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Pyroptosis represents a type of cell death mechanism notable for its cell membrane disruption and the subsequent release of proinflammatory cytokines. The Nod-like receptor family pyrin domain containing inflammasome 3 (NLRP3) plays a critical role in the pyroptosis mechanism associated with various diseases resulting from particulate matter (PM) exposure. Tert-butylhydroquinone (tBHQ) is a synthetic antioxidant commonly used in a variety of foods and products. The aim of this study is to examine the potential of tBHQ as a therapeutic agent for managing sinonasal diseases induced by PM exposure. The occurrence of NLRP3 inflammasome-dependent pyroptosis in RPMI 2650 cells treated with PM < 4 µm in size was confirmed using Western blot analysis and enzyme-linked immunosorbent assay results for the pyroptosis metabolites IL-1β and IL-18. In addition, the inhibitory effect of tBHQ on PM-induced pyroptosis was confirmed using Western blot and immunofluorescence techniques. The inhibition of tBHQ-mediated pyroptosis was abolished upon nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown, indicating its involvement in the antioxidant mechanism. tBHQ showed potential as a therapeutic agent for sinonasal diseases induced by PM because NLRP3 inflammasome activation was effectively suppressed via the Nrf2 pathway. [ABSTRACT FROM AUTHOR]

Maintaining tight junction integrity significantly contributes to epithelial barrier function. If the barrier function is destroyed, the permeability of the cells increases, and the movement of the pathogens is promoted, thereby further increasing the susceptibility to secondary infection. Ginsenoside components have multiple biological activities, including antiviral effects. In this study, we examined the protective effects of ginsenoside Re against rhinovirus-induced tight junction disruption in primary human nasal epithelial cells (HNE). Incubation with human rhinovirus resulted in marked disruption of tight junction proteins (ZO-1, E-cadherin, claudin-1, and occludin) in human nasal epithelial cells. Rhinovirus-induced disruption of tight junction proteins was strongly inhibited by the treatment of cells with ginsenoside Re. Indeed, significant amounts of reactive oxygen species (ROS) have been detected in human nasal epithelial cells co-incubated with rhinovirus. Moreover, rhinovirus-induced ROS generation was markedly reduced by the ginsenoside Re. However, ginsenosides Rb1 and Rc did not inhibit tight junction disruption or ROS generation in nasal epithelial cells following incubation with rhinovirus. Furthermore, incubation with rhinovirus resulted in a marked decrease in protein phosphatase activity and an increase in protein tyrosine phosphorylation levels in nasal epithelial cells. Treatment of cells with ginsenoside Re inhibited rhinovirus-induced inactivation of phosphatases and phosphorylation of tyrosine. Our results identified ginsenoside Re as an effective compound that prevented rhinovirus-induced tight junction disruption in human nasal epithelial cells.

Background: Airway remodeling is demonstrated in Asian patients with allergic rhinitis (AR). The epithelial–mesenchymal transition (EMT) is one of the key mechanisms underlying airway remodeling. Thymic stromal lymphopoietin (TSLP) is an important contributor to airway remodeling. Although increased TSLP is found in AR, little is known about whether TSLP is involved in airway remodeling through induction of the EMT. Objective: We investigated the effect of TSLP on the EMT in human nasal epithelial cells (HNECs) from AR patients. Methods: Human nasal epithelial cells from AR patients were stimulated with TSLP in the absence or presence of the preincubation with a selective inhibitor of transforming growth factor beta 1 (TGF-β1) receptor (SB431542). The expression of TGF-β1 in the cells was evaluated by using real-time polymerase chain reaction, Western blotting, and immunocytochemistry. Western blotting and immunocytochemistry were used to assay EMT markers including vimentin, fibroblast-specific protein 1 (FSP1) and E-cadherin, small mothers against decapentaplegic homolog2/3 (Smad2/3), and phosphorylated Smad2/3 in the cells. The levels of extracellular matrix components such as collagens I and III in supernatants were measured by enzyme-linked immunoassay. Morphological changes of the cells were observed under inverted phase-contrast microscope. Results: A concentration-dependent increase of TGF-β1 mRNA and protein was observed following stimulation with TSLP. Furthermore, TSLP decreased the expression of E-cadherin protein, but upregulated the production of FSP1 and vimentin proteins along with increased levels of collagens I and III, and the morphology of the cells was transformed into fibroblast-like shape. Additionally, a significant increase was found in phosphorylation of Smad2/3 protein. However, these effects were reversed by SB431542 preincubation. Conclusion: TSLP–induced HNECs to undergo the EMT process via TGF-β1-mediated Smad2/3 activation. TSLP is an activator of the EMT in HNECs and might be a potential target for inhibiting EMT and reducing airway remodeling in AR. [ABSTRACT FROM AUTHOR]

Pyroptosis represents a type of cell death mechanism notable for its cell membrane disruption and the subsequent release of proinflammatory cytokines. The Nod-like receptor family pyrin domain containing inflammasome 3 (NLRP3) plays a critical role in the pyroptosis mechanism associated with various diseases resulting from particulate matter (PM) exposure. Tert-butylhydroquinone (tBHQ) is a synthetic antioxidant commonly used in a variety of foods and products. The aim of this study is to examine the potential of tBHQ as a therapeutic agent for managing sinonasal diseases induced by PM exposure. The occurrence of NLRP3 inflammasome-dependent pyroptosis in RPMI 2650 cells treated with PM < 4 µm in size was confirmed using Western blot analysis and enzyme-linked immunosorbent assay results for the pyroptosis metabolites IL-1β and IL-18. In addition, the inhibitory effect of tBHQ on PM-induced pyroptosis was confirmed using Western blot and immunofluorescence techniques. The inhibition of tBHQ-mediated pyroptosis was abolished upon nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown, indicating its involvement in the antioxidant mechanism. tBHQ showed potential as a therapeutic agent for sinonasal diseases induced by PM because NLRP3 inflammasome activation was effectively suppressed via the Nrf2 pathway.

Understanding Influenza B virus infections is of critical importance in our efforts to control severe influenza and influenza-related diseases. Until 2020, two genetic lineages of influenza B virus—Yamagata and Victoria—circulated in the population. These lineages are antigenically distinct, but the differences in virus replication or the induction of host cell responses after infection have not been carefully studied. Recent IBV clinical isolates of both lineages were obtained from influenza surveillance efforts of the Johns Hopkins Center of Excellence in Influenza Research and Response and characterized in vitro. B/Victoria and B/Yamagata clinical isolates were recognized less efficiently by serum from influenza-vaccinated individuals in comparison to the vaccine strains. B/Victoria lineages formed smaller plaques on MDCK cells compared to B/Yamagata, but infectious virus production in primary human nasal epithelial cell (hNEC) cultures showed no differences. While ciliated epithelial cells were the dominant cell type infected by both lineages, B/Victoria lineages had a slight preference for MUC5AC-positive cells, and B/Yamagata lineages infected more basal cells. Finally, while both lineages induced a strong interferon response 48 h after infection of hNEC cultures, the B/Victoria lineages showed a much stronger induction of interferon-related signaling pathways compared to B/Yamagata. This demonstrates that the two influenza B virus lineages differ not only in their antigenic structure but also in their ability to induce host innate immune responses. [ABSTRACT FROM AUTHOR]

Introduction: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. Methods: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. Results: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. Conclusion: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression. [ABSTRACT FROM AUTHOR]

CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations. [ABSTRACT FROM AUTHOR]

Background: Patients with eosinophilic chronic rhinosinusitis (ECRS) respond poorly to many treatment modalities. Overproduction of periostin in the nasal mucosa is reported to contribute to polyp formation. This study examined periostin levels in patients with ECRS in comparison with levels in patients with non-ECRS. Methods: Fifty-nine patients with chronic rhinosinusitis were grouped into those with ECRS and those with non-ECRS. We compared the relationships between peripheral blood eosinophil level, serum periostin level, histopathological findings, clinical and laboratory findings, nose findings, diagnostic score of the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis Study, and postoperative recurrence of nasal polyps in each group. Results: In the ECRS group, a positive correlation was found between peripheral blood eosinophil level and serum periostin level (rs = 0.49, P

