Figure 8.

Disruption of sialic acid-siglec-E interaction by siglec-E siRNA reverses macrophage response to PA^+Sia. (A) Lysates from mock and siglec-E siRNA transfectant macrophages were analyzed by western blotting to confirm silencing of siglec-E. Western blot shown is representative of three independent experiments. (B) Mock transfectant or siglec-E knockdown macrophages were incubated with 10 MOI FITC-PA^+Sia for 30 min at 37°C. Cells were visualized by confocal microscopy to check for bacterial internalization as described in Materials and Methods. Microscopy images were representative of three independent experiments. Scale bar = 10 μm. (C) ROS generation was estimated by CM-H2DCFDA staining in these cells and analysis by flow cytometry. (D) In a similar setup, macrophages were stained with DAF-FM after PA^+Sia infection and analyzed by flow cytometry to detect levels of reactive nitrogen species. MFI data shown (C,D) are as mean ± s.e.m. from three independent experiments. (E) Mock and siglec-E siRNA transfected cells were stimulated with PMA and infected with PA^−Sia or PA^+Sia. Amount of IL-10 (TH2) and IFN-γ (TH1) cytokines secreted by macrophages in response to infection was measured by ELISA kits from culture supernatant collected after 3 h of infection. (F) Viability of bacteria in macrophages following phagocytosis was assessed using gentamicin protection assay. Macrophages were incubated with siglec-E blocking antibody at 4°C for 30 min followed by infection with PA^+Sia at 1:10 MOI for 30 min at 37°C. Infected macrophages were then incubated for 3 h with gentamicin (200 μg/mL). The viability and persistence of internalized bacteria were checked by plating the lysed macrophage contents on nutrient agar plates and checking for growing bacterial colonies (colony forming units, cfu). Data represented as mean ± s.e.m. of three independent experiments. Data from three independent experiments represented as mean ± s.e.m. Significance represented by ^nsp > 0.05, *p ≤ 0.05, **p ≤ 0.01.