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

independent experiments with n = 4-12 mice per group. (C) is pooled from 2 independent experiments with n = 6-7 mice per group. Error bars indicate SEM. p < 0.0001 by two-way ANOVA with Bonferroni post-test (B) or p < 0.01 by log-rank (Mantel-Cox) test (C). (D) Mice were intranasally inoculated with 10 4 TCID 50 of A/Anhui/1/2013 H7N9 influenza virus and monitored for survival for 12 dpi. Data represents a single experiment with n = 5 mice per group. p < 0.05 by log rank (Mantel-Cox) test. (E) Mice were intranasally inoculated with 2.0 x 10 4 plaque forming units (PFU) Sendai virus and monitored for morbidity and mortality for 12 dpi. Data is pooled from 2 independent experiments with n = 5-10 mice per group. p < 0.001 by log rank (Mantel-Cox) test. (F) Mice were inoculated with 5X the LD 100 of S. pneumoniae (D39X) and monitored for survival. Data is pooled from 3 independent experiments with n = 3-5 mice per group. p < 0.01 log rank (Mantel-Cox) test. (G) Mice were inoculated with 80 TCID 50 of A/Puerto Rico/8/34 influenza virus followed by inoculation with 100 CFU of S. pneumoniae (D39X) at 7 days post-influenza infection and monitored for survival. Data is pooled from 3 independent experiments with n = 3-5 mice per group. p < 0.05 log rank (Mantel-Cox) test. Loss of the β6 integrin leads to a modified lung microenvironment To understand the underlying mechanism(s) for the decreased viral spread within the lungs of β6 KO mice, we performed flow cytometry on BALF and lungs at different times post-infection. No significant differences were noted in numbers of TNF-α/iNOS-producing (tip)DCs, neutrophils, CD4 + T cells, CD8 + T cells, or influenza PB1-specific CD8 + T cells in either BALF (Fig 4A-4E ) or whole lung (Fig 4F-4J ). However, we noted substantial differences in the resident lung F480 + CD11c + CD11b + populations (macrophages and dendritic cells), namely that β6 KO mice lacked a conventional CD11c + alveolar macrophage (AM) population ( Fig 5A) . AMs have characteristic flow cytometry properties as compared to dendritic cells (DCs) or incoming activated bone marrow-derived inflammatory monocytes including autofluoresence in the FITC channel, high constitutive expression of CD11c, F4/80 and SiglecF and low expression of CD11b [4, 41, 42] . By contrast, incoming activated bone marrow-derived inflammatory monocytes express CD11b, providing a means to distinguish between resident AM, inflammatory bone marrow-derived monocytes and DCs [4, 43] . When we examined the lung macrophage/DC populations in uninfected β6 KO mice we found they had higher numbers of autofluorescent cells indicating AMs ( Fig 5B) but instead of the normal CD11c hi CD11bphenotype, the vast majority of the β6 KO AM were MerTK + , CD64 + , CD11c + , and uniformly CD11b + (Fig 5C; See S2 and S3 Figs for gating strategies). These cells maintained expression of F4/80 and SiglecF, suggesting they are AMs with altered properties. They were also phenotypically distinct from those of WT mice, with a 'foamy' appearance typical of activated macrophages ( Fig 5D) . Importantly, the β6 KO CD11c + CD11b + AM were present at the earliest times tested (d3 after birth), continued to be present at all points of adulthood (S4A Fig) , and were not affected by reducing neonatal exposure to airborne particulate matter by using HEPA-filtered cages or low dust cage bedding (S4B Fig). Even throughout the course of influenza infection very few CD11c hi CD11bcells were present in the lun