Airway inflammatory diseases including viral bronchiolitis and allergy are significant causes or morbidity and mortality worldwide. A recently identified lineage of leukocytes known as innate lymphoid cells (ILC) have potent inflammatory potential, but their mechanistic role in airway inflammation is not entirely understood. Group 2 ILC were identified to be activated in a mouse model of severe respiratory syncytial virus (RSV) infection and correlated with significant pathophysiologic changes in the airways including mucus production. That activation depended upon the cytokine thymic stromal lymphopoietin (TSLP). Broadly, group 1, 2, and 3 ILC responses were coordinated by signal transducer and activator of transcription 1 (STAT1), which promoted group 1 ILC and restricted group 2 and 3 ILC during RSV infection. Finally, IL-33 signaling mediated egress of ILC2 from the bone marrow during initial seeding of tissues in the post-natal period as well as in the context of allergic inflammation. These data broadly support a role for ILC2 in the pathogenesis of respiratory viral infection and provide mechanistic insights into how ILC2 egress from the bone marrow to support allergic inflammation.

Bacterial pathogens must acquire nutrients from the host during infection. The transition metal manganese (Mn) is an essential nutrient for both bacteria and vertebrates. Molecular mechanisms by which the opportunistic pathogen Acinetobacter baumannii acquires Mn and regulates Mn homeostasis were investigated. The Nramp-family transporter MumT was defined as a Mn acquisition system in A. baumannii that contributes to infection in the lung. The transcriptional regulator MumR was identified as a transcriptional activator of Mn import machinery. MumR was found to be important for defense against hydrogen peroxide. The importance of dietary Mn in Staphylococcus aureus infections was also interrogated. Excess dietary Mn enhances S. aureus infection of the heart. Mn is bioavailable to S. aureus in the heart, and S. aureus utilizes Mn to detoxify reactive oxygen species and escape neutrophil killing. The host Mn-binding protein calprotectin does not sequester Mn in the heart. Instead, calprotectin promotes S. aureus infection of the heart by modulating neutrophil recruitment. Finally, a small molecule was identified that targets metal homeostasis and is toxic to Gram-positive bacteria. Together, this Thesis establishes a role for Mn during A. baumannii infection, defines dietary Mn intake as a critical factor modulating S. aureus virulence, and presents a potential therapeutic targeting Mn homeostasis.

Oxidized low-density lipoprotein (oxLDL) is known to activate inflammatory responses in a variety of cells, especially macrophages and dendritic cells. Much of the oxLDL in circulation is complexed to antibodies, and these resulting immune complexes (ICs) are a prominent feature of chronic inflammatory diseases including atherosclerosis, systemic lupus erythematosus, and rheumatoid arthritis. Levels of oxLDL-ICs often correlate with disease severity; however, it was previously unknown how oxLDL-ICs modulate immune responses. This work demonstrates that bone marrow-derived dendritic cells (BMDCs) incubated with oxLDL-ICs secrete significantly more IL-1β compared with BMDCs treated with free oxLDL, and treatment of BMDCs with oxLDL-ICs increased expression of inflammasome-related genes. This inflammasome priming was due to oxLDL-IC signaling via multiple receptors, and signaling through these receptors converged on the adaptor protein CARD9, a component of the CARD9-Bcl10-MALT1 complex. Injection of oxLDL-ICs into LDLr-/- mice enhanced atherosclerotic lesion size and caused aortic dissection which may promote lesion instability. Finally, oxLDL IC-mediated IL-1β production resulted in increased Th17 polarization and cytokine secretion. Collectively, these data show that oxLDL-ICs induce potent and unique innate and adaptive immune responses.

Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. While the receptors and endocytic pathway utilized by TcdB for host cell intoxication are known, the mechanism of TcdA uptake remained unclear. Utilizing a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches, we show that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells. Unlike TcdB, which enters by the clathrin pathway, TcdA uptake and cellular intoxication occurs by a novel clathrin- and caveolae-independent mechanism that requires PACSIN2 and dynamin. Furthermore, our recent efforts to characterize the mechanism of neutralization of PA50, a humanized monoclonal anti-TcdA antibody, by X-ray crystallography, electron microscopy, and cell functional assays have identified regions within the C-terminus of TcdA that may promote toxin binding to the host cell surface. Current efforts are focused on identifying receptor molecules and additional host factors critical for TcdA entry and intoxication.