Background: Chronic rhinosinusitis (CRS) is an inflammatory disorder of the upper airways. MicroRNAs (miRs) are reported to regulate several diverse physiological and pathological processes. Objective: This study aimed to evaluate the impact of miR-214 on lipopolysaccharide (LPS)-mediated inflammation, and mucin 5AC (MUC5AC) expression in human nasal epithelial cells. Methods: The expression of miR-214 was detected in CRS with polyps (CRSwNP) and CRS without polyps (CRSsNP) tissues. Cells were treated with LPS and a miR-214 inhibitor. The level of miR-214 was detected by quantitative real-time reverse transcriptase-PCR (qRT-PCR). The inflammatory cytokines (IL-6, IL-8, TNF, and IL-1β) and MUC5AC production were determined by qRT-PCR and ELISA. MUC5AC protein level was detected using western blot. Similarly, we determined the relationship between miR-214 and Sirtuin 1 (SIRT1) using the Dual luciferase activity assay. Results: miR-214 was increased in CRSwNP and CRSsNP tissues. LPS triggered the expression of miR-214, while miR-214 inhibition diminished the level of miR-214. MiR-214 inhibition prevented LPS-mediated the production of inflammatory cytokines. LPS treatment augmented MUC5AC mRNA, protein levels, and secretion, whereas miR-214 loss inhibited MUC5AC production in the presence of LPS. SIRT1 is a direct target of miR-214. Impairing SIRT1 by siRNA (siSIRT1) or EX527 (a selective SIRT1 inhibitor) reversed the effects of miR-214 inhibitor on inflammation and MUC5AC expression. Furthermore, miR-214 depression inhibited the STAT3/GDF15 pathway via targeting SIRT1. Upregulation of STAT3 or GDF15 partly abolished the anti-inflammatory roles of miR-214 inhibitor. Conclusion: Taken together, miR-214 regulates LPS-mediated inflammation and MUC5AC expression via targeting SIRT1, and STAT3/GDF15 may involve in the regulation of miR-214 inhibitor on inflammation and MUC5AC expression. [ABSTRACT FROM AUTHOR]

Objectives: Allergic rhinitis (AR) is an inflammatory autoimmune disease with disorder of the nasal mucosa. Cadherin 26 (CDH26), an alpha integrin‐binding epithelial receptor, is regulated during allergic inflammation. This study aimed to investigate whether CDH26 contributes to the severity of AR. Study design: In vivo and in vitro. Methods: We investigated the effects of CDH26 knockdown by lentivirus (LV)‐mediated shRNA on ovalbumin (OVA)‐induced AR mice and IL‐13‐stimulated human nasal epithelial cells (NECs). Results: CDH26 mRNA and protein expression was significantly increased in the nasal mucosa of AR patients and mice. Intranasal instillation of LV‐shCDH26 alleviated allergic symptoms and decreased the histological changes of nasal mucosa in AR mice. Furthermore, the serum levels of OVA‐specific IgE, IgG, pro‐inflammatory factors IL‐25, IL‐33, and TSLP were decreased in AR mice with CDH26 knockdown. With regard to AR‐induced Th2 inflammation, LV‐shCDH26 intervention effectively decreased the distribution of CD4+/GATA3+ Th2 cells, and the mRNA expression of IL‐4, IL‐5, and IL‐13 in the nasal mucosa. CDH26 knockdown down‐regulated the expression of β‐catenin but not for E‐cadherin and ZO‐1 in nasal mucosa induced by AR. In vitro, CDH26 knockdown inhibited the protein expression of TSLP, GM‐CSF and eotaxin in NECs, and CDH26 overexpression remarkably promoted the production of these inflammatory factors in IL‐13‐induced NECs. Conclusions: CDH26 knockdown attenuates the AR‐induced inflammatory response both in vivo and in vitro. Level of Evidence: NA Laryngoscope, 133:1558–1567, 2023 [ABSTRACT FROM AUTHOR]

Background: Quercetin, a polyphenolic flavonoid found in various plants and foods, is known to have antioxidant, antiviral and anticancer effects. Although quercetin is well known to exert anti-inflammatory and anti-allergic effects, the precise mechanisms by which quercetin favorably modifies the clinical status of allergic diseases, such as allergic rhinitis (AR), remain unclear. The present study examined whether quercetin could modulate the production of the endogenous anti-inflammatory molecule, Clara cell 10-kD protein (CC10), in vitro and in vivo. Methods: Human nasal epithelial cells (1 × 105 cells/mL) were stimulated with 20 ng/mL of tumor necrosis factor-alpha (TNF) in the presence of quercetin for 24 h. CC10 levels in culture supernatants were examined by ELISA. Sprague Dawley rats were sensitised with toluene 2,4-diisocyanate (TDI) by intranasal instillation of 10% TDI in ethyl acetate at a volume of 5.0 μL once daily for five days. This sensitisation procedure was repeated after an interval of two days. The rats were treated with different dosages of quercetin once daily for five days starting on the 5th day following the second sensitization. Nasal allergy-like symptoms induced by the bilateral application of 5.0 μL of 10% TDI were assessed by counting sneezing and nasal-rubbing behaviours for 10 min immediately after the TDI nasal challenge. The levels of CC10 in nasal lavage fluids obtained 6 h after TDI nasal challenge were examined using ELISA. Results: The treatment of cells with low doses of quercetin (<2.5 μM) scarcely affected TNF-induced CC10 production from nasal epithelial cells. However, the ability of nasal epithelial cells to produce CC10 after TNF stimulation significantly increased on treatment with quercetin doses (>5.0 μM). The oral administration of quercetin (>25 mg/kg) for five days significantly increased the CC10 content in nasal lavage fluids and attenuated the nasal symptoms induced by the TDI nasal challenge. Conclusions: Quercetin inhibits AR development by increasing the ability of nasal epithelial cells to produce CC10. [ABSTRACT FROM AUTHOR]

Virtually all living cells are encased in glycans. They perform key cellular functions such as immunomodulation and cell–cell recognition. Yet, how their composition and configuration affect their functions remains enigmatic. Here, we constructed isogenic capsule-switch mutants harboring 84 types of capsular polysaccharides (CPSs) in Streptococcus pneumoniae. This collection enables us to systematically measure the affinity of structurally related CPSs to primary human nasal and bronchial epithelial cells. Contrary to the paradigm, the surface charge does not appreciably affect epithelial cell binding. Factors that affect adhesion to respiratory cells include the number of rhamnose residues and the presence of human-like glycomotifs in CPS. Besides, pneumococcal colonization stimulated the production of interleukin 6 (IL-6), granulocyte- macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractantprotein- 1 (MCP-1) in nasal epithelial cells, which also appears to be dependent on the serotype. Together, our results reveal glycomotifs of surface polysaccharides that are likely to be important for colonization and survival in the human airway. [ABSTRACT FROM AUTHOR]

Yue-Ying Yang,1,2,&ast; Jing Liu,2,&ast; Yi-Tong Liu,3,4 Hsiao-Hui Ong,2 Qian-Min Chen,2,4 Ce-Belle Chen,5 Mark Thong,6 Xinni Xu,6 Sui-Zi Zhou,3,4 Qian-Hui Qiu,3,4 De-Yun Wang2 1Department of Otolaryngology-Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 2Department of Otolaryngology, Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 3Department of Otolaryngology-Head and Neck Surgery, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, People’s Republic of China; 4The Second School of Clinical Medicine, Southern Medical University, Guangzhou, People’s Republic of China; 5Centre for Ion Beam Applications, Department of Physics, National University of Singapore, Singapore; 6Department of Otolaryngology-Head and Neck Surgery, National University Hospital, National University Health System, Singapore&ast;These authors contributed equally to this workCorrespondence: Qian-Hui Qiu, Department of Otolaryngology-Head and Neck Surgery, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, No. 106 Zhongshan Road II, Guangzhou, 510080, People’s Republic of China, Tel +86 20 83827812, Email qiuqianhui@hotmail.com De-Yun Wang, Department of Otolaryngology, Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, NUHS Tower Block, 1E Kent Ridge Road, 119228, Singapore, Tel + 65 6772 5373/5370/5371, Fax +65 6775 3820, Email entwdy@nus.edu.sgPurpose: Radiotherapy (RT) is the mainstay treatment for head and neck cancers. However, chronic and recurrent upper respiratory tract infections and inflammation have been commonly reported in patients post-RT. The underlying mechanisms remain poorly understood.Method and Materials: We used a well-established model of human nasal epithelial cells (hNECs) that forms a pseudostratified layer in the air-liquid interface (ALI) and exposed it to single or repeated moderate dose γ-irradiation (1Gy). We assessed the DNA damage and evaluated the biological properties of hNECs at different time points post-RT. Further, we explored the host immunity alterations in irradiated hNECs with polyinosinic-polycytidylic acid sodium salt (poly [I:C]) and lipopolysaccharides (LPS).Results: IR induced DNA double strand breaks (DSBs) and triggered DNA damage response in hNECs. Repeated IR significantly reduced basal cell proliferation with low expression of p63/KRT5 and Ki67, induced cilia loss and inhibited mucus secretion. In addition, IR decreased ZO-1 expression and caused a significant decline in the transepithelial electrical resistance (TEER). Moreover, hyperreactive response against pathogen invasion and disrupted epithelial host defense can be observed in hNECs exposed to repeated IR.Conclusion: Our study suggests that IR induced prolonged structural and functional impairments of hNECs may contribute to patients post-RT with increased risk of developing chronic and recurrent upper respiratory tract infection and inflammation.Keywords: human nasal epithelial cells, irradiation, DNA double strand breaks, epithelial barrier, host defense

