Study On The Mechanism Of MIF Regulating Post-myocardial Ischemia Inflammation

Objective:To investigate relationship between macrophage migration inhibitory factor（MIF）and myocardial ischemia injury and to detect the mechanisms of inflammation after myocardial infarction（MI）caused by MIF.First,we analyzed the association between MIF gene polymorphism and acute coronary syndrome（ACS）.Second,we used MI animal model to study how global MIF,leucocyte derived MIF and cardiac MIF mediated inflammatory responses following MI.Methods:In the case-control study,we recruited adult ACS patients confirmed by coronary angiography and healthy controls from Chinese Han population who lived in Xinjiang.Using Taq Man（?）SNP method,we tested the polymorphism of MIF gene.We also tested plasma MIF levels using enzyme linked immunosorbent assay（ELISA）method.There are two parts in animal experiments.We established MIF knock out（MIFKO）and chimeric mice,MIF deficiency in bone marrow derived-cells(WT^KO)or in somatic-cells(KO^WT)mice models with MI and tested the circulating white blood cells（WBC）and expression of monocyte chemoattractant protein（MCP-1）,CCR2（Chemokine cytokine receptor 2）,IL-6（Interleukin-6）,IL-1β（Interleukin-1β）and MMP-9（Matrix metalloproteinases-9）in peripheral blood mononuclear cells（PBMC）at different time points after MI.We also used FACS（Fluorescence-activated cell sorting）to test the circulating Ly-6C^highmonocytes,the Ly-6C^high monocytes reserved in spleen and the recruited Ly-6C^highmonocytes in the heart as well as the spleen weight and structure.Result:（1）A total of 699 ACS patients（mean age 59.1±10.1 years and 61.7%men）and1153 healthy controls（mean age 58.7±11.1 years and 55.1%men）were recruited in the present study.ACS patients were older and had greater BMI and levels of glucose and more dyslipidemia（all P<0.05）.There was no significant difference in distribution of genotypes or alleles rates in rs1007888 and rs2096525 between ACS and control groups（P>0.05）.However,we found significant differences of genotypic and allelic distributions regarding rs755622 between ACS and control groups（P<0.05）.The frequency of the CC genotype in ACS patients was higher than that in control subjects（4.4%vs.3.8%,P=0.024）.Moreover,the frequency of the C allele was also higher in the ACS than that in control group（23.9%vs.20.4%,P=0.012）.Most participants including healthy controls and ACS patients carried rs755622 GG（63.1%vs.56.7%）and CG genotypes（33.1%vs.38.9%,P=0.012）and G allele of rs755622（79.6%vs.76.1%,P=0.012 respectively）,while CC genotype（3.8%vs.4.4%,P=0.024）and C allele（20.4%vs.23.9%,P=0.024）carriers were the lowest.We first fitted a univariate logistic regression model to evaluate association between rs755622 together each of the variables.After adjusting the confounding factors,multivariate logistic regression analysis was further used to detect the association between rs755622 polymorphisms and susceptibility of ACS（CG and CC genotype vs.GG genotype,AOR=1.278,95%CI,1.042-1.567,P=0.019）.However,no difference was observed in the other two SNPs.MIF CC genotype carriers had the highest plasma levels of MIF than CG and GG genotype carriers.The MIF level in ACS patients with CC genotype was 6.5%and 15.6%higher than that in ACS patients carrying CG and GG genotypes,respectively（all P<0.05）.Our findings indicated that MIF gene rs755622 variant with C allele was associated with plasm MIF level and MIF gene rs755622 variant with CC genotype and C allele was associated with increased risk of ACS.（2）To confirm whether MIF is associated with ACS,using MI animal model,we tested the circulating leukocytes concentration in MIFKO mice and found that it was decreased compared to WT mice（4.8±0.5 vs.3.4±0.4×10⁶/m L,P=0.037）.MIFKO mice displayed less circulating Ly-6C^high monocytes at MI 72h:（0.3±0.1 vs.0.5±0.1）×10⁵/m L blood,P=0.041),lower expression of inflammatory factors and chemotactic factors such as MCP-1,CCR2,IL-1β,IL-6,MMP-9 and more splenic reserved Ly-6C^high monocytes（0.8±0.1 vs.0.4±0.1×10⁴/mg spleen,P=0.008）.Histological staining showed that the number of white pulp and leucocytes in the spleen of MIFKO mice were significantly higher than that of WT mice.FACS showed that Ly-6C^high monocyte infiltration into the heart of WT mice increased significantly after myocardial infarction at 72 hours（9.9±2.3vs.0.2±0.1×10⁴/heart,P=0.009）.Although there is no difference between the two groups,MIFKO displayed dramatically decrease in Ly-6C^high monocytes after MI compared to WT mice,only 42.2%of WT mice:（5.7±1.9 vs.9.9±2.3×10⁴/heart）.Our findings indicated that MIF could promote systemic inflammatory response after myocardial infarction,mobilized the splenic Ly-6C^high monocyte to release into the blood and infiltrate into the myocardial infarction area.（3）To investigate the effect of leucocyte derived MIF and cardiac MIF on the inflammation after MI,we established chimeric mice including MIF deficiency in bone marrow derived-cells(WT^KO)or in somatic-cells(KO^WT).Compared to KO^WT mice,WT^KO displayed fewer circulating leukocytes at MI 24 hours:（4.5±0.3 vs.3.2±0.3×10⁶/m L blood,P=0.036）,MI 72 hours:（5.6±0.4 vs.4.5±0.2×10⁶/m L blood,P=0.032）,less circulating Ly-6C^high monocytes[（0.8±0.2 vs.0.4±0.1×10⁶/m L,P=0.029）],lower expression of inflammatory factors and chemotactic factors such as MCP-1,CCR2,IL-1β,IL-6,MMP-9 and more splenic reserved Ly-6C^high monocytes at MI 24 hours,（1.3±0.5 vs.6.4±1.9×10⁴/mg spleen,P=0.032）.Histological staining showed that the number and area size of white pulp in the spleen of WT^KO mice tended to be higher than that of KO^WT mice.FACS showed that Ly-6C^high monocyte infiltration into the heart was less in WT^KO mice than that in KO^WT mice at MI 24 hours:（1.9±0.5 vs.5.5±1.4×10⁴/heart,P=0.042）.Conclusion:MIF is associated with severe myocardial ischemia.The C allele of rs755622 variant of MIF gene polymorphism is the risk factor of ACS and plasm MIF levels were higher with the greater frequency of MIF rs755622 C allele.MIF promotes systemic inflammation after myocardial ischemia.MIF promotes activation of circulating Ly-6C^highmonocytes,mobilizes splenic Ly-6C^high monocytes and recruits Ly-6C^high monocytes to the heart after myocardial infarct.MIFKO suppressed the splenic reservoir Ly-6C^highmonocytes mobilization and the decreasing of the number of white pulps.MIF regulates cardio-splenic axis after MI.Leucocyte derived MIF promotes systemic inflammation after myocardial infarction,mobilizes splenic Ly-6C^high monocytes and recruits Ly-6C^highmonocytes to the heart.Leucocyte derived MIF regulates cardio-splenic axis after MI.