EDCs are commonly thought to bind or interfere with estrogen, androgen, progesterone, thyroid, and retinoid receptors. Oxybenzone is considered to be an endocrine-disrupting chemical and approximately 97% of people in the United States were found to have BP3 in their urine. This thesis will address how BP3 affects the innate immune system, in particular myeloid cells. My Master’s thesis aims to address two main overarching questions. Does BP3 alter macrophage polarization, cytokine/chemokine secretion, the viability in vitro? Does exposure to BP3 in vivo during pregnancy/lactation affect the RNA expression of cytokines and immunosuppressant factors associated with the myeloid population? It is unknown how BP3 impacts immune subpopulations in a neoplastic setting. Additionally, it is important to consider how these effects may contribute to malignant behaviors. My thesis evaluates the effects of BP3 on the Raw 264.7 cell lines as well as tumor tissues from mice exposed to BP3 during pregnancy and lactation. We hypothesized that BP3 exposures induce changes in myeloid cell interactions in the immune system through ER-mediated mechanisms. We anticipated that BP3 would increase the growth and migration of 4T1 cells through indirect signals imparted by myeloid cell populations. We also hypothesized that there will be a decrease in T cell proliferation following BP3 exposure and an alteration in gene expression consistent with a shift from Th1 to Th2. Finally, we expected that BP3 exposure would increase the number of myeloid cells in mouse tumors. Our research shows that oxybenzone appears to enhance the pro-inflammatory state of RAW264.7 cells and may result in the release of unidentified factors that can impact 4T1 cell anchorage-independent cell growth in these pro-inflammatory conditions. BP3 may also impact the metabolic activity of recovering RAW264.7 cells following LPS-induced activity. Additionally, BP3 may impact the release of factors from macrophages that control T cell activation-induced proliferation. By using the p53-/- mouse tumors we found that exposure to 3mg/kg/day BP3 during pregnancy and lactation did alter IDO1 RNA expression but this was not associated with markers of immunosuppressive cell types.

Research Doctorate - Doctor of Philosophy (PhD) / Asthma involves chronic inflammation of the airways that is heterogeneous in nature. Eosinophilic airway responses are well described in asthma, however non-eosinophilic subtypes of asthma have been recently reported, and can involve the influx of neutrophils into the airways (neutrophilic asthma). Neutrophils are important effector cells of the innate immune system. These cells are the first to migrate to inflammatory sites, where they contain and eliminate pathogenic microorganisms. Neutrophils also release cytokines and chemokines that initiate and amplify inflammatory responses. The mechanisms of neutrophilic asthma remain largely unknown; however activation of the innate immune response is implicated, particularly increased levels of proinflammatory cytokines Interleukin (IL)-8 and IL-1beta and gene expression of Toll Like Receptor (TLR)-4 and TLR2 have been demonstrated in induced sputum samples. This thesis examines innate immune responses of airway and circulating neutrophils, with a focus on neutrophilic asthma. Innate immune neutrophil activation occurs in response to exposure to Lipopolysaccharide (LPS), which activates TLR4. The activation response consists of the release of preformed granule associated mediators such as Matrix Metalloproteinase (MMP)-9 and Oncostatin M (OSM), new gene transcription and release of inflammatory cytokines such as IL-8, IL-1beta and Tumor Necrosis Factor (TNF)-alpha, and new gene transcription of TLR2 & TLR4 which serve to amplify neutrophil responses. In addition, this thesis examines whole genome gene expression profiles of circulating neutrophils in neutrophilic and eosinophilic asthma. The aims of this thesis are based on the hypothesis that dysregulation of innate immune neutrophil responses occurs with ageing and airway disease, particularly neutrophilic asthma and chronic obstructive pulmonary disease (COPD). With advancing age, there were alterations in the innate immune responses of neutrophils, which were characterised by enhanced spontaneous activation of both airway and circulating neutrophils, and a decreased response of circulating neutrophils to LPS. There was a decreased activation of airway neutrophils in airway disease that was most pronounced in neutrophilic asthma and COPD, with decreased production and release of proinflammatory cytokines most likely due to a downregulation of TLR4. TLR2 was downregulated in resting and LPS stimulated circulating neutrophils in asthma, particularly neutrophilic asthma. Circulating neutrophils had a decreased spontaneous release of total MMP-9, and downregulation of OSM, TLR2 and TLR4 at rest in COPD. However when stimulated with LPS, subjects with COPD had an enhanced proinflammatory cytokine release, with increases in IL-8 and TNF-alpha compared to subjects with asthma or healthy controls. Analysis of whole genome gene expression of circulating neutrophils in asthma revealed distinct gene profiles relating to asthma subtype. There was upregulation of genes relating to cell motility, inhibition of apoptosis and the NF-kB in neutrophilic asthma, which would contribute to their accumulation in the airways. The innate immune response is critical in controlling infections by bacteria and viruses. The reduced innate immune response of airway neutrophils in airway disease could contribute to impaired local defense, which may lead to an increased susceptibility to infection by invading pathogens. Systemically, the molecular mechanisms of neutrophilic asthma are distinct from eosinophilic asthma and may involve the enhancement of neutrophil chemotaxis and survival, contributing to their accumulation in the airways.

