Hsiao Hui Ong

Human parainfluenza viruses (HPIVs) commonly cause croup and lower respiratory tract infections in young children. Despite typically self‐limiting with mild symptoms, the innate immune responses to HPIVs remain poorly understood, especially across all four types in human nasal epithelial cells (hNECs), the primary infection site. This study aims to investigate and compare viral replication kinetics and host immune responses in hNECs infected with HPIV types 1 to 4.The hNECs were infected in vitro, and viral replication kinetics, mucociliary function, cell tropism, and innate immune responses were assessed over 72 h. The viral RNA and progeny of HPIV types 1 to 4 were detected by 8 h postinfection (hpi). All HPIV types predominantly targeted ciliated cells of which a significant proportion displayed a unique apical distribution. HPIV‐3 generated the highest level of progeny virus and could infect a fraction of nasal goblet cells. RIG‐I and MDA5 expression was delayed during HPIV‐2 and HPIV‐3 infection (24 hpi), whereas HPIV‐1 and HPIV‐4 induced gradual upregulation from 8 hpi. Minimal upregulation of IFN‐α1 mRNA was induced across all HPIV types. Expression of type I IFN‐β was generally subdued during the earlier stage of infection (24 to 48 hpi) with the four HPIV types. Notably, IFN‐β mRNA expression was significantly elevated for HPIV‐1 infection at 48–72 hpi, whereas only a small increase was observed at 72 hpi for the other HPIV types. Although expression of type III IFN‐λ1 was similarly delayed for all four HPIV types during the earlier stage of infection, a greater upregulation of IFN‐λ1 was detected at 48–72 hpi for all HPIV types (especially HPIV‐1) as compared to type I IFN expression. For HPIV‐1 and HPIV‐4, mRNA expression of CXCL10 and MX1 appeared as early as 8 hpi and gradually increased until 72 hpi. However, for HPIV‐2 and HPIV‐3, mRNA expression of CXCL10 and MX1 was delayed at 8–16 hpi, but subsequently increased significantly at 24–72 hpi. In comparison, IFITM1 mRNA was expressed at relatively weaker levels for the four HPIV types. In conclusion, all four HPIV types exhibit high infectivity and replicative capacity in hNECs while initially evading interferon responses to varying extents. Their innate immune modulation appears to be type‐specific, potentially influencing viral virulence, pathogenesis, and progression of airway disease in high‐risk patients.

Background: Club cells, originating from basal cells, can differentiate into ciliated or goblet cells in the lower respiratory tract, but their role in nasal epithelium during chronic rhinosinusitis with nasal polyps (CRSwNP) is not well understood. We aim to investigate the role of club cells in CRSwNP and their biological mechanism in an IL‐13‐induced inflammation model of human nasal epithelial cells (HNECs). Methods: CRSwNP was classified into four endotypes (N ⁻ E ⁻ , N ⁺ E ⁻ , N ⁻ E ⁺ , and N ⁺ E ⁺ ) based on neutrophil and eosinophil infiltration. The expression of basal (TP63 ⁺ ), club (SCGB1A1 ⁺ ), ciliated (βIV‐tubulin ⁺ ), and goblet cells (MUC5AC ⁺ ) was assessed in healthy controls ( n = 23) and CRSwNP ( n = 73) using RT‐qPCR, western blot (WB), and immunofluorescence. The effects of IL‐13, Dupilumab, and STAT6 inhibitor (AS1517499) on cell differentiation were investigated in HNECs ( n = 9) through RT‐qPCR, WB, and flow cytometry (FCM). Results: Club and ciliated cells significantly decreased in four endotypes of CRSwNP (all p < 0.001), while basal and goblet cells increased in neutrophilic and eosinophilic CRSwNP, respectively (all p < 0.05). In vitro, IL‐13‐induced significant cilia loss and MUC5AC secretion compared to IFN‐γ and IL‐17A (all p < 0.05), and FCM revealed a shift from SCGB1A1 ⁺ βIV‐tubulin ⁺ to SCGB1A1 ⁺ MUC5AC ⁺ cells by IL‐13‐treated (all p < 0.05). Furthermore, Dupilumab mitigated IL‐13‐induced changes more effectively than AS1517499 (all p < 0.05). Conclusions: Club cells are crucial in nasal epithelial repair, which is notably compromised in CRSwNP. This impairment is highlighted in an IL‐13‐induced inflammation model of nasal epithelium and further underscored by the therapeutic effect of Dupilumab in restoring epithelial repair mechanisms.

The nasal epithelium is the primary site for entry of respiratory viruses. In comparison to oral administration, nasal drug applications directed locally to the site of infection can serve as early interventional barriers against respiratory virus pathogenesis by limiting viral spread in the upper airway. Experiments on the diffusion of methylene blue and nanoparticles in both water and low pH conditions revealed that hydroxypropyl methylcellulose (HPMC) can act as an effective physical barrier. This study also evaluated the activity of HPMC as a barrier against common respiratory viruses, i.e., rhinovirus (RV) and influenza A virus (IAV) using the in vitro human nasal epithelial cell (hNEC) model. Utilizing the hNEC infection model, we assessed the protective effects of HPMC in pH 3.5 and pH 7 buffers against RV and IAV. Acidic and pH-neutral buffers and HPMC dissolved in acidic and pH-neutral buffers were administered for 4 h prior to virus infection and at 4 h post-infection (hpi). The apical supernatant was harvested at 24 hpi to determine the viral loads of RV and IAV (H1N1 and H3N2). HPMC was demonstrated to exert protective effects in the infected hNECs independent of acidic pH. Pre-treatment with HPMC in acidic buffer significantly diminished viral loads for both RV and IAV infections of hNECs. Similarly, direct treatment of HPMC in acidic buffer after infection (4 hpi) also effectively decreased viral loads of both RV and IAV. Moreover, treatment using HPMC in acidic buffer before or after infection did not affect the epithelial integrity and ciliary function of hNECs. This study demonstrates the protective effects of HPMC in acidic buffer against RV and IAV infections of the human nasal epithelium.

