Ningwen Tai

The role of gut microbiota in the development of type 1, type 2 diabetes mellitus and obesity

Diabetes is a group of metabolic disorders characterized by persistent hyperglycemia and has become a major public health concern. Autoimmune type 1 diabetes (T1D) and insulin resistant type 2 diabetes (T2D) are the two main types. A combination of genetic and environmental factors contributes to the development of these diseases. Gut microbiota have emerged recently as an essential player in the development of T1D, T2D and obesity. Altered gut microbiota have been strongly linked to disease in both rodent models and humans. Both classic 16S rRNA sequencing and shot-gun metagenomic pyrosequencing analysis have been successfully applied to explore the gut microbiota composition and functionality. This review focuses on the association between gut microbiota and diabetes and discusses the potential mechanisms by which gut microbiota regulate disease development in T1D, T2D and obesity.

Cellular and humoral immune responses in the early stages of diabetic nephropathy in NOD mice

This study was designed to examine immunopathology of diabetic nephropathy in non-obese diabetic (NOD) mice and to investigate the involvement of cellular and humoral immunity at various time points after diabetes onset. We found that the glomeruli of diabetic NOD mice were infiltrated with T and B cells, as well as CD11c+ dendritic cells, which had close contact with CD4+ and CD8+ T cells in the infiltrates. We also found that IgG deposits in the glomeruli of diabetic NOD mice were accompanied by the presence of complement C3. Moreover, the serum from diabetic mice contained autoantibodies directed towards components of the glomeruli and these antibodies were not present in non-diabetic NOD mice. The immune changes in the kidney occurred together with increasing kidney weight and urinary albumin excretion along with duration of diabetes. We provide evidence that infiltrating lymphocytes and anti-kidney autoantibodies may be involved in diabetic nephropathy in autoimmune diabetes in the NOD mouse. Understanding the role that the immune system plays in the pathogenesis of diabetic nephropathy could lead to identification of new strategies and/or additional therapeutic targets for prevention and treatment of diabetic nephropathy.

TLR4 regulates cardiac lipid accumulation and diabetic heart disease in the nonobese diabetic mouse model of type 1 diabetes

Toll-like receptor (TLR)4 regulates inflammation and metabolism and has been linked to the pathogenesis of heart disease. TLR4 is upregulated in diabetic cardiomyocytes, and we examined the role of TLR4 in modulating cardiac fatty acid (FA) metabolism and the pathogenesis of diabetic heart disease in nonobese diabetic (NOD) mice. Both wild-type (WT) NOD and TLR4-deficient NOD animals had increased plasma triglyceride levels after the onset of diabetes. However, by comparison, TLR4-deficient NOD mouse hearts had lower triglyceride accumulation in the early stages of diabetes, which was associated with a reduction in myeloid differentiation primary response gene (88) (MyD88), phosphorylation of p38 MAPK (phospho-p38), lipoprotein lipase (LPL), and JNK levels but increased phospho-AMP-activated protein kinase (AMPK). Oleic acid treatment in H9C2 cardiomyocytes also led to cellular lipid accumulation, which was attenuated by TLR4 small interfering RNA. TLR4 deficiency in the cells decreased FA-induced augmentation of MyD88, phospho-p38, and LPL, suggesting that TLR4 may modulate FA-induced lipid metabolism in cardiomyocytes. In addition, although cardiac function was impaired in both diabetic WT NOD and TLR4-deficient NOD animals compared with control nondiabetic mice, this deficit was less in the diabetic TLR4-deficient NOD mice, which had greater ejection fraction, greater fractional shortening, and increased left ventricular developed pressure in the early stages after the development of diabetes compared with their diabetic WT NOD counterparts. Thus, we conclude that TLR4 plays a role in regulating lipid accumulation in cardiac muscle after the onset of type 1 diabetes, which may contribute to cardiac dysfunction.

