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Comparative Study
. 2009 Jan 15;182(2):1146-54.
doi: 10.4049/jimmunol.182.2.1146.

Differential use of CARD9 by dectin-1 in macrophages and dendritic cells

Helen S Goodridge  1 , Takahiro Shimada, Andrea J Wolf, Yen-Michael S Hsu, Courtney A Becker, Xin Lin, David M Underhill
Affiliations
Comparative Study

Differential use of CARD9 by dectin-1 in macrophages and dendritic cells

Helen S Goodridge et al. J Immunol. .

Abstract

The pattern recognition receptors TLR2 and Dectin-1 play key roles in coordinating the responses of macrophages and dendritic cells (DC) to fungi. Induction of proinflammatory cytokines is instructed by signals from both TLR2 and Dectin-1. A recent report identified a role for CARD9 in innate anti-fungal responses, demonstrating CARD9-Bcl10-mediated activation of NF-kappaB and proinflammatory cytokine induction in murine bone marrow-derived DC stimulated via Dectin-1. We now report that Dectin-1-CARD9 signals fail to activate NF-kappaB and drive TNF-alpha induction in murine bone marrow-derived macrophages. However, priming of bone marrow-derived macrophages with GM-CSF or IFN-gamma permits Dectin-1-CARD9-mediated TNF-alpha induction. Analysis of other macrophage/DC populations revealed further variation in the ability of Dectin-1-CARD9 signaling to drive TNF-alpha production. Resident peritoneal cells and alveolar macrophages produce TNF-alpha upon Dectin-1 ligation, while thioglycollate-elicited peritoneal macrophages and Flt3L-derived DC do not. We present data demonstrating that CARD9 is recruited to phagosomes via its CARD domain where it enhances TLR-induced cytokine production even in cells in which Dectin-1 is insufficient to drive cytokine production. In such cells, Dectin-1, CARD9, and Bcl10 levels are not limiting, and data indicate that these cells express additional factors that restrict Dectin-1-CARD9 signaling for TNF-alpha induction.

