Periodontal disease is a chronic inflammation of the gingiva and periodontium that leads to progressive destruction and irreversible damage to the supportive structures of the teeth. It affects nearly half of the United States population and is a particular risk factor in adults older than 65 years of age. Oral microorganisms assemble in plaque as a polymicrobial biofilm and Porphyromonas gingivalis, an important secondary colonizer in oral biofilms, has been implicated in periodontal disease. Although the protective functions of various salivary molecules such as antimicrobial proteins have been delineated, lipids present in saliva and on the oral mucosa have been largely ignored and there is growing evidence that the role of lipids in innate immunity is more important than previously realized. In fact, recent studies suggest that sphingoid bases and fatty acids, which exhibit potent broad spectrum antimicrobial activity against a variety of bacteria and fungi, are likely important innate immune molecules involved in the defense against oral bacterial and fungal infections. However little is known about their spectrum of activity or mechanisms of action. In addition, the effects of these lipids that are endogenous to the oral cavity have not been explored against oral bacteria. In this study I hypothesized that oral mucosal and salivary lipids exhibit dose-dependent antimicrobial activity against P. gingivalis and alter cell morphology and metabolic events. To test this hypothesis, I first examined the effects of two fatty acids: sapienic acid and lauric acid, and three sphingoid bases: sphingosine, dihydrosphingosine, and phytosphingosine, against a variety of gram-positive and gram-negative bacteria including P. gingivalis. Using broth microdilution assays to determine minimum inhibitory and minimum bactericidal concentrations, I show that antimicrobial activity against bacteria is dose-dependent, lipid specific, and microorganism specific. Kill kinetics were also variable across each bacteria-lipid combination. Upon examination of select bacteria-lipid combinations via scanning and transmission electron microscopy, different morphologies were evident across all treatments, demonstrating differential activity of each lipid for a particular bacterium as well as for each bacterium across different lipids. In addition, all sphingoid bases and fatty acids were taken up and retained in association with P. gingivalis cells and could be extracted along with bacterial lipids and separated using thin layer chromatography. Using a combination of two-dimensional in-gel electrophoresis and Western blots followed by mass spectroscopy and n-terminus degradation sequencing, I show that sapienic-acid treatment induces a unique stress response in P. gingivalis, as evidenced by the ability of P. gingivalis to upregulate a set of proteins involved in fatty acid biosynthesis metabolism and energy production, protein processing, cell adhesion, and virulence. Finally, utilizing flow cytometry and confocal microscopy, I assessed the effects of oral antimicrobial lipids against a representative host cell and describe oral lipid concentrations that are both antimicrobial to P. gingivalis cells and non-cytotoxic to the representative host cells tested. Combined, these data strongly suggest that sphingoid bases and fatty acids found within the saliva and on oral mucosa likely do contribute to the innate antimicrobial activity of saliva, mucosal surfaces, and skin and this dose-dependent activity is both lipid specific and bacteria specific. This information adds to current knowledge of the innate functions of endogenous lipids in the oral cavity. With bacterial resistance to current antibiotics increasing, the exploration of new antimicrobial agents is important and these lipid treatments may be beneficial for prophylactic treatments or therapeutic intervention of infection by supplementing the natural immune function of endogenous lipids on skin and other mucosal membranes.

