Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance.

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Infection of primary nasal epithelial cells differentiates among lethal and seasonal human coronaviruses.

The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary human nasal epithelial cells grown at air-liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal [Severe acute respiratory syndrome (SARS)-CoV-2 and Middle East respiratory syndrome-CoV (MERS-CoV)] and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures, though replication is differentially modulated by temperature. Infections conducted at 33 °C vs. 37 °C (reflective of temperatures in the upper and lower airway, respectively) revealed that replication of both seasonal HCoVs (HCoV-NL63 and -229E) is significantly attenuated at 37 °C. In contrast, SARS-CoV-2 and MERS-CoV replicate at both temperatures, though SARS-CoV-2 replication is enhanced at 33 °C late in infection. These HCoVs also diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV receptor availability as well as replication. MERS-CoV receptor DPP4 expression increases with IL-13 treatment, whereas ACE2, the receptor used by SARS-CoV-2 and HCoV-NL63, is down-regulated. IL-13 treatment enhances MERS-CoV and HCoV-229E replication but reduces that of SARS-CoV-2 and HCoV-NL63, reflecting the impact of IL-13 on HCoV receptor availability. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.

Differential regulation of viral entry-associated genes modulated by inflammatory cytokines in the nasal epithelium.

This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry-associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), and beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) were selected as key entry-associated genes for SARS-CoV-2, HCoV-229E, MERS-CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air-liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real-time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry-associated genes showed distinct regulation patterns in response to each interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL-4 and IL-13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF-α and IFN-γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN-γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS-CoV-2 and HCoV-229E infections but higher susceptibility to MERS-CoV and influenza infections.

Interleukin-19 enhances eosinophil infiltration through upregulation of epithelium-derived RANTES expression via the ERK/NF-κB signalling pathway in patients with eosinophilic CRSwNP.

BACKGROUD: The recurrence rate of chronic rhinosinusitis with nasal polyps (CRSwNP) is positively correlated with eosinophil infiltration. Increased interleukin (IL)-19 and eosinophil chemokine RANTES levels have been reported in patients with CRSwNP. This study aimed to clarify the role of IL-19 in mediating RANTES expression and eosinophilic infiltration in eosinophilic CRSwNP (Eos CRSwNP). METHODS: Nasal tissue samples were obtained from patients with CRSwNP and controls. The expression of IL-19, its receptors, ECP, and RANTES in tissues was investigated. Primary human nasal epithelial cells (HNECs) and nasal polyp tissue blocks were cultured, then stimulated by IL-19; ERK phosphorylation, NF-κB pathway activation, RANTES level, eosinophils migration and infiltration were detected using RT-qPCR, ELISA, western blotting, HE, immunohistochemistry, immunofluorescence staining, confocal microscopy, and transwell migration assay. RESULTS: The expression of IL-19 and its receptors (IL-20R1/IL-20R2), eosinophil cationic protein, and RANTES in nasal tissues from patients with Eos CRSwNP was significantly increased compared to that in non-Eos CRSwNP and control subjects. IL-19 co-localized with RANTES in nasal tissues and significantly elevated RANTES expression in HNECs. IL-19-blocking antibody and siRNA knockdown of IL-20R1 ameliorated the effect of IL-19 on RANTES secretion in HNECs. Moreover, IL-19-induced RANTES upregulation was associated with the activation of the ERK and NF-κB pathways. NF-κB activation was mediated by the ERK pathway in IL-19-treated HNECs, and IL-19 enhanced eosinophil infiltration in nasal polyp tissue blocks. CONCLUSIONS: Our findings indicate that IL-19 promotes RANTES expression via the ERK/NF-κB pathway in HNECs and is implicated in eosinophil infiltration in patients with Eos CRSwNP.

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Cis- and trans-eQTM analysis reveals novel epigenetic and transcriptomic immune markers of atopic asthma in airway epithelium.

