Search

Mehak Passi,1,* Sadia Shahid,2,* Sankarakuttalam Chockalingam,2 Isaac Kirubakaran Sundar,3 Gopinath Packirisamy1,2 1Nanobiotechnology Laboratory, Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India; 2Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India; 3Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY 14623, USA*These authors contributed equally to this workCorrespondence: Isaac Kirubakaran Sundar; Gopinath Packirisamy Tel +1-585-273-3034; +91-1332-285650Email isaac_sundar@urmc.rochester.edu; genegopi@gmail.comAbstract: Chronic obstructive pulmonary disease (COPD) is the most prevalent obstructive lung disease worldwide characterized by decline in lung function. It is associated with airway obstruction, oxidative stress, chronic inflammation, mucus hypersecretion, and enhanced autophagy and cellular senescence. Cigarette smoke being the major risk factor, other secondary risk factors such as the exposure to air pollutants, occupational exposure to gases and fumes in developing countries, also contribute to the pathogenesis of COPD. Conventional therapeutic strategies of COPD are based on anti-oxidant and anti-inflammatory drugs. However, traditional anti-oxidant pharmacological therapies are commonly used to alleviate the impact of COPD as they have many associated repercussions such as low diffusion rate and inappropriate drug pharmacokinetics. Recent advances in nanotechnology and stem cell research have shed new light on the current treatment of chronic airway disease. This review is focused on some of the anti-oxidant therapies currently used in the treatment and management of COPD with more emphasis on the recent advances in nanotechnology-based therapeutics including stem cell and gene therapy approaches for the treatment of chronic airway disease such as COPD and asthma.Keywords: chronic obstructive pulmonary disease, COPD, nanotechnology, anti-oxidant therapy, stem cell therapy, gene therapy

Cadmium (Cd) is a well-known toxic heavy metal that poses significant health risks, particularly through inhalation, smoking, and the consumption of contaminated food. Exposure to cadmium is linked to the development and exacerbation of chronic lung diseases such as pulmonary fibrosis and chronic obstructive pulmonary disease (COPD). This study investigated the systemic effects of intratracheal cadmium chloride (0.5 mg/kg) instillation in C57BL/6 mice. All parameters, including inflammation assessment, lung function evaluation (using Flexi-vent), and immunophenotyping of T-cells in secondary lymphoid organs (mediastinal lymph nodes and spleen), were analyzed 14 days after cadmium exposure. The results demonstrated that cadmium exposure led to significant immune cell infiltration in bronchoalveolar lavage (BAL) fluid, altered pro-inflammatory cytokine levels, and was associated with impaired lung function, characterized by increased lung resistance and Newtonian resistance. Analysis of T-cell populations revealed no significant changes in total T-cells in mediastinal lymph nodes and spleen, but a decrease in CD4 + T-cells and an increase in CD8 + T-cells were observed. These findings suggest that cadmium disrupts T-cell homeostasis in secondary lymphoid organs. Further research is crucial to elucidate the mechanisms underlying cadmium-induced lung injury and immune dysregulation, essential for developing effective therapeutic interventions against chronic lung diseases caused by cadmium exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Allergens and Th2 cytokines affect the homeostatic environment in the airways, leading to increased mucus production by goblet cells associated with altered adherens junctional complex (AJC) and tight junction (TJ) proteins responsible for maintaining epithelial barrier function. Circadian clock-dependent regulatory mechanisms such as inflammation and epithelial barrier function are gaining more attention due to their therapeutic potential against allergic inflammatory lung diseases. Currently, there are no studies to support whether REV-ERBα activation can attenuate Th2 cytokine-induced epithelial barrier dysfunction in human bronchial epithelial cells. We hypothesized that Th2 cytokine-induced epithelial barrier dysfunction may be protected by activating REV-ERBα. Treatment with Th2 cytokines or HDM significantly reduced the cell impedance, as confirmed by transepithelial electrical resistance (TEER). However, pre-treatment with SR10067 attenuated Th2 cytokine-induced barrier dysfunction, such as decreased permeability, improved TEER, localization of AJC and TJ proteins, and mRNA and protein levels of selected epithelial barrier and circadian clock targets. Overall, we showed for the first time that REV-ERBα activation regulates altered epithelial barrier function that may have direct implications for the treatment of asthma and other allergic diseases., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Environmental tobacco smoke (ETS) is known to cause lung inflammatory and injurious responses. Smoke exposure is associated with the pathobiology related to lung fibrosis, whereas the mechanism that ETS exposure augments pulmonary fibrogenesis is unclear. We hypothesized that ETS exposure could exacerbate fibrotic responses via collagen dynamic dysregulation and complement activation. C57BL/6J and p16-3MR mice were exposed to ETS followed by bleomycin administration. ETS exposure exacerbated bleomycin-induced collagen and lysyl oxidase overexpression in the fibrotic lesion. ETS exposure also led to augmented bleomycin-induced upregulation of C3 and C3AR, which are pro-fibrotic markers. Moreover, overexpressed collagens and C3 levels were highly significant in males than females. The old mice (17 months old) were exposed to ETS and treated with bleomycin to induce fibrogenesis which is considered as an aging-associated disease. Fewer gene and protein dysregulations trends were identified between ETS exposure with the bleomycin group and the bleomycin alone group in old mice. Based on our findings, we suggested that ETS exposure increases the risk of developing severe lung fibrotic responses via collagen overexpression and lysyl oxidase-mediated collagen stabilization in the fibrotic lesion, and potentially affected the complement system activation induced by bleomycin. Further, male mice were more susceptible than females during fibrogenesis exacerbation. Thus ETS and bleomycin induced lung fibrotic changes via collagen-lysyl oxidase in an age-dependent mechanism., (© 2024. The Author(s).)

