Indiana University-Purdue University Indianapolis (IUPUI) / The obligate intracellular protozoan parasite Toxoplasma gondii is a medically relevant pathogen that has infected a third of the world’s population. Toxoplasma is the causative agent of toxoplasmosis, which can have severe repercussions such as encephalitis and even death in immunocompromised patients. Current treatments for toxoplasmosis only target acute infection and can be toxic to patients, resulting in complications including allergy and bone marrow suppression. Thus, the identification of novel drug targets and therapies for toxoplasmosis is vital. Epigenetic modulators of lysine acetylation, including ‘writers,’ ‘erasers,’ and ‘readers,’ have been identified as promising drug targets for protozoan parasites. The lysine acetyltransferase (KAT) GCN5b appears to be an essential gene for Toxoplasma viability. The KAT domain of GCN5b is essential to GCN5b function and is targetable by small molecule inhibitors. While the acetyltransferase activity of this gene is well-characterized, the functionality of its C-terminal bromodomain (BRD) remains to be understood. Bromodomains are readers of lysine acetylation, and recently, bromodomain inhibitors have shown promise in a number of human diseases, as well as in protozoan parasites. We hypothesized that the GCN5b bromodomain is critical for Toxoplasma viability. The data reported herein suggest that the GCN5b bromodomain is important for tachyzoite viability and may serve as a novel therapeutic target in Toxoplasma.

Terminally differentiated B cells are responsible for antibody generation, a key component of adaptive immunity. IgG antibodies play an important role in defence against infection but can be pathogenic in some autoimmune diseases and in solid organ transplantation. In addition to antibody generation, there is increasing interest in the antibody-independent functions of B cells, including their ability to regulate immune responses via the production of IL10. In this thesis I firstly explored the therapeutic potential of belimumab, an anti-BLyS antibody, in an experimental medicine study in kidney transplant recipients. The rationale for this study was based on published studies showing that B cells activate alloreactive T cells and secrete human leukocyte antigen (HLA) and non-HLA antibodies that negatively affect graft function and survival, but may also play a protective role by regulating alloimmune responses promoting transplant tolerance. B-Lymphocyte Stimulator (BLyS) is a cytokine that promotes B cell activation and survival. We performed the first randomized controlled trial using belimumab as early maintenance immunosuppression in kidney transplantation. In belimumab-treated subjects, we demonstrate a reduction in naïve and activated memory B cells, plasmablasts, IgG transcripts in peripheral blood and new antibody formation as well as evidence of reduced CD4 T cell activation and of a skewing of the residual B cell compartment towards an IL10-producing regulatory phenotype. This experimental medicine study highlights the potential of belimumab as a novel therapeutic agent in transplantation. In the second part of my project I performed a preclinical study investigating the potential efficacy of bromodomain inhibitors in reducing antibody-mediated immune cell activation. Immune complexed antigen can activate mononuclear phagocytes (MNP), comprising macrophages and dendritic cells (DCs), via ligation of Fc gamma receptors (FcγR), that bind the Fc region of IgG. FcγR-dependent MNP activation results in profound changes in gene expression that mediate antibody effector function in these cells. The resulting inflammatory response can be pathological in the setting of autoimmune diseases, such as systemic lupus erythematosus and in antibody-mediated rejection in transplantation. BET proteins are a family of histone modification 'readers' that bind acetylated lysine residues within histones and function as a scaffold for the assembly of complexes that regulate gene transcription. Bromodomain inhibitors (I-BET) selectively inhibit the transcription of a subset of inflammatory genes in macrophages following toll-like receptor stimulation. Since MNPs make a key contribution to antibody-mediated pathology, we sought to determine the extent to which I-BET inhibits macrophage and DC activation by IgG. We show that I-BET delays phagolysosome maturation associated with build-up of immune complex (IC) whilst selectively inhibiting IC induced cytokine production. I-BET changed MNP morphology, resulting in a less adherent phenotype, prompting an assessment of its impact on DC migration. In vitro, in a three-dimensional collagen matrix, IgG-IC induced augmentation of DC chemotaxis to chemokine (C-C motif) ligand 19 (CCL19) was abrogated by the addition of I-BET. In vivo, two photon imaging showed that systemic I-BET treatment reduced IC-induced dermal DC mobilisation. Tissue DCs and transferred DC also had reduced migration to draining lymph nodes following I-BET treatment. These observations provide mechanistic insight into the potential therapeutic benefit of I-BET in the setting of antibody-associated inflammation.