| Part Ⅰ The role of TRIF in the development of T1D in NOD MiceAim:(1) To determine the role of TRIF in the development of spontaneous and induced T1D in NOD mice;(2) To investigate the influence of TRIF on islet β cell function in NOD mice.Methods:WT NOD mice and TRIF-/-NOD mice were involved in this study.(1) By the detection of urine glucose, we observed the spontaneous T1D development;(2) By adoptively transferring the splenocytes from age, sex, diabetic duration paired WT NOD mice and TRIF-/-NOD mice to three different strains of recipient mice (semi-lethal X-ray dose treated WT NOD mice, TRIF-/-NOD mice and NOD.SCID mice);(3)The anti-insulin antibody in the serum was detected by Luminex;(4) Intraperitoneal glucose tolerance test (IPGTT) was applied to compare the glucose tolerence and the insulin in the serum was measured by radioimmunoassay;(5) Glucose stimulation assay in vitro was used to compare the insulin secretion from the islet upon glucose challenge; the insulin was also measured by radioimmunoassay;(6) Pancreata were fixed in buffered formalin. Paraffin-embedded tissues were sectioned and stained with H&E. Insulitis was scored under light microscopy.Results: (1) Compared to the female WT NOD mice (n=15), the spontaneous T1D incidence decreased in female TRIF-/-NOD mice (n=10); similarly, compared to the male TRIF+/-mice (n=17), the spontaneous T1D incidence decreased in male TRIF-/-NOD mice (n=12).(2) In the adoptive transfer model, the recipient mice (semi-lethal X-ray dose treated WT NOD mice, TRIF-/-NOD mice and NOD.SCID mice) which received the diabetic splenoctyes from TRIF-/-NOD mice could delay the onset of induced T1D (P<0.01-0.05).(3) The positivity of anti-insulin antibody in the serum significantly decreased in the female TRIF-/-NOD mice compared with female WT NOD mice (P＜0.05).(4) IPGTT result indicated the glucose tolerance did not impaired in TRIF-/-NOD mice compared to WT NOD mice (P＜0.01); and the insulin level in the serum were much higher than WT NOD mice (P＜0.05).(5) Glucose stimulation assay in vitro revealed that islet from TRIF-/-NOD mice produced more insulin upon glucose challenge (P＜0.05).(6) The insulitis in TRIF-/-NOD mice were less severer than in WT NOD mice (P＜0.05).Conclusions:(1) TRIF exerts an important role in the development of T1D and TRIF deficiency could delay the T1D onset.(2) TRIF could aggravate the immune injury of isletβ cell via promoting insulitis, thus less to a decrease in islet β cell function. Part II The impact of TRIF on the immunophenotype of APCs and T cells in NOD miceAim:To study the impact of TRIF on the immunophenotype of APCs and T cells in NOD mice.Methods:12-weeks-old, female, non-diabetic WT NOD mice and TRIF-/-NOD mice were involved in this study.(1) Lymphocytes from spleen, PLN, islet and bone marrow derived cells from WT NOD mice and TRIF-/-NOD mice were collected. Splenic APCs were isolated and purified by magnetic beads.(2) Lymphocytes from spleen, BMDCs and splenic APCs were cultured O/N in the medium without any stimuli, with TLR3stimuli PolyI:C, with TLR4stimuli LPS. Next morning, cells were washed and resuspended for use.(3) Cell surface markers, intracellular cytokines and Tregs were measured by FACS.Results:(1) Splenic B cells, macrophages, DCs from TRIF-/-NOD mice expressed less MHCII molecule (IAK) than the corresponding cells from WT NOD mice (splenic B cells:upon the stimulation of PolyI:C, P<0.01; upon the stimulation of LPS, P<0.05; upon the stimulation of anti-CD40, P<0.05. Splenic macrophages and DCs:without any stimuli, P<0.05; upon the stimulation of PolyI:C, P<0.05; upon the stimulation of LPS, P<0.05). Islet DCs from TRIF-/-NOD mice also expressed less IAK than WT NOD mice (P＜0.001).