A major feature of intestinal macrophages in the normal gut is inflammatory anergy, a state of tolerance essential for intestinal homeostasis, changes in which lead to inflammatory bowel disease (IBD). Intestinal macrophages undergo a specific process of differentiation. Under homeostatic conditions, cytokines in the local environment drive functional differentiation of newly recruited monocytes into noninflammatory intestinal macrophage. This process is associated with downregulation of proinflammatory cytokines. Growing evidence supports the idea that epigenetic changes contribute to macrophage reprogramming, and lead to tailored gene expression in response to gut environmental factors. However, current knowledge on how chromatin modification drives genes expression in human intestinal macrophages is still limited. This project aimed to define the relationship between chromatin modification (histone methylation) and the repression of inflammatory genes in intestinal macrophages isolated from mucosa of control subjects and IBD patients. It was of particular interest to understand if the anergic state of macrophages in normal gut is associated with repressive marks. Also in IBD, if there are any differences in epigenetic modifications between resident and infiltrating macrophages. Finally, if by blocking histone methylation, it is possible to prevent/reduce TNFα production by macrophages from IBD mucosa. TNF-α is an inflammatory cytokine that plays a critical role in innate and adaptive immune responses and its dysregulation has been implicated in the pathology of IBD. Considering its central role in IBD pathology, the TNFA gene was selected and different repressive and permissive histone modifications were investigated. Silencing marks H3K27me3, H3K9me3 and H3K9me1, as well as activating marks H3K4me3, H3K4me1 and also RNAPII were selected and analysed using chromatin immunoprecipitation (ChIP) assays. Based on data collected, it was speculated that a break of anergic phenotype in IBD macrophages might be associated with changes in level of silencing marks. Macrophages isolated from mucosa of CD patients showed decreased enrichment of H3K27me3 and H3K9me3, with H3K27me3 having the greatest reduction. Additional analysis of peripheral blood monocytes suggested that in healthy gut, the differentiation of blood monocytes into resident intestinal macrophages is associated with deposition of H3K27me3 and H3K9me3 silencing marks at the TNFA TSS, and that this process fails in IBD environment.

T cells discriminate self from foreign peptides presented in the context of self-major histocompatibility complex (pMHC) molecules via clonotypic T cell receptors (TCRs). CD8⁺ T cell recognition and responsiveness to foreign pMHC is known to diminish over the lifespan, which is consistent with gradual thymic involution over time. It is further supported by experimental evidence that restricting the diversity of class I MHC peptides during positive and negative selection results in selection of fewer CD8⁺ T cells that are highly specific for pMHC. How the affinity of the CD4⁺ T cell compartment for self-pMHC, and its capacity to bind foreign-pMHC change over the lifespan are fundamental aspect of T cell biology that remain largely unexplored. Experimentally restricting thymic selection is known to allow degenerate CD4⁺ T cells to develop. This suggests that they might accumulate in the CD4⁺ T cell compartment over time. We report that, while old mice (18-22 months) contain fewer CD4⁺ T cells than adults (8-12 weeks), those that remain have a higher intrinsic affinity for self-pMHC. Old mice also have more cells that bind distinct foreign-pMHCs, either alone or in combination. The numerical increase of these subsets with age directly correlates with their affinity for self-pMHC. However, no relationship was observed between affinity for self-pMHC and responsiveness to foreign-pMHC. These data demonstrate that the CD4⁺ T cell compartment preferentially accumulates promiscuous constituents with age as a consequence of higher affinity T cell receptor interactions with self-pMHC. These results have important implications for the design of immunotherapeutics targeting CD4⁺ T cells to improve immunity in older adults.

