Pulmonology
Abstract
Pulmonary fibrosis (PF) is a life-threatening disease that requires effective and well-tolerated therapeutic modalities. Previously, the distinct pathogenic roles of cannabinoid receptor 1 (CB1R) and inducible nitric oxide synthase (iNOS) in the lungs and their joint therapeutic targeting were highlighted in PF. However, the cell-specific role of CB1R in PF has not been explored. Here, we demonstrate that CB1R in alveolar macrophages (AMs) mediates the release of anandamide into the alveoli, which promotes PF by inducing profibrotic macrophages that are accessible to locally delivered antifibrotic therapy. A multitargeted therapy may improve therapeutic efficacy in PF. Pulmonary delivery of 0.5 mg/kg/day MRI-1867 (zevaquenabant), a peripherally acting hybrid CB1R/iNOS inhibitor, is as effective as systemic delivery of 10 mg/kg/day, and also matches the efficacy of nintedanib in mitigating bleomycin-induced PF. A systems pharmacology approach reveals that zevaquenabant and nintedanib treatments reverse pathologic changes in both distinct and shared PF-related pathways, which are conserved in human and mouse. Moreover, zevaquenabant treatment also attenuated fibrosis and profibrotic mediators in human precision-cut lung slices. These findings establish CB1R-expressing AMs as a therapeutic target and support local delivery of dual CB1R/iNOS inhibitor zevaquenabant by inhalation as an effective, well-tolerated, and safer strategy for PF.
Authors
Abhishek Basu, Muhammad Arif, Kaelin M. Wolf, Madeline Behee, Natalie L. Johnson, Lenny Pommerolle, Ricardo H. Pineda, John Sembrat, Charles N. Zawatsky, Szabolcs Dvorácskó, Nathan J. Coffey, Joshua K. Park, Seray B. Karagoz, Grzegorz Godlewski, Tony Jourdan, Judith Harvey-White, Melanie Königshoff, Malliga R. Iyer, Resat Cinar
Abstract
Alveolar epithelial type II (AT2) cell dysfunction is implicated in the pathogenesis of familial and sporadic idiopathic pulmonary fibrosis (IPF). We previously demonstrated that expression of an AT2 cell exclusive disease-associated protein isoform (SP-CI73T) in murine and patient-specific induced pluripotent stem cell (iPSC)-derived AT2 cells leads to a block in late macroautophagy and promotes time-dependent mitochondrial impairments; however, how a metabolically dysfunctional AT2 cell results in fibrosis remains elusive. Here, using murine and human iPSC-derived AT2 cell models expressing SP-CI73T, we characterize the molecular mechanisms governing alterations in AT2 cell metabolism that lead to increased glycolysis, decreased mitochondrial biogenesis, disrupted fatty acid oxidation, accumulation of impaired mitochondria, and diminished AT2 cell progenitor capacity manifesting as reduced AT2 self-renewal and accumulation of transitional epithelial cells. We identify deficient AMP-kinase signaling as a critical component of AT2 cell dysfunction and demonstrate that targeting this druggable signaling hub can rescue the aberrant AT2 cell metabolic phenotype and mitigate lung fibrosis in vivo.
Authors
Luis R. Rodríguez, Konstantinos-Dionysios Alysandratos, Jeremy Katzen, Aditi Murthy, Willy Roque Barboza, Yaniv Tomer, Sarah Bui, Rebeca Acín-Pérez, Anton Petcherski, Kasey Minakin, Paige Carson, Swati Iyer, Katrina Chavez, Charlotte H. Cooper, Apoorva Babu, Aaron I. Weiner, Andrew E. Vaughan, Zoltan Arany, Orian S. Shirihai, Darrell N. Kotton, Michael F. Beers
Abstract
The pathobiology of pulmonary hypertension (PH) is complex and multiple cell types contribute to disease pathogenesis. We sought to characterize the molecular crosstalk between endothelial and mesenchymal cells that promote PH in the tumor necrosis factor alpha transgenic (TNF-Tg) model of PH. Pulmonary endothelial and mesenchymal cells were isolated from WT and TNF-Tg mice underwent single-cell RNA sequencing. Data were analyzed using clustering, differential gene expression and pathway analysis, ligand-receptor interaction, transcription factor binding, and RNA velocity assessments. Significantly altered ligand-receptor interactions were confirmed with immunofluorescent staining. TNF-Tg mice had increases in smooth muscle cells and Col14+ fibroblasts, and reductions in general capillary (gCAP) endothelial cells, Col13+ fibroblasts, pericytes, and myofibroblasts. Pathway analysis demonstrated NF-kB, JAK/STAT, and interferon mediated inflammation, endothelial apoptosis, loss of vasodilatory pathways, increased TGF-beta signaling, and smooth muscle cell proliferation. Ligand-receptor analysis demonstrated a loss of BMPR2 signaling in TNF lungs and establishment of a maladaptive BMP signaling cascade which functional studies revealed stems from endothelial NFkB activation and subsequent endothelial SMAD2/3 signaling. This system highlights a complex set of changes in cellular composition, cell communication, and cell fate driven by TNF signaling which lead to aberrant BMP signaling which is critical for development of PH.