BACKGROUND: Expression quantitative trait methylation (eQTM) analyses uncover associations between DNA methylation markers and gene expression. Most eQTM analyses of complex diseases have focused on cis-eQTM pairs (within 1 megabase). OBJECTIVES: This study sought to identify cis- and trans-methylation markers associated with gene expression in airway epithelium from youth with and without atopic asthma. METHODS: In this study, the investigators conducted both cis- and trans-eQTM analyses in nasal (airway) epithelial samples from 158 Puerto Rican youth with atopic asthma and 100 control subjects without atopy or asthma. The investigators then attempted to replicate their findings in nasal epithelial samples from 2 studies of children, while also examining whether their results in nasal epithelium overlap with those from an eQTM analysis in white blood cells from the Puerto Rican subjects. RESULTS: This study identified 9,108 cis-eQTM pairs and 2,131,500 trans-eQTM pairs. Trans-associations were significantly enriched for transcription factor and microRNA target genes. Furthermore, significant cytosine-phosphate-guanine sites (CpGs) were differentially methylated in atopic asthma and significant genes were enriched for genes differentially expressed in atopic asthma. In this study, 50.7% to 62.6% of cis- and trans-eQTM pairs identified in Puerto Rican youth were replicated in 2 smaller cohorts at false discovery rate-adjusted P < .1. Replicated genes in the trans-eQTM analysis included biologically plausible asthma-susceptibility genes (eg, HDC, NLRP3, ITGAE, CDH26, and CST1) and are enriched in immune pathways. CONCLUSIONS: Studying both cis- and trans-epigenetic regulation of airway epithelial gene expression can identify potential causal and regulatory pathways or networks for childhood asthma. Trans-eQTM CpGs may regulate gene expression in airway epithelium through effects on transcription factor and microRNA target genes.

Integrating 16S rRNA Sequencing, Microflora Metabolism, and Network Pharmacology to Investigate the Mechanism of SBL in Alleviating HDM-Induced Allergic Rhinitis.

Allergic rhinitis (AR) is a series of allergic reactions to allergens in the nasal mucosa and is one of the most common allergic diseases that affect both children and adults. Shi-Bi-Lin (SBL) is the modified formula of Cang Er Zi San (CEZS), a traditional Chinese herbal formula used for treating AR. Our study aims to elucidate the anti-inflammatory effects and mechanisms of SBL in house dust mite-induced AR by regulating gut microflora metabolism. In vivo studies showed that nasal allergies and the infiltration of inflammatory cells in the nasal epithelium were significantly suppressed by SBL. Moreover, SBL restored the impaired nasal epithelial barrier function with an increased tight junction protein expression and reduced the endothelial nitric oxide synthase (eNOS). Interestingly, SBL significantly reconstituted the abundance and composition of gut microbiota in AR mice; it increased the relative abundance of potentially beneficial genera and decreased the relative abundance of harmful genera. SBL also restored immune-related metabolisms, which were significantly increased and correlated with suppressing inflammatory cytokines. Furthermore, a network analysis and molecular docking indicated IL-6 was a possible target drug candidate for the SBL treatment. SBL dramatically reduced the IL-6 level in the nasal lavage fluid (NALF), suppressing the IL-6 downstream Erk1/2 and AKT/PI3K signaling pathways. In conclusion, our study integrates 16S rRNA sequencing, microflora metabolism, and network pharmacology to explain the immune mechanism of SBL in alleviating HDM-induced allergic rhinitis.

Symbiotic microbiome Staphylococcus epidermidis restricts IL-33 production in allergic nasal epithelium via limiting the cellular necroptosis.

BACKGROUND: Allergic rhinitis (AR) is characterized by airway inflammation in nasal mucosa from inhaled allergens and interleukin (IL)-33 is the potent inducer of Th2 inflammation in allergic nasal epithelium. Staphylococcus epidermidis is one of the most abundant colonizers of the healthy human nasal mucosa and might impact the allergen-induced inflammatory responses in the nasal epithelium. Thus, we sought to characterize the mechanism of S. epidermidis regulating Th2 inflammation and IL-33 production in AR nasal mucosa. RESULTS: The AR symptoms were alleviated and eosinophilic infiltration, serum IgE levels, and Th2 cytokines were significantly decreased in OVA-sensitized AR mice in response to human nasal commensal S. epidermidis. The inoculation of S. epidermidis to normal human nasal epithelial cells reduced IL-33 and GATA3 transcriptions and also reduced IL-33 and GATA3 expression in AR nasal epithelial (ARNE) cells and the nasal mucosa of AR mice. Our data exhibited that the cellular necroptosis of ARNE cells might be involved in IL-33 production and inoculation of S. epidermidis decreased the phosphorylation of necroptosis enzymes in ARNE cells, which was related to the reduction of IL-33 production. CONCLUSIONS: We present that human nasal commensal S. epidermidis reduces allergic inflammation by suppressing IL-33 production in nasal epithelium. Our findings indicate that S. epidermidis serves a role in blocking allergen-induced cellular necroptosis in allergic nasal epithelium which might be a key mechanism of reduction of IL-33 and Th2 inflammation.

