Circulating monocytes have the capacity to mature into either macrophages or dendritic cells in tissue, both of which play important roles in the induction and effector phase of immune response. In TB, the macrophages are the central player in the host-bacteria interaction as the main mycobacterial reservoir. In HIV disease, monocyte lineage cells are one of the two main cell types (along with CD4+ T-cells) in sustaining intracellular HIV infection. Monocytes are heterogeneous population with three functionally distinct subsets namely classical, intermediate and non-classical monocytes. The three subsets exist in a continuum, and have a certain plasticity or flexibility to develop into multiple roles depending on the local and tissue environment. In the current study we sought to evaluate the frequencies of these three subsets in participants with TB, HIV and TB/HIV co-infection. While previous studies had shown that the intermediate and non-classical monocyte subsets were elevated relative to classical monocytes, very little had been done in these disease groups regarding more comprehensive characterization of these subsets. In particular, we wished to quantitate the expression of multiple sets of cell surface and intracellular molecules of high relevance using multi-parameter flow cytometry from a functional point of view. In publication I, we evaluated Toll-like receptors (TLRs) expression in each of the study cohorts. TLRs are vital pattern recognition receptors by monocyte lineage cells and signal the induction of crucial functions. We focused on three such TLRs (TLR2, TLR4 and TLR9) which have been shown to be involved in many monocyte lineage cell interactions with mycobacterial and HIV infections. We observed enhanced expression of TLR2 and TLR4, but not TLR9 in TB and HIV. TLR4 was particularly high in patients with TB, but also in HIV. We observed comparable increase of TLR4 irrespective of monocyte subset. However, TLR2 expression exhibited a different pattern. Levels among the most prominent classical monocyte subsets were identical in all four cohorts, healthy controls (HC), HIV, TB, and TB/HIV co-infection. In contrast, TLR2 expression was significantly elevated in both participants with HIV and TB, but not with participants with TB/HIV co-infection in the intermediate monocyte subset. We also observed correlations between TLRs and plasma cytokines that were disease and TLR specific. In publication II, we observed elevated chemokine receptors (CRs) expression which above healthy controls and exhibit a pattern of disease preference. Thus, CCR2 and CX3CR1 were the highest in participants with TB, followed by HIV and TB/HIV co-infection, whereas CCR4 and CCR5 were highest in participants with HIV, and less elevated in TB. CCR2 and CX3CR1 are critical for migration of monocytes to sites of TB infection, as determined by murine models. CCR4 and especially CCR5 have been implicated in migration of cells to distant organs but more as co-receptors for HIV infection. Thus, the observed pattern of CRs expression in these monocytes in different disease states would predict greater availability of these cells or their receptors for interaction with either TB or HIV organisms. From the perspective of the pathogen this would lead to enhanced “substrate”, whereas from the perspective of the host, this could lead to greater immune potential. As a final point, we also observed that the pattern of disease association of CRs was independent of the monocyte subset. In publication III, we explored the expression of Programmed cell death-ligand 1 (PDL1) on the three monocyte subsets. Like many of the other molecules we have addressed in this thesis, PDL1 expression was enhanced in participants with HIV, TB, and TB/HIV co-infection. Among participants with HIV, PDL1 was correlated with HIV-1 viral load. The enhanced expression was apparent in all three subsets, but it was particularly prominent in the intermediate monocyte subset. Moreover, PDL1 expression was the highest in participants with TB/HIV co-infection. The implications behind these observations is that the subset thought to have the greatest potential for T cell antigen presentation had the highest levels of the T cell down-regulatory PDL1 molecule in the cohort of patients particularly participants with TB/HIV co-infection. Participants with TB/HIV co-infection have the greatest potential to be immuno-compromised and as a result the very need for enhanced not depressed APCs function. In addition, we also observed the PDL1 levels were correlated with multiple plasma, mostly pro-inflammatory,cytokines. We analyzed cytokine mRNA levels of total monocytes to address the source of the cytokines but mRNA levels did correlate with neither plasma cytokine nor PDL1 levels. Considering all the phenotype analysis in each of the three studies together we could see two patterns emerging. In one scenario,surface molecules expression patterns were disease specific but independent of monocyte subset expression. In other words, whatever the underlying mechanism(s) involved in their regulation, those mechanisms apparently acted similarly in all three subsets. In another scenario, expression of surface molecules showed disease specific patterns, but molecules were particularly enhanced in the intermediate monocyte subsets. These findings imply that there exist mechanisms to modulate surface phenotypes and functions that are unique to a given subset. In conclusion, we have comprehensively defined the density of multiple molecules expressed by different subsets of monocytes and explored their differences in participants with TB, HIV and TB/HIV co-infection, as well as their correlations with microbial indices and plasma cytokines. Many molecules levels were elevated to some extent in all disease cohorts, but we observed patterns of expression which were particularly elevated in TB (CCR2, CX3CR1, and TLR2), those