Fig 1.
CR3 and Dectin-1 collaborate to intensify Syk activation and subsequent cytokine response in macrophage.
(A and B) Macrophages from WT, Itgam-/-, Clec7a-/- and Itgam-/-Clec7a-/- mice were stimulated with or without (0 h) heat-killed (HK) H. capsulatum. (A) Supernatants were collected at 6, 12, and 24 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA (n = 6 from 3 independent experiments). (B) Cell lysates were collected at 30 min after stimulation and analyzed by Western blotting using antibodies against p-Syk and Syk. The intensity of p-Syk was normalized against the corresponding Syk. Data shown in the lower panel are relative intensity of p-Syk (n = 3). (C and G) Macrophages from WT mice were treated with Syk inhibitors (SkyI, 10 μM; Bay 61-3606, 3 μM) starting at 1 h prior to stimulation with HK H. capsulatum (C) or with the combination of iC3b-coated beads (iC3b-LB) and depleted zymosan (dZ) (G). Culture supernatants were harvested 6 h later and analyzed for cytokine production (n = 5 from 2 independent experiments). (D) Fetal liver-derived macrophages (FLDMs) from Syk+/+, Syk-/- and Syk+/- embryos were stimulated with HK H. capsulatum for 6 h. TNF levels in the culture supernatants were evaluated (n = 9 from 3 independent experiments). (E) Macrophages from WT, Itgam-/-, and Clec7a-/- mice were stimulated with iC3b-LB, dZ, uncoated Latex beads (LB), or the combination of iC3b-LB and dZ. Culture supernatants were collected 6 h later and analyzed for cytokine production (n = 6 from 3 independent experiments). (F) Macrophages from WT mice were stimulated with or without iC3b-LB, dZ, LB, or the combination of iC3b-LB and dZ for 30 min. Cell lysates were subjected to Western blotting. Data are representative of 3 independent experiment with similar results. The bars represent the mean ± SD. In (A),* represent comparisons with WT and # with Itgam-/-Clec7a-/-. * or # p ≦ 0.05, ** or ## p ≦ 0.01, *** or ### p ≦ 0.001. NS, not significant [one-way ANOVA with Tukey post-hoc test analysis (A, B, D and E); 2-tailed t-test (C and G)].
Fig 2.
Clustering of CR3 and Dectin-1 on lipid rafts is required for their collaboration in cytokine production and signaling activation.
(A and C) Macrophages were stimulated with or without (0 min) HK H. capsulatum for 15 or 30 min. Cells were fixed and stained for CR3 (red) and Dectin-1 (green) (A), or for p-Syk (green) (C). Cells were viewed under confocal microscope. Lipid raft was identified by staining with cholera toxin B (CTB) (violet). Nuclear compartment was stained by DAPI (blue). Arrowheads in the DIC/DAPI fields point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged images is shown in the histogram on the right. (B) Macrophages from WT mice were stimulated with or without (control) HK H. capsulatum for 30 min. Cell lysates were subjected to sucrose gradient ultracentrifugation. The fractions were collected and subjected to Western blotting and probed with anti-CD11b, anti-Dectin-1 and anti-flotillin-1 antibodies. (D and E) Macrophages were untreated or treated with methyl-β-cyclodextrin (MβCD, 10 mM) for 30 min. To reconstitute cholesterol, MβCD-treated cells were cultured in medium containing water-soluble cholesterol (MβCD-CHO, 400 μg/ml) for 1 h. After washing, macrophages were stimulated with HK H. capsulatum for 6 h (D), or 30 min (E). The concentrations of TNF and IL-6 in culture supernatants were analyzed by ELISA. Mean ± SD are shown (n = 10 from 4 independent experiments) (D). Cell lysates were analyzed by Western blotting by using antibodies against p-Syk, Syk, and β-actin. Data are representative of 2 (A and C) and 3 (B and E) independent experiments with similar results. ** p ≦ 0.01, *** p ≦ 0.001. NS, not significant [one-way ANOVA with Tukey post-hoc analysis (D)].
Fig 3.
Micro-Western Array to screen for signaling molecules involved in H. capsulatum-induced macrophage response.
Macrophages from WT mice were stimulated with or without (0 min) HK H. capsulatum for 15, 30, 60, 90, and 120 min. (A) Heat map chart shows MWA results. Protein abundance was normalized against the mean of β-actin and GAPDH. Black color indicates no change, while red and green indicate increase and decrease, respectively, of the levels of protein compared to unstimulated control. Proteins below the level of detection are in grey. (B) Cell lysates were subjected to Western blotting. Beta-actin was used as an internal control. Data shown are representative of 3 independent experiments with similar results.
Fig 4.
CR3 and Dectin-1 collaborate to enhance JNK activation.