HIV-1 vertical transmission is the predominant cause of AIDS in children. In addition, HIV-1 infected infants have a higher viral load and progress to AIDS more rapidly than infected adults. However, the molecular mechanisms of HIV-1 vertical transmission and pathogenesis are not known. Work performed in this laboratory has shown transmission of minor genotypes with R5 phenotypes, more heterogeneity associated with transmission and a higher replication and gene expression of HIV-1 in neonatal than adult cells. In this dissertation, I have made advancements by characterizing the HIV-1 gag nucleocapsid gene, that plays a pivotal role in HIV-1 lifecycle, from six mother-infant pairs and found that there was a low degree of viral heterogeneity and a high conservation of functional domains for biological activity and CTL response. With respect to differential mechanisms of HIV-1 infection in neonatal vs. adults cells, 468 HIV-1 integration sites were characterized in the T-lymphocytes and monocyte-derived-macrophages from 5 donors of infant and adult blood. Several functional classes of genes were identified by gene ontology to be over represented, including genes for cellular components, maintenance of intracellular environment, enzyme regulation, cellular metabolism, catalytic activity and cation transport. Numerous potential transcription factors binding sites at the site of integration were identified. Furthermore, the genes at integration site, transcription factors potentially binding upstream of HIV-1 promoter and factors that assist HIV-1 integration were found to be expressed at higher levels in cord than adult cells. These results may help explain a higher HIV-1 gene expression and replication in cord compared with adult cells. Finally, I have also made progress in the development of new and novel antivirals by showing that CD4-mimetic miniproteins significantly inhibited HIV-1 entry and replication in T-cell lines and primary blood mononuclear cells. In addition, several compounds from the crude extracts of endophytic fungi found in desert plants were able to inhibit HIV-1 replication in T-cell lines. Taken together, the results from this dissertation provide new insights into understanding the mechanisms of HIV-1 vertical transmission and HIV-1 gene expression and replication in infants, as well as provide new possibilities for anti-retroviral drug development.

Parabiosis is the surgical union of two organisms resulting in the development of a single, shared circulatory system. When animals of different ages are conjoined (i.e. heterochronic parabiosis), blood-borne factors from the parabionts can affect the physiology of the other parabiont. This is manifested sometimes by beneficial, rejuvenating impact upon the older animal's tissues and organs (anti-geronic effect), and sometimes by younger animal's tissues regressing and appearing old-like (pro-geronic effect). These effects, and the ability to identify individual factors that could recapitulate pro- and anti-geronic effects, have made heterochronic parabiosis a very attractive approach to studying biology of aging and rejuvenation.cHowever, heterochronic parabiosis has not been widely used to investigate the aged immune system. An important question to be answered is whether the cellular defects involved in the aged immune system are due to intrinsic defects or if they can be rescued by extrinsic factors. Heterochronic parabiosis is ideal to test cellular migration patterns, interrogate the mechanisms driving migration defects that occur with aging, establish if these defects can be rejuvenated and identify molecules that are targets for intervention. Here, we provide evidence of the importance of reducing differences in the background genetics of different C57BL/6 substrains prior to parabiosis. This improvement allowed us to improve survival and confirm robust lymphocyte equilibration across secondary, but not primary, lymphoid tissues. We found no evidence for rejuvenation of the old immune cells, whereas results suggested that adult peripheral lymph nodes (pLN) lost mass and cellularity, potentially indicating the presence of a pro-geronic factor(s) in the old circulation that affects pLN function. Adult and old immune cells were present in equal frequencies in both adult and old secondary lymphoid tissues, indicating that there was no restriction of cellular migration due to the age of the cell or age of the tissue. The propensity of adult immune cells (i.e. large naïve compartment) to occupy lymph nodes and old immune cells (i.e. large memory compartment) to occupy bone marrow was retained following heterochronic parabiosis. Finally, parabiosis separation experiments illuminated the peripheral survival advantage of old T cells over adult T cells. These results highlight the power of heterochronic parabiosis in studying immune aging and provide hypothesis-generating data for future mechanistic studies of peripheral T cell maintenance with aging.