House dust mite (HDM) is a common aeroallergen that can disrupt the airway epithelial barrier leading to dysregulated immune response, resulting in allergic lung diseases such as asthma. Cryptochrome (CRY), a circadian clock gene, plays an important role in the regulation of metabolism, and immune response. It remains unclear whether stabilizing CRY using KL001 can attenuate HDM/Th2 cytokine-induced epithelial barrier dysfunction in 16-HBE cells. We evaluate the effect of KL001 (20 µM) pre-treatment (4 hrs) in HDM/Th2 cytokine (IL-4 or IL-13)-mediated change in epithelial barrier function. HDM and Th2 cytokine-induced changes in transepithelial electrical resistance (TEER) were determined by an xCELLigence real-time cell analyzer and delocalization of adherens junction complex (AJC: E-cadherin and β-catenin) and tight junction proteins (TJP: Occludin and Zonula occludens-1) by immunostaining and confocal microscopy. Finally, quantitative real-time PCR (qRT-PCR) and Western blotting were used to measure altered gene expression and protein abundance of the epithelial barrier function and core clock genes, respectively. HDM and Th2 cytokine treatment significantly decreased TEER associated with altered gene expression and protein abundance of the selected epithelial barrier function and circadian clock genes. However, pre-treatment with KL001 attenuated HDM and Th2 cytokine-induced epithelial barrier dysfunction as early as 12-24 hrs. KL001 pre-treatment showed attenuation of HDM and Th2 cytokine-induced alteration in the localization and gene expression of AJP and TJP ( Cdh1, Ocln, and Zo1 ) and core clock genes ( Clock, Arntl/Bmal1, Cry1/2, Per1/2, Nr1d1/Rev-erbα, and Nfil3 ). We demonstrate, for the first time, the protective role of KL001 in HDM and Th2 cytokine-mediated epithelial barrier dysfunction.

Circadian rhythms have an established role in regulating physiological processes, such as inflammation, immunity, and metabolism. Ozone, a common environmental pollutant with strong oxidative potential, is implicated in lung inflammation/injury in asthmatics. However, whether O 3 exposure affects the expression of circadian clock genes in the lungs is not known. In this study, changes in the expression of core clock genes are analyzed in the lungs of adult female and male mice exposed to filtered air (FA) or O 3 using qRT-PCR. The findings are confirmed using an existing RNA-sequencing dataset from repeated FA- and O 3 -exposed mouse lungs and validated by qRT-PCR. Acute O 3 exposure significantly alters the expression of clock genes in the lungs of females (Per1, Cry1, and Rora) and males (Per1). RNA-seq data revealing sex-based differences in clock gene expression in the airway of males (decreased Nr1d1/Rev-erbα) and females (increased Skp1), parenchyma of females and males (decreased Nr1d1 and Fbxl3 and increased Bhlhe40 and Skp1), and alveolar macrophages of males (decreased Arntl/Bmal1, Per1, Per2, Prkab1, and Prkab2) and females (increased Cry2, Per1, Per2, Csnk1d, Csnk1e, Prkab2, and Fbxl3). These findings suggest that lung inflammation caused by O 3 exposure affects clock genes which may regulate key signaling pathways., (© 2023 Wiley-VCH GmbH.)

Globalization and the expansion of essential services over continuous 24 h cycles have necessitated the adaptation of the human workforce to shift-based schedules. Night shift work (NSW) causes a state of desynchrony between the internal circadian machinery and external environmental cues, which can impact inflammatory and metabolic pathways. The discovery of clock genes in the lung has shed light on potential mechanisms of circadian misalignment in chronic pulmonary disease. Here, the current knowledge of circadian clock disruption caused by NSW and its impact on lung inflammation and associated pathophysiology in chronic lung diseases, such as asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and COVID-19, is reviewed. Furthermore, the limitations of the current understanding of circadian disruption and potential future chronotherapeutic advances are discussed., (© 2023 Wiley-VCH GmbH.)

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that impairs lung mechanical properties due to dysregulated extracellular matrix remodeling. Lung function assessment is an important physiological endpoint in the mouse model of pulmonary fibrosis (PF) that has gained a broader scientific acceptance in the field. IPF pathophysiology shows sex-based differences, disproportionately affecting more men compared to women. Prior reports suggest that the circadian clock is perturbed during the pathogenesis of PF. We have comprehensively assessed the sex-based differences and time-of-day response (at Zeitgeber time: ZT0/6:00 a.m. or ZT12/6 p.m.) in lung mechanics among sham (control) versus bleomycin (BLM)-challenged female and male (C57BL/6: WT) mice using Flexi-vent. BLM challenge altered lung function significantly in males in both total lung (reduced dynamic compliance, and increased resistance and elastance) as well as lung tissue-specific parameters (increased tissue elastance and tissue damping) but less pronounced in females. BLM-challenged mice showed a time-of-day response in lung function at ZT0 versus ZT12, which was pronounced in the ZT0 BLM group. Overall, these findings provide a comprehensive analysis of altered lung function in female and male mice and the time-of-day difference in lung function parameters following BLM-induced lung fibrosis., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by excessive scarring of the lungs that can lead to respiratory failure and death. Lungs of patients with IPF demonstrate excessive deposition of extracellular matrix (ECM) and an increased presence of pro-fibrotic mediators such as transforming growth factor-beta 1 (TGFβ1), which is a major driver of fibroblast-to-myofibroblast transition (FMT). Current literature supports that circadian clock dysfunction plays an essential role in the pathophysiology of various chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and IPF. The circadian clock transcription factor Rev-erbα is encoded by Nr1d1 that regulates daily rhythms of gene expression linked to immunity, inflammation, and metabolism. However, investigations into the potential roles of Rev-erbα in TGFβ-induced FMT and ECM accumulation are limited. In this study, we utilized several novel small molecule Rev-erbα agonists (GSK41122, SR9009, and SR9011) and a Rev-erbα antagonist (SR8278) to determine the roles of Rev-erbα in regulating TGFβ1-induced FMT and pro-fibrotic phenotypes in human lung fibroblasts. WI-38 cells were either pre-treated/co-treated with or without Rev-erbα agonist/antagonist along with TGFβ1. After 48 h, the following parameters were evaluated: secretion of COL1A1 (Slot-Blot analysis) and IL-6 (ELISA) into condition media, expressions of α-smooth muscle actin (αSMA: immunostaining and confocal microscopy), and pro-fibrotic proteins (αSMA and COL1A1 by immunoblotting), as well as gene expression of pro-fibrotic targets (qRT-PCR: Acta2, Fn1, and Col1a1). Results revealed that Rev-erbα agonists inhibited TGFβ1-induced FMT (αSMA and COL1A1), and ECM production (reduced gene expression of Acta2, Fn1, and Col1a1), and decreased pro-inflammatory cytokine IL-6 release. The Rev-erbα antagonist promoted TGFβ1-induced pro-fibrotic phenotypes. These findings support the potential of novel circadian clock-based therapeutics, such as Rev-erbα agonist, for the treatment and management of fibrotic lung diseases and disorders., Competing Interests: Declaration of competing interest The authors report no declarations of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Circadian rhythms and sex differences are involved in the pathophysiology of asthma. Yet, there are no reports that simultaneously address the role of the circadian clock and sex-based differences in chronic house dust mite (HDM)-induced asthma. Here, we sought to determine if chronic HDM exposure during the resting phase (zeitgeber time: ZT0/6:00 a.m.) versus the active phase (ZT12/6:00 p.m.) differentially affects the circadian clock and alters asthma pathobiology in female and male mice. HDM exposure at ZT12 exaggerated infiltration of eosinophil subtypes and associated chemokines in females compared to males. Furthermore, HDM exposure augmented eosinophil chemokines, Th2 gene expression and cytokine release, and humoral immune response in females compared to males at ZT12. Concurrently, histopathological evaluation confirmed increased airway inflammation at ZT12 in both females and males. Overall, we showed a time-of-day response and sex-based differences in HDM-induced exaggerated asthmatic phenotypes (inflammation/remodeling) and circadian clock disruption in females compared to males., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Asthma is a chronic inflammatory disease that shows a time-of-day response to variations in symptoms/severity. However, how the lung circadian clock influences time-of-day response and sex-based differences in house dust mite (HDM)-induced airway inflammation and remodeling has not been thoroughly investigated., Objective: We sought to determine whether acute HDM exposure in wild-type mice shows time-of-day response and sex-based differences in allergic airway inflammation and circadian clock disruption in the lungs., Methods: Wild-type (C57BL/6J) and Rev-erbα knockout (KO) mice were exposed to either PBS or HDM (for 10 days) intranasally at Zeitgeber time (ZT0: 6 am; ZT12: 6 pm) and euthanized 48 hours after the last exposure. Acute HDM-induced time-of-day response and sex-based differences in lung inflammation, gated cytokines/chemokines, humoral and hormonal responses, and circadian clock gene expression were analyzed., Results: Acute HDM-exposed mice showed a time-of-day response and sex-based differences in exaggerated lung inflammation (inflammatory eosinophils and interstitial macrophages) at ZT12 when compared with ZT0. HDM-exposed female mice showed increased inflammatory response at ZT12, but HDM-exposed male mice showed comparatively lower inflammation with no time-of-day response. HDM-exposed female and male mice showed augmented IgE levels at ZT12 when compared with ZT0. Myeloid innate immunity panel, cytokines/chemokines, and mucin genes showed a time-of-day gating response at ZT0 and ZT12 in the HDM group. In addition, HDM exposure altered the expression of circadian clock genes in the lung, which was evident in female mice at ZT12. Overall, female mice showed significant time-of-day responses to all these parameters compared with male mice. Rev-erbα KO mice exposed to acute HDM showed exaggerated lung inflammation associated with increased IgE and proinflammatory cytokines in bronchoalveolar lavage fluid. Interestingly, HDM exposure causes reduced expression of clock genes in flow-sorted resident eosinophils but not alveolar macrophages. Acute HDM exposure reduced the nocturnal locomotor activity in mice 5 days post-HDM exposure until day 10., Conclusions: This study shows a time-of-day response to acute HDM exposure and sex-based differences in the severity of lung inflammation and humoral immune response associated with circadian clock disruption. Our findings support the use of separate female and male mice cohorts for preclinical studies to understand the molecular heterogeneity in asthma pathophysiology., (© 2023 The Authors.)

