Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

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.

Venous networks in the upper airways of bats: A histological and diceCT study.

Our knowledge of nasal cavity anatomy has grown considerably with the advent of micro-computed tomography (CT). More recently, a technique called diffusible iodine-based contrast-enhanced CT (diceCT) has rendered it possible to study nasal soft tissues. Using diceCT and histology, we aim to (a) explore the utility of these techniques for inferring the presence of venous sinuses that typify respiratory mucosa and (b) inquire whether distribution of vascular mucosa may relate to specialization for derived functions of the nasal cavity (i.e., nasal-emission of echolocation sounds) in bats. Matching histology and diceCT data indicate that diceCT can detect venous sinuses as either darkened, "empty" spaces, or radio-opaque islands when blood cells are present. Thus, we show that diceCT provides reliable information on vascular distribution in the mucosa of the nasal airways. Among the bats studied, a nonecholocating pteropodid (Cynopterus sphinx) and an oral-emitter of echolocation sounds (Eptesicus fuscus) possess venous sinus networks that drain into the sphenopalatine vein rostral to the nasopharynx. In contrast, nasopharyngeal passageways of nasal-emitting hipposiderids are notably packed with venous sinuses. The mucosae of the nasopharyngeal passageways are far less vascular in nasal-emitting phyllostomids, in which vascular mucosae are more widely distributed in the nasal cavity, and in some nectar-feeding species, a particularly large venous sinus is adjacent to the vomeronasal organ. Therefore, we do not find a common pattern of venous sinus distribution associated with nasal emission of sounds in phyllostomids and hipposiderids. Instead, vascular mucosa is more likely critical for air-conditioning and sometimes vomeronasal function in all bats.

Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNß or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

LBO-EMSC Hydrogel Serves a Dual Function in Spinal Cord Injury Restoration via the PI3K-Akt-mTOR Pathway.

It is critical to obtain an anti-inflammatory microenvironment when curing spinal cord injury (SCI). On the basis of this, we prepared Lycium barbarum oligosaccharide (LBO)-nasal mucosa-derived mesenchymal stem cells (EMSCs) fibronectin hydrogel for SCI restoration via inflammatory license effect and M2 polarization of microglias. LBO exhibited remarkable M2 polarization potential for microglia. However, EMSCs primed by LBO generated enhanced paracrine effects through the inflammatory license-like process. The observed dual function is likely based on the TNFR2 pathway. In addition, LBO-EMSC hydrogel possesses a synergistic effect on M2 polarization of microglia through the PI3K-Akt-mTOR signaling pathway. The obtained findings provide a simple approach for MSC-based therapies for SCI and shed more light on the role of TNFR2 on bidirectional regulation in tissue regeneration.

Exercise duration modulates upper and lower respiratory fluid cellularity, antiviral activity, and lung gene expression.

Exercise has substantial health benefits, but the effects of exercise on immune status and susceptibility to respiratory infections are less clear. Furthermore, there is limited research examining the effects of prolonged exercise on local respiratory immunity and antiviral activity. To assess the upper respiratory tract in response to exercise, we collected nasal lavage fluid (NALF) from human subjects (1) at rest, (2) after 45 min of moderate-intensity exercise, and (3) after 180 min of moderate-intensity exercise. To assess immune responses of the lower respiratory tract, we utilized a murine model to examine the effect of exercise duration on bronchoalveolar lavage (BAL) fluid immune cell content and lung gene expression. NALF cell counts did not change after 45 min of exercise, whereas 180 min significantly increased total cells and leukocytes in NALF. Importantly, fold change in NALF leukocytes correlated with the post-exercise fatigue rating in the 180-min exercise condition. The acellular portion of NALF contained strong antiviral activity against Influenza A in both resting and exercise paradigms. In mice undergoing moderate-intensity exercise, BAL total cells and neutrophils decreased in response to 45 or 90 min of exercise. In lung lobes, increased expression of heat shock proteins suggested that cellular stress occurred in response to exercise. However, a broad upregulation of inflammatory genes was not observed, even at 180 min of exercise. This work demonstrates that exercise duration differentially alters the cellularity of respiratory tract fluids, antiviral activity, and gene expression. These changes in local mucosal immunity may influence resistance to respiratory viruses, including influenza or possibly other pathogens in which nasal mucosa plays a protective role, such as rhinovirus or SARS-CoV-2.