in HIV (CCR4, CCR5) and those on both (TLR4, PDL1). Molecule-disease associations were either independent of monocyte subset, or most readily revealed in a single monocyte subset. TB/HIV co-infection did not follow a consistent pattern in association with monocytes markers, in some cases more resembling TB, in others HIV, in others neither. Finally, to proof one possible mechanism of association between disease and monocyte phenotype, we explored correlations between monocyte markers and plasma cytokines. We observed significant positive and negative associations, frequently unique to a single monocyte subset or disease cohort, such as TB/HIV co-infected cohort. Collectively, the results imply that there are likely multiple mechanisms involved at many levels regulating the phenotype and function of monocytes, and these differ in different disease states.:Abbreviations ............................................................................................................ 3 Abstract ..................................................................................................................... 4 1. Introduction ........................................................................................................... 6 1.1 Epidemiology of Tuberculosis and Human Immunodeficiency virus ................... 6 1.2 The immunological response to TB and HIV ....................................................... 7 1.2.1 Innate immunity of TB ...................................................................................... 7 1.2.2 Innate immunity of HIV .................................................................................... 11 1.2.3 Immune checkpoint regulation in TB and HIV.................................................. 13 1.3 The role of monocytes in TB, HIV and TB/HIV ................................................... 14 1.3.1 Monocytes ....................................................................................................... 14 1.3.2 Abnormalities of monocytes in TB ................................................................... 15 1.3.3 Abnormalities of monocytes in HIV ................................................................. 16 1.3.4 Abnormalities on monocytes in TB/HIV co-infection ....................................... 17 1.4 The rationale for the thesis ................................................................................ 17 2. Objectives ............................................................................................................ 19 3. Publications .......................................................................................................... 20 4. Summary .............................................................................................................. 65 References ............................................................................................................... 69 Annex I: Author contribution ..................................................................................... 76 Annex II: Declaration of independent writing of the work ......................................... 77 Annex III: Curriculum Vitea ....................................................................................... 78 Annex V: Acknowledgment........................................................................................ 82 Annex VI ................................................................................................................... 84

Renibacterium salmoninarum, the etiological agent of bacterial kidney disease (BKD) of salmonid fish, is a pathogen of great concern among fisheries and the aquaculture industry worldwide. Previous investigations have indicated the pathogenesis of BKD is complex. It is a chronic, multisystemic, granulomatous disease with a number of potential immunomodulatory effects on the host. Given the current limitations for treatment and control of BKD, it is imperative that the pursuit of development of methods of prevention, namely management strategies and vaccination, be continued. To do so, the immunology of BKD must be elucidated in order to better understand and manipulate the associated immune responses to our advantage. This dissertation is composed of four chapters which relate to BKD and the associated immune responses of three species of susceptible salmonid fish as follows: Exogenous stress factors, through stress-induced immunosuppression, have been shown to influence BKD development in cultured salmonids. Chapter 1 examines the effects of two environmental stressors common to fish culture, overcrowding and overfeeding, as they affect BKD development and R. salmoninarumantigen prevalence among juvenile chinook salmon (Oncorhynchus tshawytscha) Immunomodulatory interaction between pathogen and host in BKD is widely reported and merits further investigation. Particularly, the immunological parameters affected and the role of the extracellular protein (ECP) of R. salmoninarum are of interest. Chapter 2 examines the in vivo immune response of rainbow trout (Oncorhynchus mykiss) following exposure to the ECP in terms of both humoral and cell-mediated immunological parameters, including the immune response against another bacterial pathogen. Chapter 3 addresses the in vitro effects of the ECP upon specific splenic immunocyte functions, phagocytosis and respiratory burst activity, in brook trout (Salvelinus fontinalis). The immune-complex mediated hypersensitivity reported to occur with BKD has considerable ramifications for control measures involving immunostimulation via antigen exposure. Further investigation is warranted to discern the significance and consistency of immunological hypersensivity in BKD pathogenesis. Chapter 4 examines the renal lesions, including immunopathologic changes and indications of immune-mediated disease, of brook trout exposed to R. salmoninarum. / Ph. D.

