TLR9 overexpression in lung immune cells post-IAV infection.

(A) Relative gene expression of TLRs (9, 7, 3, 2, 4) by RTqPCR and (B) western blotting of TLR9 and β-actin. RNA and protein were isolated from lung leukocytes post-collagenase digestion in mice infected with 100 PFUs of H1N1 (PR8) for 5 days or placebo (PBS). β-actin was used to normalize RNA in samples. (C) Frequency of TLR9+ cells measured by flow cytometry in lung immune cells post-collagenase digestion. Gating was as follows: CD4+ T cells (CD45+,CD90.2+, CD3+, CD4+), CD8+ T cells (CD45+,CD90.2+, CD3+, CD4-, CD8+), natural killer cells (CD45+,CD90.2+, NKp46+), B cells (CD45+,CD90.2-, CD19+), neutrophils (CD45+, CD11b+, LY6G+), interstitial macrophages (CD45+, CD64+, CD11b+, F4/80+), alveolar macrophages (CD45+, CD64+, CD11C+, Siglec F+) and dendritic cells (CD45+, CD64-, CD11c+, MCHIIhigh). Staining for TLR9 was performed on cells which were fixed and permeabilized. (D) Relative expression of TLRs (9, 7, 3, 2, 4) after lung macrophage isolation by adherence selection from PBS or IAV-infected mice. (E) Relative expression of TLRs (9, 7, 3, 2, 4) in alveolar macrophages infected or not ex-vivo with H1N1 (MOI:0.01) for 24 hours. (F) BMDMs from Balb/c mice infected with H1N1 at MOI:0.01 and measured for TLR9 mRNA after 48h. Statistics are student T test between comparative groups. *P<0.05,**P<0.01, ***P<0.001, ****P<0.0001. Experiments in panels A, D, E and F were repeated at least 2 times with similar results. Panels B and C are single experiments on n = 4 (panel B) or n = 5 (panel C) mice.