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Mucus secretion and its composition are vital in the maintenance of airway health, among which hypoxia-inducible factors (HIFs) are thought to be involved in the regulation of mucin synthesis and regulation. Nasal mucus composition difference between healthy individuals and chronic rhinosinusitis (CRS) patients may contribute to the pathology of chronic nasal diseases, but so far, their role has yet to be completely understood. Nasal biopsy specimens were obtained from 24 healthy subjects and 99 patients with CRS without (CRSsNP, n=36) or with (CRSwNP, n=63) nasal polyps. Immunohistochemical (IHC) and immunofluorescent (IF) staining, quantitative real-time PCR, and western blot were performed to compare the nasal mucus composition between the subjects. Areas of the serous gland and mucous gland were both significantly increased in CRSsNP patients. In CRSwNP patients, a decrease in submucosal gland density and a marked increase in goblet cells were observed. The major gel-forming mucins in the sinonasal mucosa of CRSsNP and CRSwNP are MUC5B and MUC5AC respectively. Mucous cells are found in a higher proportion in both CRSsNP and CRSwNP. The proportion of MUC5AC-positive goblet cells was increased in CRSwNP. The mRNA level of HIF-2α was significantly increased in CRS, and both HIF-1α and HIF-2α were expressed in serous cell but not mucous cell. Over secretion and altered composition of mucus are observed in sinonasal mucosa of CRS, which was mainly associated with glandular hyperplasia in CRSsNP and goblet cell hyperplasia in CRSwNP. Mucus abnormality compromised both non-specific and specific antimicrobial capabilities in the sinonasal mucosa. HIF expression may contribute to differences in mucin synthesis and serous gland regulation, which needs further investigation to understand the pathology of CRS., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells., Competing Interests: The authors declare no conflict of interest.

The human nasal epithelium is the primary site of exposure to influenza virus, the initiator of host responses to influenza and the resultant pathologies. Influenza virus may cause serious respiratory infection resulting in major complications, as well as severe impairment of the airways. Here, we elucidated the global transcriptomic changes during H3N2 infection of human nasal epithelial cells from multiple individuals. Using RNA sequencing, we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. We used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. Statistical analysis highlighted strong transcriptomic signatures significantly associated with 24 and 48 h after infection, but not at the earlier 8-h time point. In particular, we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling, B-cell signaling, apoptosis, necrosis, smooth muscle proliferation, and metabolic alterations. These molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway, and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management.

Background: We established an in vitro co-culture model involving H3N2-infection of human nasal epithelium with peripheral blood mononuclear cells (PBMC) to investigate their cross-talk during early H3N2 infection. Methods: Nasal epithelium was differentiated from human nasal epithelial stem/progenitor cells and cultured wtih fresh human PBMC. PBMC and supernatants were harvested after 24 and 48 h of co-culture with H3N2-infected nasal epithelium. We used flow cytometry and Luminex to characterize PBMC subpopulations, their activation and secretion of cytokine and chemokines. Results: H3N2 infection of the nasal epithelium associated with significant increase in interferons (IFN-α, IFN-γ, IL-29), pro-inflammatory cytokines (TNF-α, BDNF, IL-3) and viral-associated chemokines (IP-10, MCP-3, I-TAC, MIG), detectable already after 24 h. This translates into rapid activation of monocytes, NK-cells and innate T-cells (MAIT and γδ T cells), evident with CD38+ and/or CD69+ upregulation. Conclusions: This system may contribute to in vitro mechanistic immunological studies bridging systemic models and possibly enable the development of targeted immunomodulatory therapies.