Apremilast mitigates interleukin (IL)-13-induced inflammatory response and mucin production in human nasal epithelial cells (hNECs).

Interleukin (IL)-13-associated inflammatory response is important for the pathogenesis of allergic rhinitis (AR). Apremilast is a phosphodiesterase-4 (PDE4) inhibitor approved for psoriasis treatment. Here, we investigated the potential effects of Apremilast against IL-13-induced injury in human nasal epithelial cells (hNECs). Firstly, Apremilast ameliorated oxidative stress in IL-13-challenged cells by decreasing the levels of reactive oxygen species (ROS) and the production of malondialdehyde (MDA). Secondly, Apremilast inhibited the expressions of IL-6 and IL-8. Moreover, Apremilast inhibited the expressions of the chemokines colony-stimulating factor 2 (CSF2) and chemokine ligand 11 (CCL11). Interestingly, exposure to IL-13 increased the expressions of mucin 4 and mucin 5AC (MUC5AC), which was ameliorated by treatment with Apremilast. Interestingly, we found that Apremilast inhibited the phosphorylation of c-Jun-N-terminal kinase (JNK). Importantly, Apremilast reduced the levels of c-fos and c-Jun, the two AP-1 subfamilies. The luciferase reporter assay demonstrates that Apremilast reduced the transcriptional activity of activator protein 1 (AP-1). Lastly, we found that Apremilast prevented the activation of nuclear factor kappa-B (NF-κB) by decreasing the levels of nuclear NF-κB p65 and the luciferase activity of the NF-κB reporter. In summary, we conclude that Apremilast possesses a protective effect against IL-13-induced inflammatory response and mucin production in hNECs by inhibiting the activity of AP-1 and NF-κB.

Green synthesized colloidal silver is devoid of toxic effects on primary human nasal epithelial cells in vitro.

Evaluating the safety of previously fabricated and effective green synthetized colloidal silver (GSCS) on the mucosal barrier structure and function is essential prior to conduct human trials. The GSCS was applied to primary human nasal epithelial cells (HNECs) grown in an air-liquid interface (ALI) culture. Epithelial barrier integrity was evaluated by measuring the transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran paracellular permeability. Ciliary beat frequency (CBF) was quantified. Effects of the GSCS on cell viability and inflammation were examined through lactate dehydrogenase, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide viability assay and interleukin 6 (IL-6) enzyme linked immunosorbent assay. The localization and transportation of GSCS within HNECs and their HNEC-ALI cultures was assessed by transmission electron microscopy and inductively coupled plasma-mass-spectrometry, respectively. Application of GSCS to HNECs-ALI cultures for up to 2 h caused a significant reduction in the TEER values, however, it did not drop within the first 10 and 20 min for CRS and non-CRS control HNECs. The paracellular permeability, cell viability, IL-6 secretion and CBF remained unchanged. No GSCS was observed within or transported across HNECs. In conclusion, application of GSCS to HNECs is devoid of toxic effects.

Astragaloside IV suppresses histamine-induced inflammatory factors and mucin 5 subtype AC overproduction in nasal epithelial cells via regulation of inflammation-related genes.

Allergic rhinitis (AR) is a symptomatic allergic disease that leads to severe inflammation. Astragaloside IV (AS-IV) is a primary active component of Astragalus membranaceus and exerts immune-regulation and anti-inflammatory effects. However, the pharmacological effect of AS-IV in the nasal epithelial cells (NECs) has not been reported. The present study aimed to assess the effect of AS-IV on inflammatory cytokines and mucin 5 subtype AC (MUC5AC) overproduction in histamine (His)-stimulated NECs and its underlying mechanism. NECs were stimulated with or without His for 24 h in the absence or presence of AS-IV. The levels of inflammatory cytokines including IL-6, IL-8, MCP-1, IL-1ß, granulocyte-macrophage colony-stimulating factor (GM-CSF), eotaxin, and MUC5AC were assayed. Our findings indicated that AS-IV inhibited His-evoked release and expression of inflammatory cytokines and MUC5AC in NECs. RNA-seq analyses indicated the significant changes in expression levels involved in inflammation genes upon treatment of His-induced NECs with AS-IV. Our findings indicated that AS-IV inhibited His-evoked inflammatory cytokines secretion and MUC5AC overproduction in NECs, which were partly mediated by regulation of inflammation-related genes. Therefore, our findings provided a scientific basis for the development of AS-IV as an effective agent for clinical therapeutic strategy in the treatment of AR.

