The Function Of B10 Cells In Patients With Kawasaki Disease And Its Microrna Regulation Mechanism Research

PARTⅠSTUDY ON THE NUMBER AND FUNCTION OF B10 CELLS IN PATIENTS WITH KAWASAKI DISEASEBackground:Kawasaki disease（KD）is an acute systemic vasculitis and activation of monocytes plays a central role in the pathogenesis of it.Regulatory B cells（Bregs）is a B cell subset with negative regulatory properties independent of secretion of immunoglobulin.Current evidence suggests that IL-10 is essential for its negative regulatory role.For clarity,this regulatory B cell subset are generally functionally identified by their ability to express cytoplasmic IL-10,named as B10 cells（IL-10-producing Bregs）.B10 cells have been found to play an important role in the pathogenesis of many diseases,but their role in KD has not been fully illuminated.Objective:We measured expression of IL-10 by different B cell subsets in samples from KD patients during the acute stage and after treatment with IVIG,and identified B cell subsets with the capacity to differentiate into IL-10-producing B cells.In addition,the inhibitory effect of B10 cells on monocytes was further evaluated to explore the causes of abnormal negative regulation in KD.We aimed to preliminarily elucidate the pathogenesis of excessive activation of monocytes.Methods:Twenty-three children diagnosed with acute KD upon admission to Shenzhen Children’s Hospital were included in the study.Patients were divided into coronary artery lesions group(KD^CAL+)and non-coronary artery lesions group(KD^CAL-).Thirty-three healthy children were included as a normal control group（Ctrl）.The expression levels of IL-10 in peripheral blood B cells（CD19+）,transitional B cells(CD19⁺CD24^hiCD38^hi),na?ve B cells(CD19⁺CD27^-CD24^intCD38^int)and memory B cells(CD19⁺CD24^hiCD27⁺)from patients with KD and healthy children were detected by flow cytometry（FCM）.Purified untouched CD19⁺B cells or CD14⁺monocyte cells were isolated from PBMCs using microbeads.Expression of mRNA of IL-10 in B cells was quantitated by real-time PCR.CD19⁺B cells were activated with sCD40L plus CpG and co-cultured with CD14⁺monocytes.Inhibition of monocyte-derived TNF-αby activated B cells were measured by FCM.Inhibition of cytoplasmic TNF-αexpression by monocytes was calculated as follows:1-（TNF-αexpression in co-cultures of monocytes cells plus activated B cells/TNF-αexpressed by monocytes cultured alone）×100.Statistical analysis was performed using IBM SPSS version 20.0 software for Windows.The Kolmogorov-Simonov test was used to assess normal distribution of data.One-way analysis of variance was used to compare multiple groups,and Student’s t-test was used to compare two groups.A P-value<0.05 was considered significant.Results:（1）Twenty-three children（13 male and 10 female;age,6–107 months[mean,39.43±25.38 months]）diagnosed with acute KD were included in the study.There were 13 cases in KD^CAL+group（56.5%）and 10 cases in KD^CAL-group（43.5%）.Clinical manifestations included fever（range 5 to 13[7.48±1.93]days）,skin rash（95.7%）,lymphadenopathy（87.0%）,conjunctival congestion（100.0%）,Oral mucosal changes（100.0%）,arthritis（13.0%）,peeling（69.6%）,pyuria（43.5%）,jaundice（13.0%）and thrombocytosis（82.6%）.（2）The results showed that all of these B cell subsets produced IL-10,but that transitional and memory B cells produced more than na?ve B cells.（3）During the acute stage of KD,IL-10 expression by all B cell subsets was decreased,especially in memory B cells（P<0.05）.After IVIG treatment,the expression of IL-10 in B cell subsets was partially recovered;the increase in CD19⁺B cells（total B cell）was statistically significant（P<0.05）.（4）Compared with the healthy control and KD^CAL-groups,the KD^CAL+group showed a more pronounced reduction in expression of IL-10.（5）Compared with the healthy control group,the mRNA expression level of IL-10 in B cells from patients with KD was significantly decreased（P<0.05）.After IVIG treatment,the mRNA expression of IL-10 in B cells was partially recovered（P=0.061）.（6）Expression of TNF-αby monocytes from KD patients was significantly higher than that by monocytes from healthy controls.After culture with autologous activated B10 cells,the inhibition level of TNF-αexpression by KD monocytes was four times less than that by control monocytes（P<0.05）.（7）The results showed that the expression of TNF-αwithin the healthy control CD14⁺cells co-cultured with activated B10 cells from KD patients was significantly higher than that in the CD14⁺cells co-cultured with activated B10 cells from healthy controls（P<0.05）.Conclusion:（1）B cells at different stages of differentiation have the potential ability to produce IL-10,and the ability varies with disease status.（2）It is more appropriate to functionally define these B cells as cells with capacity to produce IL-10,rather than trying to assign a particular phenotype.