Changyun Hu

Innate immunity and intestinal microbiota in the development of Type 1 diabetes

Type 1 diabetes (T1D) is a debilitating autoimmune disease that results from T-cell-mediated destruction of insulin-producing β-cells. Its incidence has increased during the past several decades in developed countries, suggesting that changes in the environment (including the human microbial environment) may influence disease pathogenesis. The incidence of spontaneous T1D in non-obese diabetic (NOD) mice can be affected by the microbial environment in the animal housing facility or by exposure to microbial stimuli, such as injection with mycobacteria or various microbial products. Here we show that specific pathogen-free NOD mice lacking MyD88 protein (an adaptor for multiple innate immune receptors that recognize microbial stimuli) do not develop T1D. The effect is dependent on commensal microbes because germ-free MyD88-negative NOD mice develop robust diabetes, whereas colonization of these germ-free MyD88-negative NOD mice with a defined microbial consortium (representing bacterial phyla normally present in human gut) attenuates T1D. We also find that MyD88 deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of specific pathogen-free MyD88-negative NOD donors attenuates T1D in germ-free NOD recipients. Together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a critical epigenetic factor modifying T1D predisposition.

Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice

The precise roles of B cells in promoting the pathogenesis of type 1 diabetes remain undefined. Here, we demonstrate that B cell depletion in mice can prevent or delay diabetes, reverse diabetes after frank hyperglycemia, and lead to the development of cells that suppress disease. To determine the efficacy and potential mechanism of therapeutic B cell depletion, we generated a transgenic NOD mouse expressing human CD20 (hCD20) on B cells. A single cycle of treatment with an antibody specific for hCD20 temporarily depleted B cells and significantly delayed and/or reduced the onset of diabetes. Furthermore, disease established to the point of clinical hyperglycemia could be reversed in over one-third of diabetic mice. Why B cell depletion is therapeutic for a variety of autoimmune diseases is unclear, although effects on antibodies, cytokines, and antigen presentation to T cells are thought to be important. In B cell-depleted NOD mice, we identified what we believe is a novel mechanism by which B cell depletion may lead to long-term remission through expansion of Tregs and regulatory B cells. Our results demonstrate clinical efficacy even in established disease and identify mechanisms for therapeutic action that will guide design and evaluation of parallel studies in patients.

The Role of Toll-Like Receptors 3 and 9 in the Development of Autoimmune Diabetes in NOD Mice

Innate immunity is mediated, at least in part, through a number of receptors known as Toll‐like receptors (TLRs), which are activated by different microbial stimuli. Adaptive immunity, including autoimmunity, follows the innate response in a more specific manner. To investigate the roles of TLR3 and TLR9 in the development of type 1 diabetes, we generated NOD mice that are deficient in TLR3 and 9, respectively. There was no obvious difference in the incidence of spontaneous diabetes between TLR3‐deficient mice and TLR3 heterozygous mice. However, TLR9‐deficient mice were markedly protected from the disease compared to TLR9 heterozygous mice. Our results suggest that different TLRs play a varying role in autoimmune diseases.

NLRP3 deficiency protects from type 1 diabetes through the regulation of chemotaxis into the pancreatic islets

Significance Our study demonstrated that the nucleotidebinding oligomerization domain, leucine-rich repeat and pyrin domaincontaining protein 3 (NLRP3) pathway plays an important role in type 1 diabetes (T1D) using a mouse model. NLRP3 is critical for chemokine receptors CCR5 and CXCR3 expression on T cells, influencing pathogenic T-cell migration to the islets. It also affects the chemokines CCL5 and CXCL10 expression in the islets, preventing pathogenic T cells from infiltration. Targeting this pathway may be useful in prevention and treatment of T1D as it affects both immune cells and pancreatic beta cells. Studies in animal models and human subjects have shown that both innate and adaptive immunity contribute to the pathogenesis of type 1 diabetes (T1D). Whereas the role of TLR signaling pathways in T1D has been extensively studied, the contribution of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NLRP) 3 inflammasome pathway remains to be explored. In this study, we report that NLRP3 plays an important role in the development of T1D in the nonobese diabetic (NOD) mouse model. NLRP3 deficiency not only affected T-cell activation and Th1 differentiation, but also modulated pathogenic T-cell migration to the pancreatic islet. The presence of NLRP3 is critical for the expression of the chemokine receptors CCR5 and CXCR3 on T cells. More importantly, NLRP3 ablation reduced the expression of chemokine genes CCL5 and CXCL10 on pancreatic islet cells in an IRF-1–dependent manner. Our results suggest that molecules involved in chemotaxis, accompanied by the activation of the NLRP3 inflammasome, may be effective targets for the treatment of 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.

