An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens
Fig 8
β6 KO mice have increased type I IFN activation.
(A) Whole lung homogenates from WT or β6 KO mice were probed for pSTAT1 and total STAT1. GAPDH was used as a loading control. (B) β6 KO were crossed to an IFN-β-YFP reporter. Staining for YFP and CD45 is shown. (C) Lung sections of uninfected WT or β6 KO YFP reporter mice were analyzed for YFP expression (green). Cells were counterstained with WGA (red). (D) Quantification of YFP+ cells in WT versus β6 KO lung sections. Ten fields were imaged for each mouse. p < 0.0001 by t test. (E) Heat map of selected interferon-regulated genes from sorted WT autofluorescent CD11c+CD11b- AM and isolated β6 KO autofluorescent CD11c+CD11b+ ‘AM’ from two independent experiments (yellow indicates higher expression; blue indicates lower expression) by z-score from -1.5 to +1.5. (F) Western blot analysis of lysates from sorted alveolar macrophage populations were probed for pIRF3, total IRF3 used as a loading control, and pSTAT1. (G) RNA was isolated from macrophage populations described in (E) and expression of interferon-related transcripts Irf7, Ifit1, and Oas1g determined by quantitative real-time PCR. ****p < 0.0001 and *** p < 0.001 by two-way ANOVA with Bonferroni post-test. Error bars indicate SEM. (H) Immunoblot analysis of primary mTECs at 24 hpi compared to uninfected. Cells were grown to a confluent monolayer and infected with CA/09 virus (MOI 0.1). Membranes were probed for phosphorylated and total STAT1 or IRF3. β-actin was used as a loading control. Data is representative of 2 independent experiments with n = 2–3 samples probed. (I) Immunoblot analysis of whole lung homogenates from WT and β6 KO were probed for phosphorylated STAT1 and total STAT1. GAPDH was used as a loading control. Data is representative of 2 independent experiments with n = 4–5 samples probed.