Systemic lupus erythematosus is an autoinflammatory disorder driven by the development of autoantibodies to self-nucleic acids, in particular to DNA and to chromatin. Loss-of-function mutations of the secreted deoxyribonuclease DNASE1L3 have been implicated in the development of aggressive familial lupus. In addition, recent genome-wide association studies have linked a hypomorphic variant of DNASE1L3 to sporadic lupus. Studies in the lab determined that Dnase1l3-deficient mice develop rapid autoantibody responses against dsDNA and chromatin, and at older ages this leads to a lupus-like inflammatory disease. These disease manifestations were completely independent of the intracellular DNA sensor STING, which has been implicated in other examples of self-DNA driven autoinflammatory diseases. My project focused on developing assays to track the activity of DNASE1L3, as well as identifying the endogenous source of self-DNA normally processed by DNASE1L3. Using mouse models that allow the depletion of specific cell populations, we found that circulating DNASE1L3 is produced by hematopoietic cells, in particular by CD11c+ dendritic cells and by tissue macrophages. Taking into account the unique properties of DNASE1L3, we discovered that this enzyme is uniquely able to digest chromatin contained within and on the surface of apoptotic microparticles. Loss of DNASE1L3 activity in circulation results in elevated levels of DNA in plasma, in particular within microparticles. Microparticles are extensively bound by anti-chromatin autoantibodies isolated from both murine models of lupus as well as prototypical human clones. In addition, Dnase1l3-deficient mice have high levels of circulating IgG that bind to microparticles from young ages, and these titers increased as disease progressed in aged animals. Pretreatment of microparticles with DNASE1L3 largely abrogated this binding, demonstrating that DNASE1L3 directly reduces the immunogenicity of microparticles. We also studied two human patients with null mutations in DNASE1L3, and observed increased DNA circulating in plasma and, in particular, in their microparticles, demonstrating a conserved role for DNASE1L3 in mice and humans. Finally, we obtained plasma samples from a cohort of patients with sporadic SLE, and found that roughly 80% had circulating IgG that avidly bound microparticles. Roughly half of this group failed to bind to microparticles that had been pretreated with DNASE1L3, and this DNASE1L3-sensitive group also presented with lower levels of DNASE1L3 activity. We conclude that extracellular chromatin associated with microparticles acts as a potential self-antigen capable of causing loss of tolerance to self-DNA and inflammatory disease in both mice and humans. The secretion of a DNA-processing enzyme thus represents a novel, conserved tolerogenic mechanism by which dendritic cells restrict autoimmunity.

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Includes a list of publications co-authored by the author during the preparation of this thesis. Thesis amendments in back leaf. Includes bibliographical references (leaves 123-168). The studies described concentrate on the cloning and characterisation of the two Drosophila caspases, DRONC and DECAY

