Respiratory syncytial virus disrupts the airway epithelial barrier by decreasing cortactin and destabilizing F-actin.

Respiratory syncytial virus (RSV) infection is the leading cause of acute lower respiratory tract infection in young children worldwide. Our group recently revealed that RSV infection disrupts the airway epithelial barrier in vitro and in vivo. However, the underlying molecular pathways were still elusive. Here, we report the critical roles of the filamentous actin (F-actin) network and actin-binding protein cortactin in RSV infection. We found that RSV infection causes F-actin depolymerization in 16HBE cells, and that stabilizing the F-actin network in infected cells reverses the epithelial barrier disruption. RSV infection also leads to significantly decreased cortactin in vitro and in vivo. Cortactin-knockout 16HBE cells presented barrier dysfunction, whereas overexpression of cortactin protected the epithelial barrier against RSV. The activity of Rap1 (which has Rap1A and Rap1B forms), one downstream target of cortactin, declined after RSV infection as well as in cortactin-knockout cells. Moreover, activating Rap1 attenuated RSV-induced epithelial barrier disruption. Our study proposes a key mechanism in which RSV disrupts the airway epithelial barrier via attenuating cortactin expression and destabilizing the F-actin network. The identified pathways will provide new targets for therapeutic intervention toward RSV-related disease. This article has an associated First Person interview with the first author of the paper.

Electronic cigarette exposure disrupts airway epithelial barrier function and exacerbates viral infection.

The use of electronic cigarettes (e-cigs), especially among teenagers, has reached alarming and epidemic levels, posing a significant threat to public health. However, the short- and long-term effects of vaping on the airway epithelial barrier are unclear. Airway epithelial cells are the forefront protectors from viruses and pathogens. They contain apical junctional complexes (AJCs), which include tight junctions (TJs) and adherens junctions (AJs) formed between adjacent cells. Previously, we reported respiratory syncytial virus (RSV) infection, the leading cause of acute lower respiratory infection-related hospitalization in children and high-risk adults, induces a "leaky airway" by disrupting the epithelial AJC structure and function. We hypothesized chemical components of e-cigs disrupt airway epithelial barrier and exacerbate RSV-induced airway barrier dysfunction. Using confluent human bronchial epithelial (16HBE) cells and well-differentiated normal human bronchial epithelial (NHBE) cells, we found that exposure to extract and aerosol e-cig nicotine caused a significant decrease in transepithelial electrical resistance (TEER) and the structure of the AJC even at noncytotoxic concentrations. Western blot analysis of 16HBE cells exposed to e-cig nicotine extract did not reveal significant changes in AJC proteins. Exposure to aerosolized e-cig cinnamon or menthol flavors also induced barrier disruption and aggravated nicotine-induced airway barrier dysfunction. Moreover, preexposure to nicotine aerosol increased RSV infection and the severity of RSV-induced airway barrier disruption. Our findings demonstrate that e-cig exposure disrupts the airway epithelial barrier and exacerbates RSV-induced damage. Knowledge gained from this study will provide awareness of adverse e-cig respiratory effects and positively impact the mitigation of e-cig epidemic.NEW & NOTEWORTHY Electronic cigarette (e-cig) use, especially in teens, is alarming and at epidemic proportions, threatening public health. Our study shows that e-cig nicotine exposure disrupts airway epithelial tight junctions and increases RSV-induced barrier dysfunction. Furthermore, exposure to aerosolized flavors exaggerates e-cig nicotine-induced airway barrier dysfunction. Our study confirms that individual and combined components of e-cigs deleteriously impact the airway barrier and that e-cig exposure increases susceptibility to viral infection.

RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration.

The airway epithelium's ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract's normal functions. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions. Inhibition of Rho-associated kinase, a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain 2 following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed reepithelialization. There were increased concentrations of phospho-myosin light chain 2 in day 7 naphthalene + RSV animals, which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.

RSV infection potentiates TRPV1-mediated calcium transport in bronchial epithelium of asthmatic children.