(2) Splenic macrophages from TRIF-/-NOD mice expressed less CD80(without any stimuli, P＜0.05; upon the stimulation of LPS, P<0.05), and CD86especially upon the stimulation (upon the stimulation of PolyI:C, P<0.001; upon the stimulation of LPS, P<0.01) compared with the corresponding cells from WT NOD mice. Splenic DCs from TRIF-/-NOD mice expressed less CD86upon the stimulation (upon the stimulation of PolyI:C, P＜0.001; upon the stimulation of LPS, P＜0.01) compared with the corresponding cells from WT NOD mice. Islet macrophages expressed less CD80and CD86(both P＜0.05). Bone marrow derived macrophages and DCs expressed less CD86especially upon the stimulation (upon the stimulation of PolyI:C, P＜0.001; upon the stimulation of LPS, P＜0.001).(3) Splenic macrophages and DCs from TRIF-/-NOD mice secreted less TNF-a especially upon the stimulation (splenic macrophages:upon the stimulation of PolyI:C, P＜0.05; upon the stimulation of LPS, P=0.06; DCs:without any stimuli, P＜0.05; upon the stimulation of PolyI:C, P＜0.01; upon the stimulation of LPS, P＜0.01) compared with WT NOD mice. Splenic macrophages and DCs from TRIF-/-NOD mice secreted less IFN-y especially upon the stimulation (splenic macrophages:without any stimuli, P＜0.05; upon the stimulation of PolyI:C, P<0.001; upon the stimulation of LPS, P＜0.01. DCs:without any stimuli, P＜0.01; upon the stimulation of PolyI:C, P＜0.001; upon the stimulation of LPS, P＜0.001) compared with WT NOD mice.(4) Bone marrow derived macrophages and DCs from TRIF-/-NOD mice expressed less CCR7especially upon the stimulation (bone marrow derived macrophages:without any stimuli, P＜0.05; upon the stimulation of PolyI:C, P=0.06; upon the stimulation of LPS, P＜0.01; bone marrow derived DCs:upon the stimulation of PolyI:C, P＜0.01; upon the stimulation of LPS, P＜0.05) compared with WT NOD mice.(5) Splenic (P＜0.05), PLN (P＜0.05) and islet (P＜0.001) CD4+T cells from TRIF-/-NOD mice expressed less CD69compared with WT NOD mice. Splenic CD4+T cells from TRIF-/-NOD mice expressed less ICOS and CD40L compared with WT NOD mice (both P＜0.05). (6) Splenic CD4+T cells from TRIF-/-NOD mice secreted less TNF-a, IFN-y and IL-17upon the stimulation (TNF-α:without any stimuli, P＜0.01; IFN-γ:without any stimuli, P＜0.05; upon the stimulation of PolyI:C, P＜0.05; IL-17:without any stimuli, P<0.05; upon the stimulation of LPS, P＜0.01).Conclusion:TRIF plays a key role in the upregulation the level of MHC II and co-stimulatory molecule on APCs and activation marker on T cells especially CD4+T cells. Part Ⅲ The impact of TRIF on the function of APCs and T cells in NOD mice.Aim:To study the impact of TRIF on the function of APCs and T cells in NOD mice.Methods:12-weeks-old, female, non-diabetic WT NOD mice and TRIF-/-NOD mice were involved in this study.