The Role Of Nucleotide-binding Oligomerization Domain-containing Protein 2(Nod2) In The Development Of Type 1 Diabetes In Nonobese Diabetic Mice

Background: Type 1 diabetes mellitus(T1DM)is an autoimmune disease characterized by the destruction of insulin-producing ? cells by T cells.Both genetic and environmental factors involved in T1 DM development.In the past few decades,the incidence of T1 DM has significantly increased worldwide and environmental factors are thought to play a critical role in this,but the mechanism is not completely clear.Recently,more and more studies have shown that gut microbiota is closely related to the development of T1 DM.Innate immunity protects hosts from bacterial invasion by recognizing bacterial components through their pattern recognition receptors(PRRs).These PRRs include Toll-like receptors(TLRs)and nucleotidebinding oligomerization domain-like receptors(NOD-like receptors,NLRs).TLR family has been shown to regulate the development of T1 DM by interacting with gut microbiota.However,much less is known about the role of NLR family members in T1 DM susceptibility,in humans and mice.Nucleotide-binding oligomerization domain-containing protein 2(Nod2)is a cytosolic bacterial sensor of muramyl dipeptide that induces antimicrobial peptide release and inflammatory signaling required for maintenance of the homeostasis of gut microbiota.Nod2 mutations are associated with high susceptibility to Crohn's disease,a common inflammatory disorder of the bowel.Furthermore,Nod2 deficiency in mice correlates with increased bacterial susceptibility.However,its role in spontaneous T1 DM remains unclear.Aim: To investigate the role of Nod2,one of the pattern recognition receptors in susceptibility to the autoimmune disease,T1 DM.Methods: 1.Subjects we generated Nod2-/-NOD mice,and use Nod2+/+ NOD mice as control.2.Short term and long term co-housing Three to 4-week-old female Nod2-/-NOD or Nod2+/+NOD littermates were divided into groups,whereby Nod2+/+NOD and Nod2-/-NOD mice were housed in separate groups or co-housed in cages where the genotypes were mixed.Experiments were terminated when the mice were aged 9-10 weeks or 30 weeks old.3.Histopathology and insulitis Pancreata were fixed in 10% buffered formalin and embedded in paraffin.Tissues were sectioned and stained with hematoxylin and eosin.Insulitis was scored under light microscopy.4.Feces microbiota detection Extraction of gut bacterial DNA from freash collected feces of co-house or non co-housed female Nod2-/-NOD and Nod2+/+NOD mice.16 S r RNA sequencing and quantitative real-time PCR(q PCR)were used for microbiota classification.5.Immune cells and cytokines detection Lymphatic organs and lymph nodes were prepared as a single-cell suspension,treated with or without stimulus,and detected by flow cytometry after the cells were incubated with antibodies.Secreted cytokines were detected using an enzyme-linked immunosorbent assay(ELISA).6.Interaction of gut bacteria and immune cells in co-culture with splenocytes The large intestine was harvested from 2-month old female Nod2-/-NOD and Nod2+/+NOD mice and collected the gut contents.After washing,resuspend in PBS.Bacterial concentration was measured with a spectrophotometer and heatinactivated at 90 ? for 20 min.108 heat-inactivated bacteria were co-cultured with splenocytes(2×106/ml)from Nod2-/-NOD and Nod2+/+NOD mice for 14 h.7.Treg suppression assays Splenic and pancreatic lymph node(PLN)-derived Treg cells were isolated using Treg magnetic isolation kits and mitomycin-ctreated.Total NOD splenic APCs were used as alloantigen-stimulators.Purified splenic T cells from C57BL/6 mice were used as responders.Treg function was examined by suppression of the mixed lymphocyte reaction(MLR).8.Colonization of bacteria in grem free mice Fresh fecal pellets were harvested from 4 to 5 weeks old Nod2-/-NOD or Nod2+/+NOD mice housed separately.Bacteria solution were prepared.Germ-free WT NOD mice were then colonized with 2×108 CFU by oral gavage.Successful colonization was evaluated by 16 S r RNA bacterial sequencing of fecal pellets from the ex-GF mice.9.Statistical analysis Statistical analysis was performed using Graph Pad Prism 7 software.Diabetes incidence was compared using log-rank test.In vitro assays were analyzed with Student's t-test or ANOVA and P<0.05 was considered significant.Results: 1.The development of T1 DM depended on the gut microbiota of Nod2-/-NOD mice.The Nod2-/-NOD mice were significantly protected from diabetes,but only when Nod2-/-NOD mice had housed separately from Nod2+/+NOD mice.2.Absence of Nod2 in NOD mice did not affect the phenotype and antigen presentation ability of dendritic cells and macrophages.However,Nod2 and housing conditions significantly affected the number and function of Ig A-secreting B lymphocytes and Treg cells with immunoregulatory functions.3.In vitro experiments confirmed that the gut bacteria of Nod2-/-NOD mice co-housed with Nod2+/+NOD affected the production of pro-inflammatory cytokines of CD8+ and CD4+T cells.4.Grem free NOD mice colonized with gut microbiota from Nod2-/-NOD mice showed significantly reduced secretion of proinflammatory cytokines from immune cells,but significantly increased the number of Treg cells.Conclusion: 1.The influence of Nod2 on susceptibility to T1 DM depends on the change of gut microbiota.2.Nod2 mainly affects the number and function of Treg and Ig A secreting B cells.But,not affected the phenotype and antigen-presenting ability of dendritic cells and macrophages.3.The gut microbiota of non co-housed Nod2-/-NOD mice can induce more Treg cells in grem free mice,further supporting that the protective effect of Nod2 on T1 DM under none co-housed is mediated by the immune system nodulated by gut microbiota.These data provide further evidence that gut microbiota modulates the immune system and T1 DM susceptibility.4.Importantly,this study raises a critical question about the housing mode in the interpretation of the disease phenotype of genetically-modified mouse strains in T1 DM studies.