TLR9^-/- lung macrophages have increased phagocytosis, bacterial killing, and iNOS expression post-IAV infection.

(A) Cytokine measurement in BALF from BALB/c and TLR9-/- mice that were infected with IAV (100 PFUs, H1N1) for 5 days n = 5/group (some values are overlapping in the dot plots) for all but IL-10 which was from an experiment with n = 3 TLR9-/- mice and 5 Balb/c mice. (B) Ex vivo MRSA phagocytosis by macrophages isolated by collagenase digestion and adherence purification from mock or H1N1 infected mice on day 5, n = 3. (C) SRA expression analyzed by real-time RT-PCR in monocyte/macrophages isolated on day 5 from H1N1-infected mice, n = 3. (D) Ex vivo MRSA killing assay using adherence selected lung macrophages from BALB/c and TLR9-/- mice that were infected with IAV (100 PFUs H1N1) for 5 days or treated with PBS; n = 8/group in mock infections and n = 3/group in H1N1 infected mice. (E) Quantitative reverse transcriptase-PCR measurement of relative gene expression of iNOS from adherence selected lung macrophages from BALB/c and TLR9-/- mice that were infected with IAV for 5 days or treated with PBS; RNA samples were normalized to their β-actin levels and setting mock-infected Balb/c to 1; n = 3-5/group. (F) BMDMs from Balb/c or TLR9-/- mice were mock-infected for 24 h or were infected with MOI:0.01 H1N1 for 24 or 48 h before RNA was prepared and analyzed for expression of iNOS normalized to their β-actin levels and setting mock-infected Balb/c to 1; n = 3/group. Panels A and C were analyzed by student’s t-test. Panels B, D, E and F were analyzed by two-way ANOVA with Sidak’s multiple comparison test.; Non-significant (ns), *P<0.05, ***P<0.001, ****P<0.0001. Panels A-D are single experiments using multiple mice, panels E and F were repeated two times.