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CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

Huang JH, Lin CY, Wu SY, Chen WY, Chu CL, Brown GD, Chuu CP, Wu-Hsieh BA - PLoS Pathog. (2015)

Bottom Line: Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production.Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response.Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT
Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.

No MeSH data available.


Related in: MedlinePlus

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)].
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ppat.1004985.g004: 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)].

Mentions: We used pharmacological kinase inhibitors to identify the signaling molecule(s) that participates in macrophage cytokine response to H. capsulatum. Treatment with PI3K, JNK and ERK inhibitors greatly diminished TNF and IL-6 production, yet inhibiting Raf-1 enhanced TNF and IL-6 responses (Fig 4A). Interestingly, p38 inhibitor had disparate effects on TNF and IL-6 production (Fig 4A). LDH assay showed that pharmacological inhibitors at the concentrations we used did not have cytotoxic effect on the cells (S6 Fig). Interestingly, Syk deficiency abrogated phosphorylation of Raf-1 and JNK but not that of Akt, ERK or p38 (Fig 4B) although inhibiting their activation diminished TNF and IL-6 production (Fig 4A). Results in Fig 4C show that inhibition of JNK activation in cells stimulated with iC3b-coated beads and depleted zymosan significantly reduced TNF and IL-6 production yet inhibition of Raf-1 did not affect the production of either cytokine. These results indicate that JNK, a signaling molecule downstream of Syk, plays an important role in the coordinated CR3 and Dectin-1cytokine response.


CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

Huang JH, Lin CY, Wu SY, Chen WY, Chu CL, Brown GD, Chuu CP, Wu-Hsieh BA - PLoS Pathog. (2015)

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)].
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ppat.1004985.g004: 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)].
Mentions: We used pharmacological kinase inhibitors to identify the signaling molecule(s) that participates in macrophage cytokine response to H. capsulatum. Treatment with PI3K, JNK and ERK inhibitors greatly diminished TNF and IL-6 production, yet inhibiting Raf-1 enhanced TNF and IL-6 responses (Fig 4A). Interestingly, p38 inhibitor had disparate effects on TNF and IL-6 production (Fig 4A). LDH assay showed that pharmacological inhibitors at the concentrations we used did not have cytotoxic effect on the cells (S6 Fig). Interestingly, Syk deficiency abrogated phosphorylation of Raf-1 and JNK but not that of Akt, ERK or p38 (Fig 4B) although inhibiting their activation diminished TNF and IL-6 production (Fig 4A). Results in Fig 4C show that inhibition of JNK activation in cells stimulated with iC3b-coated beads and depleted zymosan significantly reduced TNF and IL-6 production yet inhibition of Raf-1 did not affect the production of either cytokine. These results indicate that JNK, a signaling molecule downstream of Syk, plays an important role in the coordinated CR3 and Dectin-1cytokine response.

Bottom Line: Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production.Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response.Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT
Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.

No MeSH data available.


Related in: MedlinePlus