Molecular clock REV-ERBα is central to regulating lung injuries, and decreased REV-ERBα abundance mediates sensitivity to pro-fibrotic insults and exacerbates fibrotic progression. In this study, we determine the role of REV-ERBα in fibrogenesis induced by bleomycin and Influenza A virus (IAV). Bleomycin exposure decreases the abundance of REV-ERBα, and mice dosed with bleomycin at night display exacerbated lung fibrogenesis. Rev-erbα agonist (SR9009) treatment prevents bleomycin induced collagen overexpression in mice. Rev-erbα global heterozygous (Rev-erbα Het) mice infected with IAV showed augmented levels of collagens and lysyl oxidases compared with WT-infected mice. Furthermore, Rev-erbα agonist (GSK4112) prevents collagen and lysyl oxidase overexpression induced by TGFβ in human lung fibroblasts, whereas the Rev-erbα antagonist exacerbates it. Overall, these results indicate that loss of REV-ERBα exacerbates the fibrotic responses by promoting collagen and lysyl oxidase expression, whereas Rev-erbα agonist prevents it. This study provides the potential of Rev-erbα agonists in the treatment of pulmonary fibrosis., (© 2023. The Author(s).)

The circadian clock is a biochemical oscillator that rhythmically regulates physiological and behavioral processes such as inflammation, immunity, and metabolism in mammals. Circadian clock disruption is a key driver for chronic inflammatory as well as fibrotic lung diseases. While the mechanism of circadian clock regulation in the lung has been minimally explored, some evidence suggests that the transforming growth factor β (TGFβ) signaling pathway and subsequent extracellular matrix (ECM) accumulation in the lung may be controlled via a clock-dependent mechanism. Recent advancements in this area led us to believe that pharmacologically targeting the circadian clock molecules may be a novel therapeutic approach for treating chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Here, we update the current perspective on the circadian clock role in TGFβ1 signaling and extracellular matrix production during chronic lung diseases.

The circadian clock is the biochemical oscillator that orchestrates the observable circadian rhythms in physiology and behavior. Disruption of the circadian clock in the lungs during chronic pulmonary diseases is considered one of the key etiological risk factors that drive pathobiology. Preclinical studies support that pharmacological manipulation of the circadian clock is a conceivable approach for the development of novel clock-based therapeutics. Despite recent advances, no effort has been undertaken to integrate novel findings for the treatment and management of chronic lung diseases. We, therefore, recognize the need to discuss the candidate clock genes that can be potentially targeted for therapeutic intervention. Here, we aim to create the first roadmap that will advance the development of circadian- clock-based therapeutics that may provide better outcomes in treating chronic pulmonary diseases., Competing Interests: Declaration of interests The authors have declared that no conflict of interest exists., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The circadian clock is the biochemical oscillator with a near 24-h period that is responsible for generating the circadian rhythms in peripheral organs including the lung. Mounting evidence suggests that circadian clock disruption during chronic lung diseases plays an essential role in augmented oxidative stress, inflammatory response, metabolic imbalances, hypoxia/hyperoxia, mucus secretion, dysregulated autophagy, and alters pulmonary function. Here, we review circadian clock disruption and discuss candidate clock genes that are altered at the transcriptional or translational level in chronic pulmonary diseases. This review aims to provide the current knowledge and understanding of the circadian molecular clock disruption in chronic pulmonary diseases which will further advance the development of novel clock-based therapeutics in the future., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Circadian rhythms have a profound effect on lung function and immune-inflammatory response in chronic airway diseases. Thus, understanding the molecular mechanisms of circadian gene expression of core clock-controlled genes (CCGs) may help better understand how it contributes to the physiology and pathology of lung diseases. Ongoing studies have been analyzing gene expression levels of CCGs in mouse lungs using quantitative real-time PCR (qRT-PCR). However, to date, there are no reports on the most stable reference gene in the mouse lung for circadian studies. Herein, we utilized an acute house dust mite (HDM)-sensitization mouse model to evaluate the stability of 10 reference genes commonly used for qRT-PCR normalization using 5 unique algorithms: GeNorm, NormFinder, BestKeeper, RefFinder and Qbase+. Rn18s was determined as the most stable reference gene across all samples evaluated, and Actb, the least stable reference gene. Furthermore, CircWave analysis showed no diurnal variation in the expression pattern for Rn18s but Actb showed strong diurnal changes in the lungs of both PBS (control) and HDM groups. We demonstrate systematically how using Actb as a housekeeping gene offsets the diurnal expression patterns of the CCGs and leads to statistically significant results which may not be the true reflection of the qRT-PCR analysis., (© 2022. The Author(s).)