The transient receptor potential vanilloid 1 (TRPV1) channel is expressed in human bronchial epithelium (HBE), where it transduces Ca2+ in response to airborne irritants. TRPV1 activation results in bronchoconstriction, cough, and mucus production, and may therefore contribute to the pathophysiology of obstructive airway disease. Since children with asthma face the greatest risk of developing virus-induced airway obstruction, we hypothesized that changes in TRPV1 expression, localization, and function in the airway epithelium may play a role in bronchiolitis and asthma in childhood. We sought to measure TRPV1 protein expression, localization, and function in HBE cells from children with versus without asthma, both at baseline and after RSV infection. We determined changes in TRPV1 protein expression, subcellular localization, and function both at baseline and after RSV infection in primary HBE cells from normal children and children with asthma. Basal TRPV1 protein expression was higher in HBE from children with versus without asthma and primarily localized to plasma membranes (PMs). During RSV infection, TRPV1 protein increased more in the PM of asthmatic HBE as compared with nonasthmatic cells. TRPV1-mediated increase in intracellular Ca2+ was greater in RSV-infected asthmatic cells, but this increase was attenuated when extracellular Ca2+ was removed. Nerve growth factor (NGF) recapitulated the effect of RSV on TRPV1 activation in HBE cells. Our data suggest that children with asthma have intrinsically hyperreactive airways due in part to higher TRPV1-mediated Ca2+ influx across epithelial membranes, and this abnormality is further exacerbated by NGF overexpression during RSV infection driving additional Ca2+ from intracellular stores.

Titanium dioxide nanoparticles exaggerate respiratory syncytial virus-induced airway epithelial barrier dysfunction.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide. While most develop a mild, self-limiting illness, some develop severe acute lower respiratory infection and persistent airway disease. Exposure to ambient particulate matter has been linked to asthma, bronchitis, and viral infection in multiple epidemiological studies. We hypothesized that coexposure to nanoparticles worsens RSV-induced airway epithelial barrier dysfunction. Bronchial epithelial cells were incubated with titanium dioxide nanoparticles (TiO2-NP) or a combination of TiO2-NP and RSV. Structure and function of epithelial cell barrier were analyzed. Viral titer and the role of reactive oxygen species (ROS) generation were evaluated. In vivo, mice were intranasally incubated with TiO2-NP, RSV, or a combination. Lungs and bronchoalveolar lavage (BAL) fluid were harvested for analysis of airway inflammation and apical junctional complex (AJC) disruption. RSV-induced AJC disruption was amplified by TiO2-NP. Nanoparticle exposure increased viral infection in epithelial cells. TiO2-NP induced generation of ROS, and pretreatment with antioxidant, N-acetylcysteine, reversed said barrier dysfunction. In vivo, RSV-induced injury and AJC disruption were augmented in the lungs of mice given TiO2-NP. Airway inflammation was exacerbated, as evidenced by increased white blood cell infiltration into the BAL, along with exaggeration of peribronchial inflammation and AJC disruption. These data demonstrate that TiO2-NP exposure exacerbates RSV-induced AJC dysfunction and increases inflammation by mechanisms involving generation of ROS. Further studies are required to determine whether NP exposure plays a role in the health disparities of asthma and other lung diseases, and why some children experience more severe airway disease with RSV infection.

Disruption of the airway epithelial barrier in a murine model of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of hospitalization for infants and young children worldwide. RSV is known to infect epithelial cells and increase the permeability of model airway epithelial monolayers in vitro. We hypothesized that RSV infection also induces airway barrier dysfunction in vivo. C57BL/6 mice were intranasally inoculated with RSV, and on day 4 post-inoculation were examined for viral replication, lung inflammation, and barrier integrity as well as the structure and molecular composition of epithelial junctions. In parallel, primary mouse tracheal epithelial cells (mTEC) were cultured for in vitro studies. RSV-infected mice lost weight and showed significant peribronchial inflammation compared with noninfected controls and UV-inactivated RSV-inoculated animals. RSV infection increased the permeability of the airway epithelial barrier and altered the molecular composition of epithelial tight junctions. The observed RSV-induced barrier disruption was accompanied by decreased expression of several tight-junction proteins and accumulation of cleaved extracellular fragments of E-cadherin in bronchoalveolar lavage and mTEC supernatants. Similarly, in vitro RSV infection of mTEC monolayers resulted in enhanced permeability and disruption of tight-junction structure. Furthermore, incubation of mTEC monolayers with a recombinant fragment of E-cadherin caused tight-junction disassembly. Taken together, these data indicate that RSV infection leads to airway barrier dysfunction in vivo, mediated by either decreased expression or cleavage of junctional proteins. Our observations provide further insights into the pathophysiology of RSV infection and provide a rationale for development of barrier-protecting agents to alleviate the pathogenic effects of RSV infection.

