Identification Of The Levels And Function Of B10 Cells In NOD Mice And Its Mechanisms

PART ONE Study on the levels of B10 cells in NOD miceObjective: To study the alterations of B10 cells,proinflammatory Th1 and Th17 cells in non-obese diabetic(NOD)mice and the correlation between B10 cells and type 1 diabetes(T1D)in NOD mice.Methods: Hematoxylin-Eosin staining of pancreatic tissues was performed for histopathological assessment of the development of insulitis in NOD mice;The spleen,pancreatic lymph node,peripheral lymph nodes,mesenteric lymph nodes of NOD or WT C57BL/6 mice were collected for flow cytometric;Flow Cytometry(FCM)was used to measure the levels of CD19+CD5+CD1dhiB,CD19+IL-10+B,CD4 +IFN-?+Th1,CD4+ IL-17+Th17 and CD4+CD25+Foxp3+ T cells in NOD mice(4 weeks old NOD mice: group A;8 weeks old NOD mice: group B;NOD mice with diabetes: group C)and age-matched C57BL/6 mice(control group).Results:(1)Histopathological analysis showed that mice from A,B and C groups showed no insulitis,insulitis and obvious insulitis with no intact islets,respectively.(2)The frequency of B10 cells: The highest levels of B10 cells in NOD mice were observed in group B,followed by those in group C,group A and age-matched C57BL/6 mice(P<0.01),the frequency of B10 cells in 8 weeks old WT C57BL/6 mice was higher than 4 weeks old WT C57BL/6 mice(P>0.05);The distribution of B10 cells: no significant differences with the levels of B10 cells were found among different tissues of 4 weeks old NOD and WT C57BL/6 mice(P>0.05).The level of B10 cells in 8 weeks old WT C57BL/6 mice were highest in spleen,however,more B10 cells were detected in pancreatic lymph nodes than in other tissues of 8 weeks old NOD mice(P<0.05),the level of which were also higher than those in pancreatic lymph nodes of mice form group C(P<0.01).The highest levels of B10 cells were detected in peripheral lymph nodes among all tissue samples collected from NOD micewith diabetes(P<0.01).(3)The levels of proinflammatory Th1 and Th17 cells in mice from group C were remarkably increased as compared with those in mice from group A and group B(P<0.01),and the ratio of Th1/B10 and Th17/B10 in mice from group C were significant higher than those in mice from A and B groups(P<0.01).(4)The percentages of CD4+CD25+Foxp3+ T cells in mice from group C showed no differences with those in mice from group A,group B and age-matched C57BL/6 mice.Conclusion: The frequency and distribution B10 cells changed with age and the development of insulitis.Diabetic NOD mice have obvious disbanlance among B10 cells and proinflammatory Th1/Th17 cells,the decrease of B10 cells might participate in the development of T1 D in NOD mice.PART TWO Identification of the B10 cells regulating function in NOD mice and its mechanismsObjective: To identify the function of B10 cells in NOD mice,and explore whether B10 cells were dysfunction during the development and progressive of T1 D in NOD mice.Methods: Splenic B10 cells and CD19+CD5-CD1 dlow B cells in 4-wk/8-wk/diabetic NOD mice,WT C57BL/6 mice(positive control group)and IL-10-/-mice(negative control group)were sorted by FACS;MACS was used to purify CD4+CD25-T cells,CFSE-labeled CD4+CD25-T cells or unlabeled CD4+CD25-T cells were cultured alone,with B10 cells or with CD19+CD5-CD1 dlow B cells.Their proliferative response was determined 72 hours later based on the CFSE staining;the immune response of Th1 cells,Th2 cells,Th17 cells and Treg cells was determined by the level of IFN-?,IL-4,IL-17 and Foxp3.Results:(1)B10 cells of WT C57BL/6 mice can significantly inhibit the proliferation of CD4+CD25-T cells(P<0.01)compared to CD19+CD5-CD1 dlow B cells,while B10 cells in IL-10-/-mice and NOD mice could not depress the proliferation of CD4+CD25-T cells(P>0.05);B10 cells in WT C57BL/6 mice,IL-10-/-mice and NOD mice promoted the proliferation of CD4+CD25-T cells when compared to control group(P>0.05);(2)B10 cells in WT C57BL/6 mice could significantly inhibit the secretion of IFN-?,promote the secretion of IL-4 and the expression of Foxp3(P<0.01),while B10 cells in IL-10-/-mice and NOD mice had no significantly influence on the secretion of IFN-? and IL-4 and the expression of Foxp3(P>0.05),but B10 cells of WT C57BL/6 mice and IL-10-/-mice could not inhibit the secretion of IL-17(P>0.05);(3)In Transwell co-culture system,B10 cells of WT C57BL/6 mice could completely inhibit the proliferation of CD4+CD25-T cells(P<0.01),while B10 cells in NOD mice could not(P>0.05);(4)After 72 h ex vivo mixed lymphocyte reaction,while the secretion of IL-10 of B10 cells in NOD mice were significantly lessthan that in WT C57BL/6 mice(P<0.01);(5)After 72 h ex vivo mixed lymphocyte reaction,the expression of MHC-class II molecules and costimulatory molecules CD80,CD86 in B10 cells of NOD mice had no difference with that in WT C57BL/6 mice(P>0.05)Conclusion:(1)B10 cells subset in NOD mice has obvious dysfunction,and the dysfunction of B10 cell may participate in the occurrence and progression of T1 D in NOD mice;(2)B10 cells in WT and NOD mice had no influence on the secretion of IL-17,while B10 cells in NOD mice were dysfunction on regulation of the secretion of IFN-? and IL-4 and the expression of Foxp3;(3)The dysfunction of B10 cells in NOD mice may due to the apparently decrease of IL-10;(4)B10 cells in WT and NOD mice could be activated and express costimulatory molecules CD80 and CD86,while the dysfunction of B10 cells in NOD mice may be irrelevant with the level of CD80 and CD86.