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

Young-Su Yi,Deok Jeong,Narayanan Parameswaran,Jae Youl Cho,Nur Aziz,Suk Kim,Jae Gwang Park,Han Gyung Kim,Woo Seok Yang,Ji Hye Kim,Eunji Kim

doi:10.3389/fimmu.2021.767366

Abstract

BackgroundInflammation, 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.MethodsHere, 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.ResultsSyk 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.ConclusionsThese 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).

Highlights

Inflammation is an innate immune response that protects the body from pathogen infection (1)
Our findings show that myeloid differentiation primary response 88 (MyD88)-spleen tyrosine kinase (Syk) axis is a key player in the activation of inflammatory responses through molecular interactions and functional cooperation in macrophages, and that the function of the MyD88-Syk axis is regulated by upstream Src kinase activated by Rac1-induced filamentous actin (F-actin) formation
Automated DNA sequencing of mutant plasmids was performed at Bionics (Seoul, Korea). Small-guide RNA (sgRNA) of Syk and MyD88 and primers used for quantitative real-time polymerase chain reaction (PCR) amplification were synthesized at Macrogen Inc. (Seoul, Korea)

Summary

Introduction

Inflammation is an innate immune response that protects the body from pathogen infection (1). Activation of the NF-kB signaling pathway is induced by the triggering of fast-signal transduction cascades that stimulate receptor-associated intracellular adaptor molecules, such as myeloid differentiation primary response 88 (MyD88) and TRIF, and numerous intracellular signaling molecules, such as Src, spleen tyrosine kinase (Syk), phosphoinositide 3-kinase (PI3K), Akt, phosphoinositide-dependent kinase 1 (PDK1), inhibitors of kB (IkB) kinase a/b (IKKa/b), and IkB in the NF-kB signaling pathway (6, 8, 10) Activation of these molecules in the NF-kB signaling pathway induces inflammatory responses by facilitating the generation of inflammatory mediators, including reactive oxygen species (ROS), nitric oxide (NO) and prostaglandin E2; increasing the expression and the activity of inflammatory enzymes and pro-inflammatory cytokines, including caspases, ROSgenerating enzymes, inducible NO synthase, cyclooxygenase-2, matrix metalloproteinases, tumor-necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6, interferon (IFN)-b, and interferongamma inducible protein 10; and promoting the phagocytic activity of macrophages (6–18). 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

Results

Conclusion