Pulmonology
Abstract
Type 2 (T2) immune cells dominate the airways of mild-moderate asthma (MMA) patients with a more complex Type 1 (T1)-T2 mixed immune response evident in treatment-refractory severe asthma (SA). We hypothesized that comparing the transcriptomes of the airway epithelium of SA and MMA patients would reveal molecular signatures associated with more severe disease in the context of a complex immune response. Using our novel interpretable machine learning tool, SLIDE, meaningful latent factors (context-specific gene co-expression networks) were revealed that distinguished SA from MMA. Unexpectedly, an aberrant high expression of normally host-protective, membrane-tethered and IFN-inducible mucins, MUC1 and MUC4, was identified in SA. Gene networks in the significant latent factors discriminating SA from MMA corresponded to enrichment of a keratinization program in SA airways. Keratinization was marked by increased expression of the stress keratin KRT16, signifying squamous metaplasia suggesting adaptive reprogramming of the airway epithelium in response to chronic stress. These mucins and KRT16 were inversely associated with lung function in two separate asthma cohorts. Imaging of endobronchial biopsies revealed significantly higher KRT16 protein expression in SA compared to MMA that strongly correlated with MUC1 protein expression. Our study identifies dysregulated host-protective and maladaptive repair responses in SA distinguishing from MMA.
Authors
Sagar L. Kale, Augusta M. Vincent, Mark A. Ross, Isha Mehta, Michael J. Calderon, Richard P. Ramonell, Himanshu Setya, Jessica C. McCreary-Partyka, Huijuan Yuan, Stephanie A. Christenson, Prescott G. Woodruff, Mario Castro, Kaharu Sumino, Nizar N. Jarjour, Loren C. Denlinger, Benjamin Gaston, Eugene R. Bleecker, Deborah A. Meyers, Wendy C. Moore, Elliot Israel, Bruce D. Levy, David Mauger, Serpil Erzurum, Anthony Newbrough, Taylor J. Nee, Prabir Ray, Claudette M. St. Croix, Sally E. Wenzel, Jishnu Das, Anuradha Ray, Marc C. Gauthier
Abstract
Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. It is known that adaptive immunity and specifically CD4 T cells are necessary for experimental disease pathogenesis. The lectin complement system is activated in those infected with schistosomiasis. We tested the hypothesis that lectin complement promotes Th2 CD4 T cell activation, leading to PH in a schistosomiasis exposure model. Wildtype and transgenic mice lacking mannose binding lectin (MBL), and bone marrow chimeras, were experimentally exposed to Schistosoma mansoni eggs. PH severity was assessed by hemodynamics and vascular remodeling, and CD4 T cell density and phenotype was assessed by flow cytometry. Wildtype recipients of MBL knockout bone marrow (BM) were protected from Schistosoma-induced PH. The protection from PH was associated with fewer Th2 CD4 T cells. In wildtype mice exposed to Schistosoma, CD4 T cells expression of MBL increased. MBL-deficient CD4 T cells had a suppressed Th2 phenotype when exposed to Schistosoma antigens. Mice with deficiency of C4, which functions downstream of MBL in the lectin complement pathway, were not protected from Schistosoma-PH. Mice lacking MBL were not protected from PH caused by hypoxia exposure. MBL in CD4 T cells promotes Schistosoma-induced PH.
Authors
Claudia Mickael, Dara C. Fonseca Balladares, Rahul Kumar, Michael H. Lee, Kevin Nolan, Linda Sanders, Katie J. Tuscan, Ramraj Prasad, Pilar Londono, Fernanda P. Oliveira, Kennedi B. Pyper, Ari B. Molofsky, Rubin M. Tuder, Kurt R. Stenmark, Brian B. Graham
Abstract
Pulmonary alveolar proteinosis (PAP) is a rare pulmonary syndrome characterized by impaired surfactant clearance, driven by dysfunctional cholesterol efflux in alveolar macrophages (AMs). However, the molecular determinants governing AM cholesterol homeostasis remain incompletely defined. Here, through a genome-wide CRISPR screen in foamy macrophages and bulk RNA sequencing of AMs from PAP patients, we identify DTX4 as a pivotal regulator of cholesterol efflux in AMs. In mice, AAV-mediated silencing of DTX4 led to excessive AM lipid accumulation, exacerbated proteinosis, increased lung opacities, and deteriorated pulmonary function. Similarly, DTX4 depletion in primary AMs impaired cholesterol efflux and promoted intracellular lipid deposition. Conversely, AM-specific overexpression of DTX4 in the Csf2ra–/– PAP model markedly alleviated lipid accumulation, mitigated alveolar proteinosis, restored lung densities, and rescued pulmonary function. Mechanistically, DTX4 stabilizes the GM-CSF receptor via an E3-independent interaction to sustain JAK2/STAT5 signaling, which reciprocally maintains DTX4 transcription. This positive-feedback loop drives PPARγ expression, and its disruption in PAP impairs cholesterol efflux, a defect partially reversible by ectopic PPARγ expression. Collectively, our findings identify DTX4 as a central orchestrator of AM cholesterol efflux and surfactant homeostasis, positioning it as a promising therapeutic target for PAP.