Patients with eosinophilic chronic rhinosinusitis (ECRS) respond poorly to many treatment modalities. Overproduction of periostin in the nasal mucosa is reported to contribute to polyp formation. This study examined periostin levels in patients with ECRS in comparison with levels in patients with non-ECRS. Fifty-nine patients with chronic rhinosinusitis were grouped into those with ECRS and those with non-ECRS. We compared the relationships between peripheral blood eosinophil level, serum periostin level, histopathological findings, clinical and laboratory findings, nose findings, diagnostic score of the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis Study, and postoperative recurrence of nasal polyps in each group. In the ECRS group, a positive correlation was found between peripheral blood eosinophil level and serum periostin level (r s = 0.49, P < 0.01: Spearman's rank correlation coefficient). ROC curve analysis was used to evaluate the serum periostin level that could predict postoperative recurrence of nasal polyps in the ECRS group: the area under the curve (AUC) was 0.95, sensitivity was 92%, and specificity was 100%; the serum periostin cutoff value for postoperative recurrence of nasal polyps was 130 ng/ml. In ROC curve analysis to evaluate peripheral blood eosinophil level, the AUC was 0.73, sensitivity was 69.2%, and specificity was 85.0%; the cutoff value was 8.8%. periostin was implicated in the pathophysiology of ECRS. Periostin shown to be a more useful biomarker than eosinophils in ECRS. Periostin was shown to likely be an important biomarker for pathological severity of ECRS and postoperative recurrence of nasal polyps. [ABSTRACT FROM AUTHOR]

The pathogenesis of chronic rhinosinusitis (CRS) is largely unknown, but accumulating evidence supports the role of the airway epithelium in its pathophysiology. In our study here, we evaluated whether epidermal growth factor (EGF) regulates a hypoxia‐inducible factor 1α (HIF‐1α)–microRNA‐21 (miR‐21)–aquaporin 4 (AQP4) axis in nasal epithelial cells from CRS patients. We found that, compared with normal sinus mucosa, EGF, HIF‐1α, and miR‐21 were upregulated and AQP4 was downregulated in sinus mucosa from patients with CRS and in a CRS mouse model. It was established that EGF upregulated HIF‐1α and miR‐21 expression, that HIF‐1α regulated miR‐21 transcription, and that the AQP4 gene was a target of miR‐21. Knockdown of EGF and HIF‐1α mRNAs and of miR‐21, or overexpression of AQP4 mRNA, inhibited proliferation and promoted apoptosis of hypoxia‐exposed human nasal epithelial cells, effects that were associated with reduced levels of α‐SMA, fibronectin, and vimentin, as well as promoted caspase‐3 activity and E‐cadherin levels. In the mouse CRS model, EGF elevation increased in vivo production of inflammatory IL‐4 and IFN‐γ to promote CRS, which was reversed by AQP4 elevation. Collectively, EGF upregulates HIF‐1α and miR‐21 expression to inhibit AQP4 expression, thereby promoting the proliferation of nasal epithelial cells and the development of CRS. [ABSTRACT FROM AUTHOR]

Understanding Influenza B virus infections is of critical importance in our efforts to control severe influenza and influenza-related diseases. Until 2020, two genetic lineages of influenza B virus—Yamagata and Victoria—circulated in the population. These lineages are antigenically distinct, but the differences in virus replication or the induction of host cell responses after infection have not been carefully studied. Recent IBV clinical isolates of both lineages were obtained from influenza surveillance efforts of the Johns Hopkins Center of Excellence in Influenza Research and Response and characterized in vitro. B/Victoria and B/Yamagata clinical isolates were recognized less efficiently by serum from influenza-vaccinated individuals in comparison to the vaccine strains. B/Victoria lineages formed smaller plaques on MDCK cells compared to B/Yamagata, but infectious virus production in primary human nasal epithelial cell (hNEC) cultures showed no differences. While ciliated epithelial cells were the dominant cell type infected by both lineages, B/Victoria lineages had a slight preference for MUC5AC-positive cells, and B/Yamagata lineages infected more basal cells. Finally, while both lineages induced a strong interferon response 48 h after infection of hNEC cultures, the B/Victoria lineages showed a much stronger induction of interferon-related signaling pathways compared to B/Yamagata. This demonstrates that the two influenza B virus lineages differ not only in their antigenic structure but also in their ability to induce host innate immune responses.

Small Extracellular Vesicles (sEVs) are 50–200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu‐sEVs) produced by human nasal epithelial cells (HNECs) in SARS‐CoV‐2 infection. We show that uninfected HNECs produce mu‐sEVs containing SARS‐CoV‐2 receptor ACE2 and activated protease TMPRSS2. mu‐sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an 'open' conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu‐sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS‐CoV‐2 virions prone to entry into target cells using the 'early', TMPRSS2‐dependent pathway instead of the 'late', cathepsin‐dependent route. These results indicate that prefusion Spike priming by mu‐sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS‐CoV‐2 infection, but instead facilitates it. [ABSTRACT FROM AUTHOR]

Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression. [ABSTRACT FROM AUTHOR]

Influenza B Viruses (IBV) have caused an increasing number of cases over the last 15 years. The focus of this study was to assess the role of egg adapted mutants in IBV vaccines on the reactivity of serum from vaccinated or IBV infected individuals. We focused on the 2017-2018 IBV season as this was a significant influenza year with reported low vaccine effectiveness by the CDC. Patient samples were obtained from Johns Hopkins Adult Emergency Room for virus isolation and antigenic characterization. Antigenic characterization was evaluated using neutralizing antibody assays. Viral characterization was carried out using viral genome sequencing and structural modeling, MDCKSIAT1 growth curves, MDCK Plaque assays and human primary nasal epithelial cell (hNEC) growth curves. In our analysis, we found that in the vaccine strains of both IBV lineages, there was an amino acid change at position 197 (B/Brisbane HA Numbering) that leads to a loss of glycosylation. Our antigenic evaluation shows that there is a significant difference in neutralizing antibody titers between the egg adapted vaccine for the B/Yamagata lineage compared to representative clinical isolates from that season and the cell cultured vaccine. We propose that this loss of a glycosylation site is an important site for propagation in the allantois and that this common site change may play a role in antigenic recognition and therefore immune protection from circulating viruses. Screening egg cultured vaccine viruses for egg adapted mutants, further transitioning vaccine production to mammalian culture models (MDCK) or investigating new models of influenza vaccination may be necessary to improve efficacy of the seasonal influenza vaccine for protection from IBV. [ABSTRACT FROM AUTHOR]

Abstract Small Extracellular Vesicles (sEVs) are 50–200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu‐sEVs) produced by human nasal epithelial cells (HNECs) in SARS‐CoV‐2 infection. We show that uninfected HNECs produce mu‐sEVs containing SARS‐CoV‐2 receptor ACE2 and activated protease TMPRSS2. mu‐sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an ‘open’ conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu‐sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS‐CoV‐2 virions prone to entry into target cells using the ‘early’, TMPRSS2‐dependent pathway instead of the ‘late’, cathepsin‐dependent route. These results indicate that prefusion Spike priming by mu‐sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS‐CoV‐2 infection, but instead facilitates it.