Isolation, expansion, differentiation, and histological processing of human nasal epithelial cells.

This protocol is intended as a guide for implementing or refining the usage of the air-liquid interface (ALI) model system to generate airway mucociliary tissue in vitro. We present a streamlined protocol for isolating the stem cells from inferior nasal turbinates of donors, allowing for a simple and low-cost supply of primary cells for research. We also provide our detailed protocols for ALI tissue processing and immunofluorescence to aid in the standardization of these techniques between research groups. For complete details on the use and execution of this protocol, please refer to Hussain et al., (2014)Yang et al., (2016)Im et al., (2019).

Effects of HMGB1 on Tricellular Tight Junctions via TGF-ß Signaling in Human Nasal Epithelial Cells.

The airway epithelium of the human nasal mucosa acts as a physical barrier that protects against inhaled substances and pathogens via bicellular and tricellular tight junctions (bTJs and tTJs) including claudins, angulin-1/LSR and tricellulin. High mobility group box-1 (HMGB1) increased by TGF-ß1 is involved in the induction of nasal inflammation and injury in patients with allergic rhinitis, chronic rhinosinusitis, and eosinophilic chronic rhinosinusitis. However, the detailed mechanisms by which this occurs remain unknown. In the present study, to investigate how HMGB1 affects the barrier of normal human nasal epithelial cells, 2D and 2.5D Matrigel culture of primary cultured human nasal epithelial cells were pretreated with TGF-ß type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. Knockdown of angulin-1/LSR downregulated the epithelial barrier. Treatment with EW-7197 decreased angulin-1/LSR and concentrated the expression at tTJs from bTJs and increased the epithelial barrier. Treatment with a binder to angulin-1/LSR angubindin-1 decreased angulin-1/LSR and the epithelial barrier. Treatment with HMGB1 decreased angulin-1/LSR and the epithelial barrier. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen. Pretreatment with EW-7197 prevented the effects of HMGB1. HMGB1 disrupted the angulin-1/LSR-dependent epithelial permeability barriers of HNECs via TGF-ß signaling in HNECs.

Immune cell residency in the nasal mucosa may partially explain respiratory disease severity across the age range.

Previous studies focusing on the age disparity in COVID-19 severity have suggested that younger individuals mount a more robust innate immune response in the nasal mucosa after infection with SARS-CoV-2. However, it is unclear if this reflects increased immune activation or increased immune residence in the nasal mucosa. We hypothesized that immune residency in the nasal mucosa of healthy individuals may differ across the age range. We applied single-cell RNA-sequencing and measured the cellular composition and transcriptional profile of the nasal mucosa in 35 SARS-CoV-2 negative children and adults, ranging in age from 4 months to 65 years. We analyzed in total of ~ 30,000 immune and epithelial cells and found that age and immune cell proportion in the nasal mucosa are inversely correlated, with little evidence for structural changes in the transcriptional state of a given cell type across the age range. Orthogonal validation by epigenome sequencing indicate that it is especially cells of the innate immune system that underlie the age-association. Additionally, we characterize the predominate immune cell type in the nasal mucosa: a resident T cell like population with potent antiviral properties. These results demonstrate fundamental changes in the immune cell makeup of the uninfected nasal mucosa over the lifespan. The resource we generate here is an asset for future studies focusing on respiratory infection and immunization strategies.

Th17/1-Biased Inflammatory Environment Involved in the Response of Epithelial Cells to Antigen Stimuli in Nasal Polyps.

