CXCL17 is a proinflammatory chemokine and promotes neutrophil trafficking.

CXCL17, a novel member of the CXC chemokine class, has been implicated in several human pathologies, but its role in mediating immune response is not well understood. Characteristic features of immune response include resident macrophages orchestrating successive and structured recruitment of neutrophils and monocytes to the insult site. Here, we show that Cxcl17 knockout (KO) mice, compared with the littermate wild-type control mice, were significantly impaired in peritoneal neutrophil recruitment post-lipopolysaccharide (LPS) challenge. Further, the KO mice show dysregulated Cxcl1, Cxcr2, and interleukin-6 levels, all of which directly impact neutrophil recruitment. Importantly, the KO mice showed no difference in monocyte recruitment post-LPS challenge or in peritoneal macrophage levels in both unchallenged and LPS-challenged mice. We conclude that Cxcl17 is a proinflammatory chemokine and that it plays an important role in the early proinflammatory response by promoting neutrophil recruitment to the insult site.

Chemokine Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant.

Microbial infection is characterized by release of multiple proinflammatory chemokines that direct neutrophils to the insult site. How collective function of these chemokines orchestrates neutrophil recruitment is not known. Here, we characterized the role for heterodimer and show that the Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant in mice and can recruit more neutrophils than the individual chemokines. Chemokine-mediated neutrophil recruitment is determined by Cxcr2 receptor signaling, Cxcr2 endocytosis, and binding to glycosaminoglycans. We have now determined heterodimer's Cxcr2 activity using cellular assays and Cxcr2 density in blood and recruited neutrophils in heterodimer-treated mice. We have shown that the heterodimer binds glycosaminoglycans with higher affinity and more efficiently than Cxcl1 or Cxcl2. These data collectively indicate that optimal glycosaminoglycan interactions and dampened receptor activity acting in concert in a dynamic fashion promote heterodimer-mediated robust neutrophil recruitment. We propose that this could play a critical role in combating infection.

SARS-CoV-2 Inhibits NRF2-Mediated Antioxidant Responses in Airway Epithelial Cells and in the Lung of a Murine Model of Infection.

Several viruses have been shown to modulate the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of redox homeostasis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, also seems to disrupt the balance between oxidants and antioxidants, which likely contributes to lung damage. Using in vitro and in vivo models of infection, we investigated how SARS-CoV-2 modulates the transcription factor NRF2 and its dependent genes, as well as the role of NRF2 during SARS-CoV-2 infection. We found that SARS-CoV-2 infection downregulates NRF2 protein levels and NRF2-dependent gene expression in human airway epithelial cells and in lungs of BALB/c mice. Reductions in cellular levels of NRF2 seem to be independent of proteasomal degradation and the interferon/promyelocytic leukemia (IFN/PML) pathway. Furthermore, lack of the Nrf2 gene in SARS-CoV-2-infected mice exacerbates clinical disease, increases lung inflammation, and is associated with a trend toward increased lung viral titers, indicating that NRF2 has a protective role during this viral infection. In summary, our results suggest that SARS-CoV-2 infection alters the cellular redox balance by downregulating NRF2 and its dependent genes, which exacerbates lung inflammation and disease, therefore, suggesting that the activation of NRF2 could be explored as therapeutic approach during SARS-CoV-2 infection. IMPORTANCE The antioxidant defense system plays a major function in protecting the organism against oxidative damage caused by free radicals. COVID-19 patients often present with biochemical characteristics of uncontrolled pro-oxidative responses in the respiratory tract. We show herein that SARS-CoV-2 variants, including Omicron, are potent inhibitors of cellular and lung nuclear factor erythroid 2-related factor 2 (NRF2), the master transcription factor that controls the expression of antioxidant and cytoprotective enzymes. Moreover, we show that mice lacking the Nrf2 gene show increased clinical signs of disease and lung pathology when infected with a mouse-adapted strain of SARS-CoV-2. Overall, this study provides a mechanistic explanation for the observed unbalanced pro-oxidative response in SARS-CoV-2 infections and suggests that therapeutic strategies for COVID-19 may consider the use of pharmacologic agents that are known to boost the expression levels of cellular NRF2.

