Syk-MyD88 Axis Is a Critical Determinant of Inflammatory-Response in Activated Macrophages

Abstract

Background: Inflammation, a vital immune response to infection and injury, is mediated by macrophage activation. While spleen tyrosine kinase (Syk) and myeloid differentiation primary response 88 (MyD88) are reportedly involved in inflammatory responses in macrophages, their roles and underlying mechanisms are largely unknown.

Methods: Here, the role of the MyD88-Syk axis and the mechanism by which Syk and MyD88 cooperate during macrophage-mediated inflammatory responses are explored using knockout conditions of these proteins and mutation strategy as well as flowcytometric and immunoblotting analyses.

Results: Syk rapidly activates the nuclear factor-kappa B (NF-κB) signaling pathway in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the activation of the NF-κB signaling pathway is abolished in Syk^-/- RAW264.7 cells. MyD88 activates Syk and Syk-induced activation of NF-κB signaling pathway in LPS-stimulated RAW264.7 cells but Syk-induced inflammatory responses are significantly inhibited in MyD88^-/- RAW264.7 cells. MyD88 interacts with Syk through the tyrosine 58 residue (Y58) in the hemi-immunoreceptor tyrosine-based activation motif (ITAM) of MyD88, leading to Syk activation and Syk-induced activation of the NF-κB signaling pathway. Src activates MyD88 by phosphorylation at Y58 via the Src kinase domain. In addition, Ras-related C3 botulinum toxin substrate 1 (Rac1) activation and Rac1-induced formation of filamentous actin (F actin) activate Src in LPS-stimulated RAW264.7 cells.

Conclusions: These results suggest that the MyD88-Syk axis is a critical player in macrophage-mediated inflammatory responses, and its function is promoted by an upstream Src kinase activated by Rac1-generated filamentous actin (F-actin).

Keywords: F-actin; MyD88; Src; Syk; inflammation.

Figure 1

Syk was a prime activator of the NF-κB signaling pathway in macrophages. (A) RAW264.7 cells co-transfected with the NF-κB-Luciferase reporter gene plasmid and either empty (pcDNA), Syk, Src, or Akt plasmids were treated with either LPS (1 μg/mL), TNF-α (20 ng/mL), or PMA (100 nM) for 24 h. Luciferase activity was measured by a luminometer and normalized to β-galactosidase activity. (B, C) Syk, p-Syk, p85, p-p85, IκBα, and p-IκBα in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analyses. (D) p65 and p50 in the nuclear lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analysis. (E) IκBα and p-IκBα in the whole-cell lysates of WT and Syk^−/− RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by Western blot analysis. (F) p65 and p50 in the nuclear lysates of WT and Syk^−/− RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analysis. (G) IκBα and p-IκBα in the whole-cell lysates of RAW264.7 cells and p65 and p50 in the nuclear lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time in the absence or presence of Pic (50 μM) and Bay (10 μM) were determined by western blot analysis. (H) WT and Syk^−/− RAW264.7 cells transfected with the NF-κB-luciferase reporter gene plasmids for 24 h were treated with LPS (1 μg/mL) for 24 h, after which the luciferase activity of these cells was measured by a luminometer and normalized to β-galactosidase activity. (I) HEK293 cells co-transfected with the NF-κB-Luciferase reporter gene plasmids and Myc-Syk plasmids for 24 h were treated with PMA (100 nM), Pic (0–50 μM), and Bay (0-10 μM), and the luciferase activity was measured by a luminometer and normalized to β-galactosidase activity. *P < 0.05, **P < 0.01 compared with controls.

Figure 2

MyD88 activated the Syk-induced NF-κB signaling pathway in macrophages. (A) IκBα,and p-IκBα were detected by western blot analysis in the peritoneal macrophages extracted from WT, MyD88⁻/⁻, and TRIF^−/− mice under LPS (1 μg/mL) treatment. (B) IκBα and p-IκBα in whole-cell lysates of RAW264.7 cells treated with Pam3CSK4 (10 μg/mL), Poly I:C (200 μg/mL), or LPS (1 μg/mL) for the indicated time were detected by western blot analysis. (C) p-Syk, Flag, and Myc were detected by western blot analysis in whole-cell lysates of HEK293 cells co-transfected with Myc-Syk and either empty (pcDNA) or Flag-MyD88 plasmid for 48 h. (D) WT and Syk^−/− RAW264.7 cells were co-transfected with the NF-κB-Luciferase reporter gene plasmid and MyD88 plasmid for 48 h. The luciferase activity of these cells was measured by a luminometer and normalized to β-galactosidase activity. (E) HEK293 cells co-transfected with the NF-κB-Luciferase reporter gene plasmid, either empty (pcDNA) or Flag-MyD88 plasmids, and empty (pcDNA) or Myc-Syk ΔKD plasmids for 48 h, and the luciferase activity was measured by a luminometer and normalized to β-galactosidase activity. (F) MyD88^−/− RAW264.7 cells were generated by CRISPR Cas9, and expression of MyD88 was detected by western blot analysis. WT RAW264.7 and MyD88^−/− RAW264.7 cells were treated with LPS (1 μg/mL) for the indicated time, and mRNA expression of the target genes was determined by quantitative real time PCR. (G) WT RAW264.7 and MyD88^−/− RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were incubated with DHR 123 (20 μM) for 20 min, and the ROS level was determined by measuring fluorescence. (H) WT RAW264.7 and MyD88^−/− RAW264.7 cells were incubated with fluorescein isothiocyanate–E. coli (10 μg/mL) for the indicated time, and fluorescence was determined by a fluorescence plate reader. *P < 0.05, **P < 0.01 compared with controls.

