Brutonâs tyrosine kinase (BTK) is a tec-family kinase present in B lymphocytes and innate immune cells. BTK is an important regulator of B cell autoreactivity. Innate-like autoreactive-prone B1, anergic An1, and transgenic anti-insulin B cells all rely upon BTK. Autoreactive B cells drive Type 1 diabetes (T1D) development and are preferentially targeted by disruption of BTK. We hypothesized that the same strategy could be extended to systemic autoimmunity, typified by rheumatoid arthritis (RA). We found that genetic deletion of BTK in a model of spontaneous arthritis results in a severe block in B cell development, subsequent reduction of germinal center formation and reduced autoantibody production, culminating in disease protection. In contrast, the development of immune-complex mediated arthritis, which relies upon the innate immune system, was not alleviated by genetic deletion of BTK. We have well established that BTK-deficiency results in loss of autoreactive B cells; however, whether these cells require BTK for maturation only, or also for their survival, remained unknown. Our group has developed the first loxP-flanked Btk mouse model, paired with an inducible Cre-ERT2 for kinetic studies of BTK function. We achieve 90% knockdown within five days after tamoxifen-induced Cre activation. We find that the B cell phenotype typical of murine Btk-deficiency emerges quickly, including a developmental block at T2, and decreased follicular B cells. Surprisingly, B1 B cells are not reduced, in contrast to their near-absence in Btk-deficient models. BTK-negative B1 cells remain able to produce natural IgM, but cannot respond to T-independent immunization. Furthermore, transgenic anergic anti-insulin B cells, which are reduced 95% by conventional Btk-deficiency, also maintain large mature populations after deletion of Btk protein, and remain able to internalize insulin. These findings suggest that autoimmune-prone B cell populations require BTK-mediated signaling for development, but not for survival or certain functions, and may have implications for the use of BTK-inhibitors currently in clinical trials for treatment of autoimmunity.

All positive sense (+)RNA viruses encode RNA-dependent RNA polymerases (RdRps), essential for replication and regulation of the fidelity of nucleotide addition to nascent genomes. The coronavirus (CoV) RdRp is encoded in nonstructural protein 12 (nsp12). CoV replication utilizes a large complex of proteins, many of which may influence replication fidelity. Specifically, nsp14 encodes a proofreading 3'-5' exoribonuclease (ExoN) in nsp14 that when inactivated (nsp14-ExoN(-)) results in decreased replication fidelity with up to a 20-fold increase in mutations across the CoV genome. Nsp12-RdRp and nsp14-ExoN interact during replication; however, the role of nsp12-RdRp in CoV replication fidelity was not known. To test the role of nsp12-RdRp, we identified residues predicted to participate in replication fidelity by homology modeling, minority variants present after treatment with mutagen, and consensus level mutations present after treatment with the mutagen 5-fluorouracil (5-FU). Mutations, at identified residues, were engineered into MHV nsp14-ExoN(+) and nsp14-ExoN(-) isogenic backgrounds. Viable recovered viruses, as well as populations of virus from passage in the presence of mutagen, were tested for sensitivity to the mutagens 5-fluorouracil (5-FU) or 5-azacytidine (5-AZC). Additionally, changes in replication kinetics, RNA synthesis, competitive fitness, reversion, and/or the accumulation of mutations were quantified. These results demonstrate that nsp14-ExoN masks the contributions of nps12-RdRp to the fidelity of genome replication for CoVs. However, fidelity determinants in nsp12-RdRp still affect replication kinetics and fitness. CoVs encode an extensive replication complex with a number of proteins likely involved in fidelity regulation. These results are essential to understanding the core of this fidelity complex.

Macrophages represent a dynamic and plastic subset of the innate immune system. Macrophage functions include immune surveillance and clearance of pathogens, but they have also been implicated in tumorigenesis. Macrophage activation along the M1 (classical, pro-inflammatory macrophage) to M2 (alternatively activated macrophage) axis is a tightly regulated process. Some pathways that regulate macrophage activation are known, but many uncertainties remain. To address questions related to macrophage activation, we chose the highly prolific human pathogen, Helicobacter pylori. H. pylori infection leads to chronic gastric inflammation and macrophages are an essential component of H. pylori-mediated gastritis. The studies outlined in this dissertation have identified two different proteins that regulate macrophage activation. Epidermal growth factor receptor (EGFR) signaling is an essential component of macrophage activation along both M1 and M2 paradigms. Deletion of Egfr in macrophages results in protection from H. pylori-mediated gastritis due to decreased pro-inflammatory M1 activation. Additionally, loss of EGFR signaling in macrophages protected mice from colitis-associated carcinogenesis, due to decreased M1 and M2 activation, and decreased angiogenesis. Conversely, ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, serves to promote immune tolerance within the gastric niche. Loss of ODC in macrophages led to increased M1 macrophage activation and increased pro-inflammatory cytokine production. The enhanced M1 macrophage activation was due to alterations in histone modifications to promote transcription. Overall, this dissertation demonstrates that increased knowledge regarding the regulation of macrophage activation can improve our understanding of macrophage biology in inflammation-mediated diseases.

Immunotherapy has been a rapidly evolving field of basic and clinical research over the past decades. Interleukin (IL)-15 and its analog IL-15 superagonist (IL-15 SA, or IL-15/IL-15 receptor α) have shown promise in cancer and viral immunotherapy as they play an essential role in development, proliferation and activation of cytotoxic lymphocytes including natural killer (NK) and memory phenotype (m) CD8+ T lymphocytes. My dissertation study showed that IL-15 SA caused dose- and time-dependent immunotoxicity in mice by expansion and activation of NK cells as well as induction of interferon γ (IFNγ) production. Additionally, although IL-15 SA is considered a candidate immunotherapeutic for reversing lymphocyte dysfunction in the late phase of sepsis, my results showed that IL-15 SA exacerbated physiological dysfunction and systemic inflammation during acute sepsis by activation of NK cells and induction of IFNγ production. Endogenous IL-15 also contributes to the pathogenesis of acute sepsis by maintenance of NK cell number and effector function. Taken together, findings from my dissertation provide mechanistic insights into IL-15 SA-mediated immunotoxicity that will be useful as IL-15 SA advances through clinical drug development in the field of cancer and sepsis. My studies further illuminate the essential role of endogenous IL-15 in the pathogenesis of sepsis and its regulatory effect on maintaining lymphocyte number and function during sepsis.

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