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

National University Health System

Hashimoto's thyroiditis was recently divided into IgG4-plasma cell-rich and IgG4-plasma cell-poor subtypes. The former, also known as IgG4 thyroiditis, is associated with clinical, serological, sonographic and morphological features that are distinctive from those of the non-IgG4 subgroup. We describe an interesting case of IgG4-positive mucosa-associated lymphoid tissue (MALT) lymphoma arising in a background of IgG4 thyroiditis.

IgG4-positive extranodal marginal zone lymphoma arising in Hashimoto's thyroiditis: clinicopathological and cytogenetic features of a hitherto undescribed condition.

Post-radiotherapy carotid blowout syndrome (CBS) of the skull base is a rare but often catastrophic complication of head and neck malignancies. The existing literature on the treatment of this condition with flow-diverting devices (FDD) is extremely limited and disappointing. We present a case of impending CBS in a patient previously irradiated for nasopharyngeal cancer that was successfully treated with use of multiple FDDs, adjunctive endonasal packing and delayed reinforcement with pedicled naso-septal flap, yielding an excellent outcome at 14-months follow-up. Notwithstanding the discouraging results in literature, our anecdotal experience suggests that endovascular reconstruction using FDD could be an option with long-term viability in post-radiotherapy CBS involving the skull base when reinforced with a vascularised naso-septal flap.

Flow-diverter in radiation-induced skull base carotid blowout syndrome: do not write it off!

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.

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.

Downregulation and Aberrant Localization of Forkhead Box J1 in Allergic Nasal Mucosa.

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.

Upper airway inflammatory diseases are associated with abnormal expression of nasal epithelial forkhead-box J1 (FOXJ1) which regulates motile cilia formation. We sought to investigate whether aberrant FOXJ1 localizations correlate with the disease severity and the co-existence of allergic rhinitis (AR) or asthma in patients with nasal polyps (NPs).

Aberrant localization of FOXJ1 correlates with the disease severity and comorbidities in patients with nasal polyps.

Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract, and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1, the roles of other mucins are still poorly understood, especially in viral infections.

Upregulation of cell-surface mucin MUC15 in human nasal epithelial cells upon influenza A virus infection.

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.

RNA Sequencing of H3N2 Influenza Virus-Infected Human Nasal Epithelial Cells from Multiple Subjects Reveals Molecular Pathways Associated with Tissue Injury and Complications.

Our understanding of the pathophysiology of chronic rhinosinusitis (CRS) is continuously evolving. The traditional description of CRS in terms of two phenotypes based on the presence or absence of nasal polyps belies the underlying intricate immunopathophysiological processes responsible for this condition. CRS is being increasingly recognized as a disease spectrum encompassing a range of inflammatory states in the sinonasal cavity, with non-type 2 inflammatory disease on one end, type 2 inflammatory, eosinophil-heavy disease on the other and an overlap of both in different proportions in between. Abundance in research on the immune mechanisms of CRS has revealed various new endotypes that hold promise as biomarkers for the development of targeted therapies in severe, uncontrolled CRS. The introduction of precision medicine to manage this chronic, complex condition is a step forward in providing individualized care for all patients with CRS. In this review, the latest research on the pathophysiology of CRS with a focus on potential novel biomarkers and treatment options over the last 2 years are summarized and integrated into a suggested model of precision medicine in CRS.

Novel findings in immunopathophysiology of chronic rhinosinusitis and their role in a model of precision medicine.

