Fig 1.
The expression of cytokines and other inducible mediators during BMDM infection by E. faecalis.
BMDM were infected with E. faecalis E99 for 1 or 5 h before mRNA extraction and analysis by RT-PCR. The values are normalized to actin and expressed as the fold change relative to uninfected cells (Control). *, p<0.05; **, p<0.01 represent statistically significant difference compared to BMDM without infection; n.s., not statistically significant compared to BMDM without infection.
Fig 2.
NF-κB and MAPKs are involved in E. faecalis-induced upregulation of cytokine expression.
(A) RAW264.7 cells were infected with E99 at MOI of 10 for indicated times, then collected for Western blot analysis. (B&C) Cells were pretreated with the inhibitors of p38 (SB239063), JNK (SP600125), MEK (PD98059) or NF-κB (MG132) or with vehicle (DMSO) alone for 0.5 h before being infected with E99 for 5 h. The mRNA levels of TNF-α, IL-1β and actin in macrophages were measured by RT-PCR and the values were normalized to actin and expressed as the fold change relative to uninfected cells (Control). (D) TNF-α in the culture supernatants was measured by ELISA. *, p<0.05; **, p<0.01 represents statistically significant difference compared to RAW264.7 cells infected with E99 alone.
Fig 3.
The effect of E. faecalis on NF-κB and MAPK activation and cytokine expression.
This experiment included the following groups: (1) RAW264.7 cells alone; (2) RAW264.7 cells infected with E. faecalis E99; (3) RAW264.7 cells infected with E. coli; (4) E. faecalis E99 were added on the top of a Transwell semipermeable filter separating the bacteria from RAW264.7 cells; (5) the RAW264.7 cells were pretreated with CytD for 0.5 h before being infected with E. faecalis E99 for 5 h. The cells were collected to analyze the activation of NF-κB and MAPK by Western blot (A), or to measure the mRNA level of TNF-α (B) and IL-1β (C) by RT-PCR. (D) The supernatant from treated cells above were used to measure the concentration of TNF-α by ELISA. *, p<0.05; **, p<0.01 represent statistically significant difference compared to RAW264.7 cells without infection; n.s., not statistically significant compared to RAW264.7 cells without infection. (E) RAW264.7 cells were infected with E99 for 1h, cells were washed thrice with PBS and further incubated with medium containing vancomycin and gentamicin to kill the extracellular bacteria for 1 h, 3 h or 12 h before stimulation with LPS (1μg/ml) for 3.5 h. The supernatants were collected for analysis of TNF-α concentration by ELISA. *, p<0.05.
Fig 4.
The roles of TLR2 and MyD88 in NF-κB and MAPK activation and cytokine secretion during E. faecalis infection.
Wild type (WT), TLR2-/- (TLR2 KO) and MyD88-/- (MyD88 KO) BMDM were infected with E99 at a MOI of 10 for 5 h, the cells were then lysed and subjected to Western blot analysis (A). The supernatants were collected to measure the concentration of TNF-α (B) and IL-6 (C). *, p<0.05; **, p<0.01 represents statistically significant difference compared to WT BMDM infected with E99.
Fig 5.
Phagocytosis of E99GFP by RAW264.7 cells is increased when E. faecalis is opsonized with serum against whole E. faecalis cells or Esp.
(A) FACS analysis of phagocytosis of E99GFP under different conditions including: RAW264.7 cell without infection (Control), RAW264.7 cells infected with E99GFP (E99GFP); RAW264.7 cells pretreated with CytD before infecting with E99GFP (E99GFP+CytD); RAW264.7 cells infected with E99GFP pretreated with rabbit preimmune sera (E99GFP+preimmune), serum against Esp (E99GFP+Anti-Esp serum) or serum against whole-cell enterococcal antigens (E99GFP+ Anti-E. faecalis serum). (B) The mean fluorescence intensity of macrophages was measured by flow cytometry from A. (C) Representative immunofluorescence images showing phagocytosis of E99GFP by RAW264.7 cells under different treatments, Blue: DAPI, Green: E99GFP and Red: antibody against extracellular E99. (D) For each treatment, the number of E99GFP bacteria in at least twenty macrophages were counted and the mean number of intracellular E99GFP per cell was calculated *, p<0.05; **, p<0.01.
Fig 6.
Impaired phagocytosis in the absence of MyD88, ERK and JNK signal pathway.
(A) WT, TLR2-/-, MyD88-/- BMDM were infected with E99GFP at a MOI of 100 for 1 h, then the cells were analyzed by FACS after washing thrice with PBS. Representative FACS histogram shows the phagocytosis of E99GFP by WT, TLR2-/- and MyD88-/- BMDM. (B) Mean fluorescence intensity of GFP from A. *, p<0.05. (C) RAW264.7 cells were pretreated with inhibitors of p38, JNK or MEK and then infected with E99GFP at MOI of 100 for 1 h. The cells were washed with PBS for three times before analysis by FACS. FACS histogram shows phagocytosis of E99GFP by RAW264.7 cells with different treatments. (D) Mean fluorescence intensity at 1 hour after phagocytosis of E99GFP by RAW264.7 cells from C. *, p<0.05;