Plants are surrounded by various threats from the environment such as pathogens, abiotic stresses, and animal attacks. Nutrient content and distribution are essential for plant growth and development as well as plant immunity. Pathogens extract nutrients from host plants to benefit their own growth and reproduction. Sulfate, amino acids, and phosphate are indispensable elements for plant growth, plant nutrition, and plant resistance/susceptibility to disease. However, the role of these nutrients in plant-oomycete interactions is an unexplored area. We developed a hydroponic system to precisely control the nutrients applied to plants. We used Arabidopsis thaliana and Nicotiana benthamiana (N. b) as model plants. Hyaloperonospora arabidopsidis as well as two Phytophthora species, Phytophothora capsici (P. cap) and Phytophothora nicotianae (P. nic) were used as model oomycete pathogens. Hpa is an obligate biotrophic pathogen that obtains nutrients directly from the host plant without causing cell death, while P. cap and P. nic are hemibiotrophic pathogens that display a biotrophic phase followed by a necrotrophic phase where they feed on dead cells. Genomic evidence suggests that these pathogens might obtain nutrients including sulfur in different forms from the host (organic and inorganic respectively). We have optimized the hydroponic system and used Taqman PCR assays and sporangiophore counts to assay the influence of sulfur nutrients on Hpa and P. cap infections. We found that (1) sulfur transporter and metabolism genes play essential roles in plant-oomycete interactions; (2) sulfur is critical components for HR responses against Hpa; (3) low sulfur induces pathogenesis related genes as a systemic acquired response. RNA-seq analysis on Phytophthora-infected Arabidopsis suggested that sulfur transport, assimilation, and metabolism play an important role in plant-oomycete interactions. A second project used RNA-seq analysis on P. nic infected N. b, to identify potential nutrition-related-plant genes that are necessary for full pathogen virulence. RNAi knockdowns of N. b AAP6 (amino acid permease 6) and PHT4 (phosphate transporter 4) genes showed an inhibition of oomycete colonization. These experiments together advance the study on the interplay between nutrient assimilation/metabolism in host plants and oomycete infection which will provide insight into the mechanisms how pathogens intercept nutrients from host. In the long-term, this research could reveal new traits applicable for disease resistance to promote crop and food production. / Doctor of Philosophy / Plants are surrounded by diverse threats from the environment such as pathogens, abiotic stresses, and animal attacks. Oomycetes are the most destructive group of pathogens, triggering severe food security issues. Phytophthora is an oomycete genus causing serious economic loss. Traditional disease control managements including pesticides, crop rotation and culture practices, are not time- or financially- efficient due to the difficulty in managing oomycete spread and oomycete resistance to chemicals. Thus, new plant genes for resistance to oomycete diseases would have a major impact. Plant nutrients are critically important for plant fitness in every aspect of plant growth and plant immunity. Cellular regulatory networks for sulfur, amino acids, and phosphate assimilation and metabolism networks connect to every aspect of plant activity such as functioning enzymes, formation of chlorophyll, synthesis of proteins, and plant immunity. These nutrients are part of the plant defense system but also can be beneficial nutrients fed to the invading pathogens. Studying how nutrients are involved in the responses to oomycete invasions will provide information to introduce resistance strategies into crops. We utilized oomycete pathogens with different lifestyles to study the interactions and found that some sulfate transporter genes, an amino acid transporter and a phosphate transporter might be manipulated by oomycete to obtain nutrients. Sufficient nutrition is a critical factor for successfully triggering plant immunity but also could be reprogrammed by pathogens for successful infection and development. Our studies gave useful information to understand which plant nutrient genes are important during plant–oomycete interactions. These findings could be useful in identifying or engineering new plant genes to control plant diseases.