Background: Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are linked to several mitochondrial alterations. Cigarette smoke (CS) alters the structure and function of mitochondria. OPA1 is the main inner mitochondrial GTPase responsible for the fusion events. OPA1 undergoes proteolytic cleavage from long to short forms during acute stress and mitophagy. However, the exact role of OPA1 isoforms and related proteins during CS-induced mitophagy and COPD is not clear., Methods: Lung tissues from non-smokers, smokers, COPD and IPF were used to determine the relative expression of OPA1 and related proteins. Additionally, we used mouse lungs from chronic (6 months) CS exposure to evaluate the status of OPA1. Primary lung fibroblasts from normal and COPD patients and naked mole rat (NMR) lung fibroblasts, human fetal lung fibroblast (HFL1), mouse embryonic fibroblast from wild type (WT), OPA1 -/- , MFN1 and MFN2 -/- were used to determine the effect of CS on OPA1 isoforms. Various mitochondrial fusion promoters/activators (BGP-15, leflunomide, M1) and fission inhibitor (DRP1) were used to determine their effect on OPA1 status and cigarette smoke extract (CSE)-induced lung epithelial (BEAS2B) cell damage, respectively. Seahorse flux analyzer was used to determine the effect of these compounds in BEAS2B cells with and without CSE exposure., Findings: Short OPA1 isoforms were predominantly detected and significantly increased in COPD subjects. Acute CSE treatment in various cell lines except NMR was found to increase the conversion of long to short OPA1 isoforms. CSE treatment significantly increased mitochondrial stress-related protein SLP2 in all the cells used. OPA1 interacting partners like prohibitins (PHB1 and 2) were also altered depending on the CS exposure. Finally, BGP-15 and leflunomide treatment were able to preserve the long OPA1 isoform in cells treated with CSE., Interpretation/conclusion: The long OPA1 isoform along with SLP2 and prohibitins play a crucial role in CS-induced lung damage, causing mitophagy/mitochondrial dysfunction in COPD, which may be used as a novel therapeutic target in COPD., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Cigarette smoke (CS) is the main etiological factor in the pathogenesis of emphysema/chronic obstructive pulmonary disease (COPD), which is associated with abnormal epithelial-mesenchymal transition (EMT). Previously, we have shown an association among circadian rhythms, CS-induced lung inflammation, and nuclear heme receptor α (REV-ERBα), acting as an antiinflammatory target in both pulmonary epithelial cells and fibroblasts. We hypothesized that molecular clock REV-ERBα plays an important role in CS-induced circadian dysfunction and EMT alteration. C57BL/6J WT and REV-ERBα heterozygous (Het) and -KO mice were exposed to CS for 30 days (subchronic) and 4 months (chronic), and WT mice were exposed to CS for 10 days with or without REV-ERBα agonist (SR9009) administration. Subchronic/chronic CS exposure caused circadian disruption and dysregulated EMT in the lungs of WT and REV-ERBα-KO mice; both circadian and EMT dysregulation were exaggerated in the REV-ERBα-KO condition. REV-ERBα agonist, SR9009 treatment reduced acute CS-induced inflammatory response and abnormal EMT in the lungs. Moreover, REV-ERBα agonist (GSK4112) inhibited TGF-β/CS-induced fibroblast differentiation in human fetal lung fibroblast 1 (HFL-1). Thus, CS-induced circadian gene alterations and EMT activation are mediated through a Rev-erbα-dependent mechanism, which suggests activation of REV-ERBα as a novel therapeutic approach for smoking-induced chronic inflammatory lung diseases.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has affected nearly 28 million people in the United States and has caused more than five hundred thousand deaths as of February 21, 2021. As the novel coronavirus continues to take its toll in the United States and all across the globe, particularly among the elderly (>65 years), clinicians and translational researchers are taking a closer look at the nexus of sleep, circadian rhythms and immunity that may contribute toward a more severe coronavirus disease-19 (COVID-19). SARS-CoV-2-induced multi-organ failure affects both central and peripheral organs, causing increased mortality in the elderly. However, whether differences in sleep, circadian rhythms, and immunity between older and younger individuals contribute to the age-related differences in systemic dysregulation of target organs observed in SARS-CoV-2 infection remain largely unknown. Current literature demonstrates the emerging role of sleep, circadian rhythms, and immunity in the development of chronic pulmonary diseases and respiratory infections in human and mouse models. The exact mechanism underlying acute respiratory distress syndrome (ARDS) and other cardiopulmonary complications in elderly patients in combination with associated comorbidities remain unclear. Nevertheless, understanding the critical role of sleep, circadian clock dysfunction in target organs, and immune status of patients with SARS-CoV-2 may provide novel insights into possible therapies. Chronotherapy is an emerging concept that is gaining attention in sleep medicine. Accumulating evidence suggests that nearly half of all physiological functions follow a strict daily rhythm. However, healthcare professionals rarely take implementing timed-administration of drugs into consideration. In this review, we summarize recent findings directly relating to the contributing roles of sleep, circadian rhythms and immune response in modulating infectious disease processes, and integrate chronotherapy in the discussion of the potential drugs that can be repurposed to improve the treatment and management of COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Giri, Srinivasan and Sundar.)