Antiviral activity of extracellular vesicles derived from respiratory syncytial virus-infected airway epithelial cells.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in children and elderly. No vaccine or effective treatment is currently available for RSV. Extracellular vesicles (EVs) are microvesicles known to carry biologically active molecules, including RNA, DNA and proteins (i.e. cargo). Viral infections can induce profound changes in EV cargo, and the cargo can modulate cellular responses of recipient cells. We have recently shown that EVs isolated from RSV-infected cells were able to activate innate immune response by inducing cytokine and chemokine release from human monocytes and airway epithelial cells, however, we did not investigate the potential antiviral contribution of EVs to a subsequent infection. The objective of this study was to assess the presence of innate immune mediators, including type I and III interferons (IFNs) in EVs released from airway epithelial cells infected with RSV, and their potential role in modulating viral replication in recipient cells. EV-derived from cells infected with RSV were associated with significant amounts of cytokine and chemokines, as well as IFN-ß and -λ, compared to EVs isolated from mock-infected cells. Cells treated with RSV-EVs showed significantly lower levels of viral replication compared to untreated or mock-EV-treated RSV infected cells. Cellular pretreatment with Cerdulatinib, an IFN receptor signaling inhibitor, inhibited the antiviral activity of RSV-EVs in recipient airway epithelial cells. Furthermore, treatment of A549 cells with RSV-EVs induced the expression of IFN-dependent antiviral genes, supporting the idea that RSV-EVs exerts their antiviral activity through an interferon-dependent mechanism. Finally, we determined the concentrations of soluble and EV-associated IFN-ß and IFN-λ in five nasopharyngeal secretions (NPS) of children with viral infections. There were significant levels of IFN-λ in NPS and NPS-derived EVs, while IFN-ß was not detected in either of the two types of samples. EVs released from RSV-infected cells could represent a potential therapeutic approach for modulating RSV replication in the airways.

NRF2 Regulates Cystathionine Gamma-Lyase Expression and Activity in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

Cystathionine-y-lyase (CSE) is a critical enzyme for hydrogen sulfide (H2S) biosynthesis and plays a key role in respiratory syncytial virus (RSV) pathogenesis. The transcription factor NRF2 is the master regulator of cytoprotective and antioxidant gene expression, and is degraded during RSV infection. While some evidence supports the role of NRF2 in CSE gene transcription, its role in CSE expression in airway epithelial cells is not known. Here, we show that RSV infection decreased CSE expression and activity in primary small airway epithelial (SAE) cells, while treatment with tert-butylhydroquinone (tBHQ), an NRF2 inducer, led to an increase of both. Using reporter gene assays, we identified an NRF2 response element required for the NRF2 inducible expression of the CSE promoter. Electrophoretic mobility shift assays demonstrated inducible specific NRF2 binding to the DNA probe corresponding to the putative CSE promoter NRF2 binding sequence. Using chromatin immunoprecipitation assays, we found a 50% reduction in NRF2 binding to the endogenous CSE proximal promoter in SAE cells infected with RSV, and increased binding in cells stimulated with tBHQ. Our results support the hypothesis that NRF2 regulates CSE gene transcription in airway epithelial cells, and that RSV-induced NRF2 degradation likely accounts for the observed reduced CSE expression and activity.

Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) can cause severe respiratory illness in infants, immunocompromised, and older adults. Despite its burden, no vaccine or specific treatment is available. RSV infection is associated with increased reactive oxygen species (ROS) production, degradation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), and decreased antioxidant enzymes (AOEs), leading to oxidative damage and lung injury. Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays a physiological role in numerous cellular processes and a protective role in multiple pathological conditions, displaying vasoactive, cytoprotective, anti-inflammatory, and antioxidant activities. H2S can promote NRF2 activation through the sulfhydration of Kelch-like ECH-associated protein 1, the cytoplasmic repressor of NRF2. Here we investigated whether increasing cellular H2S levels could rescue NRF2 and NRF2-dependent gene expression in RSV-infected primary airway epithelial cells. We found that treatment with the H2S donor GYY4137 significantly increased NRF2 levels and AOEs gene expression by decreasing KEAP1 levels, and by modulating pathways involved in RSV-induced NRF2 degradation, such as NRF2 ubiquitination, and promyelocytic leukemia (PML) protein levels. These results suggest that the administration of exogenous H2S can positively impact the altered redox balance associated with RSV infection, which represents an important determinant of RSV-induced lung disease.