The autoimmune diseases embody a diverse range of common human conditions that are caused by a loss of self-tolerance in the host immune system to a specific organ or tissue type. Approximately 5% of the general population are affected by autoimmune diseases which include type 1 diabetes (T1D), rheumatoid arthritis (RA) and Graves disease (GD). The majority of the autoimmune diseases are multifactorial in origin, brought about by a combination of both environmental and genetic factors. Numerous susceptibility loci have been identified for each autoimmune disease and a number of these loci have been shown to be shared amongst the autoimmune diseases. The fine-mapping of susceptibility loci to the underlying disease genes remains the current challenge facing complex disease genetics. This project aimed to further characterise the autoimmune disease susceptibility locus IDDM6 on chromosome 18q12-21. This was achieved by using a comparative mapping approach that incorporates the study of genetic association in human autoimmune disease alongside the consomic mapping of the orthologous region in the non-obese diabetic (NOD) mouse model of autoimmunity. Deleted in colorectal carcinomas (DCC) provided a strong candidate gene at IDDM6 and the resident R201G polymorphism was identified as a functional candidate A potential mechanism for the R201G polymorphism involvement in T1D aetiology was identified where the polymorphism may affect the ability of DCC to induce apoptosis in vitro. However, no evidence for R201G association could be detected in autoimmune disease case-control datasets from the New Zealand (NZ) population (T1D n = 428, RA n = 730, autoimmune thyroid disease n = 192 (AITD); versus n = 1246 healthy controls). In addition, no evidence for R201G involvement in T1D could be provided in a transmission disequilibrium test (TDT) incorporating 382 affected sib-pair families (54.2% transmission; P = 0.15). Significant association of R201G with GD was detected in a United Kingdom (UK) dataset (P = 0.002) from the Newcastle population (423 cases vs. 393 controls) but this was not replicated in an additional dataset from the UK Birmingham population (731 cases vs 668 controls; P = 0.81). It was concluded that the R201G polymorphism may encode susceptibility to GD but is unlikely to be the sole aetiological variant that accounts for the linkage previously observed at IDDM6 in autoimmune disease. To further investigate DCC as a positional candidate at IDDM6, five SNPs were selected from a 100 kb window surrounding a DCC-resident microsatellite that had previously been associated with T1D, called "88,21". The five SNPs were genotyped in the NZ T1D dataset, and the ascertainment of estimated haplotypes in this dataset revealed association of a rare haplotype with T1D, called haplotype H (3.31% cases vs 1.17% controls; P = 0.0044), in addition to global association of all haplotypes (P = 0.018). Haplotype H was also associated in an independent case-control dataset from the UK comprised of 400 T1D subjects and 443 healthy controls (P = 0.038). Maximum support for association of haplotype H was extended when both the UK and NZ T1D datasets were combined (P = 0.0017). Association of haplotype H could not be verified in a family-based test for association using the 382 UK T1D families (P = 0.40). However, the inclusion of the DCC SNPs in a TDT analysis of the published DCC-resident microsatellites "88,21" and "55,26", that had been used to identify IDDM6, extends support for the previously-associated 2-10 haplotype (2-10 refers to the published allele nomenclature at "88,21" and "55,26" respectively; 2-10-haplotype A; 59.6% T; P = 0.0058). There was no evidence for association of the five SNPs with RA or AITD when using either individual SNP analyses or estimated haplotypes in the NZ datasets. A similar lack of association was reported for the UK Newcastle GD dataset. Taken together, these data further support DCC, or a nearby gene, as conferring susceptibility to T1D. The human genetic data that supports IDDM6 involvement in autoimmune disease is further strengthened by consomic mapping of the orthologous region in mouse, using the non-obese diabetic mouse (NOD) model of autoimmune disease. In this thesis, the first evidence for a diabetes and thyroiditis susceptibility locus on mouse chromosome 18 is presented, which have been designated Idd21 and Sat1 respectively. This was achieved by using a chromosome-replacement strain with chromosome 18 derived from the diabetes-resistant Biozzi ABH strain on a diabetes-susceptible NOD genome, called NOD.ABH[Chr�⁸]. Mouse chromosome 18 contains orthology to both IDDM6 and the rat diabetes-susceptibility locus Iddm3. The NOD.ABH[Chr�⁸] mice showed a dramatic and significant reduction in diabetes incidence (30% of females were affected by 7 months of age versus 85% in NOD; P <0.0001) and that of thyroiditis (15.5% at 12 months compared to 37.4% in NOD; P <0.002). The comparative mapping of the chromosome 18 autoimmune susceptibility locus IDDM6 in human and mouse presented in this thesis provides further support for this locus. This research also clearly defines the next steps required to fine-map IDDM6 to the underlying disease genes, especially in regard to the DCC gene.

Johne�s disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis. An experimental infection model in sheep was developed as a prelude to the testing of new vaccines and the development of improved diagnostic assays for Johne�s disease. The final challenge model developed used four doses of 10⁹ viable organisms given at two to three day intervals. Gross and microscopic lesions were found in a high proportion of sheep (80%) at ten months post challenge. There was considerable variation in immune responses from animals challenged with different strains of M. paratuberculosis. Sheep challenged with a low passage laboratory culture of strain (W) M. paratuberculosis, produced strong lymphocyte transformation responses and Interferon gamma (IFN-γ) production at two months post challenge. Subsequent necropsy and culture from intestinal tissues showed only a low level of infection (25%). In comparison a primary tissue isolate of M. paratuberculosis (JD3) resulted in higher (60-90%) infection rates in orally challenged animals. The immune profile from these animals showed very little reactivity for the first three months post challenge, after which IFN-γ production could be detected. Antibody production and lymphocyte transformation response could not be measured until at least seven months post challenge. Sheep challenged with the primary tissue isolate instilled directly into the tonsil resulted in equivalent levels of Johne�s disease to those obtained with oral challenge. However, intratonsillar challenge resulted in higher levels of immune reactivity than oral challenge. The proprietary Johne�s vaccines; NeoparsecTM and GudairTM and an Aqueous vaccine were tested in sheep. The immunological reactions of the sheep to these vaccines showed some variations between the two separate studies, with the NeoparasecTM and GudairTM vaccines evoking high levels of CMI and humoral reactivity within two months of vaccination. Detailed immunological examination of gut associated lymphoid tissues were carried out on subgroups of animals that were either vaccinated or non-vaccinated and went on to develop disease or were immune to experimental challenge. The results showed that the diseased animals examined had multibacillary lesions and strong CMI and humoral responses. There were decreased proportions of CD4⁺, CD8⁺ and CD25⁺ T cells in peripheral blood and gut associated lymphatics of diseased animals compared with the immune or unchallenged subgroups. Profiles from the immune subgroups showed a stronger lymphocyte transformation response than case matched diseased animals. Tissues from immune animals showed increased proportions of B cells above those seen in diseased or unchallenged animals. This study has resulted in the development of a robust experimental sheep model in which Johne�s disease occurs in a high proportion of challenged animals. Critical time points for the establishment of infection or disease have been determined. It can be used in the future to evaluate protective efficacy of vaccines or to critically chart immunological profiles that are associated with infection, disease or protective immunity. Considerable research is needed to develop improved diagnostic tests to identify patterns of immunity during the early stages of infection or while the animal has subclinical disease.