Cellular senescence and lung aging are associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). COPD progresses with aging, and chronic smoking is the key susceptibility factor in lung pathological changes concurrent with mitochondrial dysfunction and biological aging. However, these processes involving cigarette smoke (CS)-mediated lung cellular senescence are difficult to distinguish. One of the impediments to studying cellular senescence in relation to age-related lung pathologies is the lack of a suitable in vivo model. In view of this, we provide evidence that supports the suitability of p16-3MR mice to studying cellular senescence in CS-mediated and age-related lung pathologies. p16-3MR mice have a trimodal reporter fused to the promoter of the p16 INK4a gene that enables detection, isolation, and selective elimination of senescent cells, thus making them a suitable model to study cellular senescence. To determine their suitability in CS-mediated lung pathologies, we exposed young (12-14 months) and old (17-20 months) p16-3MR mice to 30 day CS exposure and studied the expression of senescent genes (p16, p21, and p53) and SASP-associated markers (MMP9, MMP12, PAI-1, and FN-1) in air- and CS-exposed mouse lungs. Our results showed that this model could detect cellular senescence using luminescence and isolate cells undergoing senescence with the help of tissue fluorescence in CS-exposed young and old mice. Our results from the expression of senescence markers and SASP-associated genes in CS-exposed young and old p16-3MR mice were comparable with increased lung cellular senescence and SASP in COPD. We further showed alteration in the; (i) tissue luminescence and fluorescence, (ii) mRNA and protein expressions of senescent markers and SASP genes, and (iii) SA-β-gal activity in CS-exposed young and old p16-3MR mice as compared to their air controls. Overall, we showed that p16-3MR is a competent model for studying the cellular senescence in CS-induced pathologies. Hence, the p16-3MR reporter mouse model may be used as a novel tool for understanding the pathobiology of cellular senescence and other underlying mechanisms involved in COPD and fibrosis.

Electronic nicotine delivery systems (ENDS), or e-cigarettes, are emerging tobacco products that produce aerosols by heating e-liquids, which most often consist of propylene glycol and vegetable glycerin along with various flavoring compounds, bypassing the combustion that occurs in the use of traditional tobacco cigarettes. These products have seen a drastic increase in popularity in recent years both as smoking cessation devices as well as among younger generations, due in large part to the widespread perception among consumers that e-cigs are significantly less harmful to health than traditional tobacco cigarettes. Due to the novelty of ENDS as well as their rapidly increasing use, research into biomarkers of e-cig exposure and toxicity have lagged behind their popularity, leaving important questions about their potential toxicity unanswered. Research into potential biomarkers of acute and chronic e-cig use, and e-cigarette- or vaping-associated lung injury is necessary for informing both clinical and regulatory decision-making. We aim to provide an updated review of recent research into potential circulating, genomic, transcriptomic, and epigenetic biomarkers of exposure to and toxicity of e-cigs. We additionally highlight research areas that warrant additional study to gain a better understanding of health risks associated with ENDS use, as well as to provide validation of existing data and methods for measuring and analyzing e-cig-associated biomarkers in human and animal biofluids, tissues, and cells. This review also highlights ongoing efforts within the WNY Center for Research on Flavored Tobacco for research into novel biomarkers in extracellular vesicles that may be associated with short- and long-term ENDS use.

Asthma is a chronic inflammatory disease of the airway diagnosed with different endotypes and phenotypes, characterized by airway obstruction in response to allergens, bacterial/viral infections, or pollutants. Several cell types such as the airway epithelial cells, mesenchymal stem cells and different immune cells including dendritic cells (DCs), T and B cells and mast cells play an essential role during the pathobiology of asthma. Extracellular vesicles (EVs) are membranous nanovesicles produced by every cell type that facilitates intercellular communications. EVs contain heterogeneous cargos that primarily depend on the composition or cell type of origin and they can alter the physiological state of the target cells. EVs encompass a wide variety of proteins including Tetraspanins, MHC classes I and II, co-stimulatory molecules, nucleic acids such as RNA, miRNA, piRNA, circRNA, and lipids like ceramides and sphingolipids. Recent literature indicates that EVs play a pivotal role in the pathophysiology of allergic asthma and may potentially be used as a novel biomarker to determine endotypes and phenotypes in severe asthmatics. Based on the prior reports, we speculate that regulation of EVs biogenesis and release might be under the control of circadian rhythms. Thus, circadian rhythms may influence the composition of the EVs, which alter the microenvironment that results in the induction of an immune-inflammatory response to various environmental insults or allergens such as air pollutants, ozone, diesel exhaust particles, pollens, outdoor molds, environmental tobacco smoke, etc. In this mini-review, we summarize the recent updates on the novel role of EVs in the pathogenesis of asthma, and highlight the link between circadian rhythms and EVs that may be important to identify molecular mechanisms to target during the pathogenesis of chronic inflammatory lung disease such as asthma., Competing Interests: Conflict of interest The authors declare that this research work was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The updated review of literature and conclusions in this review article are solely those of the authors and do not represent the official views of the National Institute of Health (NIH)/National, Heart, Lung, and Blood Institute.

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

Background: Aging is one of the key contributing factors for chronic obstructive pulmonary diseases (COPD) and other chronic inflammatory lung diseases. Here, we determined how aging contributes to the altered gene expression related to mitochondrial function, cellular senescence, and telomeric length processes that play an important role in the progression of COPD and idiopathic pulmonary fibrosis (IPF)., Methods: Total RNA from the human lung tissues of non-smokers, smokers, and patients with COPD and IPF were processed and analyzed using a Nanostring platform based on their ages (younger: <55 years and older: >55 years)., Results: Several genes were differentially expressed in younger and older smokers, and patients with COPD and IPF compared to non-smokers which were part of the mitochondrial biogenesis/function ( HSPD1 , FEN1 , COX18 , COX10 , UCP2 & 3 ), cellular senescence ( PCNA , PTEN , KLOTHO , CDKN1C , TNKS2 , NFATC1 & 2 , GADD45A ), and telomere replication/maintenance ( PARP1 , SIRT6 , NBN , TERT , RAD17 , SLX4 , HAT1 ) target genes. Interestingly, NOX4 and TNKS2 were increased in the young IPF as compared to the young COPD patients. Genes in the mitochondrial dynamics and quality control mechanisms like FIS1 and RHOT2 were decreased in young IPF compared to their age matched COPD subjects. ERCC1 and GADD45B were higher in young COPD as compared to IPF. Aging plays an important role in various infectious diseases including the SARS-CoV-2 infection. Lung immunoblot analysis of smokers, COPD and IPF subjects revealed increased abundance of proteases and receptor/spike protein like TMPRSS2, furin, and DPP4 in association with a slight increase in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor ACE2 levels., Conclusions: Overall, these findings suggest that altered transcription of target genes that regulate mitochondrial function, cellular senescence, and telomere attrition in the pathobiology of lung aging in COPD and IPF is associated with alterations in SARS-CoV-2 ACE2-TMPRSS2-Furin-DPP4 axis as pharmacological targets for COVID-19., (Copyright © 2020 Maremanda, Sundar, Li and Rahman.)

