Alveolar epithelial type II (AEC2) cells strictly regulate lipid metabolism to maintain surfactant synthesis. Loss of AEC2 cell function and surfactant production are implicated in the pathogenesis of the smoking-related lung disease chronic obstructive pulmonary disease (COPD). Whether smoking alters lipid synthesis in AEC2 cells and whether altering lipid metabolism in AEC2 cells contributes to COPD development are unclear. In this study, high-throughput lipidomic analysis revealed increased lipid biosynthesis in AEC2 cells isolated from mice chronically exposed to cigarette smoke (CS). Mice with a targeted deletion of the de novo lipogenesis enzyme, fatty acid synthase (FASN), in AEC2 cells (FasniΔAEC2) exposed to CS exhibited higher bronchoalveolar lavage fluid (BALF) neutrophils, higher BALF protein, and more severe airspace enlargement. FasniΔAEC2 mice exposed to CS had lower levels of key surfactant phospholipids but higher levels of BALF ether phospholipids, sphingomyelins, and polyunsaturated fatty acid–containing phospholipids, as well as increased BALF surface tension. FasniΔAEC2 mice exposed to CS also had higher levels of protective ferroptosis markers in the lung. These data suggest that AEC2 cell FASN modulates the response of the lung to smoke by regulating the composition of the surfactant phospholipidome.
Li-Chao Fan, Keith McConn, Maria Plataki, Sarah Kenny, Niamh C. Williams, Kihwan Kim, Jennifer A. Quirke, Yan Chen, Maor Sauler, Matthias E. Möbius, Kuei-Pin Chung, Estela Area Gomez, Augustine M.K. Choi, Jin-Fu Xu, Suzanne M. Cloonan
Optimal lung repair and regeneration is essential for recovery from viral infections including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known if the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections including the need for hospitalization and post-discharge mortality; this persists even after adjusting for common confounding factors. Further, we use a combination of lung organoid assays, single cell RNA sequencing (Sc-seq) and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show for the first time that lung organoids have a functional circadian clock, and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration- in tracheal cells via the Wnt/β-catenin pathway and through IL1β in alveolar epithelial cells. We speculate, that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.
Amruta Naik, Kaitlyn M. Forrest, Oindrila Paul, Yasmine Issah, Utham Kashyap Valekunja, Soon Yew Tang, Akhilesh B. Reddy, Elizabeth J. Hennessy, Thomas G. Brooks, Fatima N. Chaudhry, Apoorva Babu, Michael P. Morley, Jarod A. Zepp, Gregory R. Grant, Garret FitzGerald, Amita Sehgal, G. Scott Worthen, David B. Frank, Edward E. Morrisey, Shaon Sengupta
Proline and its synthesis enzyme pyrroline-5-carboxylate reductase 1 (PYCR1) are implicated in epithelial-mesenchymal transition (EMT), yet how proline and PYCR1 function in allergic asthmatic airway remodeling via EMT has not yet been addressed. In the present study, increased levels of plasma proline and PYCR1 were observed in asthmatic patients. Similarly, proline and PYCR1 in lung tissues were higher in a murine allergic asthma model induced by house dust mites (HDMs). Pycr1 knockout (KO) decreased proline in lung tissues, with reduced airway remodeling and EMT. Mechanistically, loss of Pycr1 restrained HDM-induced EMT by modulating mitochondrial fission, metabolic reprogramming, and the AKT/mTOR1 and WNT3a/β–catenin signaling pathways in airway epithelial cells. Therapeutic inhibition of PYCR1 in wild-type mice disrupted HDM-induced airway inflammation and remodeling. Deprivation of exogeneous proline partially relieved HDM-induced airway remodeling to some extent. Collectively, this study illuminates that proline and PYCR1 involved with airway remodeling in allergic asthma could be viable targets for asthma treatment.
Tingting Xu, Zhenzhen Wu, Qi Yuan, Xijie Zhang, Yanan Liu, Chaojie Wu, Meijuan Song, Jingjing Wu, Jingxian Jiang, Zhengxia Wang, Zhongqi Chen, Mingshun Zhang, Mao Huang, Ningfei Ji
Reducing inflammatory damage and improving alveolar epithelium regeneration are two key approaches to promoting lung repair in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Stimulation of cholinergic-α7nAChR (α7 nicotinic acetylcholine receptor, coded by Chrna7) signaling could dampen lung inflammatory injury. However, whether activation of α7nAChR in alveolar type II (AT2) cells promotes alveolar epithelial injury repair and underlying mechanisms are elusive. Here, we found that α7nAChR was expressed on AT2 cells and was upregulated in response to LPS-induced ALI. Meanwhile, deletion of Chrna7 in AT2 cells impeded lung repair process and worsened lung inflammation in ALI. Using in vivo AT2 lineage-labeled mice and ex vivo AT2-derived alveolar organoids, we demonstrated that activation of α7nAChR expressed on AT2 cells improved alveolar regeneration by promoting AT2 cells to proliferate and subsequently differentiate toward alveolar type I (AT1) cells. Then we screened out the WNT7B signaling pathway by the RNA sequencing analysis of in vivo AT2 lineage-labeled cells, and further confirmed its indispensability for α7nAChR activation-mediated alveolar epithelial proliferation and differentiation. Thus, we have identified an unrecognized pathway that cholinergic-α7nAChR signaling determines alveolar regeneration and repair, which might provide us a novel therapeutic target for combating ALI.