Pathophysiology of SARS-CoV-2 Infection of Nasal Respiratory and Olfactory Epithelia and Its Clinical Impact.

PURPOSE OF REVIEW: While the predominant cause for morbidity and mortality with SARS-CoV-2 infection is the lower respiratory tract manifestations of the disease, the effects of SARS-CoV-2 infection on the sinonasal tract have also come to the forefront especially with the increased recognition of olfactory symptom. This review presents a comprehensive summary of the mechanisms of action of the SARS-CoV-2 virus, sinonasal pathophysiology of COVID-19, and the correlation with the clinical and epidemiological impact on olfactory dysfunction. RECENT FINDINGS: ACE2 and TMPRSS2 receptors are key players in the mechanism of infection of SARS-CoV-2. They are present within both the nasal respiratory as well as olfactory epithelia. There are however differences in susceptibility between different groups of individuals, as well as between the different SARS-CoV-2 variants. The sinonasal cavity is an important route for SARS-CoV-2 infection. While the mechanism of infection of SARS-CoV-2 in nasal respiratory and olfactory epithelia is similar, there exist small but significant differences in the susceptibility of these epithelia and consequently clinical manifestations of the disease. Understanding the differences and nuances in sinonasal pathophysiology in COVID-19 would allow the clinician to predict and counsel patients suffering from COVID-19. Future research into molecular pathways and cytokine responses at different stages of infection and different variants of SARS-CoV-2 would evaluate the individual clinical phenotype, prognosis, and possibly response to vaccines and therapeutics.

Oncostatin M Contributes to Airway Epithelial Cell Dysfunction in Chronic Rhinosinusitis with Nasal Polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a typical type-2 inflammation involving several cytokines and is associated with epithelial cell dysfunction. Oncostatin M (OSM) (belonging to the interleukin(IL)-6 family) could be a key driver of epithelial barrier dysfunction. Therefore, we investigated the presence of OSM and IL-6 and the expression pattern of tight junctions (TJs) in the nasal tissue of CRSwNP patients and controls using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Then, their potential role in the epithelial barrier was evaluated in vitro in 27 different primary cultures of human nasal epithelial cells (HNECs) by measuring TJ expression and transepithelial electric resistance (TEER) with or without OSM or IL-6 (1, 10, and 100 ng/mL). The effect on ciliary beating efficiency was evaluated by high-speed videomicroscopy and on repair mechanisms with a wound healing model with or without OSM. OSM and IL-6 were both overexpressed, and TJ (ZO-1 and occludin) expression was decreased in the nasal polyps compared to the control mucosa. OSM (100 ng/mL) but not IL-6 induced a significant decrease in TJ expression, TEER, and ciliary beating efficiency in HNECs. After 24 h, the wound repair rate was significantly higher in OSM-stimulated HNECs at 100 ng/mL. These results suggest that OSM could become a new target for monoclonal antibodies.

Woodsmoke particle exposure prior to SARS-CoV-2 infection alters antiviral response gene expression in human nasal epithelial cells in a sex-dependent manner.