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Figures

Figure 1
Figure 1. Dectin-1 signals via CARD9 and Bcl10 to activate NF-κB
HEK293 cells were transfected with ELAM- (A and B) or SRE- (C) firefly luciferase and CMV-β-galactosidase reporters (A–C), as well as plasmids encoding CARD9, Bcl10 and streptavidin binding peptide (SBPc)-tagged Dectin-1, as indicated. The following day cells were stimulated with 100 μg/ml zymosan or depleted zymosan, or 1:60 streptavidin beads for 4 h. ELAM/SRE-driven luciferase reporter activity was normalized to β-galactosidase activity and expressed as the mean plus standard deviation of triplicate culture.
Figure 2
Figure 2. CARD9 is recruited to forming phagosomes during Dectin-1-mediated phagocytosis, but is not required for phagocytosis
(A) Full length CARD9, the CARD domain of CARD9, and the CARD9 coiled-coil domain were tagged with streptavidin-binding peptide (SBPc). (B) RAW264.7 macrophages stably expressing SBPc-tagged full length CARD9, CARD domain, or coiled-coil domain were fed 100 μg/ml zymosan particles for 10 min, prior to fixing and staining with an antibody against the SBPc tag (CARD9 constructs) and TRITC-phalloidin (β-actin). Arrows identify actin-positive early phagosomes; arrowheads identify actin-negative later phagosomes. (C) Bone marrow-derived macrophages (bmM) and dendritic cells (bmDC) from wild type and CARD9-deficient mice were fed 100 μg/ml TRITC-labeled zymosan for the times indicated and phagocytosis was assessed by flow cytometry.
Figure 3
Figure 3. Dectin-1 signals directly trigger TNF-α production in bmDC, but not bmM
(A – C) bmM (A), bmDC (B), and Dectin-1-expressing RAW264.7 cells (C) were stimulated with 100 μg/ml zymosan or depleted zymosan, 1 mg/ml depleted zymosan (10× depleted zymosan), or 100 ng/ml Pam3CSK4 for 24 h, and TNF-α levels in culture supernatants were assessed by ELISA. (D) Dectin-1-expressing RAW264.7 cells were stimulated with 100 μg/ml zymosan or depleted zymosan, and reactive oxygen production was measured by luminol-ECL. (E and F) bmDC (E) and bmM (F) from wild type and CARD9-deficient mice were stimulated with 100 μg/ml zymosan or depleted zymosan, or 100 ng/ml Pam3CSK4 for 24 h, and TNF-α levels in culture supernatants were assessed by ELISA. (G) bmM from wild type and CARD9-deficient mice were stimulated with Pam3CSK4 at the indicated concentrations in the presence or absence of 100 μg/ml depleted zymosan for 24 h, and TNF-α levels in culture supernatants were assessed by ELISA. All stimulations were performed in triplicate, and are presented as means (plus standard deviation for ELISA data). ***, p<0.001.
Figure 4
Figure 4. Dectin-1-CARD9 signals trigger NF-κB activation in bmDC, but not bmM, but contribute to p38 MAP kinase activation in bmM
(A) bmM and bmDC were stimulated with 100 μg/ml zymosan or depleted zymosan, or 100 ng/ml Pam3CSK4 for the times indicated, and I-κB levels were assessed by Western blotting, with Erk1/2 as a loading control. (B) bmM and bmDC were stimulated with 100 ng/ml LPS, or 100 μg/ml zymosan or depleted zymosan for 90 min, and nuclear translocation of NF-κB was assessed by EMSA. (C and D) bmM from wild type (C and D) and CARD9-deficient (D) mice were stimulated with 100 μg/ml zymosan or depleted zymosan, or 100 ng/ml Pam3CSK4 for the times indicated, and p38 phosphorylation was assessed by Western blotting, with total p38 as a loading control.
Figure 5
Figure 5. Analysis of Dectin-1-induced TNF-α production by GM –CSF- and IFN-γ-primed bmM, and other macrophage and DC populations
(A and B) bmDC were pre-treated overnight with 50 ng/ml M-CSF (A), and bmM were pre-treated overnight with 10 ng/ml GM-CSF or 25 U/ml IFN-γ (B). bmDC and bmM were then stimulated with 100 μg/ml zymosan or depleted zymosan, or 100 ng/ml Pam3CSK4 for 24 h, and TNF-α in culture supernatants was assessed by ELISA. (C) bmM from wild type and CARD9-deficient bmM were pre-treated overnight with 10 ng/ml GM-CSF or 25 U/ml IFN-γ, prior to stimulation with 100 μg/ml zymosan or depleted zymosan, or 100 ng/ml Pam3CSK4 for 24 h, and TNF-α was assessed by ELISA. (D and E) Resident peritoneal cells (RPC; D) and thioglycollate-elicited peritoneal macrophages (TEPM; E) were stimulated with 100 μg/ml zymosan or depleted zymosan, or 100 ng/ml Pam3CSK4 for 24 h, and TNF-α was assessed by ELISA. (F) RPC, TEPM and alveolar macrophages (AvM) were stimulated with 100 μg/ml zymosan or depleted zymosan for 4 h in the presence of brefeldin A, and TNF-α production was assessed by intracellular flow cytometry, gating for CD11b+ RPC and TEPM, and CD11b− AvM. (G) DC derived from bone marrow with Flt3L (Flt3L DC) were stimulated with 100 μg/ml zymosan or depleted zymosan for 24 h and TNF-α in culture supernatants was assessed by ELISA. For ELISA measurements stimulations were performed in triplicate, and are presented as mean plus standard deviation. ***, p<0.001; ns, no significant difference.
Figure 6
Figure 6. There is no correlation between Dectin-1, CARD9 and Bcl10 expression levels and the ability of Dectin-1 to directly induce TNF-α
(A) CARD9 and Bcl10 levels in bmM and bmDC were compared by Western blotting, with GAPDH as a loading control. (B) Surface expression of Dectin-1 on bmM and bmDC was assessed by flow cytometry. (C) CARD9 expression by bmM and bmM pre-treated overnight with 10 ng/ml GM-CSF or 25 U/ml IFN-γ was assessed by Western blotting, with β-actin as a loading control. (D and E) Surface expression of Dectin-1 and TLR2 on bmM and bmM pre-treated overnight with 10 ng/ml GM-CSF (D) or 25 U/ml IFN-γ (E) was assessed by flow cytometry. (F and G) Dectin-1 surface expression by RPC, TEPM, AvM, bmDC and Flt3L DC was assessed by flow cytometry.
Figure 7
Figure 7. Overexpression of Dectin-1 in bmM overcomes blockade of direct Dectin-1-mediated TNF-α induction
(A) bmM were transfected with either Dectin-1-GFP or a GFP control plasmid, and Dectin-1 surface expression was assessed by flow cytometry of unpermeabilized cells. (B) Control or Dectin-1-overexpressing bmM were stimulated for 4 h with 100 μg/ml zymosan or depleted zymosan in the presence of brefeldin A, and TNF-α production by GFP+ cells (control and Dectin-1 plasmids), was assessed by intracellular flow cytometry.
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References

    1. Creagh EM, O’Neill LA. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol. 2006;27:352–357. - PubMed
    1. Taylor PR, Martinez-Pomares L, Stacey M, Lin HH, Brown GD, Gordon S. Macrophage receptors and immune recognition. Annu Rev Immunol. 2005;23:901–944. - PubMed
    1. Goodridge HS, Underhill DM. Fungal Recognition by TLR2 and Dectin-1. Handb Exp Pharmacol. 2008:87–109. - PubMed
    1. Taylor PR, Tsoni SV, Willment JA, Dennehy KM, Rosas M, Findon H, Haynes K, Steele C, Botto M, Gordon S, Brown GD. Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol. 2007;8:31–38. - PMC - PubMed
    1. Saijo S, Fujikado N, Furuta T, Chung SH, Kotaki H, Seki K, Sudo K, Akira S, Adachi Y, Ohno N, Kinjo T, Nakamura K, Kawakami K, Iwakura Y. Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans. Nat Immunol. 2007;8:39–46. - PubMed

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