Parent tRNA Modification Status Determines the Induction of Functional tRNA-Derived RNA by Respiratory Syncytial Virus Infection.

tRNA-derived RNA fragments (tRFs) are a recently discovered family of small noncoding RNAs (sncRNAs). We previously reported that respiratory syncytial virus (RSV) infection induces functional tRFs, which are derived from a limited subset of parent tRNAs, in airway epithelial cells. Such induction is also observed in nasopharyngeal wash samples from RSV patients and correlates to RSV genome copies, suggesting a clinical significance of tRFs in RSV infection. This work also investigates whether the modification of parent tRNAs is changed by RSV to induce tRFs, using one of the most inducible tRFs as a model. We discovered that RSV infection changed the methylation modification of adenine at position 57 in tRNA glutamic acid, with a codon of CTC (tRNA-GluCTC), and the change is essential for its cleavage. AlkB homolog 1, a previously reported tRNA demethylase, appears to remove methyladenine from tRNA-GluCTC, prompting the subsequent production of tRFs from the 5'-end of tRNA-GluCTC, a regulator of RSV replication. This study demonstrates for the first time the importance of post-transcriptional modification of tRNAs in tRF biogenesis following RSV infection, providing critical insights for antiviral strategy development.

Exchange Protein Directly Activated by cAMP 2 Enhances Respiratory Syncytial Virus-Induced Pulmonary Disease in Mice.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in young children. It is also a significant contributor to upper respiratory tract infections, therefore, a major cause for visits to the pediatrician. High morbidity and mortality are associated with high-risk populations including premature infants, the elderly, and the immunocompromised. However, no effective and specific treatment is available. Recently, we discovered that an exchange protein directly activated by cyclic AMP 2 (EPAC2) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, EPAC2 promotes RSV replication and pro-inflammatory cytokine/chemokine induction. However, the overall role of EPAC2 in the pulmonary responses to RSV has not been investigated. Herein, we found that EPAC2-deficient mice (KO) or mice treated with an EPAC2-specific inhibitor showed a significant decrease in body weight loss, airway hyperresponsiveness, and pulmonary inflammation, compared with wild-type (WT) or vehicle-treated mice. Overall, this study demonstrates the critical contribution of the EPAC2-mediated pathway to airway diseases in experimental RSV infection, suggesting the possibility to target EPAC2 as a promising treatment modality for RSV.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

The SWI/SNF-Related, Matrix Associated, Actin-Dependent Regulator of Chromatin A4 Core Complex Represses Respiratory Syncytial Virus-Induced Syncytia Formation and Subepithelial Myofibroblast Transition.

Epigenetics plays an important role in the priming the dynamic response of airway epithelial cells to infectious and environmental stressors. Here, we examine the epigenetic role of the SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin A4 (SMARCA4) in the epithelial response to RSV infection. Depletion of SMARCA4 destabilized the abundance of the SMARCE1/ARID1A SWI/SNF subunits, disrupting the innate response and triggering a hybrid epithelial/mesenchymal (E/M) state. Assaying SMARCA4 complex-regulated open chromatin domains by transposase cleavage -next generation sequencing (ATAC-Seq), we observed that the majority of cleavage sites in uninfected cells have reduced chromatin accessibility. Paradoxically, SMARCA4 complex-depleted cells showed enhanced RSV-inducible chromatin opening and gene expression in the EMT pathway genes, MMP9, SNAI1/2, VIM, and CDH2. Focusing on the key MMP9, we observed that SMARCA4 complex depletion reduced basal BRD4 and RNA Polymerase II binding, but enhanced BRD4/Pol II binding in response to RSV infection. In addition, we observed that MMP9 secretion in SMARCA4 complex deficient cells contributes to mesenchymal transition, cellular fusion (syncytia) and subepithelial myofibroblast transition. We conclude the SMARCA4 complex is a transcriptional repressor of epithelial plasticity, whose depletion triggers a hybrid E/M state that affects the dynamic response of the small airway epithelial cell in mucosal remodeling via paracrine MMP9 activity.

