Systemic lupus erythematosus (SLE) is complex autoimmune disease in which autoantibodies form against double stranded DNA (dsDNA) and nuclear antigens. Autoantigen immune complexes form, deposit in the vasculature, and cause multisystem organ damage. Both genetic and environmental factors contribute to the development of SLE. This thesis will explore three major themes found in the study of SLE: 1) Bacterial infection as an environmental trigger, 2) cytokine dysregulation in immune cells, and 3) the treatment of end organ damage in the form of lupus nephritis. Viral infections have long been associated with the development of systemic autoimmune disease, but the mechanisms by which chronic bacterial infections may promote autoimmunity remain unclear. In chapter three we show that a component of bacterial biofilms, the amyloid-like protein “curli”, irreversibly forms fibers with bacterial or eukaryotic DNA during biofilm formation. This interaction accelerates amyloid polymerization and creates potent immunogenic complexes that activate immune cells, including dendritic cells, to produce cytokines such as type I interferons, which are pathogenic in SLE. When given systemically, curli/DNA composites trigger immune activation and production of autoantibodies in lupus-prone and wild type mice. We also found that infection with curli-producing bacteria triggered higher autoantibody titers in lupus-prone mice compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity. Cytokine dysregulation is also common in SLE patients. Serum cytokines are often elevated during active disease, including type I IFNs and IL-10. In chapter four we demonstrate that Il10 is a type I IFN response gene and has increased basal expression in dendritic cells (DCs) derived from pre-disease lupus-prone Sle1,2,3 mice. We show that Sle1,2,3-derived DCs overproduce IL-10 in response to TLR ligands and that this is the result of autocrine signaling though the type I IFN receptor (IFNAR). These results suggest that dysregulation of cytokine signaling in the myeloid compartment may contribute to IL-10 dysregulation in SLE. Renal disease remains a major cause of morbidity and mortality in SLE. A number of mouse models of chronic kidney disease have implicated the EGFR-family receptors in the progression of renal fibrosis and dysfunction. In chapter five we show that renal expression of ErbB2 is increased in murine lupus. We therefore asked if EGFR-family inhibition could prevent murine lupus nephritis. To test this possibility we used lapatinib, an EGFR-ErbB2 dual kinase inhibitor, in an IFN-accelerated model of murine lupus. We found that lapatinib administration lowered autoantibody levels but worsened renal disease. Lapatinib failure to treat murine lupus nephritis despite lowered autoantibody levels suggests EGFR-family signaling is required for tissue repair in the acute phase of kidney injury. Together this thesis clearly demonstrates the complexity of systemic autoimmune disease – bringing us to the crossroads of immunity and tolerance. The combination of both environmental triggers (e.g. bacterial infection) and genetic susceptibility (e.g. intrinsic cytokine dysregulation) leads to end organ damage (e.g. lupus nephritis). Here we sought to explore each aspect of disease progression in the hopes to develop better interventions for systemic autoimmune disease. / Microbiology and Immunology

In its essence, this thesis is concerned with how the language of immunology affects eventual patient conceptions of self,. Specifically, I examine how biomedical discourse affects both the mental and physical experience of autoimmune disease. Typically described in militaristic terms as an 'attack on self,' autoimmunity, (when an individual's immune system response to 'self' tissues), encourages the following questions: what is self, in a biological sense? What is the effect of medical intervention on a patient's sense of selfhood, when the offending other to be removed is actually an integral part of one's own body? Is there a mental readjustment of what constitutes selfhood in the wake of such a diagnosis? In my attempt to answer these questions I have divided the thesis into three chapters. Chapter one is an exploration of the semantics and methodology of basic biomedical research, eventually culminating in a discussion of two different paradigms of immunity currently in operation. One of the main points of this thesis is that scientific representations of the body are indeed constructions, rather than reflections of the truth of our selves, and that these constructions are in constant flux. By comparing and contrasting two different immunological paradigms used to frame research and articulate the body, I aim to show how different the mental body could be according to which paradigm is followed. In particular, I want to show that autoimmunity is not necessarily an attack on self, or a rejection of self. This thesis is therefore also a search for a 'better' metaphor for autoimmunity that does not involve the rejection of the diseased self. Chapter two examines the language of immunology from a cultural perspective. Paradigms of the immune system have their roots in cultural ideology as much as in the laboratory. This chapter aims to show how research and sociopolitical and economic systems serve to mutually reinforce a common view of 'reality.' Chapter three then looks at personal narratives of individuals living with autoimmune disease in light of how basic research methodology and culture construct and treat disease and the diseased individual. By showing the link between supposedly objective science and the personal experience of illness, I am hoping that work such as mine affects not only patients that might be grappling with confusing diagnoses and searching for alternate ways of conceptualizing their diseased bodies, but also how scientists and medical practitioners explain the body to others. / Thesis / Master of Arts (MA)

