contact-free-co-culture-model-for-study-innate-immune-cell-activation

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

National University of Singapore

Rhinosinusitis (RS) is a symptomatic disease classification of many causes and is a major economic burden worldwide. It is widely accepted that RS is further classified into acute (ARS) and chronic (CRS) rhinosinusitis based on the duration of the symptoms, and that viral infection plays a large role in initiating or potentiating the disease. In this review, we examine the role of respiratory virus infection in the exacerbation of ARS and CRS. We explore the epidemiology of viral exacerbation of ARS and CRS and highlight key viruses that may cause exacerbation. We also review the current understanding of viral infections in the upper airway to further explain the putative underlying mechanisms of inflammatory events in ARS and CRS exacerbation. Advances in accurate diagnosis of the etiologic respiratory viruses of ARS and CRS symptoms which can lead to better disease management are also surveyed. In addition to the current treatments which provide symptomatic relief, we also explore the potential of harnessing existing antiviral strategies to prevent ARS and CRS exacerbation, especially with improved viral diagnostic tools to guide accurate prescription of antivirals against causative respiratory viruses.

Impact of Respiratory Virus Infections in Exacerbation of Acute and Chronic Rhinosinusitis.

Motile cilia impairment is a common condition in patients with chronically inflamed airways, such as is seen in nasal polyps (NPs). The mechanism underlying this pathogenic condition is complex and not fully understood.

Absence or mislocalization of DNAH5 is a characteristic marker for motile ciliary abnormality in nasal polyps.

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.

In Vitro Model of Fully Differentiated Human Nasal Epithelial Cells Infected With Rhinovirus Reveals Epithelium-Initiated Immune Responses.

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.

H3N2 influenza virus infection enhances oncostatin M expression in human nasal epithelium.

Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in  air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases.

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium.

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.

Long-term defects of nasal epithelium barrier functions in patients with nasopharyngeal carcinoma post chemo-radiotherapy.

Hypoxia-induced factor-1α induces NLRP3 expression by M1 macrophages in noneosinophilic chronic rhinosinusitis with nasal polyps.

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 p63Krt5 (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.

p63Krt5 basal cells are increased in the squamous metaplastic epithelium of patients with radiation-induced chronic Rhinosinusitis.

Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu and Gly This cleavage triggers N-glycine-mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullin complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and interleukin-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.

Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia.

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  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 λ and  but further increased the expression of  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.

Interleukin-13 Alters Tight Junction Proteins Expression Thereby Compromising Barrier Function and Dampens Rhinovirus Induced Immune Responses in Nasal Epithelium.

Role of yes-associated protein in interleukin-13 induced nasal remodeling of chronic rhinosinusitis with nasal polyps.

Respiratory viral infections are one of the main drivers of development and exacerbation for chronic airway inflammatory diseases. Increased viral susceptibility and impaired mucociliary clearance are often associated with chronic airway inflammatory diseases and served as risk factors of exacerbations. However, the links between viral susceptibility, viral clearance, and impaired mucociliary functions are unclear. Therefore, the objective of this study is to provide the insights into the effects of improper clearance of respiratory viruses from the epithelium following infection, and their resulting persistent activation of antiviral response, on mucociliary functions.

Article	Year
Impact of Respiratory Virus Infections in Exacerbation of Acute and Chronic Rhinosinusitis. Current allergy and asthma reports\| PubMed ID: 28389843	2017
Absence or mislocalization of DNAH5 is a characteristic marker for motile ciliary abnormality in nasal polyps. The Laryngoscope\| PubMed ID: 29148098	2018
In Vitro Model of Fully Differentiated Human Nasal Epithelial Cells Infected With Rhinovirus Reveals Epithelium-Initiated Immune Responses. The Journal of infectious diseases\| PubMed ID: 29228279	2018
H3N2 influenza virus infection enhances oncostatin M expression in human nasal epithelium. Experimental cell research\| PubMed ID: 30142324	2018
Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium. Frontiers in cell and developmental biology\| PubMed ID: 32161756	2020
Long-term defects of nasal epithelium barrier functions in patients with nasopharyngeal carcinoma post chemo-radiotherapy. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology\| PubMed ID: 32353641	2020
Hypoxia-induced factor-1α induces NLRP3 expression by M1 macrophages in noneosinophilic chronic rhinosinusitis with nasal polyps. Allergy\| PubMed ID: 32854144	2021
p63Krt5 basal cells are increased in the squamous metaplastic epithelium of patients with radiation-induced chronic Rhinosinusitis. Radiation oncology (London, England)\| PubMed ID: 32977822	2020
Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia. Science (New York, N.Y.)\| PubMed ID: 33093214	2020
Interleukin-13 Alters Tight Junction Proteins Expression Thereby Compromising Barrier Function and Dampens Rhinovirus Induced Immune Responses in Nasal Epithelium. Frontiers in cell and developmental biology\| PubMed ID: 33102478	2020
Role of yes-associated protein in interleukin-13 induced nasal remodeling of chronic rhinosinusitis with nasal polyps. Allergy\| PubMed ID: 33301614	2021
Host Antiviral Response Suppresses Ciliogenesis and Motile Ciliary Functions in the Nasal Epithelium. Frontiers in cell and developmental biology\| PubMed ID: 33409274	2020

Hsiao Hui Ong

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