The Detection And Clinical Significance Of Mucosal Associated Invariant T (MAIT) Cells Subsets In Necrotizing Enterocolitis Patients

Background:Neonatal necrotizing enterocolitis(NEC)is a common life-threatening gastrointestinal tract acute inflammatory disease in neonatal intensive care unit.Monitoring level in recent years gradually increased,but the NEC is a disease still with high morbidity and mortality in preterm infants.Recently,there have been several studies on the risk factors of NEC,but the pathogenesis of NEC is still not clear.And one or more risk factors does not fully explain differences of the occurrence,development speed and severity of NEC in different individual,which also limits more effective prevention and treatment methods of application on NEC.So the study of the pathogenesis of NEC from various angles need to be explored.Mucosal associated invariant T(MAIT)cells are innate-like T cells,and mainly through secretion of inflammation cytokines such as IL-17 and cytotoxicity act as immune regulator in various diseases process.It was reported that MAIT cells were involved in inflammatory bowel disease process,and IL-17 gene polymorphism also was proved associated with the susceptibility of inflammatory bowel disease.But the relationship of MAIT cells and its subsets,IL-17 gene polymorphism and NEC has not been reported.Therefore,exploration of the change of MAIT cell and its subsets in NEC preterm infants,and its correlation with clinical indicators would provide the base on the study of pathogenesis of NEC.And study of the relationship between IL-17 susceptibility gene polymorphism and NEC disease severity provides a new direction for the pathogenesis of NEC from the gene level.Objective:To study the changes in the distribution of MAIT cells and the subsets in the NEC premature infants,and analyze the correlation of the change and the clinical values;and explore the association of inflammatory cytokine IL-17 gene polymorphism with the susceptibility of NEC.In order to provide an insight of the pathogenesis of premature NEC.Methods:1.Detection of the expression of MAIT cells and the subsets in peripheral blood mononuclear cells and lamina propria mononuclear cells of NEC premature infants and the control group subjects by the flow cytometry.Stratification analysis according to the Bell stage classification was used to explore the expression change of MAIT cells and the subsets in NEC group.In order to analyze the relationship between MAIT cells and the severity of disease.2.Analysis of quantity of serum inflammatory cytokines IL-2,IL-4,IL-17 A,IL-6,IL-10,TNFα,and the levels of IFNγ in participants by cytometric bead array(CBA)technology and the correlation of MAIT cells and their subsets.3.Detection of the expression change of circulating MAIT cells and the subsets in the NEC patients after treatment by flow cytometry.To explore the relationship between the frequencies of MAIT cells and the progression of NEC.4.Analysis of the association of IL-17A(rs2275913)and IL-17F(rs763780)SNPs with the susceptibility and severity of NEC by polymerse chain reaction(PCR)and Sanger method.Results:1.Compared with the control subjects,the frequencies of circulating MAIT cells were lower and the frequencies of intestinal tissue MAIT cells were higher in NEC premature,furthermore,they showed a negative correlation.The frequencies of CD8αα+ MAIT cells subset in peripheral blood and intestinal tissues of NEC premature were both lower than that in the control subjects,moreover,the frequencies of the circulating CD8αα+ MAIT cells subset were associated with the severity of NEC.2.The levels of serum IL-2,IL-4,IL-17 A,TNFα,and IFNγ in the NEC patients were significantly lower than those in the controls.In contrast,the level of serum IL-6 in the NEC patients was significantly higher than that in the controls.The difference in the level of serum IL-10 in the two groups was not significant.Moreover,we found no significant correlation among the levels of cytokines with the percentage of MAIT cells and CD8αα+ MAIT cells.3.After treatment,peripheral blood MAIT cells percentage significantly increased before treatment,and rose to the level of control group,but peripheral blood CD8aa+ MAIT cells percentage was no significantly different before treatment,and the level was still lower than the control group.4.There were no significance of the IL-17A(rs2275913)SNP loci genotype and allele distribution frequencies in the NEC and the control group.IL-17F(rs763780)SNP loci TC+CC genotype and C allele distribution frequencies significantly increased in the NEC group than that in the control group.IL-17F(rs763780)SNP loci TC+CC genotype distribution frequencies were found increased in Bell stage Ⅲ than that in Bell stage Ⅱ NEC patients.IL-17F(rs763780)SNP loci TC+CC genotype distribution frequencies were significantly higher in the NEC patients with pneumoperitoneum.Conclusion:This study proved that MAIT cells have the characteristics that accumulating into the intestinal tissue from the peripheral blood in the process of NEC disease and played a role of immune regulation.Premature with the decreased CD8aa+ MAIT cells may decline in the ability of the MAIT cells against microbial infection,and may contribute to the development and process of NEC.It indicated that CD8aa+ MAIT cells may be a potential biology marks to evaluate the severity of NEC disease.There was no association between IL-17A(rs2275913)SNP loci and the susceptibility of NEC.IL-17F(rs763780)SNP locus mutation may be associated with NEC susceptibility and disease severity.This study offered new research direction to further explore the immunology and genetics pathogenesis of NEC.