Improvement in insulin sensitivity and prevention of high fat diet-induced liver pathology using a CXCR2 antagonist.

BACKGROUND: Liver pathology (LP) characteristic of non-alcoholic fatty acid disease (NAFLD)/non-alcoholic steatohepatitis (NASH) is a prevalent co-morbidity of type 2 diabetes (T2D). Accumulating evidence indicates that neutrophils driving insulin resistance (IR), including hepatic IR, precipitate T2D-associated NAFLD/NASH. We hypothesized that targeting neutrophil accumulation into insulin-sensitive tissues in mice using a CXCR2 antagonist under T2D-precipitating high fat diet (HFD) could improve insulin sensitivity and prevent the progression towards liver pathology reminiscent of NAFLD/NASH. METHODS: Mice were age-matched and on standard rodent chow prior to 1:1 randomization into control and HFD formulated with the CXCR2 antagonist AZD5069 or with biologically inactive substitute. They were monitored for metabolic changes including insulin sensitivity using the hyperinsulinemic-euglycemic clamp and hepatic histopathologic evaluation in H&E-stained sections as well as via immunofluorescence microscopy of liver sections for leukocyte markers, collagen 1A1 formation, α-smooth muscle actin (SMA), and galectin-3 expression, for 16 weeks. Statistical tests used to determine significant differences among study groups and outcomes include Student's t-test, one-way ANOVA, repeated measures two-way ANOVA, and Fisher's exact test, depending on the analytical question. RESULTS: Compared to mice on HFD, mice in the AZD5069-formulated HFD exhibited improved insulin sensitivity, a modest reduction in weight gain, and a significant improvement in LP and markers related to NAFLD/NASH. Mice in the AZD5069-formulated HFD also exhibited reduced neutrophil accumulation into the liver at the end of the 16 week study period. CONCLUSIONS: These results show, for the first time, the effectiveness of a selective CXCR2 antagonist to improve insulin sensitivity, concomitantly preventing the progression towards LP characteristic of NAFLD/NASH. This represents a novel approach to target IR and developing LP under T2D-susceptible conditions using a single agent. Furthermore, our data extend the growing evidence in support of neutrophils as a leukocyte population that imprints and maintains a chronic inflammatory state in the progression of dysregulated metabolism in liver-specific co-morbid conditions.

Neutrophil-Associated Inflammatory Changes in the Pre-Diabetic Pancreas of Early-Age NOD Mice.

A growing body of evidence indicates that neutrophils are the first major leukocyte population accumulating inside the pancreas even before the onset of a lymphocytic-driven impairment of functional beta cells in type 1 diabetes mellitus (T1D). In humans, pancreata from T1D deceased donors exhibit significant neutrophil accumulation. We present a time course of previously unknown inflammatory changes that accompany neutrophil and neutrophil elastase accumulation in the pancreas of the non-obese diabetic (NOD) mouse strain as early as 2 weeks of age. We confirm earlier findings in NOD mice that neutrophils accumulate as early as 2 weeks of age. We also observe a concurrent increase in the expression of neutrophil elastase in this time period. We also detect components of neutrophil extracellular traps (NET) mainly in the exocrine tissue of the pancreas during this time as well as markers of vascular pathology as early as 2 weeks of age. Age- and sex-matched C57BL/6 mice do not exhibit these features inside the pancreas. When we treated NOD mice with inhibitors of myeloperoxidase and neutrophil elastase, two key effectors of activated neutrophil activity, alone or in combination, we were unable to prevent the progression to hyperglycemia in any manner different from untreated control mice. Our data confirm and add to the body of evidence demonstrating neutrophil accumulation inside the pancreas of mice genetically susceptible to T1D and also offer novel insights into additional pathologic mechanisms involving the pancreatic vasculature that have, until now, not been discovered inside the pancreata of these mice. However, inhibition of key neutrophil enzymes expressed in activated neutrophils could not prevent diabetes. These findings add to the body of data supporting a role for neutrophils in the establishment of early pathology inside the pancreas, independently of, and earlier from the time at onset of lymphocytic infiltration. However, they also suggest that inhibition of neutrophils alone, acting via myeloperoxidase and neutrophil elastase only, in the absence of other other effector cells, is insufficient to alter the natural course of autoimmune diabetes, at least in the NOD model of the disease.