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. The genome of Hyaloperonospora arabidopsidis (Hpa), the causative agent of downy mildew of Arabidopsis, contains at least 134 candidate RXLR effector genes. These genes contain an RXLR motif required for effector entry into host cells. Only a small subset of these candidate effectors is conserved in related oomycetes. Here, we describe a comparative functional characterization of the Hpa RXLR effector HaRxL96 and a homologous gene, PsAvh163, from the soybean pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress or activate immune responses in soybean, Nicotiana, and Arabidopsis. Several SA-responsive defense genes are suppressed in Arabidopsis Col:HaRxL96 and Col:PsAvh163 during an incompatible interaction with Hpa Emoy2. Both effectors are localized to the nucleus and cytoplasm of plant cells. Nuclear localization of both effectors is required for proper virulence functions, including suppression of basal resistance and RPP4-mediated immunity to virulent and avirulent Hpa, respectively. In addition, both effectors interact with plant U-box (PUB) proteins that are conserved between diverse plant species. The targeted PUB proteins are negative regulators of plant immunity in Arabidopsis. These experiments demonstrate that evolutionarily-conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's primary host. / Ph. D.

Mosquito-borne diseases continue to be a burden to global health. The viruses that cause these diseases are maintained in nature through a biological transmission cycle involving susceptible vertebrate and mosquito hosts. While knowledge of the interactions occurring between mosquito-borne viruses and vertebrates is considerable, much less is known about the interactions of these viruses with their disease vectors. Studies with Drosophila melanogaster have been important in understanding how insects respond to viral infections. However, mosquitoes and the viruses they vector have co-evolved during a long period of time. Unfortunately, many of the genetic advantages of a fly model are not available when working with mosquitoes. Nevertheless, a sequenced genome, and molecular tools such as high-throughput sequencing and RNAi knockdown are helping to bridge these gaps. Here we describe several additional tools for the study of virus-vector interactions in the mosquito. / Ph. D.