The mammalian immune system is capable of detecting and responding to different infectious conditions with specificity at the adaptive level, however whether this ability extends to individual cells of the innate immune system is unclear. The hypothesis of this thesis is that macrophages, as individual cells, can distinguish between productive and non-productive virus infections and respond differently at the gene expression and secreted protein level. To test the hypothesis, mouse bone marrow derived macrophages (BMDMs) were infected in parallel with either a productive (live) and non-productive (attenuated) strain of murine cytomegalovirus (MCMV) and profiled temporally using a range of techniques. Both productive and non-productive MCMV infection resulted in strong type I IFN induction in BMDMs, however induction was significantly more rapid in response to productive infection. In addition, chemoattractant and pro-inflammatory cytokines TNFα, IL-6, RANTES, MIG and MIP-2 were secreted to significantly higher levels in response to productive MCMV infection, and curtailed in response to non-productive MCMV infection. Furthermore, genome-wide microarray profiling revealed a number of co-expressed gene networks regulated differentially in response to the two conditions. This consisted of macrophage gene networks targeted for modulation by de novo MCMV proteins, and late macrophage response genes regulated specifically in response to productive MCMV infection. To further explore the mechanisms of transcriptional regulation during macrophage antiviral response, BMDMs from mice lacking either the type I IFN receptor (Ifnar1) or the IFNβ (Ifnb1) gene were profiled using a similar approach. The resulting genome-wide transcriptional data provided a unique insight into the relationship between type I IFN regulation and the macrophage transcriptome in response to MCMV infection. Overall, the study utilizes a combination of genetic mutants from both host and pathogen to investigate mechanisms of virus detection and host transcriptional regulation during the innate immune response to MCMV infection in macrophages.

Plasmodium falciparum (P. falciparum) infection modulates the response of innate immune cells. The aim of this work was to study the impact of P. falciparum infection and P. falciparum-derived molecules on the response of dendritic cells (DC) and monocytes. In paper I we investigated the effects of natural hemozoin (nHZ), a P. falciparum-derived molecule, on the phenotype and functionality of DC. We found that exposure to nHZ impaired the capacity of DC to mature. Paper II is a follow-up on paper I, where the underlying transcriptional events preceding the nHZ-induced impairment of DC maturation were investigated. More specifically, we examined the involvement of certain transcription factors, subunits of chromatin remodeling complexes and histone modifications in the regulation of DC maturation. Our findings suggest that nHZ-exposure of DC does not lead to recruitment or enrichment of molecules needed for transcriptional activation. In paper III we investigated P. falciparum effects in vivo in sympatric ethnic groups with differential susceptibility towards P. falciparum infection living in Burkina Faso. The aim of this study was to establish the transcriptional networks underlying the relatively better protection against P. falciparum infection observed in the Fulani ethnic group compared to other sympatric ethnic groups. Our findings reveal differential gene expression in monocytes of infected Fulani compared to uninfected Fulani and the difference concerned multiple classes of genes including signal transduction, immunological responses and chromatin remodelers. The results provide new aspects on molecules and regulatory mechanisms that are involved in the relatively more protective response against P. falciparum infection. Taken together, the work presented in this thesis leads to a deeper understanding of the P. falciparum-induced modulation of responses of innate immune cells and the underlying mechanisms possibly regulating those responses. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p>