Patient-derived organoids grown in three-dimensional cultures provide an excellent platform for phenotypic high-throughput screening and drug-response research. Organoid technology has been applied to study stem cell biology and various human pathologies. This study investigates the characteristics and cellular morphology of organoids derived from primary human nasal epithelial cells (HNECs) of chronic rhinosinusitis (CRS) patients. Nasal organoids were cultured up to 20 days and morphological, cell composition and functional parameters were measured by immunofluorescence, RT-qPCR, western blot and FACS analysis. The results showed that nasal organoids expressed the stem cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and markers for apical junction genes, goblet cells and ciliated cells. Moreover, we were able to regrow and expand the nasal organoids well after freezing and thawing. This study provides an effective and feasible method for development of human nasal organoids, suitable for the phenotypic high-throughput screening and drug response research.

Influenza B Viruses (IBV) have caused an increasing number of cases over the last 15 years. The focus of this study was to assess the role of egg adapted mutants in IBV vaccines on the reactivity of serum from vaccinated or IBV infected individuals. We focused on the 2017-2018 IBV season as this was a significant influenza year with reported low vaccine effectiveness by the CDC. Patient samples were obtained from Johns Hopkins Adult Emergency Room for virus isolation and antigenic characterization. Antigenic characterization was evaluated using neutralizing antibody assays. Viral characterization was carried out using viral genome sequencing and structural modeling, MDCK-SIAT1 growth curves, MDCK Plaque assays and human primary nasal epithelial cell (hNEC) growth curves. In our analysis, we found that in the vaccine strains of both IBV lineages, there was an amino acid change at position 197 (B/Brisbane HA Numbering) that leads to a loss of glycosylation. Our antigenic evaluation shows that there is a significant difference in neutralizing antibody titers between the egg adapted vaccine for the B/Yamagata lineage compared to representative clinical isolates from that season and the cell cultured vaccine. We propose that this loss of a glycosylation site is an important site for propagation in the allantois and that this common site change may play a role in antigenic recognition and therefore immune protection from circulating viruses. Screening egg cultured vaccine viruses for egg adapted mutants, further transitioning vaccine production to mammalian culture models (MDCK) or investigating new models of influenza vaccination may be necessary to improve efficacy of the seasonal influenza vaccine for protection from IBV.

Background: Fine particulate matter (PM) (PM with an aerodynamic diameter <2.5 μm, PM2.5) exposure contributes to respiratory disease development and exacerbation. Objective: We sought to investigate the effect of PM2.5 exposure on mucociliary function in primary human nasal epithelial cells (HNECs) and the underlying mechanism. Methods: HNECs derived from control subjects and patients with chronic rhinosinusitis with nasal polyps were established as air-liquid interface cultures. Confluent cultures were exposed to 100 or 200 μg/mL PM2.5 for 24 h and assessed for expression of specific mucociliary-associated factors, the percentage of β-tubulin IV-positive and MUC5AC-positive cells, expression of epidermal growth factor receptor (EGFR) ligand and activation of phosphoinositide 3-kinase (PI3K)-AKT/ERK. In addition, cultures pretreated for 30 min with AG1478 (an EGFR inhibitor) or LY294002 (a PI3K inhibitor) following PM2.5 exposure were assessed for MUC5AC mRNA and protein expression. Results: PM2.5 exposure at 100 or 200 μg/mL for 24 h did not affect geminin coiled-coil domain containing, multiciliate differentiation and DNA synthesis associated cell cycle protein, FOXJ1, or DNAI2 mRNA expression or the percentage of β-tubulin IV-positive cells. However, 200 μg/mL PM2.5 exposure significantly increased mRNA expression of SAM-pointed domain-containing ETS transcription factor and MUC5AC and the percentage of MUC5AC-positive cells. PM2.5 also increased expression of EGFR ligands, including heparin-binding EGF-like growth factor and amphiregulin. Furthermore, PM2.5induced activation of PI3K, AKT, and ERK, and pretreatment of HNECs with AG1478 or LY294002 attenuated PM2.5-induced MUC5AC mRNA and protein expression. Conclusions and Clinical Relevance: This study demonstrates that short-term PM2.5 exposure increases MUC5AC expression in HNECs. Furthermore, this study shows that PM2.5-induced MUC5AC expression is likely mediated through the EGFR-PI3K pathway. [ABSTRACT FROM AUTHOR]

Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR‐related diseases (CFTR‐RDs). Here, we investigate genotype–phenotype–CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non‐CF subjects and individuals with CF and CFTR‐RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole‐cell patch‐clamp technique or grown as primary cultures at an air–liquid interface after conditional reprogramming. The properties of cAMP‐activated whole‐cell Cl− currents in freshly isolated hNE cells identified them as CFTR‐mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non‐CF > CFTR residual function > CFTR minimal function. CFTR‐mediated whole‐cell Cl− currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR‐mediated whole‐cell Cl− currents in hNE cells correlated well with CFTR‐mediated transepithelial Cl− currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl− concentration of study subjects. Thus, this study highlights the complexity of genotype–phenotype–CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. Key points: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body.This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity.CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient‐derived cells.Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype–phenotype–CFTR function relationships. [ABSTRACT FROM AUTHOR]

Background: Quercetin, a polyphenolic flavonoid found in various plants and foods, is known to have antioxidant, antiviral and anticancer effects. Although quercetin is well known to exert anti-inflammatory and anti-allergic effects, the precise mechanisms by which quercetin favorably modifies the clinical status of allergic diseases, such as allergic rhinitis (AR), remain unclear. The present study examined whether quercetin could modulate the production of the endogenous anti-inflammatory molecule, Clara cell 10-kD protein (CC10), in vitro and in vivo. Methods: Human nasal epithelial cells (1 × 105 cells/mL) were stimulated with 20 ng/mL of tumor necrosis factor-alpha (TNF) in the presence of quercetin for 24 h. CC10 levels in culture supernatants were examined by ELISA. Sprague Dawley rats were sensitised with toluene 2,4-diisocyanate (TDI) by intranasal instillation of 10% TDI in ethyl acetate at a volume of 5.0 μL once daily for five days. This sensitisation procedure was repeated after an interval of two days. The rats were treated with different dosages of quercetin once daily for five days starting on the 5th day following the second sensitization. Nasal allergy-like symptoms induced by the bilateral application of 5.0 μL of 10% TDI were assessed by counting sneezing and nasal-rubbing behaviours for 10 min immediately after the TDI nasal challenge. The levels of CC10 in nasal lavage fluids obtained 6 h after TDI nasal challenge were examined using ELISA. Results: The treatment of cells with low doses of quercetin (5.0 μM). The oral administration of quercetin (>25 mg/kg) for five days significantly increased the CC10 content in nasal lavage fluids and attenuated the nasal symptoms induced by the TDI nasal challenge. Conclusions: Quercetin inhibits AR development by increasing the ability of nasal epithelial cells to produce CC10.

