Study On The Expression And Intervention Of Neutrophil Extracellular Traps In IgA Vasculitis

Objectives:To investigate the expression and clinical significance of neutrophil extracellular traps(NETs)in peripheral blood,kidney,stomach and duodenum tissues of children with IgA vasculitis(IgAV).And then the IgAV rat model was established and intervened by using DNase I.To explore the role of NETs in IgAV rat model and observe the effect of DNase I targeted degradation NETs in IgAV rat.Methods:A case-control study,cell model of NETs induced by human neutrophils in vitro,construction of IgAV rat model and intervention with DNase I were performed.The IgAV patients was included in the case group and the healthy children were enrolled in the control group.The case group was divided into four subgroups according to their conditions at the time of inclusion:onset group,remission group,active group and withdrawal group.The inclusion criteria were according to criteria for IgAV updated by the European Union Against Rheumatism/International Experimental Organization for Rheumatology in Children/European Society for Childhood Rheumatology(EULAR/PRITO/PRES)in 2008.In the clinical study of children with IgAV,the cell-free DNA(cf-DNA)in children peripheral blood was quantified by using fluorescence quantitative kit.The myeloperoxidase-DNA(MPO-DNA),neutrophil elastase(NE),and deoxyribonuclease I(DNase I)was measured using enzyme linked immunosorbent assay(ELISA).The expression of NETs in renal tissue of patients with IgAV nephritis(IgAVN)and gastrointestinal tissue of patients without IgAVN were detected by using multiple fluorescence immunohistochemistry.The relationship between cf-DNA and MPO-DNA,NE and DNase I was analyzed.Neutrophils from peripheral blood of healthy children were isolated in vitro,and the cellular model of NETs formation induced by phorbol ester was established.The ability of sera from IgAV patients to degrade NETs was detected.In the study of IgAV rat model,the changes of cf-DNA in peripheral blood of rat were quantified by using fluorescence quantitative kit.The MPO-DNA,IgA,IgG,IgE and complement C3 were measured using ELISA.The expression of IgA,IgG and IgE in tissues of the kidney,stomach and duodenum were detected by immunohistochemistry.The changes of IgA,C3 and NETs in tissues of the kidney,stomach and duodenum were detected by multiple fluorescence immunohistochemistry.Real-time quantitative-polymerase chain reaction(RT-PCR)was used to detect the m RNA of TNF-αand MPO,and western blot(WB)was used to detect the expression of MPO and cit H3 proteins in renal tissues.Hematoxylin-eosin(HE)staining and periodic acid-schiff(PAS)staining were performed to observe the pathomorphology.Results:In the clinical study of children with IgAV:1.The one-way ANOVA result showed that there was significant differences in cf-DNA,MPO-DNA and NE in peripheral blood between IgAV subgroups and control group(all P<0.001).Comparison between two groups showed that the peripheral blood of cf-DNA,MPO-DNA and NE in onset group were significantly higher than the withdrawal group,remission group and control group(all P<0.001).The cf-DNA,MPO-DNA and NE in activity group were significantly higher than the withdrawal group,remission group and control group(all P<0.001).The cf-DNA,MPO-DNA and NE were not significant differences between the onset group and the active group(all P>0.05).In the drug withdrawal group,there were no significant differences in cf-DNA,MPO-DNA and NE compared with the remission group and the control group(all P>0.05).2.The DNase I was decreased significantly among the IgAV subgroups and control group(P<0.05).Comparison between two groups showed that DNase I in the onset group was significantly lower than the withdrawal group,remission group and control group(all P<0.05).DNase I in the active group was significantly lower than the withdrawal group,remission group and control group(all P<0.05).There was no significant difference between the initial onset group and the active group(P>0.05).3.Multiple fluorescence immunohistochemistry showed that NETs were highly expressed in the kidney,stomach and duodenum of the onset and active groups compared to the control group(P<0.05).4.The results of correlation analysis show that cf-DNA was highly correlated with MPO-DNA and NE.There was a positive correlation between cf-DNA and MPO-DNA(r~2=0.766,P<0.001).There was also a positive correlation between cf-DNA and NE(r~2=0.687,P<0.001).There