Escalating consumer concerns have placed the poultry industry under mounting pressure to reduce the use of chemotherapeutic agents as feed additives. One possible alternative receiving increased attention is the use of immunomodulators such as β-glucan. A pilot study evaluated the effects of a yeast derived β-glucan (Auxoferm YGT) on growth performance and immune response of broiler chickens. Day-old chicks were fed a diet containing 0, 0.02, or 0.1% yeast β-glucan. On days 7 and 14 post-hatch, body weight and relative immune organ weights were measured, peripheral blood was collected to determine heterophil to lymphocyte (H:L) ratios, and small intestinal sections were sampled to evaluate relative gene expression. The addition of β-glucan had no influence on growth. Dietary β-glucan supplementation modulated the expression of interleukin (IL)-8, IL-18, interferon (IFN)-γ and inducible nitric oxide synthase (iNOS) in the small intestine. A subsequent study was conducted to investigate the effects of dietary β-glucan on broiler chick (1440 birds) performance and immune response during a mixed Eimeria infection (day 8 of age). Measurements were taken and samples collected on days 4, 7, 10, 14 and 21 post-hatch. The results from this study show that β-glucan supplementation did not negatively impact performance. The addition of β-glucan to the diet resulted in reduced gross lesion severity and increased H:L ratios. The gene expression results suggest that β-glucans are capable of skewing the host immune response toward aTh1 mediated response and consequently down-regulating the Th2 mediated response. / Master of Science

Recombinant and DNA vaccines utilizing Mycobacterium sp. antigen 85A (Ag85A) were assessed for immunostimulatory and protective effects against M. marinum. Because of their known susceptibility to piscine mycobacteriosis, Morone sp. were utilized as the models for these studies. The first study evaluated a recombinant vaccine with a Brucella abortus strain RB51 vector expressing the Mycobacterium bovis Ag85A. Striped bass (M. saxatilis) were inoculated at doses equivalent to 106, 107, 108, 109, and 1010 colony-forming units/fish. Vaccinated fish demonstrated significant specific humoral and cell-mediated immune responses towards the Ag85A in a dose-dependant manner. However, vaccinated fish failed to demonstrate cross-protective responses after live Mycobacterium marinum challenge 70 days post-vaccination. A DNA vaccine was constructed utilizing the Mycobacterium marinum Ag85A gene and a commercially-available eukaryotic expression vector. Hybrid striped bass (M. saxatilis x M. chrysops) were immunized by intramuscular (i.m.) and intraperitoneal (i.p.) injection at doses of 5 μg, 25 μg, or 50 μg plasmid. These fish produced significant Ag85A-specific antibody and lymphoproliferative responses over those of control fish injected with saline or empty plasmid. Non-specific macrophage phagocytic and respiratory burst functions failed to exhibit significant upregulation after vaccination. Fish receiving the DNA vaccine developed protective responses to high-dose M. marinum challenge 90 days post-vaccination, as demonstrated by increased relative percent survival and by reduced splenic bacterial counts over control fish. Furthermore specific immunostimulatory and protective effects were significantly increased using higher vaccine doses and using the i.m. injection route. Given these promising findings, the protective responses induced by the DNA vaccine were further investigated. Hybrid striped bass were injected with 25 μg or 50 μg plasmid i.m. and developed specific protective responses to high-dose M. marinum challenge 120 days post-vaccination. The 25 μg and 50 μg groups both developed more rapidly and significantly increased immune responses post-challenge over those of the control groups. The vaccination groups also demonstrated increased survival, reduced splenic bacterial counts, and reduced granuloma formation compared to the control groups. However, though the vaccination groups did not demonstrate the same acute effects post-challenge as the control groups, the vaccination groups ultimately developed increased splenic bacterial counts and granuloma formation, and eventually experienced 100% mortalities. Because piscine mycobacteriosis can affect virtually any species of fish, a vaccine against this disease could be widely beneficial to the aquaculture and ornamental fish industries. The vaccines in these studies exhibited significant immunostimulatory capabilities in Morone sp., but only the DNA vaccine showed promise for conferring protection against M. marinum challenge. Though the DNA vaccine only provided limited protection against high challenge doses, future studies may likely find enhanced protective effects against lower, more natural exposure doses. / Master of Science