To study the interaction of human pathogens with their host target structures, human tissue models based on primary cells are considered suitable. Complex tissue models of the human airways have been used as infection models for various viral and bacterial pathogens. The Gram-negative bacterium Bordetella pertussis is of relevant clinical interest since whooping cough has developed into a resurgent infectious disease. In the present study, we created three-dimensional tissue models of the human ciliated nasal and tracheo-bronchial mucosa. We compared the innate immune response of these models towards the B. pertussis virulence factor adenylate cyclase toxin (CyaA) and its enzymatically inactive but fully pore-forming toxoid CyaA-AC-. Applying molecular biological, histological, and microbiological assays, we found that 1 µg/ml CyaA elevated the intracellular cAMP level but did not disturb the epithelial barrier integrity of nasal and tracheo-bronchial airway mucosa tissue models. Interestingly, CyaA significantly increased interleukin 6, interleukin 8, and human beta defensin 2 secretion in nasal tissue models, whereas tracheo-bronchial tissue models were not significantly affected compared to the controls. Subsequently, we investigated the interaction of B. pertussis with both differentiated primary nasal and tracheo-bronchial tissue models and demonstrated bacterial adherence and invasion without observing host cell type-specific significant differences. Even though the nasal and the tracheo-bronchial mucosa appear similar from a histological perspective, they are differentially susceptible to B. pertussis CyaA in vitro. Our finding that nasal tissue models showed an increased innate immune response towards the B. pertussis virulence factor CyaA compared to tracheo-bronchial tissue models may reflect the key role of the nasal airway mucosa as the first line of defense against airborne pathogens. [ABSTRACT FROM AUTHOR]

To study the interaction of human pathogens with their host target structures, human tissue models based on primary cells are considered suitable. Complex tissue models of the human airways have been used as infection models for various viral and bacterial pathogens. The Gram-negative bacterium Bordetella pertussis is of relevant clinical interest since whooping cough has developed into a resurgent infectious disease. In the present study, we created three-dimensional tissue models of the human ciliated nasal and tracheo-bronchial mucosa. We compared the innate immune response of these models towards the B. pertussis virulence factor adenylate cyclase toxin (CyaA) and its enzymatically inactive but fully pore-forming toxoid CyaA-AC-. Applying molecular biological, histological, and microbiological assays, we found that 1 µg/ml CyaA elevated the intracellular cAMP level but did not disturb the epithelial barrier integrity of nasal and tracheo-bronchial airway mucosa tissue models. Interestingly, CyaA significantly increased interleukin 6, interleukin 8, and human beta defensin 2 secretion in nasal tissue models, whereas tracheo-bronchial tissue models were not significantly affected compared to the controls. Subsequently, we investigated the interaction of B. pertussis with both differentiated primary nasal and tracheo-bronchial tissue models and demonstrated bacterial adherence and invasion without observing host cell type-specific significant differences. Even though the nasal and the tracheo-bronchial mucosa appear similar from a histological perspective, they are differentially susceptible to B. pertussis CyaA in vitro. Our finding that nasal tissue models showed an increased innate immune response towards the B. pertussis virulence factor CyaA compared to tracheo-bronchial tissue models may reflect the key role of the nasal airway mucosa as the first line of defense against airborne pathogens.

Objective: Recently, depleted tissue zinc levels were found in nasal mucosa from patients with chronic rhinosinusitis (CRS) in correlation with tissue eosinophilia, however, no clinical biomarkers for tissue zinc levels have been identified. Metallothionein-3 (MT3) is an intracellular zinc chelator and previous data showed MT3 mRNA levels to be reduced in CRS patients with nasal polyps (CRSwNP). In this study, we examined the correlation between MT3 expression and zinc levels in nasal mucosa and primary human nasal epithelial cells (HNECs) to investigate whether MT3 could be a clinical biomarker for tissue zinc levels.Method: Tissue was harvested from 36 patients and mounted on tissue micro-array (TMA) slides. MT3 expression and tissue zinc fluorescence intensity were measured at different areas within the mucosa (surface epithelium and lamina propria) and compared between controls, CRSwNP and CRS without nasal polyps (CRSsNP) patients. MT3 mRNA and protein expression were examined in zinc-depleted HNECs by qPCR and immunofluorescence microscopy.Results: MT3 expression in CRSwNP was significantly decreased in both surface epithelium (p<0.001 to controls) and lamina propria (p = 0.0491 to controls). There was a significant positive correlation between tissue zinc levels and MT3 expression in nasal mucosa (r = 0.45, p = 0.007). In zinc-deplete HNECs, MT3 expression was significantly decreased at mRNA (p = 0.02) and protein level (p<0.01). There was a significant positive correlation between tissue zinc levels and MT3 expression within individual HNECs (r = 0.59, p<0.001).Conclusions: MT3 expression reflects intramucosal zinc levels in both nasal mucosa and HNECs indicating MT3 could be used as a clinical biomarker for monitoring intracellular zinc levels in the nasal mucosa. [ABSTRACT FROM AUTHOR]

Background: Oxidative stress plays crucial roles in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Thioredoxin-interacting protein (TXNIP) is essential in the process of triggering oxidative stress. However, its role and mechanism in CRSwNP remain unclear. The present study sought to explore the role and mechanism of TXNIP in the pathogenesis of CRSwNP. Methods: Western blotting, real-time PCR and immunohistochemistry (IHC) were employed to assess TXNIP, thioredoxin (TRX) expression in nasal tissue samples from patients with CRSwNP and control subjects. MDA level and SOD activity in nasal tissue homogenates were measured using MDA and SOD Assay Kit. To evaluate the role and mechanism of TXNIP in CRSwNP, human nasal epithelial cells (HNECs) were cultured and stimulated using TXNIP siRNA, with or without N-acetylcysteine (NAC, an ROS scavenger). Western blotting, real-time PCR, ROS detecting dye DCFH-DA, MDA and SOD Assay Kit were performed to assess the effects and mechanisms of stimulators on the cells. Results: We found significantly increased levels of TXNIP and decreased levels of TRX protein, mRNA, positive cells, increased MDA level and decreased SOD activity in CRSwNP patients compared with control subjects. In vitro study, significantly altered levels of TXNIP, TRX, MDA, SOD and ROS in HNECs were found following treatment of TXNIP siRNA with or without NAC on HNECs. Conclusion: TXNIP expression was increased and TRX expression was decreased in CRSwNP at both protein and mRNA levels. MDA levels were increased and SOD activities were decreased in CRSwNP. TXNIP may have negative association with TRX, and then decrease SOD activities and increase MDA levels, resulting in the upregulation of ROS and oxidative stress in HNECs, which may play a pivotal role in the pathogenesis of CRSwNP. Future studies are expected to further explore the role and mechanism of TXNIP in CRSwNP. [ABSTRACT FROM AUTHOR]

Background: Glyoxal (GO), and methylglyoxal (MGO) are among the most toxic compounds emitted by electronic cigarette (E-cig) and regular tobacco cigarette smoke. Airway diseases presented mucus over production as their major pathophysiologic feature. However, the effects of GO and MGO on pro-inflammatory cytokines and mucin expression in human nasal epithelial cells, as well as the underlying signaling pathway, have not yet been studied. Objective: This study is to determine whether GO and MGO induce pro-inflammatory cytokines, and MUC5AC/5B expression via mitogen-activated protein kinase (MAPK)s and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. Methods: The effect of GO, and MGO on pro-inflammatory cytokines, mucins expression and the signalling pathway of GO and MGO were investigated using water-soluble tetrazolium salt-1, enzyme immunoassays, and immunoblot analysis with specific inhibitors and small interfering RNA. Results: GO and MGO did not affect cell viability up to 2 mM in human nasal epithelial cells. GO and MGO increased production of pro-inflammatory such as interleukin (IL)-1β and IL-6) and MUC5AC/5B. Additionally, GO and MGO significantly activated extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and NF-κB. Whether ERK1/2, p38 MAPK, and NF-κB signaling pathway were involved in GO and MGO-induced production of pro-inflammatory cytokines (IL-1β and IL-6) and MUC5AC/5B, we used specific inhibitors and siRNA transfection. These significantly repressed GO- and MGO-induced expression of pro-inflammatory cytokines (IL-1β and IL-6) and MUC5AC/5B. Conclusions: GO and MGO induced pro-inflammatory cytokines and MUC5AC/5B expression via ERK1/2, p38 MAPK, and NF-κB in human nasal epithelial cells. These results suggested that GO and MGO may be involved in mucus hypersecretion-related airway diseases. [ABSTRACT FROM AUTHOR]