Authors
Zimu Wang, Jingwei Shi, Xu Ye, Xinye Xia, Huihui Zhu, Qi Li, Min Chen, Yichao Zhao, Yingwei Zhang, Mengshu Cao, Yonglong Xiao, Xinmei Huang
Abstract
Airway structural remodeling and hyperresponsiveness (AHR), hallmarks of asthma, are influenced by genetic variations and adverse exposures. While intrauterine perturbations in lung development have been linked to adult pulmonary disease, the developmental origins of these abnormalities remain poorly understood. Here, we provide evidence of genetic background playing a key role in this process. Using A/J and C57BL/6J mice known for their distinct susceptibility to AHR, we show that A/J embryos selectively develop an aberrant airway smooth muscle (SM) program and AHR in adulthood when exposed transiently to a vitamin A/retinoic acid (RA)-disrupted intrauterine environment in vivo by maternal BMS493 administration. Single-nuclei multiomics identified a mesenchymal cell population overactivating TGFβ targets in response to BMS selectively in A/J lungs. These cells, localized to sites of airway SM initiation and pSMAD2-3, exhibited robust BMS-mediated upregulation of SMAD2-3 targets, including regulators of SM program Pdgfra and Tnc. Functional analyses in vivo and cultured lungs showed aberrant SM formation in areas of overactive TGFβ of BMS-exposed lungs. These abnormalities were prevented by inhibiting TGFβ signaling in utero in RA-deficient embryos. These findings underscore how distinct genetic backgrounds respond to intrauterine perturbations that program airway structure and function, with potential lasting consequences in postnatal pulmonary function.
Authors
Takehiro Otoshi, Benjamin D. Kotton, Ayyappa K.S. Kameshwar, Yoshinori Seki, Zachary Cardell, Xiangyi Ke, Yuta Matsuno, Pooja Rajaram, Youn-Kyung Kim, Sarah M. Sharpton, Loredana Quadro, Wellington V. Cardoso, Masako Suzuki
Abstract
Hypercapnia, elevated carbon dioxide (CO2), is common in advanced chronic obstructive pulmonary disease (COPD) and predicts poor clinical outcomes. Traditionally considered a consequence of disease severity, hypercapnia may drive disease progression by promoting airway dysfunction. Here, we show that hypercapnia acts as an active stressor, driving airway smooth muscle (ASM) constriction through a stromal interaction molecule 1 (STIM1)-dependent pathway. Hypercapnia rapidly activates ERK, triggering sarcoplasmic reticulum calcium (Ca2+) release via phosphorylation of the inositol 1,4,5-trisphosphate receptor. ERK also induces nuclear translocation of the transcription factor c-Fos, enhancing STIM1 transcription. These responses were observed under both supraphysiological (~120 mmHg) and clinically relevant (50-60 mmHg) hypercapnia. Increased STIM1 abundance sustains store-operated Ca2+ entry (SOCE), amplifying ASM signaling. In mice, hypercapnia increased ASM and airway contractility in a STIM1-dependent manner. Human genetic analyses revealed noncoding STIM1 variants associated with reduced lung expression that were enriched in COPD patients. These variants correlated with lower airway resistance under normocapnia; however, this benefit was lost during hypercapnia, indicating a potential gene–environment interaction. Together, our findings position STIM1 as a key mechanistic node linking hypercapnia to Ca2+ dysregulation and airway obstruction, defining a CO2–ERK–STIM1–SOCE axis with translational relevance to chronic lung disease.