(A and C) Macrophages from WT mice were treated with vehicle (control) or different kinase inhibitors (PI3K inhibitor, LY294002 20 μM; Raf-1inhibitor, GW5074 1 μM; JNK inhibitor, SP600125 20 μM; ERK inhibitor, U0126 20 μM; p38 inhibitor, SP203580 20 μM) for 1 h prior to stimulation with HK H. capsulatum (A) or with the combination of iC3b-LB and dZ (C). Culture supernatants were collected 6 h later and the levels of TNF and IL-6 were quantified by ELISA. Mean ± SD of the relative (A) or absolute cytokine levels (C) of TNF and IL-6 are shown. [n = 5 from 3 independent experiments (A); n = 5 from 2 independent experiments (C)]. (B) FLDMs from Syk+/+, Syk-/- and Syk+/- embryos were stimulated with or without (0 min) HK H. capsulatum for 30 or 60 min. Cell lysates were subjected to Western blotting. (D) Macrophages from WT, Itgam-/-, Clec7a-/- and Itgam-/-Clec7a-/- mice were stimulated with or without (0 min) HK H. capsulatum for 30 min. Cell lysates were analyzed by Western blotting with the use of antibodies against p-JNK and JNK. (E) Macrophages from WT mice were treated with isotype control or blocking antibodies against CR3, Dectin-1, or both for 1 h before stimulation with HK H. capsulatum. Cell lysates were collected 30 min later and subjected to Western blotting. (F and G) Macrophages from WT (F) and Itgam-/- and Clec7a-/- (G) mice were stimulated with or without iC3b-LB, dZ, LB, or the combination of iC3b-LB and dZ for 30 min. Cell lysates were collected at 30 min after stimulation and subjected to Western blotting analysis. Data are representative of at least 3 independent experiments with similar results (B and D-G). * p ≦ 0.05, ** p ≦ 0.01, *** p ≦ 0.001. NS, not significant [2-tailed t-test (A and C)].
Fig 5.
AP-1, but not NF-κB, mediates the collaborative cytokine response upon CR3 and Dectin-1 ligation.
FLDMs from Syk+/+, Syk-/- and Syk+/- embryos (A) and macrophages from WT, Itgam-/-, Clec7a-/- and Itgam-/-Clec7a-/- mice (B and C) were stimulated with or without (0 min) HK H. capsulatum for 30 and 60 min. Cell lysates were analyzed by Western blotting. The blot shown in (A) is the same one shown in Fig 4B. Macrophages from WT mice (D and E), and Itgam-/- and Clec7a-/- mice (F) were stimulated with or without iC3b-LB, dZ, LB, or the combination of iC3b-LB and dZ for 30 min (E) or 60 min (D and F). Cell lysates were analyzed by Western blotting. Data are representative of at least 3 independent experiments (A-F). (G and H) Macrophages from WT mice were transfected with small interfering RNA (siRNA) against c-Fos or c-Jun, followed by stimulation with HK H. capsulatum 48 h later. Cell lysates and culture supernatants were collected at 6 h after stimulation. Silencing of c-Fos and c-Jun was confirmed by Western blotting (G). TNF and IL-6 levels in culture supernatants were quantified by ELISA and are presented as the mean ± SD of relative levels of TNF and IL-6 (n = 3 from 3 independent experiments) (H). ** p ≦ 0.01, *** p ≦ 0.001 [2-tailed t-test].
Fig 6.
CR3 and Dectin-1 coordinately orchestrate host defense against H. capsulatum infection.
(A-C) WT, Itgam-/-, Clec7a-/-, and Itgam-/-Clec7a-/- mice were infected with 2.5 × 105 H. capsulatum intravenously. Infected mice were killed on day 7 after infection. (A) Spleen fungal burdens are shown as CFU per spleen. Data were pooled from 3 independent experiments. Each symbol represents one individual mouse. (B) Splenocytes were cultured for 48 h and the levels of TNF, IL-6, IL-17A and IFN-γ in the supernatants were quantified by ELISA. (C) Splenocytes were cultured for 24 h and stained for surface CD4 and CD8, and intracellular IFN-γ. Cells were analyzed by flow cytometry. Bar graphs show the percentage of IFN-γ + CD4+ and IFN-γ + CD8+ cells in total CD4+ and CD8+ T cell populations. Mean ± SD are shown (n = 6 from 3 independent experiments) (B and C). (D-G) WT, Itgam-/-, Clec7a-/-, and Itgam-/-Clec7a-/- mice were infected with 5 × 106 H. capsulatum intravenously. Survival of infected mice is expressed as a Kaplan-Meier plot (D). Infected mice were killed on day 9 after infection (E-G). (E) The fungal burdens in the spleen and lung are shown as CFU per organ. (F) The total number of cells in the spleen was enumerated. (G) The percentage and the number of F4/80+ cells was analyzed by flow cytometry. Mean ± SD are shown (n = 3-4). * p ≦ 0.05, ** p ≦ 0.01, *** p ≦ 0.001 [one-way ANOVA with Duncan post-hoc analysis was used in data presented in (A-C and E-G); Generalized Wilcoxon test was used for data in (D)].