BLP Tolerance Enhances The Phagocytosis Of BMDM Through The Upregulation Of MARCO

Sepsis was a syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection,with high morbidity,high mortality and high costs.However,there was no definitely effective treatment.Since innate immune system plays a significant role in the process of sepsis,uncovering its effect to the infectious disease is important for designing new treatment.Bacterial lipoprotein(BLP)was a protein existed in the bacterial membrane.Previous studies showed that the mortality would be decrease under high dose BLP treatment on the animals that have been administrated for low dose BLP in advance,which phenomenon was regarded as BLP tolerance.Mice and cells which received BLP tolerance not only showed decreased inflammatory response,similar with the endotoxin tolerance,but also performed enhanced phagocytosis and microbicidal effect.Phageocytosis related receptors on the cell membrance recognize and bind the microbial were the key process of the cell phageocytosis and bacterial clearance.Class A scavenger receptor macrophage receptor with collagenous Structure(MARCO)could recognize and combine both Gram-positive bacterial and Gram-negative bacterial,which were important to the bacterial clearance and the endogenous anti-bacterial response activation.However,the effect of MARCO on the BLP tolerance is still unclear.In present study,we use bone marrow derived macrophages as the cell model,investigating the effect of MARCO on the cell phageocytosis and bacterial clearance of the cells undere the BLP tolerance.Primary macrophages were isolated and cultured from bone marrow.After the differentiation and maturation,cells in BLP tolerance group were treated with 100 ng/ml BLP,while cells in Naive group received same volume PBS.Gram-negative bacterial E.coli and Gram-positive bacterial staphylococcus aureus were used to stimulate the cells respectively after 24 hours tolerance treatment.Sterilization experiment research showed that the microbicidal effect of macrophages were enhanced in the BLP tolerance group.The phagocytosis of macrophages were measured by flow cytometry and confocal microscope with GFP labeled E.coli stimuli.The macrophages in BLP tolerance group showed higher ability of phagocytosis,compared with the PBS group.Fluorogenic quantitative PCR were used to measure the mRNA expression of MARCO.It is showed that BLP tolerance could up-regulate the mRNA of MARCO.In addition,the cells stimulated by E.coli or staphylococcus aureus expressed higher mRNA of MARCO,compared with the macrophages without any stimuli.Small RNA interference technology was applied to down regulate the expression of MARCO.Results showed that the phagocytosis was decreased after down-regulating MARCO measured by flow cytometry plus fluorescent technique.However,sterilization experiment showed that the microbicidal effect showed no changes on the macrophages with or without MARCO knock down.In conclusion,our results showed that BLP tolerance enhances the phagocytosis of macrophages by upregulating MARCO.However,the enhanced microbicidal effect of the BLP tolerant macrophages was independent with the MARCO expression.