Authors
Masahiko Shigemura, Vitalii Kryvenko, Jennifer A. Pacheco, Megan J. Puckelwartz, Milos Aleksic, Natalia D. Magnani, Emma E. Thompson, Francisco Javier Martin-Romero, Eoin P. Cummins, Werner Seeger, Andreas Bräuninger, Lynn C. Welch, G.R. Scott Budinger, Emilia Lecuona, Laura A. Dada, Ankit Bharat, István Vadász, Murali Prakriya, Jacob I. Sznajder
Abstract
Authors
Sarah W. Robison, Jindong Li, Kristopher R. Genschmer, Liliana Viera, Jeremy B. Foote, Landon Wilson, W. Edward Swords, J. Edwin Blalock, Amit Gaggar, Xin Xu
Abstract
Pulmonary Arterial Hypertension (PAH) is a rare vascular disorder characterized by elevated pressure in pulmonary arteries, eventually leading to right ventricular failure. Approximately 50% of pediatric disease and 20% of adult disease can be linked to a genetic mutation, with nearly 70% of these cases involving mutations in the bone morphogenetic protein receptor type 2 (BMPR2) locus. Investigations using rodent models have made substantial advances in our understanding of BMPR2 signaling; however, limited data exist regarding the onset and course of PAH, and etiologies for phenotypic expression in these patients remain unknown. In this work, we describe the development of an ovine model of heritable PAH. Because homozygous disruption of BMPR2 is embryonic lethal, we developed heterozygous BMPR2 sheep by using a PAM-disrupting synonymous single stranded oligodeoxyribonucleotide alongside a single guide RNA and Cas9 mediated gene editing strategy. The resulting BMPR2(+/-) lambs demonstrated cardiac and pulmonary vascular pathology that are consistent with BMPR2 mutation-driven PAH observed in humans. Given the genetic and physiological similarities of BMPR2(+/-) sheep to humans with heritable PAH, this large animal model will serve as a vital platform for mechanistic molecular studies and will provide a much-needed pre-clinical model for extensive treatment evaluations.
Authors
Sanjeev A. Datar, Nicholas Werry, Austin R. Brown, Devon S. Fitzpatrick, Oluwafemi Falade, Josephine F. Trott, Rachel Hutchings, Elena K. Amin, Jessica M. Morgan, Hythem Nawaytou, Gail H. Deutsch, Eric G. Johnson, Omar A. Gonzales Viera, Thomas F. Bishop, Tara Urbano Beach, Bret R. McNabb, Eric D. Austin, Jeffrey R. Fineman, Alison L. Van Eenennaam
Abstract
BACKGROUND There are no known serum biomarkers that provide mechanistic insight or prognostic enrichment for post–COVID-19 pulmonary fibrosis.METHODS We tested associations of serum biomarkers with radiographic fibrosis-like abnormalities (reticulation, traction bronchiectasis, or honeycombing) on thoracic computed tomography (CT) scans 4 months, 15 months, and 3 years after hospitalization in an American discovery cohort of severe-to-critical COVID-19 survivors, and externally validated findings in 2 Canadian cohorts of moderate-to-critical COVID-19 survivors. In the discovery cohort, we investigated the dose-response relationship of the biomarker with CT-derived airway-to-lung ratio. We performed single-cell RNA sequencing (scRNA-seq) of transbronchial lung biopsies from COVID-19 survivors obtained 3 years after COVID-19 hospitalization and conducted immunofluorescence analysis of COVID-19 lung explants.RESULTS Among 150 discovery cohort participants, only higher levels of circulating club cell secretory protein-16 (CC16, encoded by the SCGB1A1 gene) at hospital discharge, 4 months, 15 months, and 3 years were associated with thoracic CT fibrosis-like abnormalities in cross-sectional and longitudinal analyses. Higher CC16 levels were associated with thoracic CT fibrosis-like abnormalities in 2 validation cohorts (n = 56 and n = 37). CC16 levels were linearly associated with increased airway-to-lung ratio. scRNA-seq revealed increased proportions of epithelial cells expressing SCGB1A1 and SCGB1A1/MUC5B in COVID-19 survivors with fibrosis. Immunofluorescence analysis of COVID-19 lung explants demonstrated increased numbers of SCGB1A1-expressing epithelial cells only in small (<100 μm) airways, with 3-fold more CC16/MUC5B-coexpressing cells in respiratory bronchioles..CONCLUSION. Higher CC16 levels are associated with CT fibrosis-like abnormalities for up to 3 years following moderate-to-critical COVID-19. Increased CC16 reflects dysregulated small airway epithelial progenitor cell remodeling and increased expansion of CC16+MUC5B+ epithelial cells in respiratory bronchioles after COVID-19.TRIAL REGISTRATION Not applicable.FUNDING Department of Defense, NIH, and Japan Society for the Promotion of Science for Young Scientists.