Xiaoyan Chen, Cuiping Zhang, Tianchang Wei, Jie Chen, Ting Pan, Miao Li, Lu Wang, Juan Song, Cuicui Chen, Yan Zhang, Yuanlin Song, Xiao Su
During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat type 1 alveolar epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administered E. coli lipopolysaccharide (LPS) by intratracheal injection to mice with a post-developmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells co-expressing type 2, type 1, and mixed intermediate cell state markers, with few mature type 1 cells. β1-deficient AT2 cells showed persistently increased proliferation after injury, which was blocked by inhibiting NF-kB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature type 1 alveolar epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.
Jennifer M.S. Sucre, Fabian Bock, Nicholas M. Negretti, John T. Benjamin, Peter M. Gulleman, Xinyu Dong, Kimberly T. Ferguson, Christopher S. Jetter, Wei Han, Yang Liu, Seunghyi Kook, Jason J. Gokey, Susan H. Guttentag, Jonathan A. Kropski, Timothy S. Blackwell, Roy Zent, Erin J. Plosa
Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4-mediated inflammation. However, the role of CD4 T-cells in promoting LTS fibrosis is unknown. The mechanistic target of rapamycin (mTOR) signaling pathways have been shown to regulate T-cell phenotype. Here we sought to investigate the influence of mTOR signaling in CD4 T-cells on LTS pathogenesis. In this study, human LTS specimens revealed an increased population of CD4-cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and TH17-cells. Selective deletion of mTOR in CD4+-cells reduced TH17-cells and attenuated fibrosis, demonstrating CD4-cells’ pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased TH17-cells. In-vitro, TH17-cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of TH17-cells. Collectively, mTOR signaling drives pathologic CD4 T-cell phenotypes in LTS, and targeting mTOR with sirolimus is effective at treating LTS through inhibition of pro-fibrotic TH17-cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.
Kevin M. Motz, Ioan A. Lina, Idris Samad, Michael K. Murphy, Madhavi Duvvuri, Ruth J. Davis, Alexander Gelbard, Liam Chung, Yee Chan-Li, Samuel Collins, Jonathan D. Powell, Jennifer H. Elisseeff, Maureen R. Horton, Alexander T. Hillel
DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.
Amjad Horani, Deepesh Gupta, Jian Xu, Huihui Xu, Lis del C. Puga Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody
Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell–mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.
Aaditya Khatri, Jamie L. Todd, Fran L. Kelly, Andrew Nagler, Zhicheng Ji, Vaibhav Jain, Simon G. Gregory, Kent J. Weinhold, Scott M. Palmer
Emerging data indicates an association between environmental heavy metal exposure and lung disease, including lower respiratory tract infections (LRTIs). Here, we show by single cell RNA-sequencing an increase in Pparg gene expression in lung macrophages from mice exposed to cadmium and/or infected with S. pneumoniae. However, the heavy metal cadmium or infection mediated an inhibitory post-translational modification of peroxisome proliferator-activated receptor ɣ (PPARɣ) to exacerbate LRTIs. Cadmium and infection increased ERK activation to regulate PPARɣ degradation in monocyte-derived macrophages. Mice harboring a conditional deletion of Pparg in monocyte-derived macrophages had more severe S. pneumoniae infection after cadmium exposure, showed greater lung injury, and had increased mortality. Inhibition of ERK activation with BVD-523 protected mice from lung injury after cadmium exposure or infection. Moreover, subjects residing in areas of high air cadmium levels had increased cadmium concentration in their BAL fluid, increased barrier dysfunction, and showed PPARɣ inhibition that was mediated, at least in part, by ERK activation in isolated BAL cells. These observations suggest that impaired activation of PPARɣ in monocyte-derived macrophages exacerbates lung injury and the severity of LRTIs.
Jennifer L. Larson-Casey, Shanrun Liu, Jennifer M. Pyles, Suzanne E. Lapi, Komal Saleem, Veena B. Antony, Manuel Lora Gonzalez, David K. Crossman, A. Brent Carter
Mitochondrial dysfunction at birth predicts bronchopulmonary dysplasia (BPD) in extremely low birth weight (ELBW) infants. Recently, nebulized thyroid hormone (TH), given as triiodothyronine (T3) was noted to decrease pulmonary fibrosis in adult animals through improved mitochondrial function. We hypothesized that TH may have similar effects on hyperoxia-induced neonatal lung injury and mitochondrial dysfunction. To determine whether intranasal T3 decreases neonatal hyperoxic lung injury in newborn mice, T3 improves mitochondrial function in lung homogenates, neonatal murine lung fibroblasts (NMLF) and umbilical cord-derived mesenchymal stem cells (MSCs) obtained from ELBW infants, and whether neonatal hypothyroxinemia is associated with BPD in ELBW infants. Inhaled T3 (given intranasally) attenuated hyperoxia-induced lung injury and mitochondrial dysfunction in newborn mice. T3 also reduced bioenergetic deficits in UC-MSCs obtained both from infants with no/mild BPD and those with moderate/severe BPD. T3 also increased PGC1α content in lung homogenates of mice exposed to hyperoxia as well as mitochondrial potential in both NMLF and UC-MSCs. ELBW infants who died or developed moderate/severe BPD had lower TT4 compared to survivors with no/mild BPD. TH signaling and function may play a critical role in neonatal lung injury and inhaled T3 supplementation may be useful as a therapeutic strategy for BPD.
Bianca M. Vamesu, Teodora Nicola, Rui Li, Snehashis Hazra, Sadis Matalon, Naftali Kaminski, Namasivayam Ambalavanan, Jegen Kandasamy
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