Fine Tuning Inflammation at the Front Door: Macrophage Complement Receptor 3-mediates Phagocytosis and Immune Suppression for Francisella tularensis
Figure 5
C3-mediated immune suppression is not due to a difference in the kinetics of Schu S4 phagocytosis.
(A) In the presence of serum, there were more Ft Schu S4 attachment and phagocytosis. hMDMs were infected with non-opsonized or serum pre-opsonized Ft Schu S4 at an MOI of 50∶1. Infections were synchronized by centrifugation at 4°C for 10 min at 250×g. At 5, 15 and 30 min post infection samples were washed extensively, fixed with 2% paraformaldehyde (PFA), and followed with inside/outside differential staining. The numbers of attached (extracellular) and phagocytosed (intracellular) bacteria were counted under an epi-fluorescence microscope. At least 300 cells were counted for every sample. Data are representative of 3 independent experiments performed in triplicate (mean ± SD). Non-opsonized vs. serum-opsonized, * p<0.05, ** p<0.005 Student t-test. (B) hMDMs were infected with Schu S4 (Ft)or F. novicida (Fn) at MOIs of 5, 20 and 100 in the presence (RHS with 10% autologous serum) or absence (RHH) of serum. Cell-free culture supernatants were collected after 16 h and cytokine levels were measured by ELISA. Data are representative of 3 independent experiments performed in triplicate (mean ± SD). (C) hMDMs were infected with non-opsonized or serum pre-opsonized Schu S4 (Ft) or F. novicida (Fn) at MOIs of 10, 50 or 250 for 30 min. Cell lysates were subjected to Western blot analysis. Data are representative for 3 independent experiments. (D) Schu S4 bacteria were either killed with paraformaldehyde or incubated with PBS (control) for 10 min at room temperature. hMDMs were then infected with non-opsonized or serum pre-opsonized live or PFA-killed Schu S4 at MOI of 50∶1 for 30 min. Cell lysates were subjected to Western blot analysis. Data are representative of 3 independent experiments.