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Influenza H5N1 and H1N1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation.

Chan RW, Yuen KM, Yu WC, Ho CC, Nicholls JM, Peiris JS, Chan MC - PLoS ONE (2010)

Bottom Line: We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells.The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells.Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

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The (A) IFN-β, (B) RANTES and (C) IP-10 gene expression of the ud- and wd-NHBE cells at 6 h and 24 h post infection of HK98/H1N1 and VN04/H5N1.The chart showed the mean and the standard error from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01 and triple asterisks indicated statistically significant differences of means with p<0.001.
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pone-0008713-g006: The (A) IFN-β, (B) RANTES and (C) IP-10 gene expression of the ud- and wd-NHBE cells at 6 h and 24 h post infection of HK98/H1N1 and VN04/H5N1.The chart showed the mean and the standard error from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01 and triple asterisks indicated statistically significant differences of means with p<0.001.

Mentions: We next investigated the influence of the differentiation state of NHBE cells on the cytokine and chemokine responses induced by HK98/H1N1 and VN04/H5N1 viruses. Specifically, we wanted to determine whether the difference in NHBE cell differentiation (ud-NHBE Vs wd-NHBE) led to qualitative or quantitative differences in the profile of cytokines induced. Cytokine (TNF-α, IFN-β) and chemokine (RANTES, IP-10) gene expression profiles were evaluated by qPCR. TNF-α mRNA was not expressed in either mock or both influenza A virus (HK98/H1N1 and VN04/H5N1) infected ud- and wd-NHBE cells (data not shown). In general, IFN-β, RANTES and IP-10 gene was induced following infection with influenza H1N1 and H5N1 virus in both the ud-NHBE and wd-NHBE cells. VN04/H5N1 virus led to significantly higher IFN-β (Figure 6A, left panel) and IP-10 (Figure 6C, left panel) mRNA expression in ud-NHBE cells compared with HK98/H1N1 or mock infection at both 6 h and 24 h post infection. VN04/H5N1 virus induced a significantly higher RANTES expression than HK98/H1N1 virus infected cells at 6 h post infection but not at 24 hours post infection (Figure 6B).


Influenza H5N1 and H1N1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation.

Chan RW, Yuen KM, Yu WC, Ho CC, Nicholls JM, Peiris JS, Chan MC - PLoS ONE (2010)

The (A) IFN-β, (B) RANTES and (C) IP-10 gene expression of the ud- and wd-NHBE cells at 6 h and 24 h post infection of HK98/H1N1 and VN04/H5N1.The chart showed the mean and the standard error from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01 and triple asterisks indicated statistically significant differences of means with p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806912&req=5

pone-0008713-g006: The (A) IFN-β, (B) RANTES and (C) IP-10 gene expression of the ud- and wd-NHBE cells at 6 h and 24 h post infection of HK98/H1N1 and VN04/H5N1.The chart showed the mean and the standard error from three independent experiments. Single asterisk indicated statistically significant difference of means with p<0.05, double asterisks indicated statistically significant differences of means with p<0.01 and triple asterisks indicated statistically significant differences of means with p<0.001.
Mentions: We next investigated the influence of the differentiation state of NHBE cells on the cytokine and chemokine responses induced by HK98/H1N1 and VN04/H5N1 viruses. Specifically, we wanted to determine whether the difference in NHBE cell differentiation (ud-NHBE Vs wd-NHBE) led to qualitative or quantitative differences in the profile of cytokines induced. Cytokine (TNF-α, IFN-β) and chemokine (RANTES, IP-10) gene expression profiles were evaluated by qPCR. TNF-α mRNA was not expressed in either mock or both influenza A virus (HK98/H1N1 and VN04/H5N1) infected ud- and wd-NHBE cells (data not shown). In general, IFN-β, RANTES and IP-10 gene was induced following infection with influenza H1N1 and H5N1 virus in both the ud-NHBE and wd-NHBE cells. VN04/H5N1 virus led to significantly higher IFN-β (Figure 6A, left panel) and IP-10 (Figure 6C, left panel) mRNA expression in ud-NHBE cells compared with HK98/H1N1 or mock infection at both 6 h and 24 h post infection. VN04/H5N1 virus induced a significantly higher RANTES expression than HK98/H1N1 virus infected cells at 6 h post infection but not at 24 hours post infection (Figure 6B).

Bottom Line: We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells.The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells.Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

Show MeSH
Related in: MedlinePlus