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

Mice lacking the β6 integrin are protected from disease caused by influenza infection. Compared to WT controls, β6 KO mice develop less severe ALI, as characterized by edema, inflammatory cytokine expression, and vascular permeability of the lung epithelium. β6 KO mice also have altered baseline homeostasis possessing high baseline CD11b + CD11c + macrophages as 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. [18] and productively replicate in alveolar macrophages [50] . It is possible that HPAI H5N1 viruses are more resistant to type I IFN, that mice succumb due to direct viral cytopathic effect, or complications of systemic infection. Further studies are needed to understand why β6 KO mice are not protected against HPAI H5N1 infection. Taken together, these data suggest β6-mediated modulation of IFN responses affects responses to multiple respiratory pathogens whereas protective responses depend on IFN signaling. The airway epithelium is a complex barrier comprised of multiple cell types that acts as an interface between the external environment and the internal lung milieu. It serves three main functions in a healthy lung; providing a tight mechanical barrier that rapidly repairs upon insult, mediating innate immune activity to limit foreign antigen invasion, and initiating an inflammatory response through production of cytokines and chemokines [51] . A variety of experimental models and human studies demonstrate the host response to respiratory infection involves initiation, resolution, and restoration phases, all of which must be tightly regulated to prevent disease [52] . However, little is known about how interactions between epithelial cells, which are often the primary targets of respiratory pathogens, and alveolar ma