B Cells Regulate The Development Of Type 1 Diabetes By TLR9-TIMP1 Signal Pathway And TLR9 In Beta Cells Regulates Obesity By Gut Micro Biota

B Cells Regulate the Development of Type 1 Diabetes by TLR9-TIMP1 Signal PathwayBackgroundType 1 diabetes(T1D)is an autoimmune disease,which is a main disease occurred in adolescents and children.According to the epidemiological evidences,the incidence of T1D in China is significantly incresesd.T1D is characterized with the destruction of pancreatic cells mediated by T cell immune response.Due to the high incidence and severe complications of T1D,the treatment of T1D has become a major public health challenge in human.T1D is related to genetic factors and environmental factors,which ultimately leading to the loss of functional islet beta cells.Therefore,the key point is to prevent or reverse total function of beta cells.The traditional treatments of T1D are insulin and oral medications,which can not prevent the progressive failures of beta cells function.More and more studies showed that specific targeted molecular therapy can effectively maintain and repair the damaged islet beta cells function,and efficiently delay the development of diabetes complications.Toll-like receptor 9(TLR9)mainly involves in the innate immunity and adaptive immunity response.According to our preliminary research,we revealed the deficiency of TLR9 can reduce the incidence of T1D,which participated in the regulation of immune response in spleen,lymph nodes and other important immune organs,involved in regulating the immune response in islet.However,it is still not known if TLR9 specifically in B cells contributes to the beta cell autoimmunity.ObjectiveThrough specific knocking out TLR9 in B cells in mouse models,we aim to explore the fuction of TLR9 in B cells during the occurrence and development of T1D.Methods1.The establishment of mice model and diabetes incidence monitoring.By buiding up mouse models,we set up TLR9fl/fl CD19Cre+NOD mice and control(TLR9fl/fl CD19Cre-)NOD mice.Diabetes development in TLR9fl/fl CD19-Cre+NOD mice and TLR9fl/fl CD 19-Cre-NOD mice was monitored weekly for glycosuria with glucose strips from 10 weeks of age to 32 weeks of age.2.Flow cytometry staining and intracellular staining.Cells were harvested from peripheral immune tissues and stained for antibodies to CD19,B220,CD40,TCR-beta,CD5,CD1d,CD21,CD23.Cells were gated by different markers.3.Adoptive transfer into Rag-/-NOD mice.Purified splenic T cells from diabetic NOD mice with sorted splenic B cells from TLR9fl/fl CD19-Cre+or TLR9fl/fl CD19-Cre-NOD mice were injected into female Rag-/-NOD mice.All the recipients were monitored for glycosuria weekly.4.Microarray hybridization and data analysis.B cells from TLR9fl/fl CD19-Cre+or TLR9fl/fl CD19-Cre-NOD mice were sorted for whole genome Illumina microarray chip analysis to study the effect of TLR9 in B cells in mRNA level.Results1.B cells-specific TLR9 deficiency protects against T1D development.The results showed that B cells specificity deficenicy of TLR9 can significantly delay the onset of T1D.2.TLR9 intrinsically regulates the frequency and function of B subsets.B cells-specific TLR9 deficiency NOD mice showed significantly higher percentage of CD1d+CD5+ regulatory B cells(Bregs),higher percentage of marginal zone B cells and lower percentage of transitional B cells.3.B cells without TLR9 expression reduced activation and hypo-responsive to innate and adaptive immune stimuli.4.TLR9 regulates the fate of IL-10-producing B cells and suppressed T cell responses.5.TLR9 regulates the cross talk of tissue inhibitor of metalloproteinase 1(TIMP1)and IL-10 in B cells,by down-redulating IL-10 expression to aggravate the progresses of T1D.Conclusion1.TLR9 plays a critical role in immune tolerance to islet beta cell autoimmunity,as TLR9 deficiency in B cells strongly protected NOD mice from T1D development2.TLR9 modulated T1D susceptibility by in a number of ways,which included effects on B cell development and differentiation into various subsets,especially Bregs subsets.3.TLR9 in B cell can directly affect the incidence of T1D.4.A novel regulatory network of TLR9 with MMP19,TIMP1,CD40 and IL-10 signal pathway was revealed.Our study confirmed an immune tolerance(or lack of)function of B cells,mediated by TLR9 in T1D.TLR9 in Beta Cells Regulates Obesity by Gut Micro biotaBackgroundObesity is a kind of chronic-metabolic disease caused by a variety of factors,characterized with systemic,chronic and low-grade inflammation.Chronic low-grade inflammation has been proved to be the key link in the onset of obesity and related metabolic complications.The damage of intestinal barrier and intestinal dysbacteriosis,causes the imbalance of microbes or low-grade inflammation by the circulatory system of bacterial endotoxinTLR9 can identify microbe and activate immune cells response.TLR9 can be expressed in islet beta