No / It has long been appreciated in science that correlation does not imply causation. However, with any logical fallacy, simply spotting that the reasoning behind an argument is faulty does not imply that the resulting conclusion is false. Thus, I begin the tricky business of exploring the basis upon which researchers and clinicians are often tempted to conclude that two medical conditions (here alopecia areata and vitiligo), with some striking resemblances, are in fact related. This is relevant, particularly if assumptions of shared aetiology (and to some extent shared pathomechanism) encourage a common strategy to finding a treatment or cure.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

<p>Macrophages are an important key effector cell in the immune system which can practically be found in every tissue. Macrophages have for a long time been considered a population of cells only responsible for pro-inflammatory responses and anti-microbial activities. But over the past decade, many have come to realize the amazing plasticity of macrophages in response to different stimulations. The anti-microbial and pro-inflammatory macrophage is known as classically activated macrophages but newly discovered phenotypes have been revealed named wound-healing macrophages and regulatory macrophages. Through systematic screening we have identified an inducible macrophage activation state which has both wound-healing and regulatory capabilities activated by the novel cytokine combination IL-4/IL-10 with or without TGF-β.</p>

B lymphocytes are an important type of immune cell that contributes to pathogen clearance. When dysregulated, these cells contribute significantly to diseases such as autoimmunity, allergy and B cell cancers. Here we examine an important regulatory circuit that involves the lipid phosphatase SHIP, a key regulator of the PI3K signaling pathway. SHIP was first described as the major effector of inhibitory IgG receptor FcγRIIB, which downregulates B cell antigen receptor (BCR) signaling pathways when co-engaged. However, it is also known to inhibit signaling downstream of several other receptors, both activating and inhibitory. Here we examine the regulation and function of SHIP in B cells, focusing on the inter-related influences of binding partners, tyrosine phosphorylation and subcellular localization dynamics. First, we assess interaction of SHIP to selected candidate binding partners using an in vitro screening approach. The two most robust interactions were further characterized with respect to dissociation constant. These were: a novel interaction between SHIP phospho-Tyr944 and the SH2 domain of Nck, and a known interaction between the SH2 domain of SHIP and FcγRIIB phospho-Y292. Next, we perform the first examination of SHIP Tyr944. We provide evidence that it contributes to interaction with Nck after BCR engagement and is required for inhibition of actin turnover by SHIP. Finally, we perform the first detailed examination of the mechanisms controlling SHIP localization in human B cells stimulated through the BCR with and without co-engagement of FcγRIIB. We discover that SHIP is recruited to the plasma membrane equally in both stimulation contexts, however FcγRIIB co-ligation results in reduced mobility of SHIP molecules at the cell periphery. We identify a novel and essential role for Syk kinase in promoting SHIP membrane localization, tyrosine phosphorylation, and interaction with known binding partner Shc1. Together, these results provide significant and exploitable insight into the molecular control of a clinically important regulator of B cell responses. / February 2017