Neutrophil recruitment by chemokines Cxcl1/KC and Cxcl2/MIP2: Role of Cxcr2 activation and glycosaminoglycan interactions.

Chemokines play a crucial role in combating microbial infection by recruiting blood neutrophils to infected tissue. In mice, the chemokines Cxcl1/KC and Cxcl2/MIP2 fulfill this role. Cxcl1 and Cxcl2 exist as monomers and dimers, and exert their function by activating the Cxcr2 receptor and binding glycosaminoglycans (GAGs). Here, we characterized Cxcr2 G protein and ß-arrestin activities, and GAG heparan sulfate (HS) interactions of Cxcl1 and Cxcl2 and of the trapped dimeric variants. To understand how Cxcr2 and GAG interactions impact in vivo function, we characterized their neutrophil recruitment activity to the peritoneum, Cxcr2 and CD11b levels on peritoneal and blood neutrophils, and transport profiles out of the peritoneum. Cxcl2 variants compared with Cxcl1 variants were more potent for Cxcr2 activity. Native Cxcl1 compared with native Cxcl2 and dimers compared with native proteins bound HS with higher affinity. Interestingly, recruitment activity between native Cxcl1 and Cxcl2, between dimers, and between the native protein and the dimer could be similar or very different depending on the dose or the time point. These data indicate that peritoneal neutrophil recruitment cannot be solely attributed to Cxcr2 or GAG interactions, and that the relationship between recruited neutrophils, Cxcr2 activation, GAG interactions, and chemokine levels is complex and highly context dependent. We propose that the ability of Cxcl1 and Cxcl2 to reversibly exist as monomers and dimers and differences in their Cxcr2 activity and GAG interactions coordinate neutrophil recruitment and activation, which play a critical role for successful resolution of inflammation.

Lack of Type I Interferon Signaling Ameliorates Respiratory Syncytial Virus-Induced Lung Inflammation and Restores Antioxidant Defenses.

Respiratory syncytial virus (RSV) infection in mouse and human lung is associated with pathogenic inflammation and oxidative injury. RSV impairs antioxidant responses by increasing the degradation of transcription factor NF-E2-related factor 2 (NRF2), which controls the expression of several antioxidant enzymes (AOEs). In addition to its protective effects, type I IFNs have been increasingly recognized as important mediators of host pathogenic responses during acute respiratory viral infections. We used a mouse model of RSV infection to investigate the effect of lack of type I interferon (IFN) receptor on viral-mediated clinical disease, airway inflammation, NRF2 expression, and antioxidant defenses. In the absence of type I IFN signaling, RSV-infected mice showed significantly less body weight loss and airway obstruction, as well as a significant reduction in cytokine and chemokine secretion and airway inflammation. Lack of type I IFN receptor was associated with greatly reduced virus-induced promyelocytic leukemia lung protein expression, which we showed to be necessary for virus-induced NRF2 degradation in a cell model of infection, resulting in restoration of NRF2 levels, AOE expression, and airway antioxidant capacity. Our data support the concept that modulation of type I IFN production and/or signaling could represent an important therapeutic strategy to ameliorate severity of RSV-induced lung disease.

Selective Blockade of TNFR1 Improves Clinical Disease and Bronchoconstriction in Experimental RSV Infection.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infants and young children. Although some clinical studies have speculated that tumor necrosis factor (TNF)-α is a major contributor of RSV-mediated airway disease, experimental evidence remains unclear or conflicting. TNF-α initiates inflammation and cell death through two distinct receptors: TNF-receptor (TNFR)1 and TNFR2. Here we delineate the function of TNF-α by short-lasting blockade of either receptor in an experimental BALB/c mouse model of RSV infection. We demonstrate that antibody-mediated blockade of TNFR1, but not TNFR2, results in significantly improved clinical disease and bronchoconstriction as well as significant reductions of several inflammatory cytokines and chemokines, including IL-1α, IL-1ß, IL-6, Ccl3, Ccl4, and Ccl5. Additionally, TNFR1 blockade was found to significantly reduce neutrophil number and activation status, consistent with the concomitant reduction of pro-neutrophilic chemokines Cxcl1 and Cxcl2. Similar protective activity was also observed when a single-dose of TNFR1 blockade was administered to mice following RSV inoculation, although this treatment resulted in improved alveolar macrophage survival rather than reduced neutrophil activation. Importantly, short-lasting blockade of TNFR1 did not affect RSV peak replication in the lung. This study suggests a potential therapeutic approach for RSV bronchiolitis based on selective blockade of TNFR1.

