Reg3g Overexpression Promotes β Cell Regeneration And Induces Immunetolerance In Nonobese-diabetic (NOD) Mouse Model

PARTⅠ Differentiation of DC subsets and the interactions in nonobese-diabetic (NOD) mouse modelBackgroundType 1 diabetes (T1D) is an autoimmune disease characterized by immune system mediated breakdown of the islet-β cells in the pancreas. Dendritic cells (DC) is recognized as the initiator and modulator of the immune battle, taking up a diverse array of antigens and present them to self-reactive T cells as peptides bound to both MHC class Ⅰ and Ⅱ products. There are two main classes of conventional or classical DCs (cDCs):CD8a+DC and CDllb+DC, both of which arise from a common precursor characterized by expression of the C-type lectin receptor DNGR-1. To date, there is another CD8a’DC subset, merocytic DC (mcDC) with the surface phenotype of CD11c+CD11b-CD8α-PDCA-1 observed in NOD mice, based on their capacity to potently (cross-) prime both CD4+and CD8+T cells to cell-associated antigens. Therefore, investigating the interactions among the three subsets and their special properties may benefit understanding the characteristics of development of T1D in NOD mice.ObjectiveTo observe the development, the percentages, and the function of CD8α+DC (CD11c+CD8a+CD11b-PDCA-1-), CD11b+DC(CD11c+CD11b+CD8α-PDCA-1-), and mcDC(CD11c+CD11b+CD8αPDCA-1-) in spleen and bone marrow of NOD miceduring the development of T1D, and to observe the influence of ex-vivo culture on the differentiation and function of DC subsets in bone marrow cells.MethodsSpleen DC subsets and bone marrow cells were FACS-assessed from the three groups of female NOD mice aged 6～8 wk,10～12 wk, and 16～18 wk, including measuring the subsets of phenotypes CD11b+DC, CD8α+DC, and mcDC. Spleen DC subsets were isolated for mixed lymphocyte reaction. After LPS stimulation, serum cytokines were measured in both spleen and bone marrow subsets by ELISA.ResultsThere was a significant decrease in percentage of spleen CD8α+DC subset at 12 or 16 weeks, whereas percentage of mcDC was increased in spleens in the kinetic observation by 16 week. Spleen CD11b+DC subset generally remained the normal level during 8-16 weeks.mcDC featured the great power of stimulating T cell proliferation from 12 weeks, and secreted the highest level of IFN-y which was parrelling with the weeks. CD8α+DC subset significantly decreased the production of cytokines, including IL-12p70, IL-10, and TGF-β. CD11b+DC subset secreted the augmented level of TNF-α whereas a reverse trend in TGF-β level.In ex vivo cell culture, CD11b took the advantages of expression both in bone marrow cells and CD11c+DC subsets at 0 or 7 day, especially a 20-fold augment in CD11c+bone marrow cells. There was an approximately 4-5 fold increase in mcDC phenotype of bone marrow cells. After LPS stimulation, there were varying increases in IL-12p70, TNF-α, IFN-γ, TGF-β of the supernatants, whereas an increase in IL-10 was not evident.ConclusionIn the development of T1D, the loss of self-immune tolerance resulted from DC aspects of a decrease in the number and weakened function of tolerogenic CD8α+DC, the pro-inflammation bias of CD11b+DC subset, and an increased number of mcDC with strengthened capacity to stimulate T cell proliferationand increased pro-inflammatory cytokines. The findings in bone marrow cells may confer abnormality of DC subsets in the spleens in varying weeks. Significantly, mcDC may be induced in vitro for further investigation.PART Ⅱ Reg3g overexpression mediated by lentivirus vectors promotes β cell regeneration in nonobese-diabetic (NOD) mouse modelBackgroundReg gene encodes an endogenous lectin involved in the regeneration or the growth of β cells, and constitutes a Reg family (Regl-4), categorized by primary structures of the proteins; all share a conservative sequence with well-characterized C-type lectins. It is well-established that Reg protein, such as Reg I or Regll, is indispensable in the pathophysiology of various human inflammatory diseases, especially dominating the regeneration of islet-β cells in pancreatic inflammation damage.Reg3A also showed the protection of STZ-induced T1D model, and induced the significant proliferation of pancreatic tumor cells in vitro. According to the similar structure of Reg3 subtypes, we speculate that Reg3g could also benefit the protection of β cell regeneration mediated by lentivirus vectors.ObjectiveTo observe the expression of Reg3g in NOD mice mediated by lentiviral vectors plus encoded eGFP, and to investigate the potential mechanism that regulates the regeneration of β cells initiated by Reg3g.Methods6 week-old NOD mice were randomLy divided into three groups:PBS-control group, lentiviral vectors group (eGFP-Lv), and Reg3g-lentiviral vector group (Reg3g-Lv). At week of 8, mice were injected i.p. with one injection (200 μL) of Reg3g-Lv, eGFP-Lv, or PBS per week for the consecutive 4 wks, respectively. Before injection, lentvirus encoding mouse Reg3g (1.29×109 copies/mL) were diluted to 1.29×107 copies/mL with