Arrest in the Progression of Type 1 Diabetes at the Mid-Stage of Insulitic Autoimmunity Using an Autoantigen-Decorated All-trans Retinoic Acid and Transforming Growth Factor Beta-1 Single Microparticle Formulation.

Type 1 diabetes (T1D) is a disorder of impaired glucoregulation due to lymphocyte-driven pancreatic autoimmunity. Mobilizing dendritic cells (DC) in vivo to acquire tolerogenic activity is an attractive therapeutic approach as it results in multiple and overlapping immunosuppressive mechanisms. Delivery of agents that can achieve this, in the form of micro/nanoparticles, has successfully prevented a number of autoimmune conditions in vivo. Most of these formulations, however, do not establish multiple layers of immunoregulation. all-trans retinoic acid (RA) together with transforming growth factor beta 1 (TGFß1), in contrast, has been shown to promote such mechanisms. When delivered in separate nanoparticle vehicles, they successfully prevent the progression of early-onset T1D autoimmunity in vivo. Herein, we show that the approach can be simplified into a single microparticle formulation of RA + TGFß1 with surface decoration with the T1D-relevant insulin autoantigen. We show that the onset of hyperglycemia is prevented when administered into non-obese diabetic mice that are at the mid-stage of active islet-selective autoimmunity. Unexpectedly, the preventive effects do not seem to be mediated by increased numbers of regulatory T-lymphocytes inside the pancreatic lymph nodes, at least following acute administration of microparticles. Instead, we observed a mild increase in the frequency of regulatory B-lymphocytes inside the mesenteric lymph nodes. These data suggest additional and potentially-novel mechanisms that RA and TGFß1 could be modulating to prevent progression of mid-stage autoimmunity to overt T1D. Our data further strengthen the rationale to develop RA+TGFß1-based micro/nanoparticle "vaccines" as possible treatments of pre-symptomatic and new-onset T1D autoimmunity.

Tolerogenic Dendritic Cells and T-Regulatory Cells at the Clinical Trials Crossroad for the Treatment of Autoimmune Disease; Emphasis on Type 1 Diabetes Therapy.

Tolerogenic dendritic cells and T-regulatory cells are two immune cell populations with the potential to prevent the onset of clinical stage type 1 diabetes, and manage the beginning of underlying autoimmunity, at the time-at-onset and onwards. Initial phase I trials demonstrated that the administration of a number of these cell populations, generated ex vivo from peripheral blood leukocytes, was safe. Outcomes of some of these trials also suggested some level of autoimmunity regulation, by the increase in the numbers of regulatory cells at different points in a network of immune regulation in vivo. As these cell populations come to the cusp of pivotal phase II efficacy trials, a number of questions still need to be addressed. At least one mechanism of action needs to be verified as operational, and through this mechanism biomarkers predictive of the underlying autoimmunity need to be identified. Efficacy in the regulation of the underlying autoimmunity also need to be monitored. At the same time, the absence of a common phenotype core among the different dendritic cell and T-regulatory cell populations, that have completed phase I and early phase II trials, necessitates a better understanding of what makes these cells tolerogenic, especially if a uniform phenotypic core cannot be identified. Finally, the inter-relationship of tolerogenic dendritic cells and T-regulatory cells for survival, induction, and maintenance of a tolerogenic state that manages the underlying diabetes autoimmunity, raises the possibility to co-administer, or even to serially-administer tolerogenic dendritic cells together with T-regulatory cells as a cellular co-therapy, enabling the best possible outcome. This is currently a knowledge gap that this review aims to address.

Co-Stimulation-Impaired Bone Marrow-Derived Dendritic Cells Prevent Dextran Sodium Sulfate-Induced Colitis in Mice.

