We then employed H5N1 infection as

a model to study the antiviral activity of α-defensin-induced MxA. The viral plaque assay in Fig. 4A shows that, similar to IFN-α-pretreated HGECs, α-defensin-1, -2, and -3-pretreated cells significantly inhibited H5N1 replication, suggesting a functional MxA protein. On the other hand, β-defensin-1, -2, -3, and LL-37-pretreated HGECs poorly inhibited viral replication. These findings AZD9291 ic50 were confirmed by microscopically observed cytopathic effects (data not shown). To confirm the antiviral activity of MxA against H5N1, we transfected HGECs with MxA-targeted siRNA, treated the cells with α-defensin-1 overnight, and then infected them with H5N1 virus. MxA-targeted siRNA greatly reduced levels of MxA mRNA expression by 95%, (Fig. 4B) and effectively abolished inhibition of viral replication by 93% in H5N1-infected HGECs (Fig. 4C). These findings were supported by microscopically observed cytopathic effects (Fig. 4D). α-defensins are known as major proteins secreted by PMNs [[32]]. In the physiological condition of healthy gingiva, PMNs and their products are present in the tissue and the crevicular fluid in the gingival sulcus [[33, 34]]. In vitro culture of PMNs (5 × 106 cells/mL)

for 6 h led to secretion of α-defensins in supernatants (which ranged from 90 479 to 98 714 pg/mL). To investigate the role of the PMN-derived α-defensins Ku0059436 in MxA expression, we cultured HGECs with 6 h PMN supernatants. Under this condition, expression of MxA at both mRNA and protein levels in HGEC was observed after 6 h and 24 h treatment, Avelestat (AZD9668) respectively (Fig 5A and B). The MxA-inducing activity was diminished when neutralizing antibody against

α-defensins was added to the culture, whereas neutralizing antibodies against type I IFN (IFN-α and IFN-β) had no effect (Fig. 5B). These data suggest that PMN-derived α-defensins were responsible for the observed MxA expression. The immunostaining results to detect epithelial MxA were obtained using the oral, but not the sulcus, side of periodontal tissue (Fig. 2) because the epithelium at the sulcus side, especially for the junctional epithelium, is generally lost or torn during the surgical procedure. Fig. 6A depicts anatomic landmarks of the gingival sulcus. In this study, we were able to obtain two specimens of gingival sulcus area from healthy periodontal tissue. We then investigated localization of MxA protein in the healthy sulcus and also in relation to α-defensin. Fig. 6C shows that MxA protein was consistently expressed throughout epithelial cells of periodontal tissues. MxA staining was especially intense in the junctional epithelium (Fig. 6C). α-defensins were identified in small round cells with PMN morphology, most of which were found in the connective tissue layer (Fig. 6E). Migratory PMNs in junctional epi-thelium were also observed and highlighted in Fig. 6D.