Enhanced susceptibility of nasal polyp tissues to avian and human influenza viruses

Abstract

Background: Influenza viruses bind and infect respiratory epithelial cells through sialic acid on cell surface. Differential preference to sialic acid types contributes to host- and tissue-tropism of avian and seasonal influenza viruses. Although the highly pathogenic avian influenza virus H5N1 can infect and cause severe diseases in humans, it is not efficient in infecting human upper respiratory tract. This is because of the scarcity of its receptor, α2,3-linked sialic acid, in human upper airway. Expression of sialic acid can be influenced by various factors including inflammatory process. Allergic rhinitis and nasal polyp are common inflammatory conditions of nasal mucosa and may affect expression of the sialic acid and susceptibility to influenza infection.

Methodology/principal finding: To test this hypothesis, we detected α2,3- and α2,6-linked sialic acid in human nasal polyp and normal nasal mucosal tissues by lectin staining and infected explants of those tissues with avian influenza viruses H5N1 and seasonal influenza viruses. We show here that mucosal surface of nasal polyp expressed higher level of α2,3- and α2,6-linked sialic acid than normal nasal mucosa. Accordingly, both H5N1 avian influenza viruses and seasonal influenza viruses replicated more efficiently in nasal polyp tissues explants.

Conclusions/significance: Our data suggest a role of nasal inflammatory conditions in susceptibility to influenza infection, especially by avian influenza viruses, which is generally inefficient in infecting human upper airway. The increased receptor expression may contribute to increased susceptibility in some individuals. This may contribute to the gradual adaptation of the virus to human population.

Figure 1. Representative micrographs of nasal polyp and nasal turbinate mucosa showing distribution of α2,3- and α2,6-sialic acid.

Tissue sections were stained with FITC conjugated lectin MAA I (left panel) or SNA (right panel), specific toward α2,3- or α2,6-linked sialic acid, respectively (a). To confirm the specificity and the presence of α2,3-linked sialic acid on nasal polyp, tissues were digested with 1U/ml of sialidase before MAA I staining. The sialidase-treated tissue (right) lost the staining signal on the apical surface, while non-treated section was positive (left) (b).

Figure 2. Infection of human and avian influenza viruses in nasal polyp and nasal turbinate mucosal explants.

Two pieces of tissue of about 3–4 mm in diameter from each specimen were cultured in a 24-well tissue culture plate. Each tissue explant was infected with 1×10⁶ TCID50 of H5N1 AIV [A/Thailand/3 (SP-83)/04] or seasonal influenza virus H1N1 [A/Thailand/Siriraj-3/06]. After two hours infection the unattached virus was removed by washing twice with PBS. At 0, 20, 24 and 48 hr post-infection, the culture supernatants were collected for virus titrating by plaque assay. The data were derived from two nasal polyps and two nasal mucosa. One or two asterisks indicate a statistical significance at a P value of <0.05 or <0.01, respectively, as determined by a t test for a comparison between viral titers from nasal polyp and nasal mucosa at the same time point.

Figure 3. Correlation between the percentages of lectin-positive cells and the viral titers.

Dot plots of percentages of lectin-positive cells versus maximum viral titers produced from the same tissue samples show linear correlation with Pearson correlation coefficient of 0.889 for SNA (p = 0.003) and 0.859 for MAA I (p = 0.006). The data were derived from the same experiments shown in Figure 1, 2 and 4.

Figure 4. Distribution of receptor and infection of nasal polyps and adjacent normal mucosa.

Distribution of sialic acid receptors (a) and outputs of viral infection (b) of tissue samples from nasal polyps and adjacent normal mucosa from the same patients. The data were derived from experiments using nasal polyps and adjacent normal nasal mucosal tissue samples from two patients.

Figure 5. In situ hybridization of H5N1 AIV-infected (upper) and non-infected (lower) polyp tissues.

The hybridization signal is shown in red-brown color in the epithelial cells on the mucosal surface of infected tissue.

Figure 6. Expression of ST3GAL1 and ST3GAL4 mRNA measured by quantitative real time RT-PCR.

The amount of total RNA was normalized using GAPDH mRNA. The data are shown as the mean ± SD from an experiment done in triplicate. The data were derived from two pieces of tissues from the same patient. The difference between mRNA expression in nasal polyp and nasal mucosa, marked by asterisks, is statistically significant (t test, P<0.01).