IFN-γ, IL-17A, or zonulin rapidly increase the permeability of the blood-brain and small intestinal epithelial barriers: Relevance for neuro-inflammatory diseases.

Breakdown of the blood-brain barrier (BBB) precedes lesion formation in the brains of multiple sclerosis (MS) patients. Since recent data implicate disruption of the small intestinal epithelial barrier (IEB) in the pathogenesis of MS, we hypothesized that the increased permeability of the BBB and IEB are mechanistically linked. Zonulin, a protein produced by small intestine epithelium, can rapidly increase small intestinal permeability. Zonulin blood levels are elevated in MS, but it is unknown whether zonulin can also disrupt the BBB. Increased production of IL-17A and IFN-γ has been implicated in the pathogenesis of MS, epilepsy, and stroke, and these cytokines impact BBB integrity after 24 h. We here report that primary human brain microvascular endothelial cells expressed the EGFR and PAR2 receptors necessary to respond to zonulin, and that zonulin increased BBB permeability to a 40 kDa dextran tracer within 1 h. Moreover, both IL-17A and IFN-γ also rapidly increased BBB and IEB permeability. By using confocal microscopy, we found that exposure of the IEB to zonulin, IFN-γ, or IL-17A in vitro rapidly modified the localization of the TJ proteins, ZO-1, claudin-5, and occludin. TJ disassembly was accompanied by marked depolymerization of the peri-junctional F-actin cytoskeleton. Our data indicate that IFN-γ, IL-17A, or zonulin can increase the permeability of the IEB and BBB rapidly in vitro, by modifying TJs and the underlying actin cytoskeleton. These observations may help clarify how the gut-brain axis mediates the pathogenesis of neuro-inflammatory diseases.

Induction of high-mobility group Box-1 in vitro and in vivo by respiratory syncytial virus.

BackgroundDespite decades that have passed since its discovery, accurate biomarkers of respiratory syncytial virus (RSV) disease activity and effective therapeutic strategies are still lacking. The high-mobility group box type 1 (HMGB1) protein has been proposed as a possible link between RSV and immune system, but only limited information is currently available to support this hypothesis.MethodsExpression of HMGB1 gene and protein was analyzed by quantitative PCR, enzyme-linked immunosorbent assay (ELISA), western blot, immunocytochemistry, and confocal microscopy in immortalized and primary human bronchial epithelial cells, as well as in rat pup lungs. The role of HMGB1 in RSV infection was explored using glycyrrhizin, a selective HMGB1 inhibitor.ResultsRSV infection strongly induced HMGB1 expression both in vitro and in vivo. Glycyrrhizin dose-dependently inhibited HMGB1 upregulation in both RSV-infected immortalized and primary human bronchial epithelial cells, and this effect was associated with significant reduction of viral replication.ConclusionOur data suggest that HMGB1 expression increases during RSV replication. This seems to have a critical pathogenic role as its selective inhibition virtually modified the infection. These observations provide further insight into the pathophysiology of RSV infection and uncover a potential biomarker and therapeutic target for the most common respiratory infection of infancy.

Detection of Sp110 by Flow Cytometry and Application to Screening Patients for Veno-occlusive Disease with Immunodeficiency.