was a negative correlation between cf-DNA and DNase I(r~2=0.433,P<0.001).5.The degradability of NETs in sera of IgAV patients in the onset group and active group were significantly lower than the control group(P<0.001).There was no significant difference between the withdrawal group and the control group(P>0.05).In the study of IgAV rat model,the results showed that:1.Leukocytes,neutrophils and platelets in IgAV model group were significantly higher than the control group(all P<0.05).The lymphocyte and hemoglobin were no significant differences between the two groups(all P>0.05).2.The IgA and IgE levels were significantly higher and C3 was decreased significantly in the IgAV model group compared to the control group(all P<0.05).The IgG was not significantly different between the two groups(P>0.05).There were abundant of IgA and C3 deposition in the vascular wall of the glomerulus,skin,stomach,duodenum,lungs,and brain of the IgAV model group.Which were negative in the control group.3.The levels of cf-DNA and MPO-DNA in IgAV model group were significantly higher than the control group(all P<0.05).Typical NETs formation was found in the involved gastric,duodenal and the glomeruli of the IgAV model group.NETs in the control group were negative.4.In the IgAV model group,HE staining showed that marginal focal necrosis,and there were fragments of necrotic cells shedding in the lumen of renal tubules,accompanied by bleeding in the renal tissue.The glomerular capillaries and interstitial capillaries showed abundant congestion.Focal necrosis was observed in gastric mucosa,submucosa edema,loose connective tissue arrangement,accompanied by inflammatory cell infiltration.A large number of intestinal villous epithelial cells were necrotic and exfoliated in the mucosa layer of intestinal tissue,capillaries were dilated in the lamina propria,and a few intestinal glands were necrotic and disappeared.All tissues were accompanied by inflammatory cell infiltration.There were abnormalities in the control group.In the intervention study of IgAV rat model,the results showed that:1.Compared with IgAV model group,the cf-DNA and MPO-DNA in intervention group were significantly decreased(all P<0.01).There was not statistically difference between the intervention group and the control group(P>0.05).2.The m RNA levels of MPO and TNF-αin intervention group were significantly decreased compared to the IgAV model group(all P<0.05).The MPO and TNF-αm RNA were no significant difference between the intervention group and the control group(all P>0.05).3.The MPO and cit H3 protein levels in the intervention group were significantly lower than the IgAV model group(all P<0.05).There was no significant difference in MPO and cit H3 protein between the intervention group and the control group(all P>0.05).4.Compared with the IgAV model group,the expression of NETs in renal,gastric and duodenal tissues of the intervention group was significantly decreased(P<0.01).5.Compared with IgAV model group,the HE staining results of intervention group showed there were no necrotic cells and bleeding,and the congestion of glomerular capillaries and interstitial capillaries was significantly reduced.Gastric mucosa cell necrosis and bleeding were significantly improved.The villous dilatation and congestion of intestinal tissue and necrosis of mucosal epithelial cells were significantly reduced,and inflammatory cell infiltration was reduced.There were no obvious pathological abnormalities in the control group.6.There were no significant differences in leukocytes,neutrophils,lymphocytes,percentage of lymphocytes,percentage of neutrophils and platelets between the IgAV model group and the intervention group(all P>0.05).Conclusions:1.NETs are significantly increased in children’s patients with IgAV and are associated with the disease activity.These results suggest that NETs are involved in the development of IgAV.The possible mechanism is that the decreased DNase I leads to the decreased degradation of NETs.NETs maybe use as a potential indicator to assess IgAV disease activity.2.The IgAV rat model can be successfully constructed by using india ink+freund complete adjuvant+ovalbumin.This study is the first to confirm the presence of NETs in IgAV rat model and involving in the development of IgAV.3.DNase I can degrade NETs and down regulated level of NETs in IgAV rat.Which can reduce the tissue damage caused by NETs.Targeted regulation of NETs may be a potential therapeutic target for IgAV.