Pre-Treatment of Recombinant Mouse MFG-E8 Downregulates LPS-Induced TNF-α Production in Macrophages via STAT3-Mediated SOCS3 Activation

Monowar Aziz,Akihisa Matsuda,Asha Jacob,Weifeng Dong,Weng-Lang Yang,Ping Wang,Mian Zhou,Rongqian Wu,Marcelo G Bonini

doi:10.1371/journal.pone.0027685

Abstract

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8−/− mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8−/− mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8−/− mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.

Highlights

The glycoprotein, Milk fat globule-epidermal growth factor factor 8 (MFG-E8) is mainly secreted by mononuclear cells, and participates in the phagocytosis of apoptotic cells by tethering between phosphatidylserine (PS) on apoptotic cells and avb3-integrin on phagocytes [1]
Similar effects were observed in peritoneal macrophages from WT mice, where the cells treated with rmMFG-E8 significantly downregulated the LPS-induced tumor necrosis factor (TNF)-a mRNA and protein levels by 24% and 23%, respectively (Fig. 1C, 1D)
Our study demonstrated the direct anti-inflammatory role of MFG-E8 in terms of attenuating TNF-a production in mouse peritoneal macrophages and RAW264.7 cells treated with LPS

Summary

Introduction

The glycoprotein, MFG-E8 is mainly secreted by mononuclear cells, and participates in the phagocytosis of apoptotic cells by tethering between phosphatidylserine (PS) on apoptotic cells and avb3-integrin on phagocytes [1]. Toll like receptor (TLR) 4 expressed in immunereactive cells can efficiently recognize bacterial lipopolysaccharide (LPS) and trigger down-stream signaling via several adaptor molecules, e.g., myeloid differentiation factor 88 (MyD88), Toll/ IL-1 receptor domain-containing adaptor (TIRAP)/MyD88adaptor-like (Mal), IL-1 receptor-associated kinase (IRAK), and TNF receptor-associated factor 6 (TRAF6) to induce NF-kB, MAP kinases and JNK pathways. This in turn upregulates the expression of pro-inflammatory cytokines, e.g., tumor necrosis factor (TNF)-a [9,10]. To maintain the fine-tuned balance of TLR4 pathway, several intracellular negative regulators, e.g., A20, IRAK-M, and Tollip are become activated under LPS-treated conditions [12,13]

Objectives

Methods

Results

Conclusion