A Study On The Mechanism Of Macrophage Activation By Exopolysaccharide From Paecilomyces Lilacinus

Objectives : Exopolysaccharide(EPS)is a kind of polysaccharide secreted by microorganisms outside the cell during growth and metabolism.It has anti-tumor activity,immunomodulatory activity and anti-inflammatory and other biological activities.Our research group isolated a strain of Paecilomyces lilacinus from a marine environment of the mangrove tropical wetland in Hainan,China.We extracted the extracellular polysaccharide(PH-EPS)from the strain.Macrophages play a vital role as the first line of defense against infection and the main effector cells of tumor immunity.Thus,this study mainly investigates the immunomodulatory activities and mechanism of PH-EPS on macrophages.Methods:Macrophage cell line RAW264.7 was used as the experimental subjects.Different concentrations of PH-EPS and RAW264.7 cells were co-cultured for 24 h,the survival rate of macrophages was detected by MTT.The different concentrations of PH-EPS were applied to RAW264.7 cells,and the supernatant of the cell culture was used to detect the content of NO by Griess kit.After different concentrations of PH-EPS were applied to RAW264.7 cells,the cells were lysed and the inducible nitric oxide synthase(iNOS),I?B-? protein and phosphorylation proteins of MAPK pathway were detected by Western blotting(WB)assays.Tumor necrosis factor(TNF-?)and interleukin-1?(IL-1?)secreted by RAW264.7 macrophages after PH-EPS treatment were detected by enzyme-linked immunosorbent assay(ELISA);the uptake of FITC-dextran,representing the phagocytic function of macrophages,NF-?B p65 transferation from cytoplasm to nucleus was observed by laser confocal microscopy.Antibodies against NF-?b,TLR2,TLR4,Dectin-1 and CR3 were used to verify whether these receptors were the target receptors of PH-EPS.Results:MTT results showed that PH-EPS had no significant effect on the survival rate of RAW264.7 cells at concentrations of 0 ?g/ml~600 ?g/ml.Significant NO release in a dose-dependent model was found in the RAW264.7 cells treated with PH-EPS.There was not change found in NO secretion in the cells co-treated with polymyxin B(PMB)and PH-EPS,indicating that the production of NO was not related to endotoxin contamination.WB assay showed that PH-EPS could promote the high expression of NO-induced enzyme iNOS in RAW264.7 cells in a dose-dependent manner,suggesting that PH-EPS may promoted NO production in RAW264.7 cells by up-regulating the expression of iNOS.ELISA results showed that PH-EPS significantly increased the levels of TNF-? and IL-1? in macrophages in a dose-dependent manner.Subsequently,flow cytometry was used to detected the uptake of FITC labeled dextran by macrophages to reflect its phagocytosis.It was found that the fluorescence intensity of PH-EPS treated cells was significantly enhanced in a dose-dependent manner,indicating that PH-EPS can enhance RAW264.7 cell phagocytic capacity.To further study the mechanism of PH-EPS activation of RAW264.7 cells,we found significant translocation of NF-?B p65 from cytoplasm to nucleus by fluorescence confocal microscopy in PH-EPS treated RAW264.7 cells.Western blotting detection revealed that the expression of I?B-? protein was down-regulated in PH-EPS treated RAW264.7 cells.Furthermore,after the NF-?B pathway was blocked by the NF-?B inhibitor PDTC and BAY11-7082,it was found that the expression levels of TNF-? and IL-1? in PH-EPS treated were significantly decreased,indicating that NF-?B signaling pathway was involved in PH-EPS mediated activation of RAW264.7 cells.In addition,Western blotting analysis also showed that the phosphorylation levels of ERK,JNK and p38 in the three major cascade proteins of MAPK pathway were significantly increased,in contrast with MAPK inhibitors,the levels of TNF-? and IL-1? in the supernatant of macrophages was also significantly reduced,those results indicated that activation of MAPK activation signaling pathway is involved in PH-EPS mediated activation of macrophages.Finally,we treated RAW264.7 cells with anti-TLR2,TLR4,Dectin-1,CR3 antibodies and PH-EPS,then detected TNF-? and IL-1? levels by ELISA.It was found that the secretion of TNF-? and IL-1? was significantly decreased after blocking the TLR4 and Dectin-1 pathways.indicating that TLR4 and Dectin-1 was involved in PH-EPS induced RAW264.7 cell activation.Conclusions: The above results indicated that the exopolysaccharide(PH-EPS)from Paecilomyces lilacinus can activated macrophages,promoted their inflammatory processes and phagocytic activity.TLR4/NF-?B/MAPK signaling pathway activation is an important molecular mechanism for PH-EPS to activate macrophages.