Combination Treatment With Anti-CD20 and Oral Anti-CD3 Prevents and Reverses Autoimmune Diabetes

Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease, although B cells also play an important role in T1D development. Both T cell– and B cell–directed immunotherapies have shown efficacy in the prevention and reversal of T1D. However, whether the combined strategy of targeting both T and B cells could further improve therapeutic efficacy remains to be explored. We show that combined treatment with intravenous antihuman CD20 (hCD20) and oral anti-CD3 significantly delays diabetes development in prediabetic hCD20 transgenic NOD mice. More importantly, the combined treatment reverses diabetes in >60% of mice newly diagnosed with diabetes. Further mechanistic studies demonstrated that the addition of oral anti-CD3 to the B-cell depletion therapy synergistically enhances the suppressive function of regulatory T cells. Of note, the oral anti-CD3 treatment induced a fraction of interleukin (IL)-10–producing CD4 T cells in the small intestine through IL-10– and IL-27–producing dendritic cells. Thus, the findings demonstrate that combining anti-CD20 and oral anti-CD3 is superior to anti-CD20 monotherapy for restoring normoglycemia in diabetic NOD mice, providing important preclinical evidence for the optimization of B cell–directed therapy for T1D.

The Role of Gr1+ Cells after Anti-CD20 Treatment in Type 1 Diabetes in Nonobese Diabetic Mice

Studies suggest that Gr1+CD11b+ cells have immunoregulatory function, and these cells may play an important role in autoimmune diseases. In this study, we investigated the regulatory role of Gr1+CD11b+ cells in protecting against type 1 diabetes in NOD mice. In this study, we showed that temporary B cell depletion induced the expansion of Gr1+CD11b+ cells. Gr1+CD11b+ cells not only directly suppress diabetogenic T cell function but also can induce regulatory T cell differentiation in a TGF-β–dependent manner. Furthermore, we found that Gr1+CD11b+ cells could suppress diabetogenic CD4 and CD8 T cell function in an IL-10–, NO-, and cell contact-dependent manner. Interestingly, single anti-Gr1 mAb treatment can also induce a transient expansion of Gr1+CD11b+ cells that delayed diabetes development in NOD mice. Our data suggest that Gr1+CD11b+ cells contribute to the establishment of immune tolerance to pancreatic islet autoimmunity. Manipulation of Gr1+CD11b+ cells could be considered as a novel immunotherapy for the prevention of type 1 diabetes.

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.

Type 1 diabetes and gut microbiota: Friend or foe?

Type 1 diabetes is a T cell-mediated autoimmune disease. Environmental factors play an important role in the initiation of the disease in genetically predisposed individuals. With the improved control of infectious disease, the incidence of autoimmune diseases, particularly type 1 diabetes, has dramatically increased in developed countries. Increasing evidence suggests that gut microbiota are involved in the pathogenesis of type 1 diabetes. Here we focus on recent advances in this field and provide a rationale for novel therapeutic strategies targeting gut microbiota for the prevention of type 1 diabetes.

Translational Mini-Review Series on B Cell-Directed Therapies: B cell-directed therapy for autoimmune diseases.

B cells play an important role in the pathogenesis of both systemic and organ‐specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but are also specialized to present specific autoantigens efficiently to T cells. Furthermore, these B cells can secrete proinflammatory cytokines and can amplify the vicious cycle of self‐destruction. Thus, B cell‐directed therapies are potentially an important approach for treating autoimmune diseases. On the other hand, like T cells, there are subsets of B cells that produce anti‐inflammatory cytokines and are immunosuppressive. These regulatory B cell subsets can protect against and ameliorate autoimmune diseases. Thus targeting B cells therapeutically will require this balance to be considered. Here we summarize the roles of pathogenic and regulatory B cells and current applications of B cell‐directed therapy in autoimmune diseases. Considerations for future development of B cell‐directed therapy for autoimmune diseases have also been discussed.