Inhalational exposure to particulate matter (PM) derived from natural or anthropogenic sources alters gene expression in the airways and increases susceptibility to respiratory viral infection. Woodsmoke-derived ambient PM from wildfire events during 2020 was associated with higher COVID-19 case rates in the western United States. We hypothesized that exposure to suspensions of woodsmoke particles (WSPs) or diesel exhaust particles (DEPs) prior to SARS-CoV-2 infection would alter host immune gene expression at the transcript level. Primary human nasal epithelial cells (hNECs) from both sexes were exposed to WSPs or DEPs (22 µg/cm2) for 2 h, followed by infection with SARS-CoV-2 at a multiplicity of infection of 0.5. Forty-six genes related to SARS-CoV-2 entry and host response were assessed. Particle exposure alone minimally affected gene expression, whereas SARS-CoV-2 infection alone induced a robust transcriptional response in hNECs, upregulating type I and III interferons, interferon-stimulated genes, and chemokines by 72 h postinfection (p.i.). This upregulation was higher overall in cells from male donors. However, exposure to WSPs prior to infection dampened expression of antiviral, interferon, and chemokine mRNAs. Sex stratification of these results revealed that WSP exposure downregulated gene expression in cells from females more so than males. We next hypothesized that hNECs exposed to particles would have increased apical viral loads compared with unexposed cells. Although apical viral load was correlated to expression of host response genes, viral titer did not differ between groups. These data indicate that WSPs alter epithelial immune responses in a sex-dependent manner, potentially suppressing host defense to SARS-CoV-2 infection.

Transcriptomics of rhinovirus persistence reveals sustained expression of RIG-I and interferon-stimulated genes in nasal epithelial cells in vitro.

BACKGROUND: Human rhinoviruses (HRVs) are frequently associated with asthma exacerbations, and have been found in the airways of asthmatic patients. While HRV-induced acute infection is well-documented, it is less clear whether the nasal epithelium sustains prolonged HRV infections along with the associated activation of host immune responses. OBJECTIVE: To investigate sustainably regulated host responses of human nasal epithelial cells (hNECs) during HRV persistence. METHODS: Using a time-course study, HRV16 persistence and viral replication dynamics were established using an in vitro infection model of hNECs. RNA sequencing was performed on hNECs in the early and late stages of infection at 3 and 14 days post-infection (dpi), respectively. The functional enrichment of differentially expressed genes (DEGs) was evaluated using gene ontology (GO) and Ingenuity pathway analysis. RESULTS: HRV RNA and protein expression persisted throughout prolonged infections, even after decreased production of infectious virus progeny. GO analysis of unique DEGs indicated altered regulation of pathways related to ciliary function and airway remodeling at 3 dpi and serine-type endopeptidase activity at 14 dpi. The functional enrichment of shared DEGs between the two time-points was related to interferon (IFN) and cytoplasmic pattern recognition receptor (PRR) signaling pathways. Validation of the sustained regulation of candidate genes confirmed the persistent expression of RIG-I and revealed its close co-regulation with interferon-stimulated genes (ISGs) during HRV persistence. CONCLUSIONS: The persistence of HRV RNA does not necessarily indicate an active infection during prolonged infection. The sustained expression of RIG-I and ISGs in response to viral RNA persistence highlights the importance of assessing how immune-activating host factors can change during active HRV infection and the immune regulation that persists thereafter.

Chronic rhinosinusitis with nasal polyps is associated with impaired TMEM16A-mediated epithelial chloride secretion.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most common chronic disorders with limited therapeutic options. However, the pathogenesis of CRSwNP remains poorly understood. OBJECTIVE: We sought to determine the role of abnormalities in nasal epithelial ion transport in primary epithelial cultures and patients with CRSwNP. METHODS: We studied epithelial ion transport and transcript levels of the Cl- channels cystic fibrosis transmembrane conductance regulator and transmembrane protein 16A (TMEM16A) in human primary nasal epithelial cultures of patients with CRSwNP and healthy controls. Furthermore, we determined expression levels of proinflammatory cytokines that have been implicated in the regulation of epithelial ion channels (IL-1ß, INF-γ, TNF-α, IL-13) and studied effects of the key TH2 signaling molecule IL-13 in CRSwNP and control nasal epithelial cultures. Finally, we measured in vivo nasal potential difference to compare epithelial ion transport in patients with CRSwNP and controls. RESULTS: Bioelectric studies demonstrated that Ca2+-activated Cl- secretion was reduced in CRSwNP versus control nasal epithelial cultures. Transcript levels of IL-13 and the Ca2+-activated Cl- channel TMEM16A were increased in CRSwNP cultures. Stimulation with IL-13 increased TMEM16A expression further and restored Ca2+-activated Cl- secretion in CRSwNP cultures. Nasal potential difference measurements demonstrated reduced Ca2+-activated Cl- transport in patients with CRSwNP versus controls. CONCLUSIONS: This study demonstrates that TMEM16A-mediated Ca2+-activated Cl- secretion is reduced in primary nasal epithelial cultures and nasal epithelia of patients with CRSwNP. Our data suggest that the Ca2+-activated Cl- channel TMEM16A may be implicated in the pathogenesis and serve as a novel therapeutic target in patients with CRSwNP.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

Drug-Free Nasal Spray as a Barrier against SARS-CoV-2 and Its Delta Variant: In Vitro Study of Safety and Efficacy in Human Nasal Airway Epithelia.