Figure 3

MyD88 interacted with Syk in macrophages. (A) Whole-cell lysates of RAW264.7 cells and peritoneal macrophages were immunoprecipitated with either non-specific control immunoglobulin G or Syk antibodies, followed by detection of Syk and MyD88 by western blot analysis. (B) Schematic illustration of WT Myc-Syk and Syk deletion mutants, Myc-Syk(ΔSH2-N), Myc-Syk(ΔSH2-C), and Myc-Syk(ΔKD). Whole-cell lysates of HEK293 cells co-transfected with Flag-MyD88 and either Myc-Syk or Syk-deletion mutant plasmids for 48 h were immunoprecipitated with Myc antibodies, followed by detection of Myc and Flag by western blot analysis. (C) Schematic illustration of WT Flag-MyD88 and MyD88 deletion mutants, Flag-MyD88(ΔDD), Flag-MyD88(ΔID), and Flag-MyD88(ΔTIR). Whole-cell lysates of HEK293 cells co-transfected with Myc-Syk and either Flag-MyD88 or MyD88 deletion mutant plasmids for 48 h were immunoprecipitated with Myc antibodies, followed by detection of Myc and Flag by western blot analysis. (D) Schematic illustration of a WT Flag-MyD88, a MyD88 deletion mutant, Flag-MyD88(ΔITAM), and a point mutant. Whole-cell lysates of HEK293 cells co-transfected with Myc-Syk and either Flag-MyD88, Flag-MyD88(ΔITAM) or Flag-MyD88(Y58F) plasmids for 48 h were immunoprecipitated with Myc antibodies, followed by detection of Myc and Flag by western blot analysis. (E) p-Syk, Flag, and Myc in the whole-cell lysates of HEK293 cells co-transfected with Myc-Syk and either Flag-MyD88, Flag-MyD88(ΔITAM), or Flag-MyD88(Y58F) plasmids for 48 h were detected by Western blot analysis. (F) HEK293 cells co-transfected with the NF-κB-luciferase reporter gene plasmid and either empty (pcDNA), Flag-MyD88, Flag-MyD88(ΔITAM) or Flag-MyD88(Y58F) plasmids for 48 h, and the luciferase activity was measured by a luminometer and normalized to β-galactosidase activity. *P < 0.05, **P < 0.01 compared with controls.

Figure 4

Src activated MyD88 by phosphorylating 58 tyrosine residue in macrophages. (A) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time was detected by immunoprecipitation and western blot analysis. (B) p-Tyr MyD88 and Flag in whole-cell lysates of RAW264.7 cells transfected with empty (pcDNA), Flag-MyD88, Flag-MyD88(ΔITAM) or Flag-MyD88(Y58F) plasmids for 48 h were detected by immunoprecipitation and western blot analysis. (C) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells co-transfected with Flag-MyD88 and empty (pcDNA), Src, Fyn, Mal, or TLR4 for 48 h was detected by immunoprecipitation and western blot analysis. (D) Src and p-Src in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analysis (E) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells co-transfected with Flag-MyD88 and empty (pcDNA), Src, or Src(ΔKD) was detected by immunoprecipitation and western blot analysis. (F) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the 1 min with absence or presence of PP2 (20 μM) was detected by immunoprecipitation and western blot analysis (G) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells co-transfected with Src and either empty (pcDNA), Flag-MyD88, or Flag-MyD88(Y58F) plasmids for 48 h were detected by immunoprecipitation and western blot analysis.

Figure 5

Rac1 activated Src by promoting actin filament production in macrophages. (A) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time was detected by immunoprecipitation and western blot analysis. (B) Src and p-Src in whole-cell lysates of RAW264.7 cells incubated with vehicle (DMSO) or CytoB (20 μM), followed by the treatment with LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Src/β-actin level was plotted. (C) p-Syk in whole-cell lysates of RAW264.7 cells incubated with the vehicle (DMSO) or CytoB (20 μM), followed by treatment of LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Syk/β-actin level was plotted. (D) Rac1-GTP and Rac1 in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analysis. (E) Rac1, Src, and p-Src in whole-cell lysates of RAW264.7 cells transfected with siN.C or siRac1 for 24 h, followed by the treatment of LPS (1 μg/mL) for the indicated time, were detected by western blot analysis, and the p-Src/Src level was plotted. (F) RAW264.7 cells incubated with DMSO or NSC23766 (100 μM) were treated with LPS (1 μg/mL) for the indicated time in the presence of Texas Red-X phalloidin (200 units/mL), and fluorescence was determined with a flow cytometer and plotted. (G) Rac1-GTP, Src, and p-Src in the whole-cell lysates of RAW264.7 cells incubated with DMSO or NSC23766 (100 μM), followed by the treatment of LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Src/Src level was plotted. (H) Rac1-GTP, Syk, and p-Syk in whole-cell lysates of RAW264.7 cells incubated with DMSO or NSC23766 (100 μM), followed by treatment of LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Syk/Syk level was plotted.

Figure 6

Schematic summary demonstrating the role of MyD88-Syk axis in the macrophage-mediated inflammatory responses.