In allergic airway diseases, intermediate progenitor cells (IPCs) increase in number in the surface epithelium. IPCs arise from basal cells, the origin of hallmark pathological changes, including goblet cell hyperplasia and mucus hypersecretion. Thus, targeting IPCs will benefit future treatment of allergic airway diseases. However, the lack of adequate cell surface markers for IPCs limits their identification and characterization. We now show that CD44 containing exon v3 (CD44v3) is a surface marker for IPCs that are capable of both proliferating and generating differentiated goblet cells in allergic human nasal epithelium. In primary human nasal epithelial cells that had differentiated at an air-liquid interface, IL-4 upregulated mRNA expression of three CD44v variants that include exon v3 (CD44v3-v6, CD44v3, v8-v10, and CD44v3-v10), and it induced expression of CD44v3 protein in the basal and suprabasal layers of the culture. FACS analysis revealed two subpopulations differing in CD44v3 concentrations, as follows: CD44v3low cells expressed high amounts of proliferative and basal cell markers (Ki-67 and TP63), whereas CD44v3high cells strongly expressed progenitor and immature and mature goblet cell markers (SOX2, CA2, and SPDEF). Importantly, a blocking anti-CD44 antibody suppressed IL-4-induced mucin production by human nasal epithelial cells. Furthermore, CD44v3 was coexpressed with TP63, KRT5, or SOX2 and was upregulated in the basal and suprabasal layers of the nasal surface epithelium of subjects with allergic rhinitis. Taken together, these data demonstrate that high CD44v3 expression contributes to goblet cell hyperplasia in inflammation of the allergic airway. [ABSTRACT FROM AUTHOR]

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: "detection of targeted editing of CFTR in organoids")., Competing Interests: Declaration of interests A.C. is a co-founder and holds shares of Alia Therapeutics. I.S.-G. is scientific board adviser of, received academic grants from, and supervises a study sponsored by Vertex Pharmaceuticals. I.S.-G. is a scientific board adviser of Tavanta. M.S.C. received speaker fees from Vertex Pharmaceuticals., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tight junctions (TJs) are intercellular structures which are essential for epithelial barrier function and play an important role in antimicrobial defense. Epithelium dysfunction and type-2-skewed inflammation are two main pathological phenomena of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the effect of pro-inflammatory type-2 cytokine IL-13 on TJs in CRSwNP is poorly understood. Nasal biopsies of CRSwNP patients and in vitro IL-13-matured human nasal epithelial cells (hNECs) were used to analyze epithelial markers and TJ proteins. Epithelium permeability, transepithelial electrical resistance (TEER), expression of TJs were quantified for IL-13-matured hNECs and that with RV infection. The expression of occludin, claudin-3, and ZO-1 were significantly decreased in CRSwNP biopsies and in hNECs after IL-13 treatment. IL-13 treatment increased epithelium permeability, decreased TEER and altered hNECs composition resulting in lesser ciliated cells and mucus over-secretion. Interestingly, claudin-3 is selectively expressed on ciliated cells. While RV infection induced minimal changes to TJs, the IL-13-matured hNECs has reduced capacity for upregulation of IFN-λ1 and CXCL10 but further increased the expression of TSLP upon RV infection. These findings suggested that IL-13-mediated dysfunction of TJs and compromised epithelial barrier. IL-13-induced cilia loss conferred lowered viral replication and impaired antiviral responses of nasal epithelium against RV infection.

Background/Aim: Cocaine is a widely used recreational drug and is known for its nasal complications including epithelial, cartilage and bone damage. The aim of the study was to analyze the impact of cocaine on ciliary beat frequency (CBF) of human nasal epithelial cells and therefore better understand its side effects on nasal mucosa. Materials and Methods: Nasal epithelial cells of 21 healthy subjects were harvested and exposed in vitro to cocaine hydrochloride solutions ranging from 0.875% to 7%. Highspeed video footage was acquired with phase contrast microscopy and CBF was analyzed with Sissons-Ammons Video Analysis (SAVA) software. Results: All tested concentrations led to a significant reduction in CBF compared to the control. Effects increased over time and with concentration. A mechanical inhibition of cilia by cocaine crystals was also observed. Conclusion: We assume that CBF reduction is part of the pathomechanism leading to nasal complications in cocaine abuse. Considering these results, clinical usage of cocaine should be critically evaluated and restricted to select cases only. [ABSTRACT FROM AUTHOR]

Background: Nasal topical treatments can provide an effective method of disease control for patients suffering from chronic rhinosinusitis (CRS). However, some frequently used formulations lack adequate evaluation on their safety. This study investigated the effect of 0.5% povidone‐iodine (Nasodine) on the sinonasal epithelial barrier and ciliated human nasal epithelial cells (HNECs) in vitro. Methods: Nasodine was applied to air‐liquid interface (ALI) cultures of primary HNECs from CRS patients. Epithelial barrier structure was assessed by measuring the transepithelial electrical resistance (TEER), paracellular permeability, and immunolocalization of the zona occludens‐1 (ZO‐1) tight junction protein. Toxicity and ciliary beat frequency (CBF) were also studied. Results: Nasodine was not toxic and did not have detrimental effects on the paracellular permeability or CBF. Nasodine did not show a significant reduction in TEER with a 5‐minute exposure; however, with a 30‐minute exposure there was a significant reduction in TEER at 1 hour and at 4 hours after exposure. Conclusion: Application of Nasodine to HNEC‐ALI cultures in vitro for up to 30 minutes was not toxic and did not affect the paracellular permeability or CBF. [ABSTRACT FROM AUTHOR]

Maintaining tight junction integrity significantly contributes to epithelial barrier function. If the barrier function is destroyed, the permeability of the cells increases, and the movement of the pathogens is promoted, thereby further increasing the susceptibility to secondary infection. Ginsenoside components have multiple biological activities, including antiviral effects. In this study, we examined the protective effects of ginsenoside Re against rhinovirus-induced tight junction disruption in primary human nasal epithelial cells (HNE). Incubation with human rhinovirus resulted in marked disruption of tight junction proteins (ZO-1, E-cadherin, claudin-1, and occludin) in human nasal epithelial cells. Rhinovirus-induced disruption of tight junction proteins was strongly inhibited by the treatment of cells with ginsenoside Re. Indeed, significant amounts of reactive oxygen species (ROS) have been detected in human nasal epithelial cells co-incubated with rhinovirus. Moreover, rhinovirus-induced ROS generation was markedly reduced by the ginsenoside Re. However, ginsenosides Rb1 and Rc did not inhibit tight junction disruption or ROS generation in nasal epithelial cells following incubation with rhinovirus. Furthermore, incubation with rhinovirus resulted in a marked decrease in protein phosphatase activity and an increase in protein tyrosine phosphorylation levels in nasal epithelial cells. Treatment of cells with ginsenoside Re inhibited rhinovirus-induced inactivation of phosphatases and phosphorylation of tyrosine. Our results identified ginsenoside Re as an effective compound that prevented rhinovirus-induced tight junction disruption in human nasal epithelial cells., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Seon Tae Kim reports financial support was provided by 10.13039/501100003710Korea Health Industry Development Institute. Seon Tae Kim reports a relationship with 10.13039/501100003710Korea Health Industry Development Institute that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Background: Viral entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) via the spike protein enables endocytosis into host cells using the ACE2 receptor and TMPRSS2. The frequent upper respiratory tract symptoms of COVID-19 and the localization of the virus to the nasopharynx, the most common site of swabbing, indicate that the sinonasal mucosa may play an important role in SARS-CoV2 infection and viral replication. Methods: This paper investigates the presence of ACE2 receptor and TMPRESS2 expression in the primary human nasal epithelial cells (HNECs) from the following: chronic rhinosinusitis without nasal polyps (CRSsNP), CRS with nasal polyps (CRSwNP) and control (non-CRS) patients, and maps the expression changes when exposed to Th1, Th2, Th17-associated cytokines. Results: We found that ACE2 and TMPRSS2 expression was higher in control HNECs than CRSwNP HNECs, and that both ACE2 and TMPRSS2 were downregulated further by Th2 cytokines in CRSwNP HNECs. Conclusions: This indicates an immune dysregulated state of CRSwNP mucosa, which normally contributes to a chronic inflammatory state, and might support an altered susceptibility to SARS-CoV2 infection and transmission.