Tight junctions (TJs) alteration is commonly seen in airway inflammatory diseases. Oncostatin M (OSM) is an inflammatory mediator associated with chronic rhinosinusitis with nasal polyps (CRSwNP). We have previously shown that human nasal epithelial cells (hNECs) are highly permissive cells for influenza A virus (IAV). However, its role in TJs alteration and the effects of IAV on inducing OSM expression in nasal epithelium remains to be further investigated. In this study, OSM and TJs expression was measured and compared between inferior turbinate from healthy controls and nasal polyps from CRSwNP. Additionally, hNECs cultured at air-liquid interface (ALI) were infected with H3N2 influenza virus to study the role of influenza virus in inducing epithelial OSM expression as a possible means of exacerbation. The expression of ZO-1, claudin-1, and occludin was markedly decreased and correlated negatively with that of OSM in CRSwNP. By using the in vitro hNEC model, H3N2 infection resulted in significantly increased OSM expression (2.2-, 4.7- and 3.9-fold higher at 8, 24, and 48 h post-infection vs. mock infection). Furthermore, OSM is found to co-localize with ciliated and goblet cells in hNECs infected with H3N2 influenza virus. Our findings demonstrated that increased OSM expression is implicated in CRSwNP as a possible mechanism of TJs' impairment, which can be further augmented following influenza infection via epithelial OSM expression, possibly contributing to exacerbations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

Background: Forkhead box J1 (FOXJ1) plays pivotal roles in motile cilia formation. However, it remains unclear whether abnormal expression or localization of FOXJ1 in nasal mucosa tissues is associated with allergic rhinitis (AR), in which impaired mucociliary clearance is implicated., Objective: We sought to investigate the expression and localization of FOXJ1 in inferior turbinate from patients with AR and controls., Methods: We assayed mRNA levels of FOXJ1, DNAI1, DNALI1, and DNAH9 by using whole-genome expression array and quantitative real-time polymerase chain reaction. We elucidated the localization of FOXJ1 by using immunofluorescence assays in paraffin sections and primary single cells. Four patterns of FOXJ1 localization (normal, N; intermediate, I; mislocalization, M; absence, A) were defined. We developed a semiquantitative scoring system to elucidate their localization in 5 areas per paraffin section, with individual sections being assigned a score between 0 and 2., Results: The mRNA levels of FOXJ1, DNAI1, DNALI1, and DNAH9 were significantly reduced in patients with AR compared with controls (all p < 0.05). The median (1st and 3rd quartile) of the FOXJ1 score was 0.4 (0.0 and 0.85) in patients with AR, and 0.2 (0.0 and 0.4) in controls (p < 0.05). For primary cytospin samples, the mean percentages of FOXJ1 localization patterns N, I, M, and A were 46.7, 10.0, 30.0, and 26.7% in patients with AR, and 82.5, 5.0, 5.0, and 7.5% in controls, respectively (p < 0.05)., Conclusion: Downregulation and aberrant localization of FOXJ1 may be crucial characteristics of the allergic nasal mucosa., (© 2018 S. Karger AG, Basel.)

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

Background: Epithelial cytokines including IL-25, IL-33 and thymic stromal lymphopoietin (TLSP) are recently established as drivers of type 2 chronic inflammatory diseases such as chronic rhinosinusitis with nasal polyps (CRSwNP). Here, we further confirmed the increased expression of IL-25 in CRSwNP and investigated potential contributors of IL-25 in CRSwNP epithelium. Methods: Sixty CRSwNP, 25 CRSsNP and 15 healthy control tissues were examined for IL-25 expression and for the accompanying type 2 inflammatory cytokines. We then tested different respiratory virus infections on human nasal epithelial cells (hNECs) for their ability to trigger IL-25 expression. In addition, we subjected hNECs generated from CRSwNP tissues to pretreatment with recombinant interferon-alpha (IFN-α) prior to viral infection to evaluate IFN effects on IL-25 induction. Results: We confirmed that significantly enhanced levels of IL-25 were observed in CRSwNP tissues, and that IL-25 expression correlated with type 2 inflammatory cytokine expression. In vitro, we observed significantly elevated IL-25 in hNECs infected with influenza A virus as early as 24 hours post-infection (hpi), regardless of tissue origin, and IL-25 correlated positively with viral load. While other respiratory viruses exhibited increasing trends of IL-25, these were not significant at the time-points tested. IFN-α treatment of CRSwNP epithelium was found to exert bimodal effects, ie IFN-α treatment alone induced moderate IL-25 expression, whereas IFN-α pretreatment of hNECs before influenza infection significantly diminished IL-25 induction by active influenza virus infection. Conclusion: We have authenticated the observation of elevated IL-25 in CRSwNP, which is correlated with type 2 inflammatory cytokines. Notably, we identified influenza virus infection as a potential contributor of IL-25 in both control and CRSwNP epithelium during active infection. This IL-25 induction can be abated by IFN-α pretreatment which ameliorated active influenza infection. Trial Registration: Chictr.org.cn ChiCTR-BON-16010179, Registered 18 December 2016, http://www.chictr.org.cn/showproj.aspx?proj=17331. The authors agree on the sharing of deidentified participant data where it pertains to request directly related to the data in this article when contacted (Haiyu Hong; [email protected]). [ABSTRACT FROM AUTHOR]