Authors
ML Garcia-Hernandez, Javier Rangel-Moreno, Qingfu Xu, Ye Jin Jeong, Soumyaroop Bhattacharya, Ravi Misra, Stacey Duemmel, Ke Yuan, Benjamin D. Korman
Abstract
IL-33 is a key driver of type-2 inflammation and implicated in pathology of COPD and asthma. However, the mechanism for IL-33 secretion and regulation in the context of chronic airway disease is poorly understood. We previously reported an airway disease-associated isoform IL-33Δ34 that escapes nuclear sequestration and is tonically secreted from epithelial cells. Here, we describe how this IL-33Δ34 isoform interacts with HSP70 within cells and is targeted to secretory organelles through coordinated binding to phosphatidylserine (PS), and delivered to compartments for unconventional protein secretion (CUPS). Once secreted, extracellular HSP70 (eHSP70) in complex with IL-33Δ34 stabilizes cytokine by inhibiting oxidation and degradation, which results in enhanced IL-33Δ34-receptor binding and activity. We further find evidence that IL-33 along with mediators of the proteostasis network HSP70, HSP90 and the Chaperonin Containing TCP1 (CCT) complex are dysregulated in human chronic airway disease. This phenomenon is reflected in the differential extracellular vesicle (EV) proteome in bronchial wash from COPD and asthma samples, which could mark disease activity and potentiate IL-33 function. This study confirms proteostasis intermediates, chiefly HSP70, as a chaperone for non-canonical IL-33 secretion and activity that may be amenable for therapeutic targeting in the chronic airway diseases COPD and asthma.
Authors
Omar A. Osorio, Heather E. Raphael, Colin E. Kluender, Ghandi F. Hassan, Lucy S. Cohen, Deborah F. Steinberg, Ella Katz-Kiriakos, Morgan D. Payne, Ethan M. Luo, Jamie L. Hicks, Derek E. Byers, Jennifer Alexander-Brett
Abstract
The gain-of-function MUC5B promoter variant is the dominant risk factor for the development of idiopathic pulmonary fibrosis (IPF). However, its impact on protein expression in both non-fibrotic control and IPF lung specimens have not been well characterized. Utilizing laser capture microdissection coupled to mass spectrometry (LCM-MS), we investigated the proteomic profiles of airway and alveolar epithelium in non-fibrotic controls (n = 12) and IPF specimens (n = 12), stratified by the MUC5B promoter variant. Through qualitative and quantitative analyses, as well as pathway analysis and immunohistological validation, we have identified a distinct MUC5B-associated protein profile. Notably, the non-fibrotic control alveoli exhibited substantial MUC5B-associated protein changes, with an increase of IL-3 signaling. Additionally, we found that epithelial cells overlying IPF fibroblastic foci cluster closely to alveolar epithelia and express proteins associated with cellular stress pathways. In conclusion, our findings suggest that the MUC5B promoter variant leads to protein changes in alveolar and airway epithelium that appears to be associated with initiation and progression of lung fibrosis.