sterile PBS. The transfer vector was also diluted with sterile PBS and i.p. injected.One week after the last injection, mice of 12 wk were randomLy sacrificed to qualify the location of Reg3g expression in liver, pancreas, spleen, small intestine and kidney by fluorescence microscopy and laser scanning microscope. The pancreata were sliced to evaluate the histopathological insulitis by HE staining, apoptosis by TUNEL staining, cell proliferation by PCNA staining, and the insulin production. Pancreatic islets were isolated for Western-blot analysis to identify the potential signal molecules involved in the hiding transduction.ResultsIn general, Reg3g-Lv treatment ameliorated the development of T1D in NOD mice, which was associated with the overexpression of Reg3g-Lv in the islet cells verified by the combination of laser scanning microscope and Wester-blot analysis. The invading insulitis was reduced, which was accompanied by the increased PCNA-positive islet cells and strengthened insulin production in situ. Apoptotic β cells were not distinct. There was also the varying increasese of JAK2, pJAK2, STAT, pSTAT, and NF-κB observed in islets. SOCS3 also modestly displayed an increase. At the end of experiment, there was a delayed onset and a significant decrease in the incidence of T1D in NOD mice of Reg3g-Lv groupConclusionReg3g was successfully overexpressed in β cells by using lentiviral vectors, leading to the proliferation of β cells through the motivated JAK2/STAT3/NF-κB pathway. This Reg3g therapy benefits the recovery of insulin production of NOD mice, and issues the inducing self-tolerance of NOD mice by Reg3g overexpression in the coming investigation.PART Ⅲ Reg3g overexpression induces immune tolerance in nonobese-diabetic (NOD) mouse modelBackgroundThe immune mediated failure of the insulin-producing β cells is a consequence of a multifocal inflammatory reaction that is described as "insulitis". Dendritic cells (DC), T cell, and even macrophages appear at the islet periphery at 4 wk of age and their appearance correlates with transient hyperinsulinemia and islet neogenesis. Thereby, to rectify the deteriorating insulitis is another chance to rebalance, remove or reduce the local inflammation. Fortunately, Expression of Reg proteinwas firstly discovered in pancreatic juice of rats after induction of acute pancreatitis, also recognized as a pancreatitis-associated protein. For Reg3, the emerging evidence is to relieve the Thl-bias response in the combination of Reg3 and PPI therapy, and to reduce the insulitis that is composed of self-reactive immunocytes in Part II. The prospective is that the sole use of Reg3g also could benefit the inflammation and induce the tolerant properties of immune cells.ObjectiveTo observe the influence of Reg3g-Lv treatment on DC or T cells and the subsequent interaction between DC and T cells. And to investigate the potential mechanism in induced tolerance of NOD mice under the circumstances of cytokines and α1-AAT.MethodsDC subsets and T cells were isolated from NOD mice of 12 wk in Part Ⅱ. Flow cytometry was used to measure subsets and phenotypes of DCs and the appearance of MHCII and co-stimulatory molecules. DC function was ex-vivo assessed in mixed lymphocyte reactions and induction of CD4+CD25+Foxp3+ thymocytes. T cells in splenocytes were isolated for T cell proliferation assay. Percentages of CD4+CD25+Foxp3+T cells were also measured by FACS in splenocytes. Cytokines, such as IL-10, TGF-β, IL-12p70, IL-2, and IFN-y, and al-AATfrom DC or T cell supernatants and serum were respectively quantified by ELISA assay. Activation of JAK2/STAT3 pathway was finally detected by Western blot.ResultsIn NOD mice aged 12 wk, the maturation of splenic DC was of significant decrease by Reg3g-Lv treatment. There was a significant decrease in the frequency of CD11b+DC, by contrast, the significant difference of splenic DC and the notable variation in both CD8a+DC and mcDC were poor among the groups. Less proliferated T cells and more CD4+CD25+Foxp3+thymocytes were observed by FACS-sorted splenic DC, respectively. In response to LPS stimulation, there were Th2 cytokines, including IL-10 and TGF-β, significantly increased, whilst IL-12p70 decreased in DC supernatants. In splenic T cells, Treg percentage was notably increased to 20% in contrast to 5-6% in PBS group. Thl cytokines, such as IFN-y and IL-2 were markedly decreased, while Th2 cytokines including TGF-β and IL-10kept an elevated level. Interestingly, a significant increased α1-AAT was only observed in serum not in DC supernatant, which was produced in Reg3g overexpressed liverwith activated JAK2/STAT3/NF-κB pathway. Most importantly, the onset and the incidence of TID were delayed and lowered in Reg3g-Lv group.ConclusionReg3g overexpression in vivo reduced the maturation of splenic DC, leadingto the tolerogenic propertiesbenefiting the Treg differnentiation in NOD mice, and probably strengthened the synthesis of al-AAT to form the positive feedback of self-tolerance, leading to the protection of T1D.