HIF-1α Modulates Core Metabolism and Virus Replication in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

Metabolic reprogramming of host cells is key to the foundation of a successful viral infection. Hypoxia inducible factors (HIFs) mediate oxygen utilization by regulating cellular metabolism and redox homeostasis. Under normoxic conditions, HIF proteins are synthesized and subsequently degraded following ubiquitination to allow for normal metabolic activities. Recent studies suggest that respiratory syncytial virus (RSV) has the ability to induce HIF-1α stabilization and accumulation through non-hypoxic mechanisms. This makes the HIF pathway a potential avenue of approach for RSV therapeutic development. Using a model of primary human small alveolar epithelial cells, we demonstrate RSV infections to greatly alter cellular metabolism in favor of the glycolytic and pentose phosphate pathways. Additionally, we show RSV infections to stabilize HIF-1α and HIF-2α expression in these cells. Inhibition of HIF-1α, but not HIF-2α, was found to significantly reduce RSV replication as well as the glycolytic pathway, as measured by the expression of hexokinase II. Our study contributes to the understanding of RSV-mediated changes to cellular metabolism and supports further investigation into anti-HIF-1α therapeutics for RSV infections.

Gestational Exposure to Cigarette Smoke Suppresses the Gasotransmitter H2S Biogenesis and the Effects Are Transmitted Transgenerationally.

Rationale: Gestational cigarette smoke (CS) impairs lung angiogenesis and alveolarization, promoting transgenerational development of asthma and bronchopulmonary dysplasia (BPD). Hydrogen sulfide (H2S), a proangiogenic, pro-alveolarization, and anti-asthmatic gasotransmitter is synthesized by cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), and 3-mercaptopyruvate sulfur transferase (3MST). Objective: Determine if gestational CS exposure affected the expression of H2S synthesizing enzymes in the mouse lung and human placenta. Methods: Mice were exposed throughout gestational period to secondhand CS (SS) at approximating the dose of CS received by a pregnant woman sitting in a smoking bar for 3 h/days during pregnancy. Lungs from 7-days old control and SS-exposed pups and human placenta from mothers who were either non-smokers or smokers during pregnancy were analyzed for expression of the enzymes. Measurements: Mouse lungs and human placentas were examined for the expression of CSE, CBS, and 3MST by immunohistochemical staining, qRT-PCR and/or Western blot (WB) analyses. Results: Compared to controls, mouse lung exposed gestationally to SS had significantly lower levels of CSE, CBS, and 3MST. Moreover, the SS-induced suppression of CSE and CBS in F1 lungs was transmitted to the F2 generation without significant change in the magnitude of the suppression. These changes were associated with impaired epithelial-mesenchymal transition (EMT)-a process required for normal lung angiogenesis and alveolarization. Additionally, the placentas from mothers who smoked during pregnancy, expressed significantly lower levels of CSE, CBS, and 3MST, and the effects were partially moderated by quitting smoking during the first trimester. Conclusions: Lung H2S synthesizing enzymes are downregulated by gestational CS and the effects are transmitted to F2 progeny. Smoking during pregnancy decreases H2S synthesizing enzymes is human placentas, which may correlate with the increased risk of asthma/BPD in children.

Respiratory Syncytial Virus Infection Induces Chromatin Remodeling to Activate Growth Factor and Extracellular Matrix Secretion Pathways.