Background: Squamous metaplasia (SM) is an irreversible form of airway epithelial remodeling. Hyperproliferation of basal cells was observed in squamous metaplastic epithelium of chronically inflamed airway. However, the association of such aberrant proliferation of basal cells with SM in the nasal epithelium after radiation damage remains unclear. The aim of this study was to investigate SM and accompanying levels of p63+Krt5+ (basal cell markers) cells in the nasal epithelium of patients with radiation-induced chronic rhinosinusitis (CRSr) and patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared to healthy controls.Methods: We assessed the prevalence of SM and the expression of p63+, Krt5+, p63+Krt5+, and Ki67+ cells through immunofluorescence(IF) staining of the inferior turbinate (IT) tissues from patients with CRSr (n = 36), CRSsNP (n = 33) and controls (n = 28).Results: The prevalence of SM and the number of p63+Krt5+ cells were both significantly increased in patients with CRSr compared to patients with CRSsNP and controls. The number of Ki67+ cells were both significantly increased in patients with CRSr and CRSsNP compared to controls, but the ratio of Ki67+ cells to p63+Krt5+ cells was significantly lower in patients with CRSr compared to patients with CRSsNP. In patients with CRSr, an increased number of p63+Krt5+ basal cells was observed in SM epithelium compared to non-SM epithelium.Conclusion: SM is increased in the nasal epithelium of patients with CRSr, in which aberrant levels of p63+Krt5+ basal cells serves as an important pathologic feature in the squamous metaplastic epithelium. [ABSTRACT FROM AUTHOR]

• Chemo-radiotherapy induces long-term defects in nasal epithelial barrier functions. • The defects in nasal epithelium cause sinonasal mucosal infection and inflammation. • Damage of nasal epithelial stem/progenitor cells were found in barrier dysfunction. • Promote nasal epithelial stem/progenitor cells may contribute to recover the barrier. Chronic and recurrent upper respiratory tract infection and inflammation is common in patients with nasopharyngeal carcinoma (NPC) post chemo-radiotherapy (CRT). Whether it is due to intrinsic (e.g., host-defense mechanisms of the epithelium), epigenetic or extrinsic factors is not fully understood. Tissue biopsies of the middle turbinate (MT) and inferior turbinate (IT) from NPC patients after CRT (mean of 3 years, n = 39) were compared with the IT biopsies from healthy subjects (n = 44). The epithelial ultrastructure was examined by transmission electron microscope (TEM). mRNA and protein expressions of epithelial stem/progenitor cells markers, as well markers of cell proliferation and differentiation markers was analyzed. Abnormal epithelial architecture was observed in all tissue samples of NPC patients. Significantly decreased expression levels of mRNA and protein levels for p63 (basal cells), Ki67 (cell proliferation), p63+/KRT5+ (epithelial stem/progenitor cells), MUC5AC and MUC5B (secretary proteins from goblet cells), alpha-tubulin, beta-tubulin and TAp73 (ciliated cells), DNAH5 and DNAI1 and RSPH4A (microtubule assemblies of motile cilia), FOXJ1 and CP110 (ciliogenesis-associated markers) were evident in MT and IT biopsies from NPC patients when compared to healthy controls. CRT causes long-term defects of epithelial barrier functions and increases the susceptibility of these patients to upper respiratory tract infection and inflammation. [ABSTRACT FROM AUTHOR]