An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens

Meliopoulos, Victoria A.; Van de Velde, Lee-Ann; Van de Velde, Nicholas C.; Karlsson, Erik A.; Neale, Geoff; Vogel, Peter; Guy, Cliff; Sharma, Shalini; Duan, Susu; Surman, Sherri L.; Jones, Bart G.; Johnson, Michael D. L.; Bosio, Catharine; Jolly, Lisa; Jenkins, R. Gisli; Hurwitz, Julia L.; Rosch, Jason W.; Sheppard, Dean; Thomas, Paul G.; Murray, Peter J.; Schultz-Cherry, Stacey

β6 KO mice have increased Type I IFN signaling β6 KO mice had an altered lung environment resulting in decreased viral spread. Type I IFNs are amongst the most potent cytokines limiting viral spread. Thus, we tested the hypothesis that β6 KO mice had increased type I IFN than WT. We used phosphorylated STAT1 levels (pSTAT1) as a marker for type I IFN signaling. Increased pSTAT1 along with increased type I IFN gene expression and unchanged expression of type II IFN-dependent targets such as IDO1 are indicative of a type I IFN dominated environment. Further, STAT1 is a robust marker of type I IFN signaling due to its weak activation by other cytokines such as IL-6 [47] . Immunoblot analysis of whole lung homogenates showed the β6 KO mice had robust type I IFN signaling at baseline that increased 3-5 dpi (Fig 8A) . We sought to determine the source of type I IFNs and how epithelial-expressed β6 regulated the IFN response. We noted that type I IFN mRNAs were not substantially altered in uninfected β6 KO mice (S6A-S6D Figs). Furthermore, ELISA measurements for type I IFNs did not show substantial differences between WT and β6 KO mice within whole lung homogenates. Therefore, we generated β6 KO mice crossed to an IFN-β-YFP reporter strain. In these mice, YFP expression in the whole lung was predominantly found in CD45 + cells and increased in the absence of β6 ( Fig 8B) ; although experiments with n = 3-6 mice per group run in triplicate. Error bars indicate SEM. Not statistically significant by two-way ANOVA with Bonferroni post-test. (B) RNA was extracted from the lungs of infected mice and viral replication was determined by real time PCR assay targeting the influenza M gene. Data are representative of 6 independent experiments with n = 3-4 mice per group run in triplicate. Error bars indicate SEM. Not statistically significant by two-way ANOVA with Bonferroni post-test. (C) Whole lung sections were paraffin-embedded and stained for influenza NP antigen at 2, 5, and 7 dpi. Bar immunofluorescent microscopy of frozen lung sections from WT and β6 KO YFP reporter mice showed increased YFP + cells scattered throughout the lung; including in the epithelia ( Fig 8C and 8D ). Further delineation of the exact cell types in this system expressing YFP is precluded by the strong autofluorescence in the FITC channel in lung macrophages [4] . Thus, we examined isolated AMs and primary epithelial cells grown at the air-liquid interface from β6 KO and WT mice for increased type I IFN signaling.