Lower respiratory tract infection (LRTI) with respiratory syncytial virus (RSV) is associated with reduced lung function through unclear mechanisms. In this study, we test the hypothesis that RSV infection induces genomic reprogramming of extracellular matrix remodeling pathways. For this purpose, we sought to identify transcriptionally active open chromatin domains using assay for transposase-accessible-next generation sequencing (ATAC-Seq) in highly differentiated lower airway epithelial cells. High confidence nucleosome-free regions were those predicted independently using two peak-calling algorithms. In uninfected cells, ~12,650 high-confidence open chromatin regions were identified. These mapped to ~8700 gene bodies, whose genes functionally controlled organelle synthesis and Th2 pathways (IL6, TSLP). These latter cytokines are preferentially secreted by RSV-infected bronchiolar cells and linked to mucous production, obstruction, and atopy. By contrast, in RSV infection, we identify ~1700 high confidence open chromatin domains formed in 1120 genes, primarily in introns. These induced chromatin modifications are associated with complex gene expression profiles controlling tyrosine kinase growth factor signaling and extracellular matrix (ECM) secretory pathways. Of these, RSV induces formation of nucleosome-free regions on TGFB1/JUNB//FN1/MMP9 genes and the rate limiting enzyme in the hexosamine biosynthetic pathway (HBP), Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2). RSV-induced open chromatin domains are highly enriched in AP1 binding motifs and overlap experimentally determined JUN peaks in GEO ChIP-Seq data sets. Our results provide a topographical map of chromatin accessibility and suggest a growth factor and AP1-dependent mechanism for upregulation of the HBP and ECM remodeling in lower epithelial cells that may be linked to long-term airway remodeling.

Cigarette Smoke Condensate Exposure Changes RNA Content of Extracellular Vesicles Released from Small Airway Epithelial Cells.

Exposure to environmental tobacco smoke (ETS) is a known risk factor for the development of chronic lung diseases, cancer, and the exacerbation of viral infections. Extracellular vesicles (EVs) have been identified as novel mediators of cell-cell communication through the release of biological content. Few studies have investigated the composition/function of EVs derived from human airway epithelial cells (AECs) exposed to cigarette smoke condensate (CSC), as surrogates for ETS. Using novel high-throughput technologies, we identified a diverse range of small noncoding RNAs (sncRNAs), including microRNA (miRNAs), Piwi-interacting RNA (piRNAs), and transfer RNA (tRNAs) in EVs from control and CSC-treated SAE cells. CSC treatment resulted in significant changes in the EV content of miRNAs. A total of 289 miRNAs were identified, with five being significantly upregulated and three downregulated in CSC EVs. A total of 62 piRNAs were also detected in our EV preparations, with five significantly downregulated and two upregulated in CSC EVs. We used TargetScan and Gene Ontology (GO) analysis to predict the biological targets of hsa-miR-3913-5p, the most represented miRNA in CSC EVs. Understanding fingerprint molecules in EVs will increase our knowledge of the relationship between ETS exposure and lung disease, and might identify potential molecular targets for future treatments.

Cystathionine γ-lyase deficiency enhances airway reactivity and viral-induced disease in mice exposed to side-stream tobacco smoke.

BACKGROUND: Environmental tobacco smoke (ETS) is a known risk factor for severe respiratory syncytial virus (RSV) infections, yet the mechanisms of ETS/RSV comorbidity are largely unknown. Cystathionine γ-lyase regulates important physiological functions of the respiratory tract. METHODS: We used mice genetically deficient in the cystathionine γ-lyase enzyme (CSE), the major H2S-generating enzyme in the lung to determine the contribution of H2S to airway disease in response to side-stream tobacco smoke (TS), and to TS/RSV co-exposure. RESULTS: Following a 2-week period of exposure to TS, CSE-deficient mice (KO) showed a dramatic increase in airway hyperresponsiveness (AHR) to methacholine challenge, and greater airway cellular inflammation, compared with wild-type (WT) mice. TS-exposed CSE KO mice that were subsequently infected with RSV exhibited a more severe clinical disease, airway obstruction and AHR, enhanced viral replication, and lung inflammation, compared with TS-exposed RSV-infected WT mice. TS-exposed RSV-infected CSE KO mice had also a significant increase in the number of neutrophils in bronchoalveolar lavage fluid and increased levels of inflammatory cytokines and chemokines. CONCLUSION: This study demonstrates the critical contribution of the H2S-generating pathway to airway reactivity and disease following exposure to ETS alone or in combination with RSV infection.

