Recombinant human KAI1/CD82 attenuates M1 macrophage polarization on LPS-stimulated RAW264.7 cells via blocking TLR4/JNK/NF-κB signal pathway

Abstract

Therefore, the aim of the present study was to investigate the effect of recombinant protein for second extracellular domain of human KAI1 (Gly 111-Leu 228, rhKAI1) on LPS-stimulated RAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM), and to identify the its underlying mechanism. Our data showed that rhKAI1 suppressed the expression of active macrophages (M1) phenotype-related surface markers F4/80+CD86+ in LPS-stimulated BMDM and RAW264.7 cells. In addition, LPS markedly up-regulated the mRNA expression and release levels of pro-inflammatory cytokines and mediators, such as interleukin (IL)-1β, IL-6, tumor necrosis factor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2, whereas this increase was substantially down-regulated by rhKAI1. Furthermore, LPS strongly promoted the fluorescence expression of NF-κB p65 in the nuclei, and increased the phosphorylation of ERK, JNK, and p38 MAPK. However, the nuclear translocation of NF-κB p65 and the phosphorylation of JNK were greatly reversed in the presence of the rhKAI1. Especially, rhKAI1 markedly suppressed the expression of -like receptor (TLR4) and it prevent the binding of LPS with TLR4 through molecular docking predict analysis. Importantly, Glu 214 of rhKAI1 residue strongly interact with Lys 360 of TLR4 residue, and the distance between the bound was 2.9 Å. Taken together, all these findings suggested that rhKAI1 had anti-inflammatory effect in LPS-polarized macrophages by interacting with TLR4 and down-regulating the JNK/NF-κB signaling pathway.