Authors
Jeremy A. Herrera, Mark Maslanka, Rachel Z. Blumhagen, Rachel Blomberg, Nyan Ye Lwin, Janna Brancato, Carlyne D. Cool, Jonathan P. Huber, Jonathan S. Kurche, Chelsea M. Magin, Kirk C. Hansen, Ivana V. Yang, David A. Schwartz
Abstract
Bacterial pneumonia is the most common cause of acute respiratory distress syndrome (ARDS), characterized by disrupted pulmonary endothelial barrier function, hyperinflammation, and impaired alveolar epithelial fluid clearance. ARDS has a high mortality rate and no proven pharmacological treatments, stressing the need for new targeted therapies. The TIP peptide, mimicking the lectin-like domain of TNF, directly binds to the α subunit of the epithelial Na+ channel, expressed in both alveolar epithelial and capillary endothelial cells, and may increase lung endothelial barrier function and alveolar fluid clearance during bacterial infection. This study tested these potential therapeutic mechanisms of the TIP peptide in a clinically relevant preparation of the ex vivo–perfused human lung injured by Streptococcus pneumoniae. Therapeutic administration of the TIP peptide reduced pulmonary barrier permeability to protein and lung edema formation, increased alveolar edema fluid clearance, and produced an antiinflammatory effect in the airspaces with reductions in IL-6 and IL-8 levels. Additionally, the TIP peptide reduced the translocation of bacteria into the circulation. These findings establish 3 mechanisms of benefit with the TIP peptide to reduce injury in the human lung and support the clinical relevance as a potential therapeutic for pneumococcal bacterial pneumonia.
Authors
Mazharul Maishan, Hiroki Taenaka, Bruno Evrard, Shotaro Matsumoto, Angelika Ringor, Carolyn Leroux, Rudolf Lucas, Michael A. Matthay
Abstract
In asthma, airway epithelial remodeling is characterized by aberrant goblet cell metaplastic differentiation accompanied by epithelial cell hyperplasia and hypertrophy. These pathologic features in severe asthma indicate a loss of control of proliferation, cell size, differentiation, and migration. mTOR is a highly conserved pathway that regulates protein synthesis, cell size, and proliferation. We hypothesized that the balance between mTOR and autophagy regulates mucous cell metaplasia. Airways from individuals with severe asthma showed increased mTOR signaling by RPS6 phosphorylation, which was reproduced using an IL-13-activated model of primary human airway epithelial cells (hAECs). mTOR inhibition by rapamycin led to a decrease of IL-13-mediated cell hypertrophy, hyperplasia, and MUC5AC mucous metaplasia. BrdU labeling during IL-13-induced mucous metaplasia confirmed that mTOR was associated with increased basal-to-apical hAEC migration. mTOR activation by genetic deletion of Tsc2 in cultured mouse AECs increased IL-13-mediated hyperplasia, hypertrophy, and mucous metaplasia. Transcriptomic analysis of IL-13-stimulated hAEC identified mTOR-dependent expression of genes associated with epithelial migration and cytoskeletal organization. In summary, these findings point to IL-13-dependent and independent roles of mTOR signaling in the development of pathogenic epithelial changes contributing to airway obstruction in severe asthma.
Authors
Katrina M. Kudrna, Luis F. Vilches, Evan M. Eilers, Shailendra K. Maurya, Steven L. Brody, Amjad Horani, Kristina L. Bailey, Todd A. Wyatt, John D. Dickinson
Abstract
There is an emerging role for Stimulator of interferon genes (STING) signaling in pulmonary hypertension (PH) development. Related, prior resesarch has demonstrated the relevance of the immune checkpoint protein Programmed death ligand 1 (PD-L1) expression by immunoregulatory myeloid cells in PH. However, there remains a need to elucidate the cell-specific role of STING expression, and the STING/PD-L1 signaling axis in PH, before readily available disease-modifying therapies can be applied to patients with disease. Here, through generation of bone marrow chimeric mice, we show that STING-/- mice receiving wild-type (WT) bone marrow are protected against PH secondary to chronic hypoxia. We further demonstrate a cellular dichotomous role for STING in PH development with STING expression by smooth muscle cells contributing to PH, and its activation on myeloid cells being pivotal in severe disease prevention. Finally, we provide evidence that a STING-PD-L1 axis modulates disease severity, suggesting future potential therapeutic applications. Overall, these data provide concrete evidence of STING involvement in PH in a cell-specific manner, establishing biologic plausibility for cell-targeted STING-related therapies in PH treatment.