Several studies showed that IL-17A was significantly increased in nasal polyps (NPs). However, the source and characteristics of IL-17A-producing cells in NPs were not fully understood. We isolated mononuclear cells from NPs and uncinate tissues and analyzed them using flow cytometry. The results indicated that IL-17A was increased in NP tissues compared to uncinate tissues. The main IL-17A-expressing cells were CD3+ T cells in NP tissues, including Th17 cells, Tc17 cells, and γδT17 cells. Not similar to those in uncinate tissues, the majority of Th17 cells highly coexpressed IFN-γ in NP tissues, such as Th17/1 cells, which highly expressed CXCR3, CCR6, RORγt, and T-bet. Furthermore, Th17/1-biased environment increased the response of nasal epithelial cells to bacterial and viral stimuli, implying that Th17/1 cells play a greater role in the pathological development of NPs than Th17 or Th1 cells.

The diverse roles of YAP in the regulation of human nasal epithelial remodeling.

Yes-associated protein (YAP) is essential in maintaining tissue size. Aberrant epithelial remodeling is a key pathological alteration in both inflammation and benign tumors in nasal mucosa. We sought to investigate the expression and localization patterns of YAP in remodeled nasal epithelium of basal cell hyperplasia, goblet cell metaplasia and squamous metaplasia. YAP expression patterns were evaluated in tissues obtained from patients with NP (n = 45) and IP (n = 27), and control subjects with septal deviation (n = 17) and tissue-derived primary cell cultures. Compared to the normal epithelium, expressions of YAP were significantly higher in basal cell hyperplasia (NP, 11.4-fold; IP, 19.6-fold), followed by squamous metaplasia (8.2-fold) and mild to moderate goblet cell metaplasia (2.9-fold); while their expression was lower in severe goblet cell metaplasia (3.3-fold). Our resultsshowed that: 1) ectopic nuclear YAP expression associated with p63+ basal cell hyperplasia and the high proliferative potential epithelial cells; 2) increase of cytoplasmic YAP correlated with mild to moderate goblet cell metaplasia; 3) increase of cytoplasmic YAP correlated with squamous cell metaplasia. The in vitro cell model also demonstrated almost concordant changes of YAP with the mucosa findings. Different YAP expression and localization patterns should play critical but differential roles in the nasal epithelial remodeling processes under mucosal inflammation and benign tumor formation.

A comparison of three mucus-secreting airway cell lines (Calu-3, SPOC1 and UNCN3T) for use as biopharmaceutical models of the nose and lung.

The aim of this work was to compare three existing mucus-secreting airway cell lines for use as models of the airways to study drug transport in the presence of mucus. Each cell line secreted mature, glycosylated mucins, evidenced by the enzyme-linked lectin assay. The secretagogue, adenylyl-imidodiphosphate, increased mucin secretion in SPOC1 (3.5-fold) and UNCN3T (1.5-fold) cells but not in Calu-3 cells. In a novel mucus-depleted (MD) model the amount of mucus in the non-depleted wells was 3-, 8- and 4-fold higher than in the mucus-depleted wells of the Calu-3, SPOC1 and UNCN3T cells respectively. The permeability of 'high mucus' cells to testosterone was significantly less in SPOC1 and UNCN3T cells (P < 0.05) but not Calu-3 cells. Mucin secretion and cytokine release were investigated as indicators of drug irritancy in the SPOC1 and UNCN3T cell lines. A number of inhaled drugs significantly increased mucin secretion at high concentrations and the release of IL-6 and IL-8 from SPOC1 or UNCN3T cells (P < 0.05). SPOC1 and UNCN3T cell lines are better able to model the effect of mucus on drug absorption than the Calu-3 cell line and are proposed for use in assessing drug-mucus interactions in inhaled drug and formulation development.

Evaluating the distribution of T-lymphocytes and S-phase proliferating cells across the nasal mucosa of dromedary camel (Camelus dromedarius).