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

The abnormal inflammatory responses due to the lung tissue damage and ineffective repair/resolution in response to the inhaled toxicants result in the pathological changes associated with chronic respiratory diseases. Investigation of such pathophysiological mechanisms provides the opportunity to develop the molecular phenotype-specific diagnostic assays and could help in designing the personalized medicine-based therapeutic approaches against these prevalent diseases. As the central hubs of cell metabolism and energetics, mitochondria integrate cellular responses and interorganellar signaling pathways to maintain cellular and extracellular redox status and the cellular senescence that dictate the lung tissue responses. Specifically, as observed in chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis, the mitochondria-endoplasmic reticulum (ER) crosstalk is disrupted by the inhaled toxicants such as the combustible and emerging electronic nicotine-delivery system (ENDS) tobacco products. Thus, the recent research efforts have focused on understanding how the mitochondria-ER dysfunctions and oxidative stress responses can be targeted to improve inflammatory and cellular dysfunctions associated with these pathologic illnesses that are exacerbated by viral infections. The present review assesses the importance of these redox signaling and cellular senescence pathways that describe the role of mitochondria and ER on the development and function of lung epithelial responses, highlighting the cause and effect associations that reflect the disease pathogenesis and possible intervention strategies., Competing Interests: Declaration of competing interest Authors declare no conflict of interest with work described in this manuscript., (Copyright © 2020. Published by Elsevier B.V.)

Background: Wheezing is a symptom of potential respiratory disease and known to be associated with smoking. Electronic cigarette use ('vaping') has increased exponentially in recent years. This study examined the cross-sectional association of vaping with wheezing and related respiratory symptoms and compare this association with smokers and dual users., Methods: The Population Assessment of Tobacco and Health study wave 2 data collected from October 2014 to October 2015 with 28 171 adults were used. The cross-sectional association of vaping with self-reported wheezing and related respiratory symptoms relative to smokers and dual users of tobacco and electronic cigarettes were studied using multivariable logistic and cumulative logistic regression models with consideration of complex sampling design., Results: Among the 28 171 adult participants, 641 (1.2%) were current vapers who used e-cigarettes exclusively, 8525 (16.6%) were current exclusive smokers, 1106 (2.0%) were dual users and 17 899 (80.2%) were non-users. Compared with non-users, risks of wheezing and related respiratory symptoms were significantly increased in current vapers (adjusted OR (aOR)=1.67, 95% CI: 1.23 to 2.15). Current vapers had significantly lower risk in wheezing and related respiratory symptoms compared with current smokers (aOR=0.68, 95% CI: 0.53 to 0.87). No significant differences were found between dual users and current smokers in risk of wheezing and related respiratory symptoms (aOR=1.06, 95% CI: 0.91 to 1.24)., Conclusions: Vaping was associated with increased risk of wheezing and related respiratory symptoms. Current vapers had lower risk in wheezing and related respiratory symptoms than current smokers or dual users but higher than non-users. Both dual use and smoking significantly increased the risk of wheezing and related respiratory symptoms., Competing Interests: Competing interests: MLG received a research grant from Pfizer and served as a member of advisory board to Johnson & Johnson, manufacturers of smoking cessation medications., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Cigarette smoke (CS) is known to cause mitochondrial dysfunction leading to cellular senescence in lung cells. We determined the mechanism of mitochondrial dysfunction by CS in lung epithelial cells. CS extract (CSE) treatment differentially affected mitochondrial function, such as membrane potential, mitochondrial reactive oxygen species (mtROS) and mitochrondrial mass as analyzed by FACS, and were associated with altered oxidative phosphorylation (OXPHOS) protein levels (Complexes I-IV) in primary lung epithelial cells (SAEC and NHBE), and (complexes I and II) in BEAS2B cells. There were dose- and time-dependent changes in mitochondrial respiration (oxygen consumption rate parameters i.e. maximal respiration, ATP production and spare capacity, measured by the Seahorse analyzer) in control vs. CSE treated BEAS2B and NHBE/DHBE cells. Electron microscopy (EM) analysis revealed perinuclear clustering by localization and increased mitochondrial fragmentation by fragement length analysis. Immunoblot analysis revealed CS-mediated increase in Drp1 and decrease in Mfn2 levels that are involved in mitochondrial fission/fusion process. CSE treatment reduced Miro1 and Pink1 abundance that play a crucial role in the intercellular transfer mechanism and mitophagy process. Overall, these findings highlight the role of Miro1 in context of CS-induced mitochondrial dysfunction in lung epithelial cells that may contribute to the pathogenesis of chronic inflammatory lung diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Cigarette smoke (CS)-induced lung inflammation and Chronic Obstructive Pulmonary disease (COPD) involves mitochondrial dysfunction. Mesenchymal stem cells (MSC) and MSC-derived exosomes (EXO) are reported to show therapeutic effects in many animal models of inflammation and injury. In the present study, we determined the role of MSC and EXO intervention in CS-induced lung inflammation with a focus on mitochondrial dysfunction., Methods: EXO were characterized using Western blot for exosomal markers, tunable resistive pulse sensing by qNano and transmission electron microscopy (TEM). Mitochondrial reporter mice (mt-Keima and mito-QC) were exposed to air or CS for 10 days. mt-Keima mice were treated with intraperitoneal injections of MSC or EXO or MSC and EXO (MSC + EXO) for 10 days. Total cell counts, differential cell counts were performed using automated cell counter and flow cytometry respectively. Further, the levels of pro-inflammatory mediators in bronchoalveolar lavage (BAL) fluid were measured using ELISA. Western blot analysis, quantitative PCR, confocal microscopy were used in the current study to determine the effects in the lungs of CS exposed mice. Seahorse flux analyzer was used to measure the oxidative-phosphorylation (OXPHOS) in the BEAS2B cells and BEAS2B - mMSC co-culture experiments., Results: CS exposure increased the inflammatory cellular infiltrations in the lungs of the mt-Keima mice. MSC + EXO treatment showed protection compared to individual treatments (MSC or EXO alone). There were no changes in the mitophagy proteins like PINK1 and Parkin, which was also found using the mito-QC mice. CS exposure led to significant increase in the mitochondrial fission protein DRP1 and other DAMPs pathway mediators like S100A4 and S100A8, HMGB1, RAGE and AGE. MSC + EXO treatment increased the gene expression of (fusion genes) mfn1, mfn2 and opa1. Additionally, the rhot1 gene expression was increased in MSC + EXO treatment group compared to Air- and CS exposed groups. BEAS2B-mMSC co-cultures showed protective response against the CSE-altered mitochondrial respiration parameters, confirming the beneficial effect of MSC towards human bronchial lung epithelial cells., Conclusion: CS affects some of early mitochondrial genes involved in the fission/fusion process, enhancing the damage response along with altered cytokine levels. MSC + EXO combination treatment showed their protective effects. MSC + EXO combination treatment may act against these early events caused by CS exposure owing to its anti-inflammatory and other mitochondrial transfer mechanisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) play a vital role in normal lung physiology to maintain homeostasis in the airways via intercellular communication. EVs include exosomes and microvesicles, and are characterized by their phospholipid bilayers. EVs have been recognized as novel circulating biomarkers of disease, which are released by different cell types. In this study, we used different EV isolation and purification methods to characterize the plasma-derived EV miRNAs from non-smokers, smokers and patients with COPD. A small RNA sequencing (RNA-seq) approach was adapted to identify novel circulating EV miRNAs. We found that plasma-derived EVs from non-smokers, smokers and patients with COPD vary in their size, concentration, distribution and phenotypic characteristics as confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of EV surface markers. RNA-seq analysis confirmed the most abundant types of small RNAs, such as miRNAs, tRNAs, piRNAs snRNAs, snoRNAs and other biotypes in plasma-derived EVs. We mainly focused on miRNAs as novel biomarkers in smokers and patients with COPD for further analysis. Differential expression by DESeq2 identified distinct miRNA profiles (up-regulated: miR-22-3p, miR-99a-5p, miR-151a-5p, miR-320b, miR-320d; and down-regulated: miR-335-5p, miR-628-3p, miR-887-5p and miR-937-3p) in COPD versus smokers or non-smokers in a pairwise comparison. Gene set enrichment analysis (GSEA) of differentially expressed miRNAs revealed the top pathways, gene ontology and diseases associated with smokers and patients with COPD. We selectively validated miRNAs in EVs isolated from BEAS-2B cells treated with cigarette smoke extract by quantitative PCR analysis. For the first time, we report that plasma-derived EV miRNAs are novel circulating pulmonary disease biomarkers. Thus, molecular profiling of EV miRNAs has great translational potential for the development of biomarkers that may be used in the diagnosis, prognosis, and therapeutics of COPD., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Nicotine inhalation via electronic cigarettes (e-cigs) is an emerging concern. However, little is known about the acute toxicity in the lungs following inhalation of nicotine-containing e-cig aerosols. We hypothesized that acute exposure to aerosolized nicotine causes lung toxicity by eliciting inflammatory and dysregulated repair responses. Adult C57BL/6J mice were exposed 2 h daily for 3 days to e-cig aerosols containing propylene glycol (PG) with or without nicotine. Acute exposure to nicotine-containing e-cig aerosols induced inflammatory cell influx (neutrophils and CD8a + T-lymphocytes), and release of pro-inflammatory cytokines in bronchoalveolar lavage fluid in a sex-dependent manner. Inhalation of e-cig aerosol containing PG alone significantly augmented the lung levels of various homeostasis/repair mediators (PPARγ, ADRP, ACTA2, CTNNB1, LEF1, β-catenin, E-cadherin, and MMP2) in a sex-dependent manner when compared to air controls. These findings were accompanied by an increase in protein abundance and altered gene expression of lipogenic markers (PPARγ, ADRP) and myogenic markers (fibronectin, α-smooth muscle actin and β-catenin), suggesting a dysregulated repair response in mouse lungs. Furthermore, exposure to nicotine containing e-cig aerosols or PG alone differentially affected the release of pro-inflammatory cytokines in healthy and COPD human 3D EpiAirway tissues. Overall, acute exposure to nicotine containing e-cig aerosols was sufficient to elicit a pro-inflammatory response and altered mRNA and protein levels of myogenic, lipogenic, and extracellular matrix markers in mouse lung in a sex-dependent manner. Thus, acute exposure to inhaled nicotine via e-cig leads to dysregulated repair and inflammatory responses, which may lead to airway remodeling in the lungs., Competing Interests: Conflicting financial interests: The authors have declared that no conflict of interest exists.