Background: Nasal irrigation (NI) is commonly used to treat several sinonasal diseases, including chronic rhinosinusitis with nasal polyps (CRSwNP); however, the effects of NI on the sinonasal epithelium are not fully known. The aim of this study was to investigate the effects of commonly used NI solutions on epithelial mucociliary and barrier functionality in primary cultured human nasal epithelial cells (HNECs). Methods: HNECs from control subjects and patients with CRSwNP were established as air‐liquid interface (ALI) cultures. Differentiated cultures were treated with different NI solutions, including isotonic 0.9% and hypertonic 3.0% saline, isotonic and hypertonic seawater, and Ringer lactate solution. The changes in ciliary beat frequency (CBF), numbers of ciliated and goblet cells, and cytotoxicity were measured. Epithelial barrier functionality was assessed by measuring the transepithelial electric resistance (TER), paracellular flux, and expression of tight junction protein zonula occludens‐1 (ZO‐1) and occludin. Results: Isotonic saline, isotonic seawater, and Ringer lactate solutions did not affect epithelial mucociliary and barrier function in either control or CRSwNP‐derived ALI cultures; however, hypertonic saline induced a significant disruption of these cell functions in both cultures. Hypertonic seawater caused a transient decrease of CBF and TER in CRSwNP‐derived ALI cultures, in contrast to inducing an obvious mucociliary and barrier dysfunction and cytotoxicity in control ALI cultures. Conclusion: Although isotonic NI solutions appear to not affect epithelial mucociliary and barrier function in control and CRSwNP‐derived ALI cultures, hypertonic saline and seawater solutions damaged sinonasal epithelial cells in ALI cultures. The safety and efficacy of these solutions requires further investigation. [ABSTRACT FROM AUTHOR]

Background: Cellulose powder (CP) has been reported as a safe and effective complementary treatment for allergic rhinitis (AR). Currently, CP has gained increasing application for clinical management worldwide, particularly in China. However, studies focusing on the effect of CP on normal human nasal epithelial cells (hNECs) and ciliary function are lacking. Here, we aimed to explore the adverse effects of CP on the activity and ciliary function of hNECs. Methods: We biopsied ethmoid sinus or middle turbinate tissues during surgical resection from control subjects who underwent endoscopic sinus surgery for diseases other than AR. Cells were isolated and passaged, followed by differentiation in an air-liquid interface (ALI). Flow cytometry and cell viability test (cell counting kit-8) were performed to detect the cytotoxicity of CP (effects on cell proliferation) on normal hNECs. By using the ALI culture model, we investigated the effects of CP on ciliary beat frequency (CBF). Results: There was a significant reduction in hNEC count at high concentrations of CP (2.5 mg/mL) at days 3 and 7 (both p < 0.05). As the concentration increased, cell death increased progressively from day 3 to day 7. However, these effects were not evident at low concentrations (0.25 mg/mL, p > 0.05). High-dose CP (2.5 mg) significantly reduced the CBF (p < 0.05). At lower concentrations (0.25–2.5 mg/mL), CP initially increased but subsequently reduced the CBF of hNECs compared with control group. Conclusions: Cytotoxicity and the suppression of ciliary beat at high concentrations justify more prudent use of CP for the management of AR. [ABSTRACT FROM AUTHOR]

Purpose: Epithelial thymic stromal lymphopoietin (TSLP) promotes Th2 inflammatory responses through induction of OX40 ligand (OX40L) on dendritic cells in allergic rhinitis (AR). Emerging evidence supports the important role of histamine H4 receptor (H4R) in allergic inflammation. This study aimed to investigate the effects of H4R in Th2-cytokine profile mediated by TSLP in AR. Methods: Human nasal epithelial cells (HNECs) from AR patients were stimulated with histamine in the presence or absence of H4R agonist (4-methylhistamine, 4-MH) and antagonist (NJ7777120, JNJ) or H1R agonist (2-pyridylethylamine). TSLP protein was measured by Western blotting and ELISA. To further elucidate the role of H4R in the in vivo situation of experimental AR, rats were sensitized and treated with JNJ or 4-MH. TSLP and OX40 ligand (OX40L) in the nasal mucosa were assayed by Western blotting. Th2 cytokines including interleukin-4, 5 and 13 in nasal lavage fluids were detected by ELISA. Results: Histamine alone did not induce TSLP production by HNECs. The pre-incubation with 4-MH prior to histamine promoted TSLP expression, which was inhibited by the stimulation with JNJ prior to histamine and 4-MH. The pre-incubation with 2-pyridylethylamine before histamine stimulation had no impact on TSLP production. In AR rats, the levels of TSLP and OX40L protein were increased as well as Th2 cytokines, which was further up-regulated by 4-MH treatment, while JNJ treatment attenuated these effects. Conclusions: H4R activation induced TSLP production by HNECs, which up-regulated OX40L expression in the nasal mucosa of sensitized rats. These factors promoted Th2-cytokine profile in AR. [ABSTRACT FROM AUTHOR]

• The expression of IL-36γ and its receptor IL-36R by eosinophils in AR were up-regulated. • IL-36γ exaggerates eosinophilic inflammation in AR by promoting the function of eosinophils. • Human nasal epithelial cells secrete IL-36γ after treated with recombinant IL-17, IL-25, IL-33. Allergic rhinitis (AR) is characterized by eosinophilic inflammation. However, the function and regulation of eosinophils in AR are largely unknown. This study aimed to explore the expression and role of interleukin-36 (IL-36) cytokines in AR. Sixty AR patients and 20 control subjects were recruited in this study. The mRNA and protein expression of serum IL-36 family cytokines and IL-36R in AR were detected by quantitative RT-PCR and enzyme-linked immunosorbent assay ELISA, respectively. IL-36R expression and regulation by eosinophils and the role of IL-36γ in the survival, adhesion, migration and activation of eosinophils were performed in purified eosinophils. Human nasal epithelial cell line was cultured and treated with different stimulators and IL-36γ was measured. The mRNA and protein expression of serum IL-36 cytokines and IL-36R were significantly higher in AR compared with control, especially in asthmatic patients. Among the IL-36 cytokines, the expression of IL-36γ was the highest. The expression of IL-36R by eosinophils were significantly increased compared with normal controls and was up-regulated by recombinant IL-17, IL-25, IL-33 and Dermatophagoides pteronyssinus group 1. The IL-36γ promote the survival, adhesion, migration and activation of eosinophils. Human nasal epithelial cells can secrete IL-36γ after treated with recombinant IL-17, IL-25, IL-33. High expression of IL-36γ exaggerates eosinophilic inflammation in AR by promoting the survival, adhesion, and activation of eosinophils. [ABSTRACT FROM AUTHOR]

In vitro and in vivo research based on cell lines and animals are likely to be insufficient in elucidating authentic biological and physiological phenomena mimicking human systems, especially for generating pre-clinical data on targets and biomarkers. There is an obvious need for a model that can further bridge the gap in translating pre-clinical findings into clinical applications. We have previously generated a model of in vitro differentiated human nasal epithelial cells (hNECs) which elucidated the nasal-initiated repertoire of immune responses against respiratory viruses such as influenza A virus and rhinovirus. To assess their clinical utility, we performed a microarray analysis of influenza virus-infected hNECs to elucidate nasal epithelial-initiated responses. This was followed by a metagenomic analysis which revealed transcriptomic changes comparable with clinical influenza datasets. The primary target of influenza infection was observed to be the initiator of innate and adaptive immune genes, leaning toward type-1 inflammatory activation. In addition, the model also elucidated a down-regulation of metabolic processes specific to the nasal epithelium, and not present in other models. Furthermore, the hNEC model detected all 11 gene signatures unique to influenza infection identified from a previous study, thus supporting the utility of nasal-based diagnosis in clinical settings. In conclusion, this study highlights that hNECs can serve as a model for nasal-based clinical translational studies and diagnosis to unravel nasal epithelial responses to influenza in the population, and as a means to identify novel molecular diagnostic markers of severity.