CD4⁺ T cells are a critical component of the adaptive immune compartment. Each T cell expresses a clonotypic T cell receptor (TCR) that must discriminate between self and foreign peptides presented in major histocompatibility molecules (pMHC) on the surface of antigen presenting cells to direct T cell fate decisions. Information regarding TCR-pMHC interactions must then be transmitted to the TCR-associated CD3 signaling modules, which contain ITAMs that serve as signaling substrates for Src kinases. The Src kinase, Lck, is recruited to the pMHC-bound TCR-CD3 complex via association with the CD4 coreceptor that binds MHCII. It is therefore through the coordinated interactions within the TCR-CD3-pMHC-CD4 macro-complex that productive TCR signaling can occur to inform T cell activation and fate decisions. While much is known regarding the structure of the individual subunits that make up the TCR-CD3-pMHC-CD4 macro-complex, there is little information regarding how these components come together to initiate TCR signaling and determine functional outcomes. Here, we have interrogated how interaction of these individual components leads to productive T cell activation. Specifically, we interrogated the nature of TCR-MHC interactions and provide evidence that there is intrinsic specificity of the TCR for MHCII. We have also built mouse models to determine the role of TCR-CD3 interactions and TCR dimerization in the transmission of information from the TCR to the CD3 subunits following TCR-pMHC engagement. Finally, we show that both the CD4 transmembrane and extracellular domains contribute to T cell activation in vitro. Overall, this work provides insight into how the constituents of the TCR-CD3-pMHC-CD4 macro-complex interact to initiate T cell fate and function.

Infectious diseases remain amongst leading causes of death in people aged 65 years and older; therefore, much research is focused on determining the immune components that contribute to age-dependent increased susceptibility to, and increased mortality from, infections. CD8⁺ T cells are critical for clearing intracellular pathogens through production of cytokines and direct killing of infected cells. Age-dependent CD8⁺ T cell alterations have been described, including decreased numbers of naïve CD8⁺ T cell precursors and decreased numbers and function during infection. This dissertation explores the mechanisms contributing to these changes. First, we demonstrated that multiple mechanisms contribute to changes in the CD8⁺ T cell pool with age. CD8⁺ T cells from unimmunized T cell receptor transgenic (TCRTg) old mice undergo massive virtual memory (VM) conversion with age; both homeostatic proliferation and cross-reactivity may contribute to the generation and accumulation of VM cells with age. These VM cells exhibit an age-dependent replicative impairment to cognate antigen, which points to possible detrimental functional consequences due to changes in the overall T cell pool. Second, we evaluated the cell intrinsic contribution to the decreased old CD8⁺ T cell response. With in vitro stimulation, old CD8⁺ T cells exhibit decreased ability to enter into late cell divisions and decreased production of effector molecules. In addition, we found that old CD8⁺ T cells have decreased expression of the master transcription factor T-bet, which correlates to decreased effector function and terminal differentiation in vivo. Collectively, these results identify possible cell-intrinsic targets for improving CD8⁺ T cell immunity. Finally, we measured whether a Listera monocytogenes live vaccine model induces protective immune responses in old mice. We found that vaccination conferred little protection in old mice upon pathogen challenge. These results contrast with other vaccine models, which may allow for pinpointing both the vaccine and immune components required for generating strong protective immunity in the elderly. Collectively, this dissertation demonstrates that CD8⁺ T cell precursors, effector cells, and memory cells exhibit profound changes with, age and identifies both possible mechanisms contributing to these alterations as well as possible therapeutic/vaccine targets for improving immunity in the elderly.

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Neisseria gonorrhoeae (Ng) encounters different microenvironments during its life-cycle. Some of these niches have different concentrations of oxygen, which influences the rate of Ng growth; as well as iron, an element essential for Ng survival. Differential expression of several proteins allows the bacteria to adapt to the diverse conditions it comes encounters. One protein affected by environmental changes during Ng growth is Gcp, a tRNA-modification enzyme essential for protein synthesis. To study the regulation of expression of Gcp, we first analyzed the sequence of its ORF, gcp. Orthologs of this gene are found in all kingdoms of life. In silico analysis shows that among Neisseria species, gcp ranges in homology from 76% to 99%, at the nucleotide level. Reverse transcription PCR indicates that gcp is expressed as part of an operon, together with three cytochrome-associated genes cyc4, resB and resC. Rapid amplification of complementary DNA ends determined the start of transcription of cyc4 (and possibly of the cyc4-gcp operon) at 95 nucleotides from the gene start codon. Transcriptional fusions determined that the promoter region upstream of cyc4 is the strongest promoter in the operon. However, the region directly upstream of gcp also has low level of promoter activity, suggesting that the gene may be expressed from two different promoters. Semi-quantitative determination of the concentration of gcp mRNA indicates that the transcription of the gene is significantly repressed when Ng is grown under low iron or low oxygen conditions. Analysis of an fnr mutant, grown under the same conditions as its parental wild type, indicates that the FNR transcriptional regulator is involved in the repression of gcp in low iron or low oxygen conditions. Contrary to expectation, the cyc4 promoter is upregulated when Ng is grown under low oxygen or low iron conditions. However, these results cannot be compared to the original promoter strength. Determination of which was performed on bacteria grown in liquid medium. Coregulation of gcp with cytochrome genes can guarantee low levels of protein synthesis when Ng encounters adverse microenvironments and needs its energy redirected to the expression of genes that would allow it to survive.