The use of emerging tobacco products, such as waterpipe or hookah and electronic cigarettes (e-cigs), has gained significant popularity and are promoted as safer alternatives to conventional cigarettes. Circadian systems are internal biological oscillations that are considered important regulators of immune functions in mammals. Tobacco induced inflammatory lung diseases frequently exhibit time-of-day/night variation in lung function and symptom severity. We investigated the impact of inhaled e-cig vapor and waterpipe smoke (WPS) on pulmonary circadian molecular clock disruption by determining the changes in expression levels and abundance of core clock component genes (BMAL1, CLOCK) and clock-controlled output genes (Rev-erbα, Per2, Rev-erbβ, Cry2, Rorα) in mouse lungs. We showed that the expression levels of these pulmonary core clock genes and clock-controlled output genes were altered significantly following exposure to WPS (Bmal1, Clock, and Rev-erbα). We further showed a significant yet differential effect on expression levels of core clock and clock-controlled genes (Bmal1, Per2) in the lungs of mice exposed to e-cig vapor containing nicotine. Thus, acute exposure to WPS and e-cig vapor containing nicotine contributes to altered expression of circadian molecular clock genes in mouse lungs, which may have repercussions on lung cellular and biological functions., Competing Interests: The authors have declared that no competing interests exist.

Histone deacetylase 2 (HDAC2), a critical determinant of chromatin remodeling, is reduced as a consequence of oxidative stress-mediated DNA damage and impaired repair. Cigarette smoke (CS) exposure causes DNA damage and cellular senescence. However, no information is available on the role of HDAC2 in CS-induced DNA damage, stress-induced premature senescence (SIPS), and senescence-associated secretory phenotype (SASP) during the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that CS causes persistent DNA damage and cellular senescence via HDAC2-dependent mechanisms. We used HDAC2 global knockout (KO) and HDAC2 lung epithelial cell-specific KO [Clara cell-specific HDAC2 deletion (HDAC2 CreCC10)] mice to determine whether HDAC2 is a major player in CS-induced oxidative stress, SIPS, and SASP. HDAC2 KO mice exposed to CS show exaggerated DNA damage, inflammatory response, and decline in lung function leading to airspace enlargement. Chronic CS exposure augments lung senescence-associated β-galactosidase activity in HDAC2 KO, but not in HDAC2 CreCC10 mice. HDAC2 lung epithelial cell-specific KO did not further augment CS-induced inflammatory response and airspace enlargement but instead caused an increase in lymphoid aggregate formation. Our study reveals that HDAC2 is a key player regulating CS-induced DNA damage, inflammatory response, and cellular senescence leading to COPD/emphysema.-Sundar, I. K., Rashid, K., Gerloff, J., Rangel-Moreno, J., Li, D., Rahman, I. Genetic ablation of histone deacetylase 2 leads to lung cellular senescence and lymphoid follicle formation in COPD/emphysema.

