title: Pre-treatment of recombinant mouse MFG-E8 downregulates LPS-induced TNF-α production in macrophages via STAT3-mediated SOCS3 activation.
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.
authors: Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, New York, United States of America.
authors: Monowar
authors: M
authors: Aziz
authors: Asha
authors: A
authors: Jacob
authors: Akihisa
authors: A
authors: Matsuda
authors: Rongqian
authors: R
authors: Wu
authors: Mian
authors: M
authors: Zhou
authors: Weifeng
authors: W
authors: Dong
authors: Weng-Lang
authors: WL
authors: Yang
authors: Ping
authors: P
authors: Wang
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000502:physiology*; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D019446:Endotoxemia / Q000188:drug therapy / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008894:Milk Proteins; D010587:Phagocytosis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D000954:Antigens, Surface; D008070:Lipopolysaccharides; C490919:Mfge8 protein, mouse; D008894:Milk Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C491411:Socs3 protein, mouse; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014409:Tumor Necrosis Factor-alpha