(1) Bone marrow chimera was performed by adoptively transferring BMDCs from WT NOD mice and TRIF-/-NOD mice to TRIF-/-NOD mice and WT NOD mice treated by lethal X-ray dose. The incidence of T1D was observed;(2) In order to compare in vitro antigen presenting function of APCs by proliferation assay, CD4+T cells and CD8+T cells isolated and purified from BDC2.5NOD mice and NY8.3NOD mice respectively by magnetic beeds co-cultured with splenic APCs either from WT NOD mice or TRIF-/-NOD mice upon different concentration of mimotope and IGRP stimulation;(3) CD4+T cells were isolated and purified from BDC2.5NOD mice by magnetic beeds, then labeled with CSFE and injected to WT NOD mice and TRIF-/-NOD mice. Mice were sacrificed and the spleen and PLN were collected3days after the injection. Ex vivo antigen presenting function of APCs was evaluated by the measurement of the proliferation of CSFE+CD4+T cells via FACS analysis;(4) In order to compare in vivo antigen presenting function of APCs, purified BDC2.5CD4+T cells together with purified APCs from WT NOD mice or TRIF-/-NOD mice were adoptively transferred to NOD.SCID mice, then the incidence of T1D was observed;(5) In order to compare in vitro antigen presenting function of BMDCs by proliferation assay, CD4+T cells isolated and purified from BDC2.5NOD mice by magnetic beeds co-cultured with BMDCs from WT NOD mice or TRIF-/-NOD mice upon mimotope stimulation;(6) So as to compare in vitro proliferative function of T cells, CD4+T cells and CD8+T cells isolated and purified from WT NOD mice or TRIF-/-NOD mice by magnetic beeds co-cultured with splenoctyes from WT NOD mice upon anti-CD3stimulation;(7) In order to compare in vivo proliferative and diabetogenic function of T cells, CD4+T cells and CD8+T cells isolated and purified from WT NOD mice and TRIF-/-NOD mice respectively were adoptively transferred to NOD.SCID mice, then the incidence of T1D was observed.Results:(1) Compared to the lethal irradiated NOD mice which received the bone marrow cells from TRIF-/-NOD mice, the onset of T1D could be accelerated in the lethal irradiated TRIF-/-NOD mice which received the bone marrow cells from NOD mice (P<0.05).(2) APCs from WT NOD mice and TRIF-/-NOD mice showed similar antigen presenting function to NY8.3CD8+T cells at different concentration of IGRP (P>0.05). APCs from TRIF-/-NOD mice showed impaired antigen presenting function to BDC2.5CD4+T cells than the APCs from WT NOD mice at the different concentration of mimotope (P＜0.001-0.05).(3) In the spleen and PLN, CSFE labeled BDC2.5CD4+T cells showed less ex vivo proliferative ability in TRIF-/-NOD mice than in WT NOD mice.(4) In adoptive transfer, compared to the NOD.SCID mice which received BDC2.5CD4+T cells and NOD APCs, the onset of T1D could be delayed in NOD.SCID mice which received BDC2.5CD4+T cells and TRIF-/-AAPCs.(5) BMDCs from TRIF-/-NOD mice showed impaired antigen presenting function to BDC2.5CD4+T cells than the BMDCs from WT NOD mice upon the mimotope stimulation (wo stimulation:P＜0.05, upon PolyI:C stimulation:P<0.05, upon LPS stimulation:P＜0.01).(6) There was no difference in CD8+T cells in vitro proliferation between WT NOD mice and TRIF-/-NOD mice. However, TRIF-/-CD4+T cells was less proliferative than WT NOD CD4+T cells in vitro (P<0.001-0.05).(7) In adoptive transfer, NOD.SCID mice which received WT NOD CD4+T cells and WT NOD CD8+T cells developed T1D fastest; the next was the NOD.SCID mice which received WT NOD CD4+T cells and TRIF-/-CD8+T cells; the third was the NOD.SCID mice which received TRIF-/-CD4+T cells and WT NOD CD8+T cells; the last was the NOD.SCID mice which received TRIF-/-CD4+T cells and TRIF-/-CD8+T cells. It indicated that TRIF deficiency suppressed the proliferation and pathopoiesis of CD4+T cells.Conclusions:TRIF is a predisposing factor by upregulation of antigen presenting function of APCs, which leads to CD4+T cells activation and proliferation, thus induce TID. |
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