Objective: Rhinoviruses (RV), which are responsible for the majority of common colds, induce mucus overproduction, increased vascular permeability, and secondary bacterial infection. These symptoms are primarily caused by barrier function disruption, which is controlled by intercellular junctions. In this study, we investigated whether reactive oxygen species (ROS) are closely involved in tight junction disruption of primary human nasal epithelial (HNE) cells induced by infection of RV .Methods and Results: Incubation with RV resulted in disruption of tight junction proteins (ZO-1, E-cadherin, claudin-1, and occludin) in HNE cells. Pretreatment with diphenylene iodonium (DPI) decreased RV-induced disruption of tight junction in HNE cells. RV-induced generation of ROS was diminished by DPI. However, rotenone was not inhibited in HNE cells following incubation with RV. Rhinoviruses resulted in a marked decrease in protein phosphatases activity and an increase in protein tyrosine phosphorylation levels in HNE cells. Diphenylene iodonium inhibited the RV-induced inactivation of phosphatases and phosphorylation of protein tyrosine. In addition, inhibition of protein tyrosine phosphatases with phenylarsine oxide resulted in a marked decrease in protein phosphatase activity and disruption of tight junction proteins in HNE cells.Conclusion: Our results suggest that ROS-mediated inhibition of phosphatases plays a crucial role in disruption of tight junctions in HNE cells by RV. The data suggest that RV infection may damage nasal epithelial barrier function.Level Of Evidence: NA Laryngoscope, 128:E393-E401, 2018. [ABSTRACT FROM AUTHOR]

In vitro and in vivo research based on cell lines and animals are likely to be insufficient in elucidating authentic biological and physiological phenomena mimicking human systems, especially for generating pre-clinical data on targets and biomarkers. There is an obvious need for a model that can further bridge the gap in translating pre-clinical findings into clinical applications. We have previously generated a model of in vitro differentiated human nasal epithelial cells (hNECs) which elucidated the nasal-initiated repertoire of immune responses against respiratory viruses such as influenza A virus and rhinovirus. To assess their clinical utility, we performed a microarray analysis of influenza virus-infected hNECs to elucidate nasal epithelial-initiated responses. This was followed by a metagenomic analysis which revealed transcriptomic changes comparable with clinical influenza datasets. The primary target of influenza infection was observed to be the initiator of innate and adaptive immune genes, leaning toward type-1 inflammatory activation. In addition, the model also elucidated a down-regulation of metabolic processes specific to the nasal epithelium, and not present in other models. Furthermore, the hNEC model detected all 11 gene signatures unique to influenza infection identified from a previous study, thus supporting the utility of nasal-based diagnosis in clinical settings. In conclusion, this study highlights that hNECs can serve as a model for nasal-based clinical translational studies and diagnosis to unravel nasal epithelial responses to influenza in the population, and as a means to identify novel molecular diagnostic markers of severity. [ABSTRACT FROM AUTHOR]

Human rhinoviruses (HRVs) are the commonest cause of the common cold. While HRV is less pathogenic than other respiratory viruses, it is frequently associated with exacerbation of chronic respiratory diseases such as rhinosinusitis and asthma. Nasal epithelial cells are the first sites of viral contact, immune initiation, and airway interconnectivity, but there are limited studies on HRV infection of nasal epithelial cells. Hence, we established a model of HRV infection of in vitro-differentiated human nasal epithelial cells (hNECs) derived from multiple individuals. Through HRV infection of hNECs, we found that HRV mainly targeted ciliated cells and preferentially induced type I and III interferon antiviral pathways. Quantitative polymerase chain reaction analysis of inflammatory genes suggested predominant type 1 immunity signaling and recruitment, with secreted CXCL9, IP-10, CXCL11, and RANTES as likely initiators of airway inflammatory responses. Additionally, we further explored HRV bidirectional release from the hNECs and identified 11 associated genes. Other HRV interactions were also identified through a systematic comparison with influenza A virus infection of hNECs. Overall, this in vitro hNEC HRV infection model provides a platform for repeatable and controlled studies of different individuals, thus providing novel insights into the roles of human nasal epithelium in HRV interaction and immune initiation. [ABSTRACT FROM AUTHOR]

Ninety years after the discovery of the virus causing the influenza disease, this malady remains one of the biggest public health threats to mankind. Currently available drugs and vaccines only partially reduce deaths and hospitalizations. Some of the reasons for this disturbing situation stem from the sophistication of the viral machinery, but another reason is the lack of a complete understanding of the molecular and physiological basis of viral infections and host−pathogen interactions. Even the functions of the influenza proteins, their mechanisms of action and interaction with host proteins have not been fully revealed. These questions have traditionally been studied in mammalian animal models, mainly ferrets and mice (as well as pigs and non-human primates) and in cell lines. Although obviously relevant as models to humans, these experimental systems are very complex and are not conveniently accessible to various genetic, molecular and biochemical approaches. The fact that influenza remains an unsolved problem, in combination with the limitations of the conventional experimental models, motivated increasing attempts to use the power of other models, such as low eukaryotes, including invertebrate, and primary cell cultures. In this review, we summarized the efforts to study influenza in yeast, Drosophila, zebrafish and primary human tissue cultures and the major contributions these studies have made toward a better understanding of the disease. We feel that these models are still under-utilized and we highlight the unique potential each model has for better comprehending virus−host interactions and viral protein function.

Ze339, an herbal extract from Petasites hybridus leaves is effective in treatment of allergic rhinitis by inhibition of a local production of IL-8 and eicosanoid LTB4 in allergen-challenged patients. However, the mechanism of action and anti-inflammatory potential in virally induced exacerbation of the upper airways is unknown. This study investigates the anti-inflammatory mechanisms of Ze339 on primary human nasal epithelial cells (HNECs) upon viral, bacterial and pro-inflammatory triggers. To investigate the influence of viral and bacterial infections on the airways, HNECs were stimulated with viral mimics, bacterial toll-like-receptor (TLR)-ligands or cytokines, in presence or absence of Ze339. The study uncovers Ze339 modulated changes in pro-inflammatory mediators and decreased neutrophil chemotaxis as well as a reduction of the nuclear translocation and phosphorylation of STAT molecules. Taken together, this study suggests that phyto drug Ze339 specifically targets STAT-signalling pathways in HNECs and has high potential as a broad anti-inflammatory drug that exceeds current indication. © 2016 BioFactors, 43(3):388-399, 2017 [ABSTRACT FROM AUTHOR]

Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic sinonasal inflammatory disease characterized histologically by hyperplastic nasal epithelium and epithelial cells proliferation. Cysteine-rich angiogenic inducer 61 (CYR61) acts as a positive regulator of cell cycle process. Cyclin D1 (CCND1) and c-Myc play key roles in the processes of cell cycle and cell growth. The purpose of our research was to explore the expression and roles of CYR61, CCND1 and c-Myc in CRSwNP. FeaturePlot and vlnPlot functions embedded in the seurat package (version 4.1.1) of R software (version 4.2.0) were applied to explore the cellular distribution of CYR61, CCND1 and c-Myc in the single-cell RNA sequencing (scRNA-seq) dataset of nasal tissue samples. CYR61, CCND1 and c-Myc immunolabeling and mRNA levels in nasal tissue samples were assessed by immunohistochemistry and real-time PCR. Co-localization of CYR61, CCND1 and c-Myc with basal epithelial cell marker P63 was assayed using double-label immunofluorescence staining. Furthermore, we collected and cultured human nasal epithelial cells (HNEC) to assess the regulation and role of CYR61 in vitro study. CYR61, CCND1 and c-Myc were primarily expressed by nasal epithelial cells. Significant upregulation of CYR61, CCND1 and c-Myc positive cells and increased levels of CYR61, CCND1 and c-Myc mRNA were found in nasal polyps in comparison to control samples. Of note, CYR61 mRNA and protein levels were altered by SEB, LPS, IFN-γ, IL-13, IL-17A and TGF-β1 in HNEC. In addition, CYR61 intervention could increase CCND1 and c-Myc mRNA and protein levels to promote HNEC proliferation, and siRNA against ITGA2 (si-ITGA2) could reverse CYR61 induced upregulation of CCND1 and c-Myc mRNA and protein levels in HNEC and cell proliferation of HNEC. CYR61, CCND1 and c-Myc were primarily expressed by epithelial cells in nasal mucosa. CYR61, CCND1 and c-Myc expression levels were increased in CRSwNP compared with controls. CYR61 could interact with ITGA2 to enhance HNEC proliferation via upregulating CCND1 and c-Myc levels in the HNEC, leading to hyperplastic nasal epithelium in CRSwNP. [Display omitted] • Increased levels of CYR61, CCND1 and c-Myc expression were found in nasal polyps. • CYR61, CCND1 and c-Myc were primarily expressed by HNEC. • CYR61 expression in HNEC could be altered by SEB, LPS and various cytokines. • CYR61 could upregulate CCND, c-Myc and cellular proliferation levels in HNEC. • CYR61 could interact with ITGA2 to enhance HNEC proliferation. [ABSTRACT FROM AUTHOR]

Objective: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1. Methods: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19. Results: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126. Conclusion: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration. [ABSTRACT FROM AUTHOR]