<p>Linker for Activation of T cells, LAT, is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals that modulate T cell development, activation, and proliferation. Upon engagement of the T cell receptor, LAT is phosphorylated and associates with Grb2, Gads, and PLC&#947;1 through its four distal tyrosine residues. Mutation of tyrosine 136 abolishes LAT binding to PLC&#947;1. This results in impaired TCR-mediated calcium mobilization and Erk activation. LATY136F knock-in mice have a severe but incomplete block in T cell development. Yet, CD4+ &#945;&#946; T cells undergo uncontrolled expansion in the periphery, resulting in a severe autoimmune syndrome characterized by Th2 skewing and resultant B cell autoreactivity. Here, we further studied the role of LAT-PLC&#947;1 signaling in T cell lineage commitment, cytokine production, and autoimmunity.</p><p>First, we investigated the importance of the LAT-PLC&#947;1 interaction in &#947;&#948; T cells by crossing LATY136F mice with TCR&#946;-deficient mice. Our data showed that the LATY136F mutation had no major effect on the homeostasis of epithelial &#947;&#948; T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4+ &#947;&#948; T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL4, resulting in an autoimmune syndrome similar to that caused by &#945;&#946; T cells in LATY136F mice. Development of these hyperproliferative &#947;&#948; T cells was not dependent on expression of MHC class II or CD4, and their proliferation could be partially suppressed by regulatory T cells. Our data indicated that a unique subset of CD4+ &#947;&#948; T cells could hyperproliferate in LATY136F mice and suggested that LAT-PLC&#947;1 signaling may function differently in various subsets of &#947;&#948; T cells. </p><p>In addition to examining &#947;&#948; and &#945;&#946; T cell development, we also were interested in further exploring the role of LAT in cytokine production. While our previous data have demonstrated that T cells in LATY136F mice are Th2 skewed, producing large amounts of IL4, we investigated other cytokines that may be important for autoimmunity and found that these CD4+ &#945;&#946; T cells could also produce the proinflammatory cytokine IL6. Analysis of whole cell lysates from CD4+ &#945;&#946; LATY136F T cells demonstrated that NF&#954;B, AKT, and p38 were constitutively phosphorylated, and inhibition of these pathways resulted in reduced IL6 production. By crossing LATY136F mice with IL6 deficient mice, we demonstrated that early T cell survival was diminished in the absence of IL6. We further showed that this reduced CD4+ T cell pool was not due to further blocks in development, or an increase in FoxP3+ regulatory T cells. Finally, we demonstrated that over time, CD4+ T cells do hyperproliferate, yet B cell class switching and autoreactivity remains low. Our data uncovered a novel role for LAT-PLC&#947;1 signaling in regulating IL6 production by T cells during autoimmunity. </p><p>Finally, we wanted to further examine IL4 production and T helper cell differentiation in LATY136F mice. We examined IL4 production using KN2 reporter mice, where huCD2 marks T cells that have recently produced IL4 protein. We demonstrated that only a small proportion of the LATY136F T cells were actively secreting IL4. This subset of T cells were Tfh cells that expressed BCL6 and localized to B cell-rich germinal centers within the spleen. Most studies to date have examined Tfh cells in infection models, and have demonstrated that Tfh cells have very low expression of GATA3. Our results revealed in a spontaneous T cell-mediated autoimmune model system, that Tfh cells express both high levels of BCL6 and GATA3. Additionally, using an inducible deletion system, where normal development occurs, we showed that Tfh cells differentiation is the result of aberrant LAT signaling, rather than autoreactive TCRs with high affinity for self-peptide-MHC. LATY136F Tfh cells did require B cells for their development. Together, these results displayed a novel role for tonic LAT-PLC&#947;1 signaling in modulating Tfh cell differentiation and BCL6 expression.</p> / Dissertation

Regulatory T cells (Treg) are critical to the maintenance of immune tolerance, partly by controlling the unwanted activation of effector T cells (Teff) and thereby enhancing the resolution of autoimmune and allergic inflammation. Recent data suggest that Treg can specialize to better control different types of inflammation by using transcriptional machinery which controls differentiation and function of Teff. This thesis addresses questions related to the efficacious use of Treg, notably their ability to adopt distinct phenotypic profiles under different inflammatory contexts and their need to recognize antigen in the inflamed organ. Two differentially mediated mouse disease models were used in this project, namely Th1/Th17-mediated experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis and Th2-mediated allergic airways inflammation (AAI) as a model of asthma. A new model of rMOG-induced AAI was developed to specifically answer the questions on the importance of cell phenotype versus antigen-reactivity for the effective Treg-mediated suppression. It was demonstrated that Treg from the inflamed CNS in EAE had an upregulated expression of Th1 master regulator T-bet and Th1-associated chemokine receptor CXCR3, whereas Treg derived from the inflamed lung in AAI had an increased expression of Th2 master regulator GATA-3, lacked expression of T-bet and displayed decreased levels of CXCR3. This specialized and activated phenotype was restricted to tissue-derived Treg. The importance of appropriate Treg phenotype for effective suppression was suggested by the observed inability of CNS-derived Treg to inhibit AAI. A different Treg subset, TGF-β-induced Treg (iTreg), was shown to express high levels of T-bet and CXCR3, but not GATA-3 upon induction in vitro. iTreg effectively suppressed both Th1 and Th2 types of inflammation and the antigenreactivity was key to this. This thesis demonstrates that Treg are capable of acquiring a distinct phenotype corresponding with a CD4+ T cell response driving inflammatory disease and identifies antigen-reactivity as key to the efficacious suppression of inflammation. It also highlights substantial phenotypic differences between iTreg and naturally-occurring Treg which could be associated with different modes of suppression.