Mutations in Sp110 are the underlying cause of veno-occlusive disease with immunodeficiency (VODI), a combined immunodeficiency that is difficult to treat and often fatal. Because early treatment is critically important for patients with VODI, broadly usable diagnostic tools are needed to detect Sp110 protein deficiency. Several factors make establishing the diagnosis of VODI challenging: (1) Current screening strategies to identify severe combined immunodeficiency are based on measuring T cell receptor excision circles (TREC). This approach will fail to identify VODI patients because the disease is not associated with severe T cell lymphopenia at birth; (2) the SP110 gene contains 17 exons, making it a challenge for Sanger sequencing. The recently developed next-generation sequencing (NGS) platforms that can rapidly determine the sequence of all 17 exons are available in only a few laboratories; (3) there is no standard functional assay to test for the effects of novel mutations in Sp110; and (4) it has been difficult to use flow cytometry to identify patients who lack Sp110 because of the low level of Sp110 protein in peripheral blood lymphocytes. We report here a novel flow cytometric assay that is easily performed in diagnostic laboratories and might thus become a standard assay for the evaluation of patients who may have VODI. In addition, the assay will facilitate investigations directed at understanding the function of Sp110.

Pre-existing tolerance shapes the outcome of mucosal allergen sensitization in a murine model of asthma.

Recent published studies have highlighted the complexity of the immune response to allergens, and the various asthma phenotypes that arise as a result. Although the interplay of regulatory and effector immune cells responding to allergen would seem to dictate the nature of the asthmatic response, little is known regarding how tolerance versus reactivity to allergen occurs in the lung. The vast majority of mouse models study allergen encounter in naive animals, and therefore exclude the possibility that previous encounters with allergen may influence future sensitization. To address this, we studied sensitization to the model allergen OVA in mice in the context of pre-existing tolerance to OVA. Allergen sensitization by either systemic administration of OVA with aluminum hydroxide or mucosal administration of OVA with low-dose LPS was suppressed in tolerized animals. However, higher doses of LPS induced a mixed Th2 and Th17 response to OVA in both naive and tolerized mice. Of interest, tolerized mice had more pronounced Th17-type inflammation than did naive mice receiving the same sensitization, suggesting pre-existing tolerance altered the inflammatory phenotype. These data show that a pre-existing tolerogenic immune response to allergen can affect subsequent sensitization in the lung. These findings have potential significance for understanding late-onset disease in individuals with severe asthma.

Epithelial barrier function: at the front line of asthma immunology and allergic airway inflammation.

Airway epithelial cells form a barrier to the outside world and are at the front line of mucosal immunity. Epithelial apical junctional complexes are multiprotein subunits that promote cell-cell adhesion and barrier integrity. Recent studies in the skin and gastrointestinal tract suggest that disruption of cell-cell junctions is required to initiate epithelial immune responses, but how this applies to mucosal immunity in the lung is not clear. Increasing evidence indicates that defective epithelial barrier function is a feature of airway inflammation in asthmatic patients. One challenge in this area is that barrier function and junctional integrity are difficult to study in the intact lung, but innovative approaches should provide new knowledge in this area in the near future. In this article we review the structure and function of epithelial apical junctional complexes, emphasizing how regulation of the epithelial barrier affects innate and adaptive immunity. We discuss why defective epithelial barrier function might be linked to TH2 polarization in asthmatic patients and propose a rheostat model of barrier dysfunction that implicates the size of inhaled allergen particles as an important factor influencing adaptive immunity.

Breaking barriers. New insights into airway epithelial barrier function in health and disease.

Epithelial permeability is a hallmark of mucosal inflammation, but the molecular mechanisms involved remain poorly understood. A key component of the epithelial barrier is the apical junctional complex that forms between neighboring cells. Apical junctional complexes are made of tight junctions and adherens junctions and link to the cellular cytoskeleton via numerous adaptor proteins. Although the existence of tight and adherens junctions between epithelial cells has long been recognized, in recent years there have been significant advances in our understanding of the molecular regulation of junctional complex assembly and disassembly. Here we review the current thinking about the structure and function of the apical junctional complex in airway epithelial cells, emphasizing the translational aspects of relevance to cystic fibrosis and asthma. Most work to date has been conducted using cell culture models, but technical advancements in imaging techniques suggest that we are on the verge of important new breakthroughs in this area in physiological models of airway diseases.

Sustained protein kinase D activation mediates respiratory syncytial virus-induced airway barrier disruption.

Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.

Lpa2 is a negative regulator of both dendritic cell activation and murine models of allergic lung inflammation.

