Figure 1.
C3 is required for Ft Schu S4 uptake by human macrophages.
hMDMs were incubated with Schu S4 for 1 hr at an MOI of 50∶1 in the presence or absence of 10% autologous serum, C3-depleted serum (C3-dpl) or C3-repleted serum (C3-rpl). (A) Extracellular bacteria were killed by gentamycin treatment for 30 min at 37°C. hMDMs were then lysed and intracellular bacteria were enumerated by CFUs recovered. Data are representative of three independent experiments. The data were analyzed by 1-Way ANOVA and Tukey's Multiple Comparison Test (*p<0.05 serum vs. no serum and C3-rpl vs. C3-dpl). (B) hMDMs were fixed after 1 hr of infection and followed by inside/outside differential staining to differentiate intracellular (red) and extracellular (yellow or green) bacteria as described in Material and Methods. Images shown are representatives of three independent experiments. In the experiment shown, quantification of the total number of Sch S4 per cell was as follows: no serum = 0.35; serum = 4.04; C3-dpl. = 0.269; C3-rpl serum = 3.757 (150 cells per experimental group).
Figure 2.
Serum components, specifically C3, have an inhibitory effect on pro-inflammatory cytokine production upon Schu S4 infection of human macrophages.
(A–C) hMDMs were infected with Ft Schu S4 at an MOI of 50∶1 in RHH or RHS with 10% autologous serum, C3-depleted serum (C3d) or C3-repleted serum (C3r) for 1 h; or (D–F) hMDMs were infected with Ft Schu S4 (Ft) or F. novicida (Fn) at an MOI of 50∶1 in RHH or RHS with 10% autologous serum. Extracellular bacteria were killed with 50 µg/ml gentamycin at 37°C for 30 min. Media were replenished and cell-free culture supernatants were collected at 16 hrs post infection. TNFα, IL-6 and IL-1β concentrations were measured by ELISAs. Uninfected resting cells (R) were included as a control. Data are representative of at least 3 independent experiments. The data were analyzed by 1-Way ANOVA and Tukey's Multiple Comparison Test. ** p<0.005.
Figure 3.
Serum components, specifically C3 opsonization, inhibit ERK1/2 and p38 activation during Schu S4 phagocytosis by macrophages.
(A) Schu S4 was either non-opsonized, or pre-opsonized with 10% autologous serum, C3-depleted serum (C3d) or C3-repleted serum (C3r), and then used to infect hMDM monolayers at an MOI of 50∶1 in RHH in the absence of serum. Infection was synchronized by centrifuging at 250×g for 10 min at 4°C, and incubated at 37°C for 30 min. (B) Schu S4 was either non-opsonized, or pre-opsonized with 10% autologous serum, and then used to infect hMDM monolayers at an MOI of 50∶1 in RHH in the absence of serum. Infection was synchronized by centrifuging at 250×g for 10 min at 4°C, and incubated at 37°C for 15 or 30 min. (C) Schu S4 (Ft) or F. novicida (Fn) were either non-opsonized or serum pre-opsonized, and then used to infect hMDMs as in (A) for 30 or 60 min. MDMs lysates were subjected to Western Blot. Uninfected resting cells (R) were included as control. Data are representative of 3 independent experiments.
Figure 4.
Serum opsonization of Schu S4 leads to inhibition of NF-κB p65 phosphorylation and translocation into nucleus during phagocytosis by macrophages.
(A) hMDMs were infected with pre-opsonized (10% autologous serum) or non-opsonized Schu S4 at an MOI of 50∶1 in RHH. Infection was synchronized by centrifuging at 250×g for 10 min at 4°C, and incubated at 37°C for different time points (15′, 30′ and 60′). MDM lysates were subjected to Western Blot using Phospho NF-κB p65 or total p65 primary antibodies. (B) Cumulative data of band intensities from n = 2 (* p<0.05). (C) hMDMs were fixed after 1 hr of infection with pre-opsonized or non-opsonized Schu S4 at an MOI of 50∶1 and NF-κB p65 translocation was examined by immunofluorescence microscopy with rabbit NF-κB p65 antibody followed by anti-rabbit AF 594 antibody. Nuclei were stained with DAPI. NF-κB p65 translocation was scored using a total of 300 cells per experiment. The graph shown represents cumulative data from two independent experiments. The data were analyzed by a two-tailed Student t-test (** p<0.005, Student t-test).
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.
Figure 6.
CR3, partially activated by TLR2 inside-out signaling, is critical for Schu S4 phagocytosis by hMDMs.