Induced and activated as part of the innate immune response, the first line of defence against bacterial or viral infections, Inhibitor of Kappa-B Kinase ε (IKKε) triggers NF-κB and IFNβ signalling. Whilst not expressed at basal levels in healthy cells and tissue, the kinase is overexpressed in roughly 30% of human breast cancer cases, driving oncogenesis through aberrant activation of NF-κB. The impracticality of therapeutic targeting of NF-κB for cancer treatment has led to a requirement for greater understanding of IKKε's oncogenic potential to treat tumours driven by the kinase. Considering that IKKε alters cellular metabolism in dendritic cells, promoting aerobic glycolysis akin to the metabolic phenotype observed in cancer, it was hypothesised that the kinase would play a similar role in breast cancer. Using a Flp-In 293 model of IKKε induction and suppressing IKKε expression in a panel of breast cancer cell lines using siRNA, IKKε-dependent changes in cellular metabolism were characterised using labelled metabolite analysis. IKKε was found to induce serine biosynthesis, an important pathway in breast cancer development that supports glutamine-fuelling of the TCA cycle and contributes to one carbon metabolism to maintain redox balance. Promotion of serine biosynthesis occurred via a dual mechanism. Firstly, PSAT1, the second enzyme of the pathway, was found to be phosphorylated in an IKKε-dependent manner, promoting protein stabilisation. Secondly, an IKKε-dependent transcriptional upregulation of all three serine biosynthesis enzymes, PHGDH, PSAT1 and PSPH, was observed, induced by the inhibition of mitochondrial activity and the subsequent induction of ATF4-mediated mitochondria-to-nucleus retrograde signalling. These data demonstrate a previously uncharacterised mechanism of metabolic regulation by IKKε and highlight new potential therapeutic targets for the treatment of IKKε-driven breast cancer in the form of the enzymes of the serine biosynthesis pathway.

Our murine model of mild traumatic brain injury (mTBI) has shown improved survival after Pseudomonas aeruginosa (Psd) challenge as compared to controls (tail trauma or sham injury). Previous work suggests an mTBI-specific involvement of the neuro-immune axis which augments the innate immune response, increasing survival. Additional factors for the enhanced mTBI survival were explored via microarray analysis of lungs harvested 48 hours post-trauma, the point prior to Psd challenge in our model. At 48 hours post-trauma, mTBI lungs have a number of upregulated ATP synthesis and mitochondrial gene sets. Increased available energy could prime the mTBI lungs, allowing an earlier and more robust response to Psd infection, possibly contributing to the increased mTBI survival. This is supported by increased neutrophil recruitment in the bronchoalveolar lavage of mTBI mice four hours after Psd instillation. Downregulated gene sets related to cellular connections suggest that neutrophils recruited to the lung have an easier extravasation pathway into the air space of mTBI lungs compared to control. Based on genetic and neutrophil recruitment data, it is possible that mTBI creates an energetically prepared and easily accessible lung better tailored for recruiting and allowing entry of neutrophils in response to an infection compared to control.

Despite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (2 mg/Kg BW; dissolved in 0.9% saline) or vehicle (0.9% saline) to stimulate the immune system. Two hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells. The data show 628 significantly upregulated transcripts in the hypothalamus, as well as 439 and 121 in the spleen and RBCs, respectively, relative to controls. Also, 134 transcripts in the hypothalamus, 517 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1β, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated, suggesting a potential trade-off in expression of genes that regulate immunity and metabolism. Unlike mammals, birds have nucleated RBCs, and these results suggest a novel transcriptomic response of RBCs to immune challenge. Lastly, molecular biomarkers could be developed to rapidly screen bird populations by simple blood sampling in the field.

