Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors

Abstract

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1alpha and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kappaB (IkappaB), enhancing NFkappaB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

Figure 1

Collaborative induction of pro-inflammatory cytokines through Dectin-1 and TLR2 using specific ligands. (A) Production of TNF by C57BL/6 macrophages stimulated with 100, 10 or 1 μg/mL particulate β-glucan, with or without 10 ng/mL Pam₃CSK₄. (B) Western blots showing phosphorylation of Syk, and its substrate SLP-76, upon stimulation of RAW macrophages with 10 μg/mL β-glucan. Data shown are representative of two independent experiments. (C) Activation of Syk in 129Sv macrophages by β-glucan (arrows), as determined by fluorescence microscopy. Left panel, phase image; right panel phospho-Syk image. Scale bar indicates 10 μm. (D) Collaborative production of TNF by C57BL/6 macrophages stimulated with 10 μg/mL particulate β-glucan occurs over a range of Pam₃CSK₄ concentrations. (E) Collaborative TNF production by 129/Sv macrophages stimulated with 10 μg/mL particulate β-glucan and 10 ng/mL Pam₃CSK₄ is dependent on Dectin-1 expression. Collaborative MIP-1α (F) and MIP-2 (G) production from C57BL/6 macrophages stimulated with 10 μg/mL particulate β-glucan occurs over a range of Pam₃CSK₄ concentrations. Data shown are mean ± SD and are representative of at least two independent experiments.

Figure 2

Collaboration of Syk kinase and TLR/MyD88 pathways sustain IkB degradation and enhance NFkB nuclear translocation. TNF production from C57BL/6 wild-type and MyD88^–/– (A) or BALB/C wild-type and Syk^–/– (B) macrophages stimulated with 10 μg/mL particulate β-glucan and 10 ng/mL Pam₃CSK₄, as indicated. (C) RAW macrophages stimulated with 10 ng/mL Pam₃CSK₄ and 10 μg/mL particulate β-glucan. Top: IkB degradation after 2 h was assayed by Western blotting. Bottom: localization of NFkB c-Rel in nuclear lysates. Numbers above each lane show fold decrease (IkB) or increase (cRel) of the relative band intensities of IkB and c-Rel, with actin and USF-2 as loading controls, versus unstimulated control. (D) IkB degradation in RAW macrophages was assayed after the indicated times. Data shown are mean ± SD and are representative of two independent experiments. (E) Nuclear translocation of c-Rel (green) following costimulation of C57BL/6 macrophages with 10 ng/mL Pam₃CSK₄ and 10 μg/mL particulate β-glucan for 1 h. Nuclei were stained with Hoechst 33258 (red). Scale bar represents 10 μm. (F) Nuclear translocation of c-Rel was quantified microscopically over time in C57BL/6 macrophages stimulated with 10 ng/mL Pam₃CSK₄ followed by 10 μg/mL particulate β-glucan. Data shown are mean ± SD and are representative of two independent experiments.

Figure 3

Collaborative responses occur with multiple TLR and are comparable to synergistic responses induced through TLR2 and TLR4. Production of TNF by human peripheral blood mononuclear cells stimulated with or without 10 μg/mL particulate β-glucan and the indicated concentrations of Pam₃CSK₄ (A), LPS (B) or flagellin (C). Data shown are mean ± SEM of pooled data from five independent donors. (D) Collaborative TNF responses from C57BL/6 macrophages stimulated with TLR7 (0.2 μg/mL CL-097) or TLR9 (1 μg/mL ODN1826) ligands and particulate β-glucan, as indicated. Data shown are the mean ± SD of one representative experiment of three. (E) Costimulation of C56BL/6 macrophages with 10 μg/mL particulate β-glucan and 10 ng/mL Pam₃CSK₄ or 1 ng/mL LPS induces TNF production that is comparable to the synergistic response following TLR2 and TLR4 co-ligation. Shown are the mean ± SEM pooled of data pooled from three independent experiments.