Authors
Ann T. Pham, Shiza Virk, Aline C. Oliveira, Matthew D. Alves, Chunhua Fu, Yutao Zhang, Jimena Alvarez-Castanon, Brian B. Lee, Keira L. Lee, Radwan Mashina, Katherine E. Ray, Patrick Donabedian, Elnaz Ebrahimi, Harsh Patel, Reeha Patel, Duncan Lewis, Zhiguang Huo, Harry Karmouty-Quintana, Li Chen, Lei Jin, Andrew J. Bryant
Abstract
Pathologic implications of dysregulated pulmonary vascular metabolism to pulmonary arterial hypertension (PAH) are increasingly recognized, but their clinical applications have been limited. We hypothesized that metabolite quantification across the pulmonary vascular bed in connective tissue disease–associated (CTD-associated) PAH would identify transpulmonary gradients of pathobiologically relevant metabolites, in an exercise stage–specific manner. Sixty-three CTD patients with established or suspected PAH underwent exercise right heart catheterization. Using mass spectrometry–based metabolomics, metabolites were quantified in plasma samples simultaneously collected from the pulmonary and radial arteries at baseline and during resistance-free wheeling, peak exercise, and recovery. We identified uptake and excretion of metabolites across the pulmonary vascular bed, unique and distinct from single vascular site analysis. We demonstrated the physiological relevance of metabolites previously shown to promote disease in animal models and end-stage human lung tissues, including acylcarnitines, glycolytic intermediates, and tryptophan catabolites. Notably, pulmonary vascular metabolite handling was exercise stage specific. Transpulmonary metabolite gradients correlated with hemodynamic endpoints largely during free-wheeling. Glycolytic intermediates demonstrated physiologic significance at peak exercise, including net uptake of lactate in those with more advanced disease. Contribution of pulmonary vascular metabolism to CTD-PAH pathogenesis and therapeutic candidacy of metabolism modulation must be considered in the context of physiologic stress.
Authors
Michael H. Lee, Thaís C. F. Menezes, Julie A. Reisz, Francesca I. Cendali, Eloara V. M. Ferreira, Jaquelina S. Ota-Arakaki, Priscila A. Sperandio, Rahul Kumar, Claudia Mickael, Martin M. Ieong, Juliana Lucena Santos, Ana Carolina B. Duarte, Dara C. Fonseca Balladares, Kevin Nolan, Rubin M. Tuder, Paul M. Hassoun, Angelo D’Alessandro, Rudolf K. F. Oliveira, Brian B. Graham
Abstract
Airway smooth muscle (ASM) hyperplasia is a hallmark of airway remodeling in asthma, which still lacks an effective treatment. Low-density lipoprotein receptor-related protein 1 (LRP1) is involved in regulating the proliferation of various cell types, and the intracellular domain of LRP1 (LRP1-ICD) also exhibits unique biological functions. However, the role of LRP1 in asthma airway remodeling remains unclear. In the present study, LRP1 was increased in ASM cells of mice with OVA-induced chronic asthma, with the elevation in LRP1-ICD protein levels being significantly greater than that of the LRP1 β-chain. In vivo experiments demonstrated that inhibiting LRP1 reduced ASM proliferation in these mice. Mechanistically, LRP1 knockdown inhibited the FGF2/ERK signaling pathway, thereby arresting cell cycle progression and suppressing ASM cell proliferation. Additionally, in vitro experiments revealed that the inhibitory effect of LRP1-ICD overexpression on ASM cell proliferation was lost after adjusting the levels of the LRP1. LRP1-ICD overexpression inhibited full-length LRP1 protein levels by promoting its protein degradation rather than by suppressing its transcription, thus preventing further exacerbation of asthma. In conclusion, this study clarifies the molecular biological mechanism by which LRP1 regulates ASM proliferation, suggesting targeting full-length LRP1 as a novel strategy for therapeutic intervention in asthma airway remodeling.
Authors
Ya Deng, Jiaying Zhao, Chen Gong, Wenqian Ding, Lulu Fang, Huaqing Liu, Ming Li, Bing Shen, Shenggang Ding
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