IL-10-conditioned dendritic cells prevent autoimmune diabetes in NOD and humanized HLA-DQ8/RIP-B7.1 mice

This study was to determine whether BMDCs cultured in the presence of IL-10 (G/10-DCs) could promote T cell tolerance and prevent autoimmune diabetes in two different animal models of T1D. Our results showed that G/10-DCs suppressed both insulitis and spontaneous diabetes in NOD and HLA-DQ8/RIP-B7.1 mice. The suppression was likely to be mediated by T cells, as we found that regulatory CD4(+)CD25(+)Foxp3(+) cells were significantly increased in G/10-DC treated animals. In vivo, the G/10-DCs inhibited diabetogenic T cell proliferation; in vitro, they had reduced expression of costimulatory molecules and produced little IL-12/23 p40 or IL-6 but a large amount of IL-10 when compared with DCs matured in the presence of IL-4 (G/4-DC). We conclude that IL-10-treated DCs are tolerogenic and induce islet-directed immune tolerance, which was likely to be mediated by T regulatory cells. This non-antigen-specific DC-based approach offers potential for a new therapeutic intervention in T1D.

Maternal Antibiotic Treatment Protects Offspring from Diabetes Development in Nonobese Diabetic Mice by Generation of Tolerogenic APCs

Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease that involves the slow, progressive destruction of islet β cells and loss of insulin production, as a result of interaction with environmental factors, in genetically susceptible individuals. The gut microbiome is established very early in life. Commensal microbiota establish mutualism with the host and form an important part of the environment to which individuals are exposed in the gut, providing nutrients and shaping immune responses. In this study, we studied the impact of targeting most Gram-negative bacteria in the gut of NOD mice at different time points in their life, using a combination of three antibiotics—neomycin, polymyxin B, and streptomycin—on diabetes development. We found that the prenatal period is a critical time for shaping the immune tolerance in the progeny, influencing development of autoimmune diabetes. Prenatal neomycin, polymyxin B, and streptomycin treatment protected NOD mice from diabetes development through alterations in the gut microbiota, as well as induction of tolerogenic APCs, which led to reduced activation of diabetogenic CD8 T cells. Most importantly, we found that the protective effect was age dependent, and the most profound protection was found when the mice were treated before birth. This indicates the importance of the prenatal environment and early exposure to commensal bacteria in shaping the host immune system and health.

Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice

Wen et al. show that there are gut microbial antigens sharing significant homology with the host pancreatic protein peptide IGRP and that can drive self-reactive T cell activation and accelerate diabetes in NOD mice.

TLR9 Deficiency Promotes CD73 Expression in T Cells and Diabetes Protection in Nonobese Diabetic Mice

TLR9-deficient (TLR9−/−) NOD mice develop a significantly reduced incidence of diabetes. This study was to investigate the molecular mechanisms of the protective role of TLR9 deficiency. Through gene screening and confirmation by both mRNA and protein expression, we found a significant increase in CD73-expressing immune cells from peripheral lymphoid tissues in TLR9−/− NOD mice. The elevated frequency of CD73-expressing immune cells seemed to be specific for TLR9 deficiency and was MyD88 independent. Moreover, the increased frequency of CD73 expression was limited to the NOD background. Increased frequency of CD73 expression was also associated with lower levels of proinflammatory cytokines and more anti-inflammatory cytokine production in CD4+ T cells in TLR9−/− NOD mice. Purified CD73+CD4+ T cells showed stronger immunosuppressive function in vitro and delayed diabetes development in vivo. The immunosuppression appeared to be mediated by TGF-β. In addition, elevated frequency of CD73-expressing cells was associated with improved β cell function. Our observations were further confirmed by protection from diabetes with similar alterations in CD73 in the NY8.3 TCR NOD mouse model crossed with TLR9−/− mice and by the use of a TLR9 inhibitor in NOD mice. Our novel findings suggest an important immune-regulatory role of CD73 in regulation of diabetes development and may offer a new therapeutic strategy for specific intervention to prevent type 1 diabetes.

A novel “humanized mouse” model for autoimmune hepatitis and the association of gut microbiota with liver inflammation

Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease characterized by interface hepatitis, the presence of circulating autoantibodies, and hyper‐gammaglobulinemia. There are two types of AIH, type 1 (AIH‐1) and type 2 (AIH‐2), characterized by distinct autoimmune serology. Patients with AIH‐1 are positive for anti–smooth muscle and/or antinuclear autoantibodies, whereas patients with AIH‐2 have anti–liver kidney microsomal type 1 and/or anti–liver cytosol type 1 autoantibodies. Cytochrome P4502D6 is the antigenic target of anti–liver kidney microsomal type 1, and formiminotransferase cyclodeaminase is the antigenic target of anti–liver cytosol type 1. It is known that AIH, both types 1 and 2, is strongly linked to the human leukocyte antigen (HLA) alleles ‐DR3, ‐DR4, and ‐DR7. However, direct evidence of the association of HLA with AIH is lacking. We developed a novel mouse model of AIH using the HLA‐DR3 transgenic mouse on the nonobese‐diabetic background by immunization of HLA‐DR3– and HLA‐DR3+ nonobese‐diabetic mice with a DNA plasmid, coding for human cytochrome P4502D6/formiminotransferase cyclodeaminase fusion protein. Immunization with cytochrome P4502D6/formiminotransferase cyclodeaminase leads to a sustained elevation of alanine aminotransferase, development of antinuclear autoantibodies and anti–liver kidney microsomal type 1/anti–liver cytosol type 1 autoantibodies, chronic immune cell infiltration, and parenchymal fibrosis on liver histology in HLA‐DR3+ mice. Immunized mice also showed an enhanced T helper 1 immune response and paucity of the frequency of regulatory T cells in the liver. Moreover, HLA‐DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion: Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. (Hepatology 2015;62:1536–1550)

The Dual Effects of B Cell Depletion on Antigen-Specific T Cells in BDC2.5NOD Mice

B cells play a critical role in the pathogenesis of autoimmune diabetes. To investigate the mechanisms by which B cell depletion therapy attenuates islet β cell loss and particularly to examine the effect of B cells on both diabetogenic and regulatory Ag-specific T cells, we generated a transgenic BDC2.5NOD mouse expressing human CD20 on B cells. This allowed us to deplete B cells for defined time periods and investigate the effect of B cell depletion on Ag-specific BDC2.5 T cells. We depleted B cells with anti-human CD20 Ab using a multiple injection protocol. We studied two time points, before and after B cell regeneration, to examine the effect on BDC2.5 T cell phenotype and functions that included antigenic response, cytokine profile, diabetogenicity, and suppressive function of regulatory T (Treg) cells. We found unexpectedly that B cell depletion induced transient aggressive behavior in BDC2.5 diabetogenic T cells and reduction in Treg cell number and function during the depletion period. However, after B cell reconstitution, we found that more regenerated B cells, particularly in the CD1d− fraction, expressed immune regulatory function. Our results suggest that the regenerated B cells are likely to be responsible for the therapeutic effect after B cell depletion. Our preclinical study also provides direct evidence that B cells regulate both pathogenic and Treg cell function, and this knowledge could explain the increased T cell responses to islet Ag after rituximab therapy in diabetic patients in a recent report and will be useful in design of future clinical protocols.

The role of the innate immune system in destruction of pancreatic beta cells in NOD mice and humans with type I diabetes.

Type 1 diabetes (T1D) is an organ-specific autoimmune disease characterized by T cell-mediated destruction of the insulin-producing pancreatic β cells. A combination of genetic and environmental factors eventually leads to the loss of functional β cell mass and hyperglycemia. Both innate and adaptive immunity are involved in the development of T1D. In this review, we have highlighted the most recent findings on the role of innate immunity, especially the pattern recognition receptors (PRRs), in disease development. In murine models and human studies, different PRRs, such as toll-like receptors (TLRs) and nucleotide-binding domain, leucine-rich repeat-containing (or Nod-like) receptors (NLRs), have different roles in the pathogenesis of T1D. These PRRs play a critical role in defending against infection by sensing specific ligands derived from exogenous microorganisms to induce innate immune responses and shape adaptive immunity. Animal studies have shown that TLR7, TLR9, MyD88 and NLPR3 play a disease-predisposing role in T1D, while controversial results have been found with other PRRs, such as TLR2, TLR3, TLR4, TLR5 and others. Human studies also shown that TLR2, TLR3 and TLR4 are expressed in either islet β cells or infiltrated immune cells, indicating the innate immunity plays a role in β cell autoimmunity. Furthermore, some human genetic studies showed a possible association of TLR3, TLR7, TLR8 or NLRP3 genes, at single nucleotide polymorphism (SNP) level, with human T1D. Increasing evidence suggest that the innate immunity modulates β cell autoimmunity. Thus, targeting pathways of innate immunity may provide novel therapeutic strategies to fight this disease.