FIGURE 1.

Influenza H1N1 activates TGFβ via the αvβ6 integrin in human bronchial epithelial cells. A, unstimulated iHBECs were incubated with either an anti-αv or an anti-β6 antibody and analyzed by flow cytometry. The data reveal that both the αv and β6 subunits (solid black lines) are expressed basally on iHBECs. The dashed lines represent the secondary antibody controls. B, confirmation of influenza A infection in iHBECs. IHBECs were infected with H1N1 (A/PR/8/34) at m.o.i. of 1 for up to 4 h, and total RNA was isolated. Viral matrix (Mx) and hemagglutinin (HA) genes were detected by PCR in all influenza-infected but not in mock-infected samples. The experiment was repeated three times, and a representative example is shown. C, iHBECs were pretreated with H1N1 (m.o.i. 1) + 1 μg/ml TPCK-trypsin or mock-infected with 1 μg/ml TPCK-trypsin only and co-cultured with TMLCs without inhibitors (black bars) and with the pan-TGFβ-neutralizing antibody 1D11 (10 μg/ml; gray bars), the αvβ6 integrin-blocking antibody 6.3G9 (15 μg/ml; striped bars), or the Rho kinase inhibitor H1152 (10 μm; white bars). Experiments were performed in duplicate and repeated three times. The mean fold change in total luciferase units compared with control (mock-infected without inhibitors) of the independent experiments was determined. Data are expressed as mean ± S.E. ***, p < 0.001. D, iHBECs were transiently transfected with the TGFβ-sensitive promoter reporter construct CAGA₁₂-MLP-Luc and Renilla luciferase pRL-SV40, prior to stimulation with 1 μg/ml TPCK-trypsin only (mock-infected) or H1N1 m.o.i. 1 + 1 μg/ml TPCK-trypsin and incubated for 4 h post-infection (hpi). Experiments were performed in duplicate and repeated three times. The mean fold change in the F:R ratio versus control (mock-infected) of the independent experiments was determined. Data are expressed as mean ± S.E. **, p < 0.01.