The lining mucosa of the nasal cavity performs important roles for the host adaptation to the external environment. Camels are unique in their adaptation to the lifestyle of nomadic deserts. The present study aimed to evaluate the distribution pattern of T lymphocytes and S-phase proliferating cells within the nasal mucosa of camel using antibodies against CD3 and PCNA, respectively. The mucosa of the rostral, middle, and caudal parts of the nasal cavity was collected and processed for immunohistochemical staining. CD3-immunoreactive (-IR) cells were observed within the epithelium and lamina propria of all examined parts. However, the numbers of these cells were significantly higher in the rostral part of the nasal mucosa compared to its middle and caudal parts (P < 0.05). Such expression of CD3-IR cells within the rostral nasal mucosa was most pronounced within its lamina propria which also revealed aggregations of lymphoid cells. The increased frequency of CD3 expressing cells at the rostral part of the nasal mucosa suggests a potential role of the nasal vestibule in limiting the infection via constant clearance of encountered pathogens. PCNA-IR cells were mainly found within the basal layers of the nasal epithelium at the rostral part of the nasal cavity, though they showed a significant decrease in their frequencies on moving caudad. The results of the present work will form a basis for assessment of various nasal pathologies affecting camels particularly those associated with increased rates of T lymphocytes infiltration and/or cell proliferation.

1α,25-Dihydroxyvitamin D3 Supplementation during Pregnancy Is Associated with Allergic Rhinitis in the Offspring by Modulating Immunity.

BACKGROUND: Maternal supplementation with 1α,25-dihydroxyvitamin D3 (VD3) has immunologic effects on the developing fetus through multiple pathways. This study was aimed at investigating the effects of VD3 supplementation on immune dysregulation in the offspring during allergic rhinitis. METHODS: Different doses of VD3 as well as control were given to pregnant female mice. Ovalbumin (OVA) challenge and aluminum hydroxide gel in sterile saline were used to induce allergic rhinitis in offspring mice. Nasal lavage fluids (NLF) were collected, and eosinophils were counted in NLF 24 hours after the OVA challenge. Th1, Th2, Th17, and Treg subtype-relevant cytokines, including IFN-γ, IL-4, IL-10, IL-17, TGF-ß, and OVA-IgE levels from the blood and NLF of offspring mice, were detected by the enzyme-linked immunosorbent assay (ELISA) method. The Treg subtype was analyzed by flow cytometry. Treg cells were purified from offspring and were adoptively transferred to OVA-sensitized allogenic offspring mice. The outcomes were assessed in allogenic offspring. RESULTS: Our data showed that VD3 supplementation significantly decreased the number of eosinophils, basophils, and lymphocytes in the peripheral blood and NLF. The proportion of CD4+CD25+FoxP3+Tregs had a positive correlation with VD3 in a dose-dependent manner. The levels of serum IgE, IL-4, and IL-17 were decreased while the expressions of IFN-γ, IL-10, and TGF-ß were significantly enhanced in VD3 supplementation groups. Adoptive transfer CD4+CD25+FoxP3+Tregs of VD3 supplementation groups promoted Th1 and suppressed Th2 responses in the offspring during allergic rhinitis. CONCLUSION: Our findings indicated that low dose VD3 supply in pregnant mice's diet suppressed Th2 and Th17 responses in allergic rhinitis by elevating the Th1 subtype and the proportion of CD4+CD25+FoxP3+Tregs in offspring. It suggested that low dose VD3 supply may have the potential to act as a new therapeutic strategy for allergic rhinitis.

Circ_0067835 regulates allergic inflammatory response in type-2 innate lymphoid cells in allergic rhinitis (AR) via miR-155/GATA3.