The nasal epithelium is a key portal for infection by respiratory viruses such as SARS-CoV-2 and represents an important target for prophylactic and therapeutic interventions. In the present study, we test the safety and efficacy of a newly developed nasal spray (AM-301, marketed as Bentrio) against infection by SARS-CoV-2 and its Delta variant on an in vitro 3D-model of the primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in pre-viral load and post-viral load application on airway epithelium. No toxic effects of AM-301 on the nasal epithelium were found. Prophylactic treatment with AM-301 significantly reduced viral titer vs. controls over 4 days, reaching a maximum reduction of 99% in case of infection from the wild-type SARS-CoV-2 variant and more than 83% in case of the Delta variant. When AM-301 administration was started 24 h after infection, viral titer was reduced by about 12-folds and 3-folds on Day 4. The results suggest that AM-301 is safe and significantly decelerates SARS-CoV-2 replication in cell culture inhibition assays of prophylaxis (pre-viral load application) and mitigation (post-viral load application). Its physical (non-pharmaceutical) mechanism of action, safety and efficacy warrant additional investigations both in vitro and in vivo for safety and efficacy against a broad spectrum of airborne viruses and allergens.

Human Respiratory Syncytial Virus-Induced Immune Signature of Infection Revealed by Transcriptome Analysis of Clinical Pediatric Nasopharyngeal Swab Samples.

Human respiratory syncytial virus (HRSV) constitutes one the main causes of respiratory infection in neonates and infants worldwide. Transcriptome analysis of clinical samples using high-throughput technologies remains an important tool to better understand virus-host complex interactions in the real-life setting but also to identify new diagnosis/prognosis markers or therapeutics targets. A major challenge when exploiting clinical samples such as nasal swabs, washes, or bronchoalveolar lavages is the poor quantity and integrity of nucleic acids. In this study, we applied a tailored transcriptomics workflow to exploit nasal wash samples from children who tested positive for HRSV. Our analysis revealed a characteristic immune signature as a direct reflection of HRSV pathogenesis and highlighted putative biomarkers of interest such as IP-10, TMEM190, MCEMP1, and TIMM23.

Current Smoking Alters Gene Expression and DNA Methylation in the Nasal Epithelium of Patients with Asthma.

Current smoking contributes to worsened asthma prognosis and more severe symptoms and limits the beneficial effects of corticosteroids. As the nasal epithelium can reflect smoking-induced changes in the lower airways, it is a relevant source to investigate changes in gene expression and DNA methylation. This study explores gene expression and DNA methylation changes in current and ex-smokers with asthma. Matched gene expression and epigenome-wide DNA methylation samples collected from nasal brushings of 55 patients enrolled in a clinical trial investigation of current and ex-smoker patients with asthma were analyzed. Differential gene expression and DNA methylation analyses were conducted comparing current smokers with ex-smokers. Expression quantitative trait methylation (eQTM) analysis was completed to explore smoking-relevant genes by CpG sites that differ between current and ex-smokers. To investigate the relevance of the smoking-associated DNA methylation changes for the lower airways, significant CpG sites were explored in bronchial biopsies from patients who had stopped smoking. A total of 809 genes and 18,814 CpG sites were differentially associated with current smoking in the nose. The cis-eQTM analysis uncovered 171 CpG sites with a methylation status associated with smoking-related gene expression, including AHRR, ALDH3A1, CYP1A1, and CYP1B1. The methylation status of CpG sites altered by current smoking reversed with 1 year of smoking cessation. We confirm that current smoking alters epigenetic patterns and affects gene expression in the nasal epithelium of patients with asthma, which is partially reversible in bronchial biopsies after smoking cessation. We demonstrate the ability to discern molecular changes in the nasal epithelium, presenting this as a tool in future investigations into disease-relevant effects of tobacco smoke.