Tight junction (TJ) proteins establish a physical barrier between epithelial cells, playing a crucial role in maintaining tissue homeostasis by safeguarding host tissues against pathogens, allergens, antigens, irritants, etc. Recently, an increasing number of studies have demonstrated that abnormal expression of TJs plays an essential role in the development and progression of inflammatory airway diseases, including chronic obstructive pulmonary disease, asthma, allergic rhinitis, and chronic rhinosinusitis (CRS) with or without nasal polyps. Among them, CRS with nasal polyps is a prevalent chronic inflammatory disease that affects the nasal cavity and paranasal sinuses, leading to a poor prognosis and significantly impacting patients' quality of life. Its pathogenesis primarily involves dysfunction of the nasal epithelial barrier, impaired mucociliary clearance, disordered immune response, and excessive tissue remodeling. Numerous studies have elucidated the pivotal role of TJs in both the pathogenesis and response to traditional therapies in CRS. We therefore to review and discuss potential factors contributing to impair and repair of TJs in the nasal epithelium based on their structure, function, and formation process.

Our previous study revealed that prolonged human rhinovirus (HRV) infection rapidly induces antiviral interferons (IFNs) and chemokines during the acute stage of infection. It also showed that expression levels of RIG-I and interferon-stimulated genes (ISGs) were sustained in tandem with the persistent expression of HRV RNA and HRV proteins at the late stage of the 14-day infection period. Some studies have explored the protective effects of initial acute HRV infection on secondary influenza A virus (IAV) infection. However, the susceptibility of human nasal epithelial cells (hNECs) to re-infection by the same HRV serotype, and to secondary IAV infection following prolonged primary HRV infection, has not been studied in detail. Therefore, the aim of this study was to investigate the effects and underlying mechanisms of HRV persistence on the susceptibility of hNECs against HRV re-infection and secondary IAV infection. We analyzed the viral replication and innate immune responses of hNECs infected with the same HRV serotype A16 and IAV H3N2 at 14 days after initial HRV-A16 infection. Prolonged primary HRV infection significantly diminished the IAV load of secondary H3N2 infection, but not the HRV load of HRV-A16 re-infection. The reduced IAV load of secondary H3N2 infection may be explained by increased baseline expression levels of RIG-I and ISGs, specifically MX1 and IFITM1, which are induced by prolonged primary HRV infection. As is congruent with this finding, in those cells that received early and multi-dose pre-treatment with Rupintrivir (HRV 3C protease inhibitor) prior to secondary IAV infection, the reduction in IAV load was abolished compared to the group without pre-treatment with Rupintrivir. In conclusion, the antiviral state induced from prolonged primary HRV infection mediated by RIG-I and ISGs (including MX1 and IFITM1) can confer a protective innate immune defense mechanism against secondary influenza infection.

Huang ZQ, et al. Allergy Asthma Immunol Res. 2023 Jul;15(4):512-525. https://doi.org/10.4168/aair.2023.15.4.512

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.

Background: Nasal inverted papilloma (NIP) is defined based on its histological characteristic of inverted epithelium growth into the stroma. The inversion can result in epithelial growth in the underlying connective tissue stroma when the basement membrane completely separates from the epithelial layer. To date, such inversion mechanism underlying NIP’s pathological phenomenon is unknown. Therefore, we hypothesized that mediators and soluble proteins released by neutrophils, the most predominant infiltrating cells in NIP, is vital in causing the epithelial changes and pathogenesis of NIP. Methods: We collected 37 NIP tissues from patients who underwent surgical removal of NIP and performed hematoxylin–eosin (HE), immunohistochemical, and immunofluorescence staining to analyze in-depth the basic characteristics of NIP, including detecting the expression and distribution of MMPs and associated factors in NIP. Western blotting and quantitative real-time PCR were further performed to analyze the protein and mRNA expression levels of specific factors including MMPs, HIF-1α, and tissue inhibitors of metalloproteinases (TIMPs). Results: We observed finger-like projections that insert into the epithelium in NIP tissue as its main characteristics. The projections contain fibroblasts, extracellular matrix, capillaries, and infiltrating inflammatory cells. We found abundant neutrophils clustered at the finger-like projection of NIP, and also noted MMP-1 and MMP-9 were up-regulated in NIP (p< 0.05), whereas TIMP-1/3 was decreased. The expression level of HIF-1α was also found to be increased in NIP tissue. We further showed that MMP-9 and HIF-1α were mainly expressed by neutrophils and were predominantly observed in the finger-like projections that contribute to the NIP pathology. Conclusion: Upregulation and release of MMP-9 and HIF-1α from infiltrating neutrophils may cause damage to the epithelial basement membrane and epithelial clefts, forming finger-like projections with angiogenesis and fibroblasts insertion, resulting in epithelial growth in the tissue stroma, a typical histo-pathological characteristic in NIP.