Negative regulation of innate immune responses is essential to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration, and activation and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G protein-coupled receptors, whose function in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. In this article, we show that DC from lpa2(-/-) mice are hyperactive compared with their wild-type counterparts and are less susceptible to inhibition by different LPA species. In transient-transfection assays, we found that lpa2 overexpression inhibits NF-κB-driven gene transcription. Using an adoptive-transfer approach, we found that allergen-pulsed lpa2(-/-) DC induced substantially more lung inflammation than did wild-type DC after inhaled allergen challenge. Finally, lpa2(-/-) mice develop greater allergen-driven lung inflammation than do their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits DC activation and allergic airway inflammation.

8-Bromo-cAMP attenuates human airway epithelial barrier disruption caused by titanium dioxide fine and nanoparticles.

Titanium dioxide fine particles (TiO2-FPs) and nanoparticles (TiO2-NPs) are the most widely used whitening pigments worldwide. Inhalation of TiO2-FPs and TiO2-NPs can be harmful as it triggers toxicity in the airway epithelial cells. The airway epithelium serves as the respiratory system's first line of defense in which airway epithelial cells are significant targets of inhaled pathogens and environmental particles. Our group previously found that TiO2-NPs lead to a disrupted barrier in the polarized airway epithelial cells. However, the effect of TiO2-FPs on the respiratory epithelial barrier has not been examined closely. In this study, we aimed to compare the effects of TiO2-FPs and TiO2-NPs on the structure and function of the airway epithelial barrier. Additionally, we hypothesized that 8-Bromo-cAMP, a cyclic adenosine monophosphate (cAMP) derivative, would alleviate the disruptive effects of both TiO2-FPs and TiO2-NPs. We observed increased epithelial membrane permeability in both TiO2-FPs and TiO2-NPs after exposure to 16HBE cells. Immunofluorescent labeling showed that both particle sizes disrupted the structural integrity of airway epithelial tight junctions and adherens junctions. TiO2-FPs had a slightly more, but insignificant impact on the epithelial barrier disruption than TiO2-NPs. Treatment with 8-Bromo-cAMP significantly attenuated the barrier-disrupting impact of both TiO2-FPs and TiO2-NPs on cell monolayers. Our study demonstrates that both TiO2-FPs and TiO2-NPs cause comparable barrier disruption and suggests a protective role for cAMP signaling. The observed effects of TiO2-FPs and TiO2-NPs provide a necessary understanding for characterizing the pathways involved in the defensive role of the cAMP pathway on TiO2-induced airway barrier disruption.

Vitamin D3 protects against respiratory syncytial virus-induced barrier dysfunction in airway epithelial cells via PKA signaling pathway.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in infants and young children globally and is responsible for hospitalization and mortality in the elderly population. Virus-induced airway epithelial barrier damage is a critical step during RSV infection, and emerging studies suggest that RSV disrupts the tight junctions (TJs) and adherens junctions (AJs) between epithelial cells, increasing the permeability of the airway epithelial barrier. The lack of commercially available vaccines and effective antiviral drugs for RSV emphasizes the need for new management strategies. Vitamin D3 is a promising intervention for viral infection due to its critical role in modulating innate immune responses. However, there is limited evidence on the effect of vitamin D3 on RSV pathogenies. Here, we investigated the impact of vitamin D3 on RSV-induced epithelial barrier dysfunction and the underlying mechanisms. We found that pre-incubation with 1,25(OH)2D3, the active form of vitamin D3, alleviated RSV-induced epithelial barrier disruption in a dose-dependent manner without affecting viability in 16HBE cells. 1,25(OH)2D3 induced minor changes in the protein expression level of TJ/AJ proteins in RSV-infected cells. We observed increased CREB phosphorylation at Ser133 during 1,25(OH)2D3 exposure, indicating that vitamin D3 triggered protein kinase A (PKA) activity in 16HBE. PKA inhibitors modified the restoration of barrier function by 1,25(OH)2D3 in RSV-infected cells, implying that PKA signaling is responsible for the protective effects of vitamin D3 against RSV-induced barrier dysfunction in airway epithelial cells. Our findings suggest vitamin D3 as a prophylactic intervention to protect the respiratory epithelium during RSV infections.