(A) hMDMs were transfected with scrambled siRNA or siRNAs targeting CD11b or TLR2. 48 hrs later, the level of CD11b and TLR2 were examined by Western blot. (B) CD11b knockdown was also examined by immunofluorescence microscopy with mouse CD11b (M1/70) antibody. Nuclei were stained with DAPI. (C) 48 h after siRNA transfection hMDMs were infected with non-opsonized or serum pre-opsonized Ft Schu S4 in the absence of serum for 15 min. Infected cells were subjected to differential staining of extracellular (yellow or green) and intracellular (red) bacteria. Representative images are shown from 3 independent experiments. (D) Bacterial uptake was quantified as the number of bacteria that are inside or attached per cell. At least 300 cells were counted for every sample. Data are representative of 3 independent experiments performed in triplicate (mean ± SD). * p<0.05, compared with control siRNA samples (1-Way ANOVA and Tukey's Multiple Comparison Test).
Figure 7.
CR3 inhibits while TLR2 activates pro-inflammatory responses in human macrophages after Schu S4 infection.
hMDMs were transfected with scrambled siRNA or siRNA targeting CD11b or TLR2. 48 hrs after transfection cells were infected with Schu S4 (Ft) or F. novicida (Fn) in the presence or absence of serum at an MOI of 50∶1. TNFα (A) and IL-1β (B) levels at 16 h post infection in the culture supernatants were measured by ELISAs. Data are representative of 3 independent experiments performed in triplicates. The data were analyzed by 1-Way ANOVA and Tukey's Multiple Comparison Test. * p<0.05, **p<0.005, n.s., not significant. (C) After siRNA transfection hMDMs were infected with non-opsonized or serum pre-opsonized Ft Schu S4 at an MOI of 250∶1. Infections were synchronized as described in the materials and methods. Cell lysates were collected at 5, 15 and 30 min post infection and subjected to Western blot analysis using antibodies against phospho-ERK1/2, phospho-p38 and β-actin. Uninfected resting cells (R) were also included as control. Results are representative of at least 3 independent experiments.
Figure 8.
The CR3 cytoplasmic tail is critical for serum opsonin-mediated immune suppression in human macrophages.
(A) CHO cells stably expressing full length CR3 or a tail less mutant form of the receptor were transiently transfected with functional TLR2 and subsequently infected with serum pre-opsonized or non-opsonized Ft Schu S4 at an MOI of 50∶1 for different time points. Cell lysates were used to examine for ERK, p38 and Akt activation by Western blot using phosphor-specific antibodies. The same membrane was re-probed with total ERK, p38 and Akt as loading controls. The lower panel shows the band intensities measured by Image J software. (B) Cell association was quantified as the number of bacteria per cell by immunofluorescence microscopy using mouse Ft LPS antibody followed by Alexa Fluor 488. At least 300 cells were counted for every sample. Data are representative of 2 independent experiments performed in triplicate.
Figure 9.
Lyn, AKT and MKP-1 are involved in CR3-mediated immune suppression during Schu S4 phagocytosis by macrophages.
(A) hMDMs were transfected with control siRNA or siRNA targeting CD11b or TLR2. 48 h later cells were infected with non-opsonized or serum pre-opsonized Ft Schu S4 in the absence of serum for 30 min at MOI of 50. Infections were synchronized as described in the materials and methods. Cell lysates were subjected to SDS-PAGE and Western blot using antibodies against phospho-Lyn (Y396), phospho-Akt (T308), MKP-1 or β-actin. (B) Lyn siRNA knockdown in hMDMs. hMDMs were transfected with control siRNA or siRNAs targeting Lyn. 48 hrs later cell lysates were subjected Western blot using an antibody against Lyn. (C) 48 h after scrambled or Lyn siRNA transfection hMDMs were infected with Schu S4 in the presence or absence of serum at an MOI of 50∶1. 16 h post infection TNFα, IL-6 and IL-1β levels in culture supernatants were measured by ELISA. Data are representative of 3 independent experiments, each performed in triplicate. The data were analyzed by a two-tailed Student t-test *** p<0.001.
Figure 10.
Model of CR3-mediated immune suppression and crosstalk with TLR2 signaling during the phagocytosis of Schu S4 by human macrophages.
In the absence of serum, pathogen associated molecular determinants such as lipoproteins will be recognized by TLR2 which activates MAP kinase (ERK1/2 and p38) and NF-κB signaling pathways, and induces host pro-inflammatory responses. Phagocytosis is limited without the optimal engagement of CR3. In the presence of serum C3bi is deposited on the Ft Schu S4 surface, which optimally engages CR3 and enhances phagocytosis. At the same time, TLR2 is activated at least to some degree, leading to inside-out activation of CR3, which contributes to enhanced outside-in CR3 signaling and increased phagocytosis. CR3-mediated signaling activates Lyn and AKT, and leads to an increased MKP-1 level which results in inhibition of MAPK activity. This allows for increased phagocytosis simultaneously with a dampened host immune response.