Ambient air pollution, including airborne particulate matter (PM) derived from combustion of fossil fuels (FF) or biomass (BM), has detrimental inflammatory effects on human health. Myeloid antigen presenting cells, including dendritic cells (DCs) regulate immune responses in the airway and sample inhaled PM. This study tests the hypothesis that PM interacts with multiple environmental sensing pathways in DCs with outcomes that depend on particle size and composition as determined by combustion source. The effects of different sized PM (< 10μm, PM10; < 2.5μm, PM2.5), derived from the combustion of FF or BM, on human monocyte-derived or ex vivo sputum DCs, were examined. DC activation status, cytokine production and aryl hydrocarbon receptor (AhR) signalling were assessed by flow-cytometry, multiplex ELISA and qRT-PCR, following exposure to PM. Pathway-specific antagonists were used to explore underlying mechanisms. Particle size and combustion source influenced the effects of PM on DCs. Irrespective of combustion source, PM10 but not PM2.5, induced MoDC maturation and stimulated production of inflammatory cytokines, including IL-1β and IL-18, indicative of inflammasome activation. These responses were dependent, at least in part, on TLR4 as was the induction of IDO by PM10. AhR signalling was induced by PM in both MoDC and ex vivo sputum DC. It was stimulated by both PM10 and PM2.5 and was induced more strongly by BM-derived PM. AhR activation was independent of DC maturation and TLR4 signalling. Additionally, BM- but not FF-derived PM increased NADH levels in DC suggestive of altered metabolism. Thus, PM induces a complex programme of DC activation, influenced by size and combustion source, which includes classical maturation, inflammasome dependent cytokine release and AhR signalling as well as potential metabolic changes. In the airway, exposure to different PM and the changes in DCs induced by them may lead to altered responses to inhaled antigen.

The rise of antimicrobial resistance (AMR) has culminated in the most pressing problem in modern medicine. The situation is most acute with regards to the management of multi- drug resistant Gram-negative infections (MDRGNB) with common infections increasingly untreatable due to rapidly dwindling therapeutic options. A solution to the problem of AMR is unlikely to be easily found, but revisiting and re-purposing existing antimicrobials is a viable approach in the medium term. This study investigated the use of unorthodox antimicrobial combination therapies for the treatment of MDRGNB, with particular focus on agents of last resort. A systematic review of clinical studies highlighted the potential for polymyxin (colistin) combination therapies (e.g. colistin-rifampicin, colistin-carbapenems), although this could not be supported in a formal meta-analysis. A systematic approach for screening MDRAB for susceptibility to novel colistin combinations using multiple methods was employed and uncovered a number that were more potent than those previously identfied. The most potent combination that was consistently identified was colistin when combined with fusidic acid, despite this drug having no useful activity against MDRGNB on its own. The combination was further evaluated in static time-kill assays against a range of Gram-negative pathogens with defined resistance mechanisms, including to polymyxins and using invertebrate (Galleria mellonella) and murine models of MDRGNB infection. Colistin and fusidic acid combination therapy was subsequently used to successfully treat a case of ventilator-associated pneumonia due to MDR A. baumannii. This work highlights how older drugs can be re-purposed to tackle the problem of AMR using a precision medicine approach. Further studies to elucidate the mechanism of action of the colistin- fusidic acid combination and a formal clinical trial are warranted to investigate the potential utility of this combination in the treatment of MDRGNB with the expressed goal of bridging the current antimicrobial development gap.