Exchange Proteins Directly Activated by cAMP and Their Roles in Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of respiratory infection in young children and high-risk adults. However, a specific treatment for this viral infection is not currently available. In this study, we discovered that an exchange protein directly activated by cyclic AMP (EPAC) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, treatment with EPAC inhibitor (ESI-09), but not protein kinase A inhibitor (H89), significantly inhibits RSV replication and proinflammatory cytokine/chemokine induction. In addition, RSV-activated transcriptional factors belonging to the NF-κB and IRF families are also suppressed by ESI-09. Through isoform-specific gene knockdown, we found that EPAC2, but not EPAC1, plays a dominant role in controlling RSV replication and virus-induced host responses. Experiments using both EPAC2 knockout and EPAC2-specific inhibitor support such roles of EPAC2. Therefore, EPAC2 is a promising therapeutic target to regulate RSV replication and associated inflammation.IMPORTANCE RSV is a serious public health problem, as it is associated with bronchiolitis, pneumonia, and asthma exacerbations. Currently no effective treatment or vaccine is available, and many molecular mechanisms regarding RSV-induced lung disease are still significantly unknown. This project aims to elucidate an important and novel function of a protein, called EPAC2, in RSV replication and innate inflammatory responses. Our results should provide an important insight into the development of new pharmacologic strategies against RSV infection, thereby reducing RSV-associated morbidity and mortality.

Central Role of the NF-κB Pathway in the Scgb1a1-Expressing Epithelium in Mediating Respiratory Syncytial Virus-Induced Airway Inflammation.

Lower respiratory tract infection with respiratory syncytial virus (RSV) produces profound inflammation. Despite an understanding of the role of adaptive immunity in RSV infection, the identity of the major sentinel cells initially triggering inflammation is controversial. Here we evaluate the role of nonciliated secretoglobin (Scgb1a1)-expressing bronchiolar epithelial cells in RSV infection. Mice expressing a tamoxifen (TMX)-inducible Cre recombinase-estrogen receptor fusion protein (CreERTM) knocked into the Scgb1a1 locus were crossed with mice that harbor a RelA conditional allele (RelAfl ), with loxP sites flanking exons 5 to 8 of the Rel homology domain. The Scgb1a1CreERTM/+ × RelAfl/fl mouse is a RelA conditional knockout (RelACKO) of a nonciliated epithelial cell population enriched in the small bronchioles. TMX-treated RelACKO mice have reduced pulmonary neutrophilic infiltration and impaired expression and secretion of NF-κB-dependent cytokines in response to RSV. In addition, RelACKO mice had reduced expression levels of interferon (IFN) regulatory factor 1/7 (IRF1/7) and retinoic acid-inducible gene I (RIG-I), components of the mucosal IFN positive-feedback loop. We demonstrate that RSV replication induces RelA to complex with bromodomain-containing protein 4 (BRD4), a cofactor required for RNA polymerase II (Pol II) phosphorylation, activating the atypical histone acetyltransferase (HAT) activity of BRD4 required for phospho-Ser2 Pol II formation, histone H3K122 acetylation, and cytokine secretion in vitro and in vivo TMX-treated RelACKO mice have less weight loss and reduced airway obstruction/hyperreactivity yet similar levels of IFN-γ production despite higher levels of virus production. These data indicate that the nonciliated Scgb1a1-expressing epithelium is a major innate sensor for restricting RSV infection by mediating neutrophilic inflammation and chemokine and mucosal IFN production via the RelA-BRD4 pathway.IMPORTANCE RSV infection is the most common cause of infant hospitalizations in the United States, resulting in 2.1 million children annually requiring medical attention. RSV primarily infects nasal epithelial cells, spreading distally to produce severe lower respiratory tract infections. Our study examines the role of a nonciliated respiratory epithelial cell population in RSV infection. We genetically engineered a mouse that can be selectively depleted of the NF-κB/RelA transcription factor in this subset of epithelial cells. These mice show an impaired activation of the bromodomain-containing protein 4 (BRD4) coactivator, resulting in reduced cytokine expression and neutrophilic inflammation. During the course of RSV infection, epithelial RelA-depleted mice have reduced disease scores and airway hyperreactivity yet increased levels of virus replication. We conclude that RelA-BRD4 signaling in nonciliated bronchiolar epithelial cells mediates neutrophilic airway inflammation and disease severity. This complex is an attractive target to reduce the severity of infection.