Authors
Matthew R. Baldwin, Ansley E. Jones, David Zhang, Chandan Gurung, Zain Khan, Anjali Saqi, Xuehan Yang, Ying Wei, Renu Nandakumar, Scarlett O. Murphy, Claire F. McGroder, Faisal Shaikh, Selim Arcasoy, Luke Benvenuto, Harpreet Grewal, Benjamin M. Smith, Eric A. Hoffman, Agnes C.Y. Yuen, Parteek Johal, Christopher Carlsten, Christopher J. Ryerson, J. Brent Richards, Alyson W. Wong, Tomoko Nakanishi, Aditi S. Shah, Christine Kim Garcia
Abstract
Post-acute sequelae of SARS-CoV-2 (PASC) occurs in subsets of individuals, including those with pre-existing lung disease. To investigate PASC pathogenesis and therapeutics in a chronic bronchitis mouse model (Scnn1b-Tg), Scnn1b-Tg and WT mice were inoculated with a mouse adapted SARS-CoV-2 virus (SARS-CoV-2MA10) and followed for 60 days. Viral titer, histology, immunohistochemistry (IHC), single-cell RNA sequencing, RNA in situ hybridization, and spatial transcriptomic profiling characterized disease pathologies. Scnn1b-Tg mice inoculated with SARS-CoV-2MA10 exhibited lower viral titers and less weight loss than WT mice. Airway epithelia of Scnn1b-Tg mice were less infected than epithelia of WT mice, reflecting increased airway mucus and enhanced epithelial antiviral activities in Scnn1b-Tg mice. However, Scnn1b-Tg mice subsequently exhibited heterogeneous airway and parenchymal disease with elevated Il33 expression characteristic of human eosinophilic pneumonia. Cohorts of infected mice were administered a monoclonal antibody targeting the IL-33 receptor (ST2) or enteral prednisone. Administration of an anti-ST2 monoclonal antibody mitigated development of eosinophilic pneumonia while enteral prednisone suppressed IL33 expression and disease. The eosinophilic pneumonia in Scnn1b-Tg mice after SARS-CoV-2MA10 infection mimics reports of eosinophilic pneumonia in humans post-SARS-CoV-2, suggesting targeting of IL-33 may be beneficial in treating post-viral eosinophilic pneumonia in humans.
Authors
Padraig E. Hawkins, Sarah R. Leist, Hong Dang, Minako Saito, Lisa C. Morton, Rodney C. Gilmore, Stephen A. Schworer, Ella F. Burns, Jason R. Rock, Robert S. Hagan, James J. Pestka, Alexandra Schäfer, Kenichi Okuda, Lauren K. Heine, Jack R. Harkema, Wanda K. O'Neal, Alessandra Livraghi-Butrico, Raymond J. Pickles, Ralph S. Baric, Richard C. Boucher
Abstract
Influenza A virus (IAV) infection is a major cause of morbidity and mortality for patients worldwide. Alveolar type 2 (AT2) cells are the preferential target of IAV as part of the pathogenesis of viral pneumonia and acute respiratory distress syndrome (ARDS). Early IAV infection of alveolar cells has been challenging to model both in vitro and in vivo. To address this challenge, we used a combination of murine and human primary alveolar organoids to define methods for robust IAV infection and evaluated cell-autonomous consequences of IAV using a temporal series of multiome paired single nuclei RNA and ATAC sequencing assays. Infected AT2 cells demonstrated conserved changes defined by early loss of surfactant secretion, decreased lipid biogenesis, a rapid burst of antiviral response, and late viral-mediated suppression. Surprisingly, uninfected AT2 cells underwent substantial transcriptional and epigenomic changes in IAV-treated cultures, leading to transition to damage-associated cell states within hours via a process driven by the inflammatory milieu of murine organoids. Together, these data provide new methods for high-fidelity modeling of IAV infection in alveolar cells and defined a conserved AT2 cell response signature to IAV with implications for ARDS pathogenesis.
Authors
Amber Elitz, Sharlene Fernandes, Kathleen C.S. Cook, Helen I Warheit-Niemi, Barbara Zhao, Andrea Toth, Amanda L. Zacharias, William J. Zacharias
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