Polyinosinic:polycytidylic acid induces protein kinase D-dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells.

BACKGROUND: Disruption of the epithelial barrier might be a risk factor for allergen sensitization and asthma. Viral respiratory tract infections are strongly associated with asthma exacerbation, but the effects of respiratory viruses on airway epithelial barrier function are not well understood. Many viruses generate double-stranded RNA, which can lead to airway inflammation and initiate an antiviral immune response. OBJECTIVES: We investigated the effects of the synthetic double-stranded RNA polyinosinic:polycytidylic acid (polyI:C) on the structure and function of the airway epithelial barrier in vitro. METHODS: 16HBE14o- human bronchial epithelial cells and primary airway epithelial cells at an air-liquid interface were grown to confluence on Transwell inserts and exposed to polyI:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of epithelial apical junctions by means of immunofluorescence microscopy. RESULTS: PolyI:C induced a profound decrease in transepithelial electrical resistance and increase in paracellular permeability. Immunofluorescence microscopy revealed markedly reduced junctional localization of zonula occludens-1, occludin, E-cadherin, ß-catenin, and disorganization of junction-associated actin filaments. PolyI:C induced protein kinase D (PKD) phosphorylation, and a PKD antagonist attenuated polyI:C-induced disassembly of apical junctions and barrier dysfunction. CONCLUSIONS: PolyI:C has a powerful and previously unsuspected disruptive effect on the airway epithelial barrier. PolyI:C-dependent barrier disruption is mediated by disassembly of epithelial apical junctions, which is dependent on PKD signaling. These findings suggest a new mechanism potentially underlying the associations between viral respiratory tract infections, airway inflammation, and allergen sensitization.

Airway Epithelial Cell Junctions as Targets for Pathogens and Antimicrobial Therapy.

Intercellular contacts between epithelial cells are established and maintained by the apical junctional complexes (AJCs). AJCs conserve cell polarity and build epithelial barriers to pathogens, inhaled allergens, and environmental particles in the respiratory tract. AJCs consist of tight junctions (TJs) and adherens junctions (AJs), which play a key role in maintaining the integrity of the airway barrier. Emerging evidence has shown that different microorganisms cause airway barrier dysfunction by targeting TJ and AJ proteins. This review discusses the pathophysiologic mechanisms by which several microorganisms (bacteria and viruses) lead to the disruption of AJCs in airway epithelial cells. We present recent progress in understanding signaling pathways involved in the formation and regulation of cell junctions. We also summarize the potential chemical inhibitors and pharmacological approaches to restore the integrity of the airway epithelial barrier. Understanding the AJCs-pathogen interactions and mechanisms by which microorganisms target the AJC and impair barrier function may further help design therapeutic innovations to treat these infections.

RSV-induced changes in a 3-dimensional organoid model of human fetal lungs.

We have shown that respiratory syncytial virus (RSV) can spread hematogenously from infected airways of a pregnant woman to the developing fetal lungs in utero. This study sought to measure RSV replication, cytopathic effects, and protein expression in human lung organoids (HLOs) reproducing architecture and transcriptional profiles of human fetal lungs during the 1st trimester of gestation. HLOs derived from human pluripotent stem cells were microinjected after 50 or 100 days in culture with medium or recombinant RSV-A2 expressing the red fluorescent protein gene (rrRSV). Infection was monitored by fluorescent microscopy and PCR. Immunohistochemistry and proteomic analysis were performed. RSV infected HLOs in a dose- and time-dependent manner. RSV-infected HLOs increased expression of CC10 (Club cells), but had sparse FOXJ1 (ciliated cells). Disruption of F-actin cytoskeleton was consistent with proteomic data showing a significant increase in Rho GTPases proteins. RSV upregulated the transient receptor potential vanilloid 1 (TRPV1) channel and, while ß2 adrenergic receptor (ß2AR) expression was decreased overall, its phosphorylated form increased. Our data suggest that prenatal RSV infection produces profound changes in fetal lungs' architecture and expression profiles and maybe an essential precursor of chronic airway dysfunction. expression profiles, and possibly be an important precursor of chronic airway dysfunction.