Cigarette smoke (CS) affects DNA damage and cellular senescence signaling pathways in the pathogenesis of chronic obstructive pulmonary disease (COPD). p16 INK4a (p16: a cyclin-dependent kinase inhibitor) is a key marker of cellular senescence, which is induced by CS in lung cells. It is thought that removal of p16 attenuates premature aging by removing senesced cells. However, the role of p16 in CS-induced stress-induced premature senescence (SIPS) and senescence-associated secretory phenotype (SASP) during the development of COPD/emphysema is not known. We hypothesize that p16 regulates cellular senescence and DNA damage/repair molecular signaling targets during chronic CS-induced inflammation and airspace enlargement in mouse models of COPD. We used p16 global knockout (KO) and p16 lung epithelial cell-specific KO (p16 CreCC10 ) mice to determine whether p16 removal in lung epithelium augments or protects against cellular senescence (SIPS and SASP) in chronic CS- and elastase-induced development of COPD/emphysema in mice. p16 KO mice exposed to chronic CS and p16 lung epithelial cell-specific KO mice exposed to elastase did not show attenuation of lung inflammation, altered lung function, or airspace enlargement. p16 KO and p16 CreCC10 exposed to CS and elastase showed increases in lung senescence-associated β-galactosidase activity. Thus, removal of p16-positive cells did not protect against airspace enlargement and decline in lung function induced in COPD mouse models. Our findings suggest that p16 is not the only key player associated with CS-induced cellular senescence phenotypes (SIPS and SASP), decline in lung function, and airspace enlargement in COPD/emphysema.

Cigarette smoke (CS) induces lung cellular senescence that plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). How aging influences cellular senescence and other molecular hallmarks, and increases the risk of CS-induced damage remains unknown. We hypothesized that aging-associated changes in lungs worsen the COPD/emphysema by CS exposure. Younger and older groups of C57BL/6J mice were exposed to chronic CS for 6 months with respective age-matched air-exposed controls. CS caused a decline in lung function and affected the lung structure of both groups of mice. No alterations were observed in the induction of inflammatory mediators between the air-exposed younger and older controls, but aging increased the severity of CS-induced lung inflammation. Aging per se increased lung cellular senescence and significant changes in damage-associated molecular patterns marker S100A8. Gene transcript analysis using the nanoString nCounter showed a significant upregulation of key pro-senescence targets by CS (Mmp12, Ccl2, Cdkn2a, Tert, Wrn, and Bub1b). Aging independently influenced lung function and structure, as well as increased susceptibility to CS-induced inflammation in emphysema, but had a negligible effect on cellular senescence. Thus, aging solely does not contribute to the induction of cellular senescence by CS in a mouse model of COPD/emphysema.

At the cellular level, circadian timing is maintained by the molecular clock, a family of interacting clock gene transcription factors, nuclear receptors and kinases called clock genes. Daily rhythms in pulmonary function are dictated by the circadian timing system, including rhythmic susceptibility to the harmful effects of airborne pollutants, exacerbations in patients with chronic airway disease and the immune-inflammatory response to infection. Further, evidence strongly suggests that the circadian molecular clock has a robust reciprocal interaction with redox signaling and plays a considerable role in the response to oxidative/carbonyl stress. Disruption of the circadian timing system, particularly in airway cells, impairs pulmonary rhythms and lung function, enhances oxidative stress due to airway inhaled pollutants like cigarette smoke and airborne particulate matter and leads to enhanced inflammosenescence, inflammasome activation, DNA damage and fibrosis. Herein, we briefly review recent evidence supporting the role of the lung molecular clock and redox signaling in regulating inflammation, oxidative stress, and DNA damage responses in lung diseases and their exacerbations. We further describe the potential for clock genes as novel biomarkers and therapeutic targets for the treatment of chronic lung diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tobacco use is associated with an increased risk of hearing loss in older individuals, suggesting cigarette smoke (CS) exposure may target the peripheral auditory organs. However, the effects of CS exposure on general cochlear anatomy have not previously been explored. Here we compare control and chronic CS exposed cochleae from adult mice to assess changes in structure and cell survival. Two-photon imaging techniques, including the imaging of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) from native molecules, were used to probe the whole cochlear organ for changes. We found evidence for fibrillar collagen accumulation in the spiral ganglion and organ of Corti, consistent with fibrosis. Quantitative TPEF indicated that basal CS-exposed spiral ganglion neurons experienced greater oxidative stress than control neurons, which was confirmed by histological staining for lipid peroxidation products. Cell counts confirmed that the CS-exposed spiral ganglion also contained fewer basal neurons. Taken together, these data support the premise that CS exposure induces oxidative stress in cochlear cells. They also indicate that two-photon techniques may screen cochlear tissues for oxidative stress.

Waterpipe smoking is emerging as a form of tobacco smoking, but its lung health/risks is not known. It has been shown that different mouse strains show differences in susceptibility to tobacco smoke. However, the effect of waterpipe smoke (WPS) exposure and strain differences in susceptibility to oxidative and inflammatory responses is not known. Here, we showed acute WPS exposure induced oxidative stress and inflammatory response in C57BL/6J and BALB/cJ mouse strains. WPS exposure induced inflammatory cell influx (neutrophils and T-lymphocytes) in bronchoalveolar lavage fluid (BAL fluid), which varied among mouse strains. Proinflammatory cytokines release differed among both the strains, but was significantly increased in C57BL/6J mice. Myeloperoxidase levels in BAL fluid were increased significantly in both the strains. Total reduced glutathione (GSH) level was decreased, whereas the level of oxidized or glutathione disulfide (GSSG) increased in lungs of both the strains. Similarly, the level of lipid peroxidation markers, 15-isoprostane (plasma), malondialdehyde and 4-hydroxy-2-nonenal (lung homogenates) were increased by WPS. Our data suggest that, oxidative stress and inflammatory responses are influenced by strain characteristics during acute WPS exposure. Overall, C57BL/6J mice showed more susceptibility to oxidative stress and inflammatory responses compared to BALB/cJ mice. Acute WPS mediated pulmonary toxicity is differentially regulated in different mouse strains., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)