The goal of this thesis was to investigate the role of the IsdG family of heme oxygenases within microbial cells. In Chapter II, I identified the first IsdG family member in a eukaryotic organism and expanded the IsdG family from 22 protein members to 866. C. reinhardtii is the first organism which contains both HO-1 and IsdG family heme oxygenases. Intriguingly, LFO1 is the only heme oxygenase whose transcription is upregulated under low iron conditions, a condition where the cytochrome associated with the photosynthetic complexes in the chloroplast are degraded and heme is released into the chloroplast cytoplasm. I have hypothesized that LFO1 is important for degradation of this heme and in iron recycling under these iron limited conditions. Additionally, during the process of trying to determine the role of the IsdG family heme catabolites within S. aureus, in Chapter III I elucidated the importance of Fur regulation of the heme oxygenases. Removing the inhibition of transcription by Fur under iron replete conditions, the heme oxygenases led to degradation of endogenously biosynthesized heme and an increased reliance on fermentation for energy production. In total, these studies have displayed the importance of the IsdG family of heme oxygenases for iron recycling and heme homeostasis within organisms from different domains of life.

The host protein cyclophilin A (CypA) can both stimulate and inhibit HIV-1 infection through its interaction with the viral capsid (CA). CypA enhances the early stages of HIV-1 infection in part by promoting nuclear import of the virus; while the details of its ability to inhibit HIV-1 infection are less clear. This thesis advances our understanding of the mechanisms underlying the ability of CypA to inhibit HIV-1 infection. I demonstrate that CypA inhibits nuclear import of HIV-1 in the presence of inhibitory capsid-binding host proteins TRIM5α and CPSF6, and that inhibition is not a consequence of increased binding of inhibitory factors to the viral capsid. My work also demonstrates that CypA-dependent inhibition depends in part, on a conserved domain of HIV-1 CA which determines interactions with host nuclear pore proteins. These results suggest a common mechanism underlies the ability of CypA to stimulate HIV-1 infectivity in some cells and to inhibit infection in others.

Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is important to study these peptides in order to use them as potential templates for new antibiotic therapeutics. The aim of this study was to determine the structure/activity relationships of selected peptides and to determine the mode of action of these peptides on Gram positive bacteria. Studies with model membrane systems using circular dichroism and fluorescence spectroscopy indicated that these active peptides were induced into a more defined structure upon binding to lipids and detergents. In general, peptide interaction with lipids and detergents were similar. The peptides entered a hydrophobic environment, with tryptophan residues inaccessible to the aqueous solution. Although there were differences between peptides in specific interactions, no correlation could be made between lipid interaction and antimicrobial specificity or activity. The activity of cationic peptides from different structural classes was determined on various Gram positive strains and the killing kinetics of these peptides were very similar at 10-fold the MIC. Electron microscopy of S. aureus and S. epidermidis treated with the peptides at 10-fold the MIC showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in S. aureus with all peptides, whereas different effects, including nuclear condensation, were seen in the case of S. epidermidis. The membrane-potential-sensitive fluorophore DiSC₃(5) was utilized to assess the interaction of antimicrobial peptides with the cytoplasmic membrane of S. aureus. Studies of the kinetics of killing and membrane depolarization showed that no correlation could be made between cytoplasmic membrane depolarization and peptide activity. Thus, although cytoplasmic membrane permeabilization was a widespread ability among peptides, it did not appear to be the killing mechanism. Macromolecular synthesis assays showed that all peptides studied had intracellular effects, and these were often seen at sub-lethal peptide concentrations. The peptides differed in their specific effects on macromolecular synthesis. In general, evidence presented here suggested that cytoplasmic membrane disruption is not the sole mechanism of action against bacteria, and that multiple targets may be involved. The specific mechanism differs between peptides of different structural classes / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

B cells play a crucial role in adaptive immunity. They perform a multitude of functions including antibody and cytokine production and antigen presentation. The human adaptive immune response would not function effectively without B cells. The genome of a B cell undergoes profound changes during maturation and differentiation. This genome manipulation is a double-edged sword that provides both adaptive immunity to a wide array of pathogens and risks creating genomic changes associated with cancer, autoimmunity, or allergic disease. Studying B cells from a healthy immune system is important for understanding natural disease course, vaccine development, and the manufacture of antibodies for research and medicine. Here, I apply a systems biology approach to understanding B cells in health and disease with the use of mass cytometry and computational tools. The goals were to 1) Characterize B cells in follicular lymphoma (FL), 2) Characterize germinal center (GC) B cell signaling in response to reactive oxygen species (ROS), and 3) Integrate and optimize computational tools such as viSNE to capture the biology of B cells. This systems biology approach produced numerous findings. The GC B cell compartment was diminished in FL tumors compared to tonsil. FL malignant B cells displayed both intra- and inter-tumoral heterogeneity in phenotype. This heterogeneity within the malignant cells was driven by cell to cell variation in expression levels of human leukocyte antigen D related antigen (HLA-DR). FL malignant B cells, which are thought to arise from a GC B cell, were phenotypically distinct from GC B cells. GC B cells were found to be more sensitive to ROS than other B cell types found in tonsil. viSNE served as a useful tool in the visualization and characterization of malignant and non-malignant cells. The systems biology approach using mass cytometry enabled simultaneous identification and characterization of B cells in health and disease settings.

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