（3）The number and function of B10 cell from patients with KD were all impaired during acute stage.PART Ⅱ THE MICRORNA REGULATION MECHANISM OF B10 CELL IN PATIENTS WITH KAWASAKI DISEASEBackground: IL-10 is a key negative regulatory cytokine produced by a variety of cell types,and the regulation of its expression has always been the focus of research.Recent studies have shown important interactions between IL-10 and micro RNAs（Mi RNAs）.Mi RNAs regulation has been shown to play an important role in autoimmune and inflammatory diseases.However,most research is based on monocyte cells and T cells.The regulation of mi RNAs on IL-10 expression by B cells has not been systematically studied.Objective: In the study,we sought to systematically study the mi RNAs mechanism in B10 cells by using hyperinflammatory KD disease model.We aimed to explore the reasons underlying aberrant expression of IL-10 in acute stage KD,as well as further clarify the relationship between abnormal regulation of B10 cell and monocyte-mediated proinflammatory response.It is conducive to providing a new theoretical basis for elucidating the pathogenesis of KD and screening new therapeutic targets for disease early intervention.Methods: Purified untouched CD19+ B cells or CD14+ monocyte cells were isolated from PBMCs using microbeads.The expression level of TNF-α in monocytes was detected by flow cytometry.Expression of micro RNAs related to IL-10 in B cells were quantitated by real-time PCR,including mi R-21-3p,mi R-98-5p/3p,mi R-27a-3p,let7b-5 and mi R-142-3p/5p.Multivariate analysis was performed to identify an association between IL-10 expression（a dependent variable）and expression of micro RNAs（independent variable）.Possible associations between IL-10 and micro RNAs were explored initially by calculating Pearson’s（normal data distribution）or Spearman’s（non-normal data distribution）correlation coefficients.Predictors of IL-10 were examined by multiple linear regression analysis.A stepwise elimination approach was used to determine the best overall model when including data with a P-value <0.05 and excluding data with a P-value >0.1.The mi RNA transfection experiments were performed using polyethylenimine.Agomir-27-3p-FAM（mimic）,Antagomir-27-3p-FAM（inhibitor）,and their respective FAM-negative controls were transfected into B cells.Intracellular localization of FAM-mi RNA was then examined under a confocal microscope.Mi RNA uptake and intracellular IL-10 expression were assessed by flow cytometer.Purified CD19+ B cells were cultured with s CD40 L plus Cp G for inducing to differentiate into IL-10-competent B10 cells.Secreted IL-10 in cultural supernatant was quantified by enzyme-linked immunosorbent assay（ELISA）.We next assessed the inhibitory effect of B10 cells on monocyte by measuring TNF-α-positive monocytes in a transfected B10 cell-monocyte co-culture system.The expression level of IL-10 in monocytes was detected by FCM.The expression of mi R-27a-3p was quantitated by real-time PCR.Results:（1）The results showed that expression of mi R-98-5p/3p,mi R-21-5p,let7b-5p,and mi R-27a-3p was significantly higher than that in controls during the acute phase of KD,and that expression of all except let7b-5p was downregulated after IVIG treatment（P < 0.05）.Increased expression of mi R-98-5p and mi R-27a-3p was more obvious in the KDCAL+ group,whereas that of mi R-21-5p and mi R-98-3p was more significant in the KDCAL-group,than in the control group（P < 0.05）.The trend in expression of mi R-142-3p/5p showed a pattern opposite to this during the acute stage（P < 0.05）.There was no significant change in expression of mi R-106 a between the control group and acute stage KD.（2）Micro RNAs showing significant changes in expression between acute KD and control subjects were selected as independent variables（mi R-21-3p,mi R-98-5p/3p,mi R-27a-3p,let7b-5p,and mi R-142-3p/5p）.The results of multivariate analysis showed that an inverse relationship between m RNA or protein expression of IL-10 and mi R-27a-3p（r =-0.599,P < 0.05;r =-0.667,P < 0.05）,but no significant relationship between IL-10 and the other micro RNAs.The final equation was given by IL-10 = 0.001-0.001 × mi R-27a-3p（depending on the m RNA level）;IL-10 = 1.508-0.999 × mi R-27a-3p（depending on the protein level）.（3）Fluorescent cells were observed under a laser confocal microscope after transfection.We found that fluorescence was mainly distributed throughout the cytoplasm.Flow cytometry analysis revealed that the average mi RNA was taken up by 12.42% ± 0.94% of B cells from patients.（4）After transfection of B cells from patients with KD for 24 h,expression of IL-10 by B cells from the antagomir-27 a group was significantly higher than that by the NC group（P < 0.05）.（5）After transfection of B cells from healthy children for 24 h,the expression of cytoplasmic and secreted IL-10 by B cells from the agomir-27 a or antagomir-27 a groups was significantly lower or higher,respectively,than that by each NC group（P < 0.05）.（6）The expression of TNF-α within the CD14+ cells co-cultured with agomir-27 a B cells was significantly higher than that in the CD14+ cell population co-cultured with antagomir-27 a B cells（P < 0.05）.（7）The IL-10 concentration in cultural supernatant of antagomir-27a-B10 cells from both patients and controls were significantly increased compared to NC groups.The expression of TNF-α within the monocytes co-cultured with antagomir-27a-B10 cells was significantly reduced compared to NC group（P < 0.05）.（8）The level of IL-10 protein expression in monocytes of KD was higher than it in the controls.The m RNA expression of mi R-27a-3p in monocytes was significantly higher than that in controls during the acute phase of KD,and that expression was downregulated after IVIG treatment.Conclusion: Upregulated mi R-27 a in B cells from patients with KD may promote monocyte-mediated inflammatory responses by inhibiting the regulatory function of B10 cells.