cell surface,and participate in immune cell infiltration.TLR9 mediates the immune response,through the production of inflammation cytokines,which linked microbes and development of obesity closely.The development of obesity is closely related to TLR9 and gut microbes.ObjectiveThrough the specific knockout of TLR9 in islet beta cells,we generated TLR9-deficiency in beta cells mouse models.Our study aims to clarify the effect of TLR9 in beta cells mediated with gut microbiota,immune response and islet function.The study will provide a new approach for the treatment of obesity.Methods1.The establishment of mice model and body weight monitoring TLR9fl/fl and Pdxlcre+C57BL/6 mice were inter-crossed to obtain beta cell-specific TLR9 deficient(TLR9fl/fl PdxlCre+)and control(PdxlCre+)C57BL/6 mice.The feeding time was lasted for 12 weeks.The body weight of mice were recorded weekly2.Flow cytometry staining and Intracellular staining.B subsets,T subsets,macrophages,beta cells and alpha cells were staining for antibodies to CD19,B220,CD40,TCR-beta,CD5,CDld,CD21,CD23,CD11c,CD11b,IAb,IgA,Gr1,Fluozin,CD45 and other specific markers3.Oral gavage of germ-free C57BL/6(GFB6)mice.Fresh fecal pellets were harvested from obese mice separately.GF B6 mice were then colonized with bacteria by oral gavage.Successful colonization was evaluated by 16S rRNA bacterial sequencing of fecal pellets from the GF B6 mice.4.16S rRNA sequencing and microbiota classification.16S rRNA gene was amplified from each DNA sample using bar-coded broadly-conserved bacterial forward and reverse primers.The sequencing data were analyzed by operational taxonomic units(OTUs).Beta-diversity was calculated to compare differences between microbial communities.shown as Principal Coordinate Analysis(PCoA).5.Enzyme-linked immunosorbent method(ELISA)to detect cytokines and polymerase chain reaction(PCR).Elisa kit uesd to detect immunoglobulins in serum and gut flush.PCR was performed with specific primers related to islet and antimicrobial peptides.Results1.Beta cells-specific TLR9 deficiency protects against obesity.We found TLR9fl/fl Pdx1Cre+ compared to Pdx1Cre+C57BL/6 mice fed with high fat diet,showed lower body weight,enhanced glucose reactivity,and enhanced insulin sensitivity.Similarly,we found beta cells-specific TLR9 deficiency obese B6 mice showed lower dry weight of adipose tisssue and liver.2.TLR9 intrinsically regulates the frequency and function of B subsets,T subsets and macrophages.Beta cells-specific TLR9 deficiency obese B6 mice showed decreased percentage of activated T cells,higher percentage of Bregs and decreased percentage of macrophage,along with significantly higher levels of IL-10 and IgM in serum.3.Beta cells without TLR9 involved in regulation of antimicrobial peptides expression in small intestine.Beta cells-specific TLR9 deficiency obese B6 mice showed changes in gene expressions of antimicrobial peptides in samll intestine,mainly with a higher expression trend of antimicrobial peptides.4.Beta cells without TLR9 involved in regulation of islet number and function.The islet of beta cells-specific TLR9 deficiency obese B6 mice showed higher expression of Insulin 1,Glucagon mRNA,along with a higher trend in the expression of antimicrobial peptides.5.Colonized with the fecal bacteria of beta cells-specific TLR9 deficiency obese B6,GF B6 mice could mediate immune response.After oral gagvage,GF B6 mice colonized with fecal from beta cells-specific TLR9 deficiency obese B6 showed similar IPGTT,insulin release results as donors.Like donors results,GF B6 mice colonized with fecal from beta cells-specific TLR9 deficiency obese B6 showed higher percentage of Tregs and Bregs,and lower percentage of pro-inflammation macrophages.6.Beta cells without TLR9 regulate the composition and diversity of gut microbiota.Component analysis of gut microbita in vitro were tested and found significant difference under the PCoA-beta diversity in both beta cells-specific TLR9 deficiency obese B6 mice and GF B6 mice colonized with fecal from beta cells-specific TLR9 deficiency obese B6 mice.7.Beta cells without TLR9 link gut microbiota,immune response and beta cell function together.GF B6 mice colonized with fecal from beta cells-specific TLR9 deficiency obese B6 mice showed higher islet numbers,higher proportion of beta cells,together with significantly higher levels of IgA in gut flush and serum.Conclusion1.TLR9 plays a critical role in beta cells in mediating obesity by immune reseponse,which related to B subsets,T subsets and macrophages.2.TLR9 modulated gut microbiota by in antimicrobial peptides,which related to the fuction of islets.3.In the absence of TLR9 in beta cells,gut microbiota and immune composition were altered by colonizing fecal bacteria in GF B6 mice.4.The cross-talk of TLR9,microbiota,immune response can regulate beta cells function,finally modulate the development of obesity.