Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Abstract

Background: Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim: To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods: We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results: We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion: The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.

Figure 1

Representation of the transwell insert-setup during the influenza virus infection experiment. Type I-like alveolar epithelial cells or HLMVEs (indicated in blue) were seeded on top of the porous membrane. During apical infection (A) virus was added into the apical compartment while the transwell was placed upside down during basolateral infection (B).

Figure 2

Lectin binding assay to determine the Sias distribution on polarized type I-like alveolar epithelial cells and HLMVEs. (A, C, E) SNA binds to Siaa2-6Gal and (B, D, F) MAA binds to Siaa2-3Gal presented on the (A-D) type I-like alveolar epithelial cells and (E-F) HLMVE cell pellet in reddish brown. En face sections were generated from selected planes in the vertical sections.

Figure 3

Viral matrix (M) gene expression in copy number normalized with β-actin gene expression (10⁵ copies) during infection of human primary type I-like alveolar epithelial cell. M gene expression in type I-like alveolar epithelial cells infected (MOI = 2) with influenza (A) A/HK/54/98 (H1N1) and (B) A/HK/483/97 (H5N1) virus. Black circles indicate gene expression after apical infection and open square indicates gene expression after basolateral infection. Asterisk indicates a greater M gene expression in apical infected cells than basolateral infected cells with statistical significance of p < 0.05.

Figure 4

A representative immunofluorescence staining of type I-like alveolar epithelial cells after (A, D) mock, (B, E) influenza H1N1 and (C, F) H5N1 virus infection and (G) chart with percentage of infection. Virus matrix protein and nucleoprotein were stained green by FITC-conjugated mouse antibody. The immunofluoresecent staining of the type I-like alveolar epithelial cells after apical (A-C) and basolateral (D-F) infection at 24 h post infection respectively. (G) Bar chart shows the mean percentage of infection and error bar represent the standard derivation, dark bar represents apical infection and open bar represents basolateral infection. Single and double asterisk indicates statistically significant difference with p < 0.05 and p < 0.01 respectively.

Figure 5

Virus titer detected in the supernatant of influenza virus infected type I-like alveolar epithelial cells. Virus titer of the (A) A/HK/54/98 (H1N1) and (B) A/HK/483/97 (H5N1) was determined after apical infection and basolateral infection of the type I-like alveolar epithelial cells at 3 h and 24 h post infection. Aa = apical release after apical infection, Ab = basolateral release after apical infection, Ba = apical release after basolateral infection, Bb = basolateral release after basolateral infection. (C) At 24 h post infection following basolateral infection of type I-like alveolar epithelial cells, the titers of influenza H5N1 virus at the apical aspect of the cells is significantly more seen with H1N1 infected cells. Single and double asterisk indicates statistically significant difference with p < 0.05 and p < 0.01, respectively. Dotted line represents the lowest detection limit of the TCID₅₀ assay.

Figure 6

Virus titer detected in the supernatant of influenza virus infected HLMVE cells. Virus titer of the (A) A/HK/54/98 (H1N1) and (B) A/HK/483/97 (H5N1) was determined after apical infection and basolateral infection of the HLMVE cells at 1 h and 8 h post infection. Aa = apical release after apical infection, Ab = basolateral release after apical infection, Ba = apical release after basolateral infection, Bb = basolateral release after basolateral infection. Single and double asterisk indicates statistically significant difference with p < 0.05 and p < 0.01 respectively. Dotted line represents the lowest detection limit of the TCID₅₀ assay.

Figure 7

Cytokine and chemokine gene expression in type I-like alveolar epithelial cells after influenza virus infection. The cytokine (A) IFN-β, (B) IL-6 and chemokine (C) RANTES, (D) IP-10 gene expression from type I-like alveolar epithelial cell after apical (black) and basolateral (grey) influenza A virus infection at 24 h post infection. The graph shows the mean and the standard error from three representative experiments. Single and double asterisk indicates statistically significant difference with p < 0.05 and p < 0.005 respectively.

Figure 8

Chemokine secretion from type I-like alveolar epithelial cells after influenza virus infection. The apical (dark bar) and basolateral release (grey bar) of (A) RANTES and (B) IP-10 protein from type I-like alveolar epithelial cell after apical infection of A/HK/54/98 (H1N1) and A/HK/483/97 (H5N1). Single asterisk indicates statistically significant difference with p < 0.05.