Allergic rhinitis (AR) is a familiar respiratory allergic inflammatory disease with higher incidence. The pathogenesis of AR is particularly complex. Therefore, a lot of work is acquired to excavate deep mechanisms, thereby providing effective strategies for AR diagnose and treatment. AR mice model was induced by recombinant murine IL-33 (0.05 µg/µl) on days 1, 3, and 5. The lentiviral vectors carrying si-circ_0067835, miR-155 mimic, si-NC or miR-NC were injected into AR mice. Thus, mice were divided into control, AR, AR + si-NC, AR + si-circ_0067835, AR + si-circ_0067835 + miR-NC, and AR + si-circ_0067835 + miR-155 mimic groups. qRT-PCR experiment was used to measure the expression of circ_0067835 and miR-155. Behavioral test result was quantified to assess AR mice model. Hematoxylin and eosin (HE) staining was performed to analyze histopathological changes. Helper T cell 2 (Th2) cytokines (IL-4, IL-5, IL-9 and IL-13) and percentage of type-2 innate lymphoid cells (ILC2s) in nasal mucosa tissues in AR mice model were evaluated needing western blot, ELISA, and flow cytometry. Besides, the targeting relationship between circ_0067835 and miR-155, or between miR-155 and GATA3, was investigated via luciferase report assay. Circ_0067835 expression levels were raised in the nasal mucosa tissues of AR mice. Inhibiting circ_0067835 could reduce Type2 cytokines and ILC2s levels in AR mice model. Furthermore, circ_0067835 targeted and positively regulated miR-155 expression, and GATA3 was a downstream target of miR-155 and adjusted by circ_0067835/miR-155 axis. In addition, silencing circ_0067835 inhibited cytokines and ILC2s levels by down-regulating miR-155. Circ_0067835 effectively inhibited AR response in ILC2s through participation of miR-155/GATA3 axis.

Calcitriol Attenuates HRV-Induced Respiratory Injury through the AMPK-mTOR-ER Stress Signaling Pathway.

Human rhinovirus (HRV) infection is one of the main causes of respiratory injury. Recently, calcitriol has been reported to have protective effect against respiratory infections. In this paper, we aimed to explore the effects and mechanisms of calcitriol on HRV-induced respiratory infection. Participants including pediatric patients diagnosed with HRV-induced respiratory infection (n = 50) and paired healthy controls (n = 40) were recruited at the Weifang People's Hospital between May 2019 and May 2020. The serum 25(OH)D3 level was measured in participants using ELISA kit. The HRV-induced respiratory infection model in human nasal mucosal epithelial cells (hNECs) was adapted, in vitro. HRV infection was measured by real-time PCR analysis of HRV expression. After HRV infection and treatment with calcitriol, the changes of cell viability were detected by MTT assay, the expression of ER stress-induced apoptosis and AMPK-mTOR related proteins by western blot, and the cell apoptosis by flow cytometry assay. In order to confirm whether AMPK-mTOR signal pathway was involved in the ER stress-induced apoptosis of hNECs, cells were pretreated with compound C which was a AMPK inhibitor. The 25-(OH)D3 concentration in serum collected in HRV-infected children was lower than that in controls. In vitro experiments showed that HRV infection decreased cell viability, and this effect was reversed when treated with calcitriol. Additionally, HRV increased levels of apoptosis and ER stress markers (including cleaved-caspase3, Bax, CHOP, nATF6, and BiP), while calcitriol significantly reversed these effects. Furthermore, calcitriol played a protective role by increasing p-AMPK and decreasing p-mTOR level. However, the protective effects of calcitriol could be abolished by compound C. Calcitriol protected HRV-infected hNECs by inhibiting the ER stress-induced apoptosis through the AMPK-mTOR signaling pathway. These protective effects of calcitriol against HRV-induced respiratory infection may provide an experimental basis for the clinical application.

Elexacaftor co-potentiates the activity of F508del and gating mutants of CFTR.

Trikafta, the combination of elexacaftor (VX-445), tezacaftor (VX-661) and ivacaftor (VX-770), was approved for therapy of cystic fibrosis (CF) patients with at least one allele of the CFTR mutation F508del. While the corrector function of VX-445 is well established, here we investigated the putative potentiator activity of VX-445 alone and in combination with VX-770. Acute addition of VX-445 increased the VX-770-potentiated F508del- and G551D-CFTR current by ~24% and >70%, respectively, in human bronchial and nasal epithelia. Combinatorial profiling and cluster analysis of G551D- and G1244E-CFTR channel activation with potentiator pairs indicated a distinct VX-445 mechanism of action that is, at least, additive to previously identified potentiator classes, including the VX-770. Since VX-770 only partially normalizes the G551D-CFTR channel function and adult G551D patients still experience progressive loss of lung function, VX-445+VX-770 combination therapy could provide clinical benefit to CF patients with the G551D and other dual potentiator responsive mutants.