Th2 cytokines-DUOX2-ROS-HMGB1 translocation axis is important in the pathogenesis of allergic rhinitis.

The function of high-mobility group box 1 (HMGB1) varies according to its location. However, the translocation mechanism behind HMGB1 remains unclear. We hypothesize that type 2 helper T cell (Th2) cytokines are involved in the translocation of HMGB1 in the upper airway epithelium. We investigated the mechanism behind HMGB1 translocation using Th2 cytokine stimulation and examined the clinical significance of HMGB1 translocation in allergic rhinitis (AR). Cytoplasmic and extracellular HMGB1 were increased in AR. Inhibiting HMGB1 translocation with glycyrrhizic acid (GA) decreased the level of antigen-specific immunoglobulin E (IgE), the degree of Periodic Acid-Schiff (PAS), and Sirius Red staining in the murine model. The in vivo reactive oxygen species (ROS) level in the nasal mucosa was higher in the mice with AR than in the controls. Th2 cytokine-induced up-regulation of the ROS and translocation of HMGB1 by Th2 cytokines was dependent on the generated ROS. The ROS level also increased in the murine model. We suggest that the Th2 cytokine-dual oxidase (DUOX)2-ROS-HMGB1 translocation axis is important in AR pathogenesis.

Is a diluted seawater-based solution safe and effective on human nasal epithelium?

PURPOSE: Nasal irrigation is an effective method for alleviating several nasal symptoms and regular seawater-based nasal irrigation is useful for maintaining nasal hygiene which is essential for appropriate functioning of the nose and for preventing airborne particles including some pollutants, pathogens, and allergens from moving further in the respiratory system. However, safety studies on seawater-based nasal irrigation are scarce. In this study, the safety and efficacy of a diluted isotonic seawater solution (Stérimar Nasal Hygiene, SNH) in maintaining nasal homeostasis were evaluated in vitro. METHODS: Safety was assessed by measuring tissue integrity via transepithelial electrical resistance (TEER). Efficacy was measured by mucociliary clearance (MCC), mucin secretion, and tissue re-epithelization (wound repair) assays. All assays were performed using a 3D reconstituted human nasal epithelium model. RESULTS: In SNH-treated tissues, TEER values were statistically significantly lower than the untreated tissues; however, the values were above the tissue integrity limit. SNH treatment significantly increased MCC (88 vs. 36 µm/s, p < 0.001) and mucin secretion (1717 vs. 1280 µg/ml, p < 0.001) as compared to untreated cultures. Faster wound closure profile was noted upon pre-SNH treatment as compared to classical isotonic saline solution pre-treatment (90.5 vs. 50.7% wound closure 22 h after wound generation). CONCLUSION: SNH did not compromise the integrity of the nasal epithelium in vitro. Furthermore, SNH was effective for removal of foreign particles through MCC increase and for enhancing wound repair on nasal mucosa.

The Communication between Ocular Surface and Nasal Epithelia in 3D Cell Culture Technology for Translational Research: A Narrative Review.

There is a lack of knowledge regarding the connection between the ocular and nasal epithelia. This narrative review focuses on conjunctival, corneal, ultrastructural corneal stroma, and nasal epithelia as well as an introduction into their interconnections. We describe in detail the morphology and physiology of the ocular surface, the nasolacrimal ducts, and the nasal cavity. This knowledge provides a basis for functional studies and the development of relevant cell culture models that can be used to investigate the pathogenesis of diseases related to these complex structures. Moreover, we also provide a state-of-the-art overview regarding the development of 3D culture models, which allow for addressing research questions in models resembling the in vivo situation. In particular, we give an overview of the current developments of corneal 3D and organoid models, as well as 3D cell culture models of epithelia with goblet cells (conjunctiva and nasal cavity). The benefits and shortcomings of these cell culture models are discussed. As examples for pathogens related to ocular and nasal epithelia, we discuss infections caused by adenovirus and measles virus. In addition to pathogens, also external triggers such as allergens can cause rhinoconjunctivitis. These diseases exemplify the interconnections between the ocular surface and nasal epithelia in a molecular and clinical context. With a final translational section on optical coherence tomography (OCT), we provide an overview about the applicability of this technique in basic research and clinical ophthalmology. The techniques presented herein will be instrumental in further elucidating the functional interrelations and crosstalk between ocular and nasal epithelia.