Regulation of type 1 diabetes development and B-cell activation in nonobese diabetic mice by early life exposure to a diabetogenic environment

Microbes, including viruses, influence type 1 diabetes (T1D) development, but many such influences remain undefined. Previous work on underlying immune mechanisms has focussed on cytokines and T cells. Here, we compared two nonobese diabetic (NOD) mouse colonies, NODlow and NODhigh, differing markedly in their cumulative T1D incidence (22% vs. 90% by 30 weeks in females). NODhigh mice harbored more complex intestinal microbiota, including several pathobionts; both colonies harbored segmented filamentous bacteria (SFB), thought to suppress T1D. Young NODhigh females had increased B-cell activation in their mesenteric lymph nodes. These phenotypes were transmissible. Co-housing of NODlow with NODhigh mice after weaning did not change T1D development, but T1D incidence was increased in female offspring of co-housed NODlow mice, which were exposed to the NODhigh environment both before and after weaning. These offspring also acquired microbiota and B-cell activation approaching those of NODhigh mice. In NODlow females, the low rate of T1D was unaffected by cyclophosphamide but increased by PD-L1 blockade. Thus, environmental exposures that are innocuous later in life may promote T1D progression if acquired early during immune development, possibly by altering B-cell activation and/or PD-L1 function. Moreover, T1D suppression in NOD mice by SFB may depend on the presence of other microbial influences. The complexity of microbial immune regulation revealed in this murine model may also be relevant to the environmental regulation of human T1D

Modulation of immune responses in experimental autoimmune encephalomyelitis

The aim of this thesis work has been to elucidate mechanisms for modulation of the immune response in experimental autoimmune encephalomyelitis (EAE), the animal model for the human disease multiple sclerosis (MS). MS is a degenerative disorder of the central nervous system (CNS) believed to be of autoimmune origin, meaning that components of the immune system attack the tissue in the CNS causing inflammation and destruction. The underlying cause for the attack against self tissue remains unclear, and probably depends on several factors acting together. Genetic as well as environmental risk factors have been suggested. To date there is no cure for MS. That several factors act together to cause MS of course makes it more difficult to solve the mystery of the disease's etiology. However, if several factors need to synergise in order for disease to occur, one should be able to prevent disease by just removing one of them. In a mouse model for MS, myelin oligodendrocyte glycoprotein (MOG)-induced EAE, we have studied very diverse ways of influencing the pathogenic immune response to CNS antigens, both in disease-preventing and diseasepromoting ways. In the first two studies, we have studied mechanisms for induction of antigen-specific tolerance. Through intraperitoneal injection of MOG adsorbed to the weak adjuvant alum, we induced a benevolent type of immunity that protected the mice from developing EAE upon later challenge with MOG in the strong adjvant complete Freund's adjuvant (CFA) normally used to induce EAE. The protective effect was mediated through antigen-specific production of TGF-beta, and T cells producing this cytokine could also down-regulate the immunostimulatory properties of antigen presenting cells (APCs) in vitro. In the third study, we investigated the impact of infection with Trypanosoma brucei brucei (Tbb) on the development of EAE. We discovered that concurrent infection abrogated development of EAE and that infected animals, in concordance with previous studies, were severely immunosuppressed. This immunosuppression was determined to be caused at least in part by parasite-induced APCs, which were deficient in their capacity to activate T cells. This in turn was in part attributed to increased production of IL-10 in the APCs from Tbb-infected hosts. IL10 from APCs from infected mice could down-regulate the function of control APCs in co-cultures, and APCs from infected mice could mitigate or prevent EAE in MOG-CFA immunised mice when transferred 7 days after immunisation. The fourth and final study in this thesis concerns the properties of the antigen itself, in our case MOG. We investigated how the formation of adducts on lysine residues through incubation with malondialdehyde (MDA), a reactive dialdehyde formed during lipid peroxiadation in the body, affects the immunogenicity and encephalitogenicity of the protein. We determined that the MDA adducts did render MOG more immunogenic, and that this increase in immunogenicity was caused by increased uptake of the modified protein into APCs. Increased uptake was mediated by scavenger receptors of class A. MDA modified MOG was more encephalitogenic than was non-modified MOG, and interestingly it was also more efficient when used in the protective protocol described in the first two studies in this thesis. Additionally, pulsing of bone marrow derived macrophages (BMMphi) with MDA-MOG resulted in upregulation of transcription of the pro-inflammatory cytokines IL-12 and IL-23 as well as their receptors, indicating yet another mechanism for the increased immunogenicity of the modified protein

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Non-Invasive Multiphoton Imaging of Islets Transplanted Into the Pinna of the NOD Mouse Ear Reveals the Immediate Effect of Anti-CD3 Treatment in Autoimmune Diabetes.