Five experiments were conducted comparing differential intestinal immune responses to two isolates of Eimeria acervulina (EA), EA1 and EA2. In three experiments, broiler chicks were divided into control (non-challenged), EA1, or EA2 challenged (14 days of age) groups. On day 6 post-challenge (PC), changes in body weight were determined, intestinal lesions were scored, and duodenal tissue was evaluated for morphometric alterations and mucosal mast cell responses. EA1 produced duodenal lesions and reduced villus height to crypt depth ratios when compared to controls; however, no differences were found in mast cell counts. EA2 produced differing results, and observed data were suggestive of an intestinal secretory response when compared to EA1 or controls. In Experiment 4, tissues were analyzed from day 2 through day 6 PC. Villus atrophy and crypt hyperplasia were heightened on day 5 PC in both challenged groups. Mast cell counts were significantly greater on days 3 and 4 PC in EA1 birds. In Experiment 5, EA2 oocysts were cleaned with 5.25% sodium hypochlorite to evaluate the possibility of a bacterial contaminant contributing to the pathogenesis of intestinal alterations. Weight gains were decreased by challenge and villus heights and crypt depths were significantly altered in challenged birds, resulting in lower villus to crypt ratios, however, there were no differences in mast cell number. These data are indicative of differential host response and immunovariability between different isolates of the same Eimeria species and are suggestive of mast cell involvement in coccidial immunity in broiler chickens. / Master of Science

Doctor of Philosophy / Department of Anatomy and Physiology / Elizabeth G. Davis / Activation of innate immunity through Toll-like receptor (TLR) signaling can also enhance antigen-specific adaptive immunity. TLR9 is an endosomal receptor for unmethylated bacterial and viral cytosine-phosphate-guanine DNA (CpG-DNA). West Nile virus (WNV) infection may result in meningitis and encephalitis in humans and horses, especially aged and immunocompromised individuals. Using flow cytometric analyses and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated equine cell-mediated immunity (CMI) to an inactivated West Nile virus vaccine in healthy yearling and adult horses. We also studied the potential of enhancing equine adaptive immunity to viruses and other pathogens by activation of innate immunity though TLR9 signaling pathway. We found vaccination with inactivated WNV vaccine induced strong WNV-specific T helper type 1 (Th1) and Th2 CMI with a Th1 bias, also effectively induced WNV-specific CTLs in yearling horses. In adult horses, the pre-existing Th1 CMI bias against WNV was enhanced following booster vaccination with inactivated WNV vaccine. Molecular characterization and flow cytometric analysis of TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in neutrophils (PMNs), CD4[superscript]+ and CD8[superscript]+ T cells and other leukocytes. Conservation of equine TLR9 and a high expression profile among leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity. Unmethylated CpG-DNA can significantly activate equine PMNs. It also induces expression of interferon (IFN)-[Alpha], IFN-[Beta], IFN-[Gamma], and interleukin (IL)-12p35 in PBMCs, as well as IFN-[alpha] and IFN-[gamma] in monocyte-derived DCs. Enhanced expression of IFNs in immune cells by CpG-DNA is not only crucial for host viral clearance, but also important in mediating host immune responses due to IFNs' anti-inflammatory effects. Compared to the relatively weaker activation of equine innate immunity by inactivated WNV, the tested CpG-DNA species showed potential as vaccine adjuvants for enhancement of CTLs and Th1 CMI against intracellular pathogens, characterized by significant induction of type I IFNs and Th1-specific cytokines such as IL-12p35 and IFN-γ. These data provide a basis for further investigation of these CpG-DNA species as potentially effective vaccine adjuvants in horses.

Malaria is a leading cause of death in some developing countries. The malaria parasite has been around for over a century, and has coevolved with humans. Coming up with an effective vaccine for P. falciparum will save millions of lives and reduce the morbidity and mortality of malaria globally. Understanding the role of exported parasite proteins i.e PfEMP1 a virulence factor and major cause of malarial pathogenesis, has been of great interest to vaccine researchers in the last decade. The focus of this review is to provide a literature review on PfEMP1s, their interaction with the human immune system, and their role in helping P. falciparum parasite to evade the immune system. This review will primarily focus on the intra-erythrocytic stage, which is the stage that results in the symptoms of malaria. A review is necessary to understand the antigenic variation of PfEMP1s, and how PfEMP1s challenge the different arms of the immune response, both the innate and adaptive. This review is unique in touching on the major parts of the immune system's interaction with the PfEMP1 antigen. Furthermore, the review explores the discussion of future research and therapeutic opportunities based on our knowledge of PfEMP1 antigens.