Indole-3-carbinol (I3C) is a naturally occurring plant alkaloid, found in significant concentrations in cruciferous vegetables such as broccoli and Brussels sprouts. I3C is an unstable compound that undergoes rapid oligomerization in an acidic environment to form higher order condensation products (I3C-ACPs), such as 3-3'-diindolylmethane (DIM). Both I3C and DIM are marketed as dietary supplements and are under investigation as potential chemopreventive agents, despite limited data on the effects of chronic exposure. Previous studies have demonstrated that the chemopreventive potential of I3C and DIM in animal studies is dependent on species, strain, tissue and timing of treatment relative to carcinogen exposure, and long-term post-initiation exposure can even promote tumors. The majority of biological effects from I3C are the result of the abilities DIM and other I3C-ACPs to bind to the aryl hydrocarbon receptor and the subsequent induction of phase I and phase II enzymes. Phase I and phase II enzyme induction in many cases leads to protection from carcinogens by increasing the rate of metabolism and excretion but in some cases enhances carcinogenicity by increasing the rate of bioactivation. It has been demonstrated that modulation of enzyme levels can also result in altered metabolism of compounds that could affect efficacy and toxicity of pharmaceuticals and xenobiotics. The current work utilizes chronic dietary I3C and DIM exposures in rodent models to further elucidate the effect these compounds might have on health, drug metabolism and carcinogenesis. The reduced weight of Fischer 344 rats treated with 2500 ppm I3C for 1 year may be indicative of adverse effects but toxicity was not confirmed by blood chemistry or histopathological examination. Furthermore, no toxicity was observed after a comparable treatment of Sprague-Dawley rats. As observed after acute and sub-chronic exposures to I3C and DIM, we documented significant induction of cytochrome P450 enzymes and a related modification to drug metabolism in liver slice incubations. Evidence is also provided that may suggest that tumor modulation in mice may occur through an estrogenic mechanism. Further studies should be completed to determine the potential for similar responses in humans. / Graduation date: 2004

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C57B1/6 mice bearing Lewis lung carcinoma CLLC) are suppressed in their ability to generate cytotoxic T lymphocytes against the LLC tumor associated antigens. Since LLC have previously been shown to secrete the immunosuppressive factor PGE2, indomethacin, a prostaglandine synthetase inhibitor, was administered to LLC-bearing mice in their drinking water to prevent the immunosuppression typical of tumor bearers. The indomethacin treated tumor-bearers displayed an increase in their cytotoxic responses to the LLC tumor associated antigens. Thus the immunosuppression found in mice bearing LLC could be due to prostaglandin synthesis. Production of PGE2 by tumor cells may be a mechanism by which the tumors escape immune mediated destruction.Ball State UniversityMuncie, IN 47306

Severe lymphopenia is a clinical feature of Severe Acute Respiratory Syndrome (SARS) patients. However, lymphocytes do not express receptor for SARS-CoV, neither the widely accepted viral receptor angiotensin converting enzyme 2 (ACE2) nor the putative receptors Dendritic Cell- and Liver/lymph-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN and L-SIGN). Our group previously showed in vitro that, SARS-CoV Spike pseudotyped particles (SARSCoVpp) could infect human B cells only when inoculated in presence of anti-SARSCoV Spike immune serum. Such observations raised concerns about the possible occurrence of antibody-dependent enhancement (ADE) of infection, a phenomenon during which a virus bounded by antibodies could gain entry into cells through mechanisms involving complement receptors or Fc receptors. Recently, we have demonstrated the participation of the human Fc gamma receptor II (hFcγRII) molecules in granting SARS-CoV an opportunity to infect human immune cells. The aim of this study was to decipher the molecular mechanism leading to antibodymediated, FcγRII-dependent infection of immune cells by SARS-CoV. By using transduction experiment, I highlighted that different members of the hFcγRII family (namely hFcγRIIA, hFcγRIIB1 and hFcγRIIB2) could confer susceptibility to ADE of SARS-CoVpp infection. I further demonstrated that purified anti-viral immunoglobulin G, but not other soluble factor(s) from heat-inactivated immune serum, was the determinant for occurrence of ADE infection. Additionally, with the development of a cell-cell fusion assay, I illustrated that in contrast to the ACE2- dependent pathway, ADE infection did not occur at the plasma membrane, but rather require internalization of virus/antibodies immune complexes by the target cells. In line with this hypothesis, my results using a panel of FcγRII-expressing mutants demonstrated that binding of immune complexes to cell surface FcγRII was a prerequisite but was not sufficient to trigger ADE infection. In these experiments, only FcγRII signaling-competent constructions conferred susceptibility to ADE of SARS-CoVpp infection. Altogether my results point toward a role of the anti-SARS-CoV Spike IgG in vitro in granting SARS-CoV an opportunity to infect cells bearing signaling-competent FcγRII receptors. If further confirmed, such observations could have implications for understanding SARS-CoV tropism and SARS pathogenesis, as well as warrant for careful design of SARS vaccines and immunotherapy based on anti-viral antibodies. / published_or_final_version / Microbiology / Master / Master of Philosophy