The Mechanism Of Th Cell Differentiation And Coronary Artery Lesions By Gut Microbiota In Kawasaki Disease

Objective:To analyse the effects of intestinal microbiota changes on Th cells and related molecular expression in children with Kawasaki disease and the possible mechanism of coronary artery injury.Methods:Children with KD in this research are choosed from Shenzhen Chilfren’s Hospital during August 2016 to December 2019,the diagnosis standard set according to the guidelines of Kawasaki disease propsed by AHA in 2017.The group was divided into KD group and KD^IVIG group before and after IVIG treatment.Refer to the report of Doppler ultrasound,divided KD group into coronary artery injury（CAL）and no coronary injury group（NCAL）before and after treatment.We explored in the following two aspects.1 Get specimen（feces）from 31 children with KD and 9 children with normal age-matched health children.24 cases were before and 7 cases were after treatment with IVIG.Observed the difference of Gut microbiota in diversity and abundance before and after IVIG treatment,and analyzed the correlation with KD and vascular lesions,the sensitivity of IVIG and screened out possible biomarkers.2 Children of 42 with KD were recruited in present study,while 36 age-matched healthy volunteer as controls.To explore the histone methylation of IL-4 gene and its role in dysfunction of Th2 cells during pediatric Kawasaki disease.Results:1.（1）According to 16SrDNA sequencing data,obtain 2352803 valid lines,614 species taxon,including 14 Phylum,24 Class,46 Order,86 Family,218 Genus and 80 Species.（2）The richness（chao1 index）of patiens with coronary artery lesions increased remarkablely than KD^IVIGgroup（P<0.05）and CAL group（P<0.05）;The variety of each group make no significant difference,and large heterogeneity show in control group.（3）The intestinal microflora of each group was mainly composed of 4phylum,Firmicates,Actinobacteria,Proteobacteria and Bacteroidetes.The larger relative abundance of Genus are Bifidobacterium、Bacteroides、Escherichia、Akkermansia、Erysipelatoclostridium decrease progressively;The proportion of Bifidobacterium in control group was higher than that in KD group,the NCAL group lower,the CAL group significantly decreased,and the KD^IVIG group recoveredafter treatment of IVIG.Akkermansia increased in varying degrees compared with the control group and KD^IVIGVIG group,while decreased in the CAL group.But there was no statistical difference.（4）LefSe analysis showed that Pseudomonadaceae can be used as biomarkers of the NCAL group,Actinobacteria can be used as biomarkers of KD^IVIG,while Betaproteobacteriales is a biomarker of CAL group.2.（1）In comparison with healthy controls,the frequencies of Th2 cells,mRNA levels and plasma concentrations of Th2 cytokines（IL-4,IL-5 and IL-13）,and methylation levels of H3K4me3 associating with IL-4 loci（CNS1,HSII and HSVa）increased significantly in patients with acute KD（P<0.05）.Of interesting,the items mentioned afore in patients with coronary artery lesions（CAL）were found to be higher than those without coronary artery lesions（NCAL）,and restored to some extent after IVIG therapy（P<0.05）.（2）ChIP results showed binding abilities of GATA-3 and MLL1 proteins with IL-4gene loci（CNS1,HSII and HSVa）,were up-regulated remarkably in CD4⁺T cells from patients with acute KD（P<0.05）,and down-regulated after IVIG treatment（P<0.05）.There are positive correlations between binding abilities of MLL1 protein with IL-4 loci（CNS1,HSII and HSVa）and transcription level of IL-4 mRNA detected during acute KD（r=0.42、0.33、0.39,P<0.05）.In parallel,binding abilities of GATA-3 and MLL1 with the three IL-4 loci in CAL group were higher than those in NCAL group（P<0.05）.（3）During acute phase of KD,plasma levels of IL-4 protein,and expression levels of IL-4signaling downstream molecules（IL-4Rα,IL-2Rγ、pSTAT6 and GATA-3）and negative regulator SOCS5 increased remarkably（P<0.05）,and all the items decrease after IVIG treatment（P<0.05）.Furthermore,the five items aforementioned in CAL group were higher than those in NCAL group,while the last items in CAL group was lower than that in NCAL group（P<0.05）.Conclusion:The intestinal microflora disordered in patients of KD,and it mainly performance in KD with CAL.After the treatment of IVIG.As a Possible biomarker,Pseudomonadaceae may interact with KD disease by participating in regulatory of Th1/Th2.The possible protective markers were Lactobacillus and actinomycetes,and the pathological markers were Pseudomonas and Clostridium.2.The overactivation of IL-4 and relatively inadequate expression of negative regulator SOCS5 in stage KD contributed to the overexpression of transcription factor GATA3.The latter binds to IL-4 and recruit histone methylase MLL1,which mediating the up-regulation of IL-4 gene histone H3K4me3 modification and activity,resulting in a positive feedback effect and IL-4 over-expression,Eventually lead to abnormal activation of Th2cells.3.Microbial community metabolites such as SCFA、PPARγmay be involved in the regulation of immune and vascular injury processes in the acute phase of the KD by affecting the epigenetic modification of host Th2 cells.