| BackgroundDuring the immunosuppressive phase of sepsis,immune cells often exhibit endotoxin tolerance.Neutrophils is the largest number of innate immune cells in the blood circulation of human.as the body's first line of defense,neutrophils in the early infection can quickly through the vascular endothelial cells into the site of infection,effectively eliminating pathogens by degranulation,respiratory outbreaks and neutrophil extracellular trap network in a variety of ways.However,as a lot of studies have shown that in severe sepsis,the response of neutrophils to chemokines released at the infected site and their ability to migrate across the endothelium is significantly reduced due to the dysfunction of the immune system.so they cannot exert their ability to remove pathogens.At the same time,the excessive accumulation and obstruction of neutrophils in the microcirculation of the organs will further lead to microcirculation disorders of the organs,thus promoting the occurrence of multiple organ failure.How to enhance and improve the migration of neutrophils in sepsis and thereby improve the organ damage caused by microcirculation disorders and the prognosis of sepsis has become a new focus of research in this field.Through the re-analysis of the results of neutrophil gene chip in patients with immunosuppression of sepsis,we also found that MIIP was significantly up-regulated in the neutrophil gene expression in patients with immunosuppression of sepsis.As a newly discovered protein that inhibits cell migration and invasion,migration and invasion inhibitory protein(MIIP)can inhibit cell migration through multiple pathways.Although current studies on MIIP mainly focus on the mechanism of tumor invasion and metastasis,several studies suggest that MIIP may be involved in the regulation of inflammatory responses.In addition,HDAC6 plays an important role in the regulation of macrophages and T cells in inflammatory responses,and MIIP can bind to HDAC6 to inhibit its deacetylase activity.We speculate that MIIP may cause sepsis neutrophil migration disorder.The purpose of this study was to investigate the effect of endotoxin tolerance on the ability of neutrophils to migrate across the endothelium and its potential mechanism,in the hope of providing new ideas and new targets for clinical treatment of patients with immunosuppression of sepsis from the perspective of improving the function of neutrophils to migrate across the endothelium.METHOD1.Considering that neutrophils are prone to apoptosis,HL-60 differentiation induced by DMSO was used to replace normal neutrophils.1.3% DMSO induced HL-60 for 5 days to differentiate into neutrophils.The obtained cells were stained with trypan blue to analyze cell activity and ensure cell activity more than 95%.2.Neutrophils were divided into three groups: Control group(PBS+PBS),LPS group(PBS+LPS)and ET group(LPS*+LPS).Medium supernatant and cells were collected after treatment.3.The cells were divided into Control siRNA Control group,Control siRNA LPS group,Control siRNA ET group,MIIP siRNA Control group,MIIP siRNA LPS group and MIIP siRNA ET group.Medium supernatant and cells were collected after treatment.4.The cells were divided into Control siRNA ET group,Control siRNA ET+Tubastatin A group,MIIP siRNA ET group and MIIP siRNA ET+Tubastatin A group.Medium supernatant and cells were collected after treatment.5.Transwell assay was performed to detect the transendothelial migration of neutrophils in each group Under the same condition as in 2/3/4.6.The expression of MIIP mRNA in cells of each group was detected by qRT-PCR Under the same condition as in 2.7.WB was used to detect the expression of MIIP protein in each group Under the same condition as in 2/3..The expression of HDAC6 protein in each group was detected by WB Under the same condition as in 3.WB was used to detect the expression of tubulin ?-tubulin and its acetylation and p65 and its phosphorylation in each group Under the same condition as in 3/4.8.Under the same condition as in 2,the expression of TNF-? in the supernatant of each group was detected by ELISA;Under the same condition as in 3/4.the expression of IL-8 and MCP-1 in the supernatant of each group was detected by ELISA.9.All data were statistically analyzed and processed using GraphPad Prism 7.0 software.Measurement data were presented in the form of mean standard deviation,and the difference between the two groups was compared by t test.P<0.05 was considered statistically significant.RESULT1.Compared with the LPS group and the Control group,the number of neutrophils in the ET group significantly decreased,and their migration ability gradually weakened with the increase of the induced endotoxin tolerance dose.The mRNA and protein content of MIIP were consistent with the change trend of cell migration ability.In the analysis of gene chip results,MIIP gene expression was increased in neutrophils of patients with immunosuppression in sepsis.2.Compared with the Control siRNA ET group,the reduced ability of endotoxin tolerant neutrophils to migrate across endothelial cells after inhibiting the expression of MIIP can be partially improved;Tubastatin A can reverse the ability of endotoxin tolerant neutrophils to migrate across the endothelium due to inhibition of MIIP expression.3.Compared with the Control siRNA ET group,inhibition of MIIP expression partially reversed the down-regulation of endotoxin-tolerant neutrophil HDAC6 protein expression.4.Compared with the Control siRNA ET group,inhibition of MIIP expression can reverse the up-regulation of the acetylation level of ?-tubulin in endotoxin-tolerant neutrophil.Tubastatin A can reverse the up-regulation of the acetylation level of ?-tubulin caused by inhibiting MIIP expression.5.Compared with the Control siRNA ET group,inhibition of MIIP expression can reverse the phosphorylation level of endotoxin tolerance neutrophil p65 and down-regulate the expression of cytokines IL-8 and MCP-1.Tubastatin A can reverse the phosphorylation level of endotoxin tolerance neutrophil p65 and the up-regulation of cytokines IL-8 and MCP-1 caused by the interference of MIIP expression.6.Compared with the Control siRNA group,although the inhibition of MIIP expression did not affect the expression of p65 and its phosphorylation,cytokines IL-8 and MCP-1,it could up-regulate the level of tubulin-tubulin acetylation in the Control group and LPS group.7.Compared with the ET group,Tubastatin A can further inhibit the transendothelial migration of endotoxin-tolerant neutrophils,up-regulate the acetylation level of ?-tubulin,and down-regulate the phosphorylation level of p65 and the expression of cytokines IL-8 and MCP-1.CONCLUSIONEndotoxin tolerance can lead to decreased migration of neutrophils across endothelial cells,which may be related to endotoxin tolerance up-regulating MIIP mRNA and protein expression,inhibiting the amount of HDAC6 protein and its deacetylase activity.In the neutrophil of Endotoxin tolerance,the inhibition of HDAC6 deacetylase activity caused by MIIP may not only upregulate the level of ?-tubulin acetylation,but also inhibit the activation of NF-?B,the expression of cytokines IL-8 and MCP-1 to suppress neutrophil transendothelial migration. |
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