lung immunity. To test this, we examined whether lung BRM cells are elicited by the respiratory pathogen pneumococcus, are present in humans, and are important in pneumonia defense. Lungs of mice that had recovered from pneumococcal infections did not contain organized tertiary lymphoid organs, but did have plasma cells and noncirculating memory B cells. The latter expressed distinctive surface markers (including CD69, PD-L2, CD80, and CD73) and were poised to secrete antibodies upon stimulation. Human lungs also contained B cells with a resident memory phenotype. In mice recovered from pneumococcal pneumonia, depletion of PD-L2+ B cells, including lung BRM cells, diminished bacterial clearance and the level of pneumococcus-reactive antibodies in the lung. These data define lung BRM cells as a common feature of pathogen-experienced lungs and provide direct evidence of a role for these cells in pulmonary antibacterial immunity.
Kimberly A. Barker, Neelou S. Etesami, Anukul T. Shenoy, Emad I. Arafa, Carolina Lyon de Ana, Nicole M.S. Smith, Ian M.C. Martin, Wesley N. Goltry, Alexander M.S. Barron, Jeffrey L. Browning, Hasmeena Kathuria, Anna C. Belkina, Antoine Guillon, Xuemei Zhong, Nicholas A. Crossland, Matthew R. Jones, Lee J. Quinton, Joseph P. Mizgerd
Severe asthma remains challenging to manage with limited treatment options. We have previously shown that targeting smooth muscle integrin α5β1 interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this paper we show that another member of the integrin superfamily, integrin α2β1, is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I, and to a lesser degree, laminin-111. The addition of an inhibitor of integrin α2β1 impaired IL-13-enhanced contraction in mouse tracheal rings and human bronchial rings, and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of α2β1, it did increase α2β1-mediated adhesion and the level of expression of an activation-specific epitope on the β1 subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using two-photon microscopy and demonstrated that inhibition of α2β1 mitigated IL-13-enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identify cell-matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma.
Sean Liu, Uyen Ngo, Xin-Zi Tang, Xin Ren, Wenli Qiu, Xiaozhu Huang, William DeGrado, Christopher D.C. Allen, Hyunil Jo, Dean Sheppard, Aparna B. Sundaram
The dynamic regulation of endothelial pathophenotypes in pulmonary hypertension (PH) remains undefined. Cellular senescence is linked to PH with intracardiac shunts; however, its regulation across PH subtypes is unknown. Since endothelial deficiency of iron-sulfur (Fe-S) clusters is pathogenic in PH, we hypothesized that a Fe-S biogenesis protein, frataxin (FXN), controls endothelial senescence. An endothelial subpopulation in rodent and patient lungs across PH subtypes exhibited reduced FXN and elevated senescence. In vitro, hypoxic and inflammatory FXN deficiency abrogated activity of endothelial Fe-S-containing polymerases, promoting replication stress, DNA damage response, and senescence. This was also observed in stem cell-derived endothelial cells from Friedreich’s ataxia (FRDA), a genetic disease of FXN deficiency, ataxia, and cardiomyopathy, often with PH. In vivo, FXN deficiency-dependent senescence drove vessel inflammation, remodeling, and PH, while pharmacologic removal of senescent cells in Fxn-deficient rodents ameliorated PH. These data offer a model of endothelial biology in PH, where FXN deficiency generates a senescent endothelial subpopulation, promoting vascular inflammatory and proliferative signals in other cells to drive disease. These findings also establish an endothelial etiology for PH in FRDA and left heart disease and support therapeutic development of senolytic drugs, reversing effects of Fe-S deficiency across PH subtypes.
Miranda K. Culley, Jingsi Zhao, Yi Yin Tai, Ying Tang, Dror Perk, Vinny Negi, Qiujun Yu, Chen-Shan C. Woodcock, Adam Handen, Gil Speyer, Seungchan Kim, Yen-Chun Lai, Taijyu Satoh, Annie M.M. Watson, Yassmin Al Aaraj, John Sembrat, Mauricio Rojas, Dmitry Goncharov, Elena A. Goncharova, Omar F. Khan, Daniel G. Anderson, James E. Dahlman, Aditi U. Gurkar, Robert Lafyatis, Ahmed U. Fayyaz, Margaret M. Redfield, Mark T. Gladwin, Marlene Rabinovitch, Mingxia Gu, Thomas Bertero, Stephen Y. Chan
Discovering dominant epitopes for T cells, particularly CD4+ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2-expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium (Be)-specific CD4+ T cells in the lung. We discovered lung resident CD4+ T cells that expressed a disease-specific public CDR3β T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligands 4 (CCL4) and 3 (CCL3). HLA-DP2-CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and 4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4+ T cells specific for the HLA-DP2-CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a vicious cycle of innate and adaptive immune activation.
Michael T. Falta, Jeremy C. Crawford, Alex N. Tinega, Laurie G. Landry, Frances Crawford, Douglas G. Mack, Allison K. Martin, Shaikh M. Atif, Li Li, Radleigh G. Santos, Maki Nakayama, John W. Kappler, Lisa A. Maier, Paul G. Thomas, Clemencia Pinilla, Andrew P. Fontenot