Dendritic cells (DC) are important in the onset and severity of inflammatory bowel disease (IBD). Tolerogenic DC induce T-cells to become therapeutic Foxp3+ regulatory T-cells (Tregs). We therefore asked if experimental IBD could be prevented by administration of bone marrow-derived DC generated under conventional GM-CSF/IL-4 conditions but in the presence of a mixture of antisense DNA oligonucleotides targeting the primary transcripts of CD40, CD80, and CD86. These cell products (which we call AS-ODN BM-DC) have demonstrated tolerogenic activity in preventing type 1 diabetes and preserving beta cell mass in new-onset type 1 diabetes in the NOD mouse strain, in earlier studies. In addition to measuring efficacy in prevention of experimental IBD, we also sought to identify possible mechanism(s) of action. Weight, behavior, stool frequency, and character were observed daily for 7-10 days in experimental colitis in mice exposed to dextran sodium sulfate (DSS) following injection of the AS-ODN BM-DC. After euthanasia, the colons were processed for histology while spleen and mesenteric lymph nodes (MLNs) were made into single cells to measure Foxp3+ Treg as well as IL-10+ regulatory B-cell (Breg) population frequency by flow cytometry. AS-ODN BM-DC prevented DSS-induced colitis development. Recipients of these cells exhibited significant increases in Foxp3+ Treg and IL-10+ Breg in MLN and spleen. Histological examination of colon sections of colitis-free mice remained largely architecturally physiologic and mostly free of leukocyte infiltration when compared with DSS-treated animals. Although DSS colitis is mainly an innate immunity-driven condition, our study adds to the growing body of evidence showing that Foxp3+ Treg and IL-10 Bregs can suppress a mainly innate-driven inflammation. The already-established safety of human DC generated from monocytic progenitors in the presence of the mixture of antisense DNA targeting the primary transcripts of CD40, CD80, and CD86 in humans offers the potential to adapt them for clinical IBD therapy.

Generation of antigen-specific Foxp3+ regulatory T-cells in vivo following administration of diabetes-reversing tolerogenic microspheres does not require provision of antigen in the formulation.

We have developed novel antisense oligonucleotide-formulated microspheres that can reverse hyperglycemia in newly-onset diabetic mice. Dendritic cells taking up the microspheres adopt a restrained co-stimulation ability and migrate to the pancreatic lymph nodes when injected into an abdominal region that is drained by those lymph nodes. Furthermore, we demonstrate that the absolute numbers of antigen-specific Foxp3+ T regulatory cells are increased only in the lymph nodes draining the site of administration and that these T-cells proliferate independently of antigen supply in the microspheres. Taken together, our data add to the emerging model where antigen supply may not be a requirement in "vaccines" for autoimmune disease, but the site of administration - subserved by lymph nodes draining the target organ - is in fact critical to foster the generation of antigen-specific regulatory cells. The implications of these observations on "vaccine" design for autoimmunity are discussed and summarized.

Involvement of suppressive B-lymphocytes in the mechanism of tolerogenic dendritic cell reversal of type 1 diabetes in NOD mice.

The objective of the study was to identify immune cell populations, in addition to Foxp3+ T-regulatory cells, that participate in the mechanisms of action of tolerogenic dendritic cells shown to prevent and reverse type 1 diabetes in the Non-Obese Diabetic (NOD) mouse strain. Co-culture experiments using tolerogenic dendritic cells and B-cells from NOD as well as transgenic interleukin-10 promoter-reporter mice along with transfer of tolerogenic dendritic cells and CD19+ B-cells into NOD and transgenic mice, showed that these dendritic cells increased the frequency and numbers of interleukin-10-expressing B-cells in vitro and in vivo. The expansion of these cells was a consequence of both the proliferation of pre-existing interleukin-10-expressing B-lymphocytes and the conversion of CD19+ B-lymphcytes into interleukin-10-expressing cells. The tolerogenic dendritic cells did not affect the suppressive activity of these B-cells. Furthermore, we discovered that the suppressive murine B-lymphocytes expressed receptors for retinoic acid which is produced by the tolerogenic dendritic cells. These data assist in identifying the nature of the B-cell population increased in response to the tolerogenic dendritic cells in a clinical trial and also validate very recent findings demonstrating a mechanistic link between human tolerogenic dendritic cells and immunosuppressive regulatory B-cells.

Phosphatidylinositol-3-kinase activity during in vitro dendritic cell generation determines suppressive or stimulatory capacity.

Modulating PI3K at different stages of dendritic cells (DC) generation could be a novel means to balance the generation of immunosuppressive versus immunostimulatory DC. We show that PI3K inhibition during mouse DC generation in vitro results in cells that are potently immunosuppressive and characteristic of CD8alpha- CD11c+ CD11b+ DC. These DC exhibited low surface class I and class II MHC, CD40, and CD86 and did not produce TNF-alpha. In allogeneic MLR, these DC were suppressive. Although in these mixed cultures, there was no increase in the frequency of CD4+ CD25+ Foxp3+ cells, the Foxp3 content on a per cell basis was significantly increased. Sustained TLR9 signaling in the presence of PI3K inhibition during DC generation overrode the cells' suppressive phenotype.