The Gram-negative bacterium Legionella pneumophila is an intracellular parasite of aquatic protozoa. It exhibits a distinct dimorphic lifecycle that alternates between vegetative replicative form (RF) and infectious cyst-like form (CLF). Inadvertent inhalation of aerosolized CLFs by immunocompromised individuals leads to an infection in alveolar macrophages causing Legionnaires' disease. To further study a Legionella infection the use of the multicellular organism Caenorhabditis elegans was done. Differential Interference Contrast (DIC) microscopy of live L. pneumophila infected nematodes shows Legionella-containing vacuoles (LCVs) with motile forms. Transmission Electron Microscopy (TEM) defined the ultrastructure of L. pneumophila forms found in the primary infection site of the intestinal lumen and the secondary infection site in the gonadal tissues. These findings suggest the possible intracellular replication cycle of Legionella occurring in the gonadal tissues of the nematode. Providing insight and a plausible evolutionary origin of the ability of L. pneumophila to manipulate the macrophage innate immune system. / October 2014

Two different feeding trials were conducted to evaluate the effects of dietary supplementation with the dairy/yeast prebiotic GroBiotic-A (GBA) to Nile tilapia diets. A nutritionally complete basal diet was supplemented with GBA at either 1 or 2% of dry weight, and all three diets were fed to triplicate groups of juvenile fish in two consecutive trials. Trial 1 continued for 8 weeks, while Trial 2 was conducted for 5 weeks to more specifically assess immunological responses, intestinal characteristics and disease resistance of tilapia. At the conclusion of Trial 1, there were no differences in weight gain (WG) or feed efficiency (FE) among fish fed the three diets. However, fish fed the diet with GBA at 2% had significantly increased survival and noticeably elevated levels of plasma lysozyme compared to fish fed the basal diet or the diet with GBA at 1%. Similarly, at the conclusion of Trial 2, WG and FE were unaffected by GBA supplementation; however, fish fed the diet with GBA at 2% also exhibited elevated plasma lysozyme as well as significantly (P < 0.05) increased levels of extracellular superoxide anion production (EX-SOAP) by macrophages. Dendrogram analysis of denaturing gradient gel electrophoresis (DGGE) images detected a significantly different microbial community within the intestine of fish fed the diet with GBA at 2% compared to fish fed the basal diet and diet with GBA at 1%. None of the experimental diets resulted in significant improvements to survival after exposure to Streptococcus iniae due to within treatment variability. However, fish fed the diet with GBA at 2% did tend to experience reduced mortality (12.5%) as compared to fish fed the basal diet (35%). Thus, supplementation of GBA at 2% of diet did alter the gut microbiota of tilapia and enhanced immunological responses and disease resistance to S. iniae.

Equine influenza virus causes a mild, self-limiting upper respiratory disease in its natural host. In stark contrast, equine influenza viruses of the H7N7 subtype produce lethal infection in BALB/c mice. This dissertation explored the mechanism underlying the differential pathogenicity of the equine H7N7 influenza virus observed in horses and BALB/c mice. Initially, a comparative study of the pathogenesis was conducted in BALB/c mice inoculated intranasally with a representative isolate of either H7N7 or H3N8 subtype equine influenza virus. All H3N8 virus-infected mice survived the infection whereas 100% mortality was documented for the mice receiving the H7N7 virus by day 8 post infection. Both viruses replicated to a similar degree in the lungs at the early stages of infection. However, after day 2 post infection until the death of the mice, the pulmonary viral loads of the H7N7 group were significantly higher than those of the control, whereas the H3N8 virus was eventually eradicated from the mice at day 7 p.i. Correspondingly, a vigorous pro-inflammatory cytokine response in the lung was induced by the H7N7 virus but not the H3N8 virus, which reflected a desperate attempt by the host immune responses to restrain the overwhelming infection. The H7N7 virus was poorly sensitive to the innate immune containment, resulting in a significant higher cumulative mortality rate than that of the control virus in chicken embryos aged 9 days and older. On the contrary, in horses, replication of the paired viruses was completely cleared by the host immune responses at day 7 p.i. and the infections produced an acute yet non-lethal illness, albeit the H3N8 virus induced generally more pronounced clinical manifestations than the H7N7 virus. The clinical severity correlated to the difference in cytokine-inducing capacity between the two viruses in horses, as evidenced by the finding that the H3N8 virus triggered significantly higher levels of gene transcription of multiple key inflammatory cytokines in the circulation than those seen for the H7N7 virus. In addition, equine peripheral monocyte-derived macrophages were found to be a target of equine influenza virus and can support the productive replication of the virus in vitro.