We present a novel and readily accessible method facilitating cellular time-resolved imaging of transplanted pancreatic islets. Grafting of islets to the mouse ear pinna allows non-invasive, in vivo longitudinal imaging of events in the islets and enables improved acquisition of experimental data and use of fewer experimental animals than is possible using invasive techniques, as the same mouse can be assessed for the presence of islet infiltrating cells before and after immune intervention. We have applied this method to investigating therapeutic protection of beta cells through the well-established use of anti-CD3 injection, and have acquired unprecedented data on the nature and rapidity of the effect on the islet infiltrating T cells. We demonstrate that infusion of anti-CD3 antibody leads to immediate effects on islet infiltrating T cells in islet grafts in the pinna of the ear, and causes them to increase their speed and displacement within 20 min of infusion. This technique overcomes several technical challenges associated with intravital imaging of pancreatic immune responses and facilitates routine study of beta islet cell development, differentiation, and function in health and disease.This work was funded by grants from the NC3Rs (NC/M001083/1) (MW), Diabetes UK (BDA 13/0004785), Diabetes Research and Wellness (SCA/OF/12/13), European Research Council 7th Frame Programme (health-f5-2009-241883) (AC and HW), Wellcome Trust (095691/Z/11/Z) (KO), NIHR (NIHR-BRTU-10027) (JRF and MRC), and pump priming funding from the Cambridge University Isaac Newton trust

Microbiological characterisation of female NOD^low and NOD^high mice.

<p>(<b>A</b>) Venn diagram depicting microorganisms consistently detected by routine health screens in sentinel mice from the NOD^low and NOD^high colonies (also includes the results of screening for SFB). None of the other viruses, bacteria or parasites that are routinely tested under FELASA guidelines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.ref031" target="_blank">31</a>] were detected in either colony. (<b>B</b> and <b>C</b>) Quantification by qPCR (normalized to EUB) of <i>H</i>. <i>hepaticus</i> (<b>B</b>) and SFB (<b>C</b>) in the feces of individual NOD^low (black circles) and NOD^high (white circles) mice; horizontal bars represent means. Species-specific primers for the 16S RNA gene were used. (<b>D</b>) Metagenomic analysis of bacterial 16S rRNA genes from NOD^low and NOD^high females at 5 weeks of age. Different bacterial clades are color-coded, and log₂-fold mean differences between the detection frequencies in NOD^high and NOD^low mice shown on the y-axis; positive values show over-representation in the NOD^high colony. The dotted horizontal lines represent tenfold differences in either direction; significant colony differences (p < 0.05 after correcting for multiple comparisons) of tenfold or greater are shown as large squares with their names given. (<b>E</b>) Weights of individual age-matched NOD^low and NOD^high females (symbols as in <b>B/C</b>); means (horizontal lines) were compared by Student’s t test.</p

Characterisation of NOD^low and NOD^high colonies.

<p>(<b>A</b>) Kaplan-Meier survival curves showing diabetes-free survival up 30 weeks of age in 85 NOD^low (black circles) and 45 NOD^high (white circles) female mice. Incidence curves were compared by the log-rank test. From [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181964#pone.0181964.ref028" target="_blank">28</a>] with permission; Copyright 2013, The American Association of Immunologists, Inc. (<b>B</b>) and (<b>C</b>) Percent of islets exhibiting no insulitis, peri- or intra-insulitis in six NOD^low vs. eight NOD^high 6-week-old female mice (<b>B</b>), or in seven NOD^low vs. five NOD^high 30-week-old female mice (<b>C</b>). Each bar represents analysis of three pancreatic sections from an individual mouse. (<b>D</b>) Examples of H&E stained pancreatic sections from 30-week-old NOD^low females showing, on the left, two islets without insulitis and, on the right, one islet each with peri-insulitis (*) and intra-insulitis (**).</p

Increased B-cell activation in mesenteric lymph nodes of NOD^high mice.

<p>(<b>A</b>) Total cell counts in the indicated lymphoid organs of NOD^low (closed circles) vs. NOD^high (open circles) mice: MLN = mesenteric lymph nodes, PLN = pancreatic lymph nodes, ILN = inguinal lymph nodes. Analysis by 2-way ANOVA with p values given for significant colony differences. (<b>B</b>) Representative flow cytometry dot plots of CD69^high B cells isolated from mesenteric lymph nodes of NOD^low (left) and NOD^high (right) females at six weeks of age. (<b>C</b>) Frequencies of CD69^high cells in gated B220⁺ B cells from mesenteric lymph nodes (MLN) or spleens (SP) of NOD^low (closed circles) vs. NOD^high (open circles) female mice. Individual mice, means and significant p values (p < 0.05, by 2-way ANOVA) are shown.</p