 Nasal polyp (NP) is a chronic upper airway inflammatory disease that is frequently triggered by defective host-defense. However, the mechanisms underlying the impaired barrier function such as cilia-mediated mucociliary clearance remain poorly understood.  To assess ciliary ultrastructural and ciliogenesis marker expression and the phenotypes of ciliated cells in NP.  NP biopsy samples were obtained from 97 NP patients and inferior turbinate from 32 healthy controls. Immunofluorescence staining, quantitative polymerase chain reaction, and single-cell cytospin staining were performed. We classified the patterns of radial spoke head protein () 1, 4A (), 9 (), and dynein axonemal heavy chain 5 () localization. A semi-quantitative scoring system was developed to assess their expression patterns and associations with ciliogenesis markers [centrosomal protein 110 () and forkhead box j1 ()].  Median scores of , ,  and  were significantly higher in NP than in healthy controls, particularly in eosinophilic NPs. Expression pattern scores of , ,  and  correlated positively with each other in both groups. In primary-cell specimens, abnormal expression patterns were significantly more common in NP. The total fluorescence intensity of  and  was significantly higher in NPs and correlated positively with expression pattern scores of , ,  and . A trend towards lengthened cilia was observed in NP.  In the chronic airway inflammatory milieu, the up-regulated ciliogenesis correlates with the abnormal expression of ciliary ultrastructural markers (i.e., ) in NP (particularly eosinophilic NP).

An Integrated Analysis of Radial Spoke Head and Outer Dynein Arm Protein Defects and Ciliogenesis Abnormality in Nasal Polyps.

The coronavirus disease (COVID-19) pandemic has led to a global struggle to cope with the sheer numbers of infected persons, many of whom require intensive care support or eventually succumb to the illness. The outbreak is managed by a combination of disease containment via public health measures and supportive care for those who are affected. To date, there is no specific anti-COVID-19 treatment. However, the urgency to identify treatments that could turn the tide has led to the emergence of several investigational drugs as potential candidates to improve outcome, especially in the severe to critically ill. While many of these adjunctive drugs are being investigated in clinical trials, professional bodies have attempted to clarify the setting where the use of these drugs may be considered as off-label or compassionate use. This review summarizes the clinical evidence of investigational adjunctive treatments used in COVID-19 patients as well as the recommendations of their use from guidelines issued by international and national organizations in healthcare.

Role of adjunctive treatment strategies in COVID-19 and a review of international and national clinical guidelines.

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.

Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.

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
IgG4-positive extranodal marginal zone lymphoma arising in Hashimoto's thyroiditis: clinicopathological and cytogenetic features of a hitherto undescribed condition. Histopathology\| PubMed ID: 26332259	2016
Flow-diverter in radiation-induced skull base carotid blowout syndrome: do not write it off! Neurosurgical review\| PubMed ID: 28712070	2017
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
Downregulation and Aberrant Localization of Forkhead Box J1 in Allergic Nasal Mucosa. International archives of allergy and immunology\| PubMed ID: 29635245	2018
H3N2 influenza virus infection enhances oncostatin M expression in human nasal epithelium. Experimental cell research\| PubMed ID: 30142324	2018
Aberrant localization of FOXJ1 correlates with the disease severity and comorbidities in patients with nasal polyps. Allergy, asthma, and clinical immunology : official journal of the Canadian Society of Allergy and Clinical Immunology\| PubMed ID: 30459817	2018
Upregulation of cell-surface mucin MUC15 in human nasal epithelial cells upon influenza A virus infection. BMC infectious diseases\| PubMed ID: 31307416	2019
RNA Sequencing of H3N2 Influenza Virus-Infected Human Nasal Epithelial Cells from Multiple Subjects Reveals Molecular Pathways Associated with Tissue Injury and Complications. Cells\| PubMed ID: 31461941	2019
Novel findings in immunopathophysiology of chronic rhinosinusitis and their role in a model of precision medicine. Allergy\| PubMed ID: 31512248	2020
An Integrated Analysis of Radial Spoke Head and Outer Dynein Arm Protein Defects and Ciliogenesis Abnormality in Nasal Polyps. Frontiers in genetics\| PubMed ID: 31798623	2019
Role of adjunctive treatment strategies in COVID-19 and a review of international and national clinical guidelines. Military Medical Research\| PubMed ID: 32370766	2020
Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles. PLoS pathogens\| PubMed ID: 33284849	2020
Host Antiviral Response Suppresses Ciliogenesis and Motile Ciliary Functions in the Nasal Epithelium. Frontiers in cell and developmental biology\| PubMed ID: 33409274	2020

Yew Kwang Ong

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