Leishmania are obligate intracellular protozoan parasites that are inoculated into human skin while a sand fly vector takes a blood meal with the resulting disease coined leishmaniasis. The twenty plus species of Leishmania known to cause human disease are found throughout tropical and subtropical regions of the world. Leishmaniasis affects at least eighty-eight countries with three hundred and fifty million people at risk for infection, resulting in an estimated seventy thousand deaths annually. Different species of Leishmania have developed distinct methods for host defense evasion, leading to a wide spectrum of pathologies within humans. Prior studies of macrophage infections with Leishmania have shown global changes in macrophage mRNA expression. We hypothesized miRNAs are important modifiers of mRNA changes during Leishmania infection. Analysis of miRNA expression patterns revealed that changes were detected primarily during macrophage infection with the low virulent logarithmically growing promastigotes. Profiling studies of mRNA and miRNA changes upon infection with promastigotes in logarithmic growth revealed a decrease in miR-200b and increase in miR-744 levels whereas infections with the highly virulent metacyclic promastigotes revealed a decrease in miR-708 levels. Furthermore, microarray studies revealed differences in macrophage mRNA levels between macrophages infected with the low virulent promastigotes verses the highly virulent promastigotes. Correlative studies between miRNA and mRNA changes suggested some of Leishmania induced changes in mRNA levels may be modified by miRNAs. The importance of Toll-like receptors (TLR) in detection of microbial products has been well-documented. Leishmania infection is known to initiate signaling through TLRs 2, 3, 4 and 9, of which TLRs 2, 4 and 9 signal through the adaptor molecule MyD88. We found that miR-200b, a microRNA decreased by infection of macrophages with the low virulent Leishmania promastigotes, regulates signaling through the TLR4 pathway by targeting and repressing MyD88 transcript levels. Furthermore, we have shown that MyD88 repression results in the decreased expression of the downstream effector molecules IL-6, CXCL9 and TNFΑ upon challenge with a TLR4 ligand. The suppression of miR-200b during Leishmania infection could serve to up-regulate inflammatory responses induced through TLR4 and other MyD88 dependent TLRs. This may be responsible, in part, for the decreased virulence of logarithmically growing compared to metacyclic promastigotes. Furthermore, low levels of inflammation may promote parasite survival by promoting the influx of inflammatory phagocytic cells to the site of infection in which the highly virulent parasites can survive. Microarray studies revealed a remarkable increase in expression of metallothionein (MT) transcripts in macrophages infected with low virulent promastigotes but not in macrophages infected with the highly virulent promastigotes. To explore a possible mechanistic role for metallothioneins in leishmaniasis, we used knock-out mice for MT-1 genes. Bone-marrow derived macrophages from MT-1 knock-out mice (MT-KO) generated higher levels of reactive oxygen species upon incubation with Leishmania promastigotes. Consistently, the initial ROS-induced killing of promastigotes, which occurs during the first hours of infection, was greater twenty-four hours after infection of MT-1 KO bone-marrow macrophages than in our wild type controls. Overall, data presented in this thesis documents changes to macrophage mRNA and miRNA expression patterns upon infection with Leishmania promastigotes that correspond to the overall parasite survival in the host macrophage.

Marine animals live and thrive in a literal sea of microorganisms, yet are often able to maintain specific associations that are largely dictated by the environment, host immunity and microbial interactions. Animal-associated microbiomes include bacteria and viruses that vastly outnumber host cells, especially in the gut environment, and are considered to be integral parts of healthy, functioning animals that act as a metaorganism. However, the processes underlying the initial establishment of these microbial communities are not very well understood. This dissertation focuses on the establishment of a well-known developmental animal model, Ciona intestinalis (sea squirt), to study the establishment and maintenance of a stable gut microbiome. Generation of a new model for studying microbial colonization of the gut requires the ability to rear Ciona in the absence of microbes (i.e., germ-free). This dissertation describes the establishment of a germ-free technique for rearing Ciona and the methods utilized for bacterial exposure and colonization. Additionally, to determine the spatial structure of the gut microbiome, viral and bacterial communities within the three main gut compartments (stomach, midgut, hindgut) of Ciona from San Diego, CA, were assessed. The viral community was dominated by phages (viruses infecting bacteria), and numerous prophages (phages integrated into bacterial genomes) matching sequences found in bacteria belonging to the Ciona microbiome were detected within the active viral fraction. To determine the prevalence of prophages within the Ciona microbiome, a total of 70 bacteria cultured from the gut were tested, and 22 isolates were found to possess inducible prophages. When co-cultured with other bacteria, these induced prophages were capable of lytic infection of other members of the microbiome, often exhibiting broad host ranges. The dynamic interactions of gut bacteria and phages were explored further with the isolation and characterization of a novel Shewanella phage-host system from the adult Ciona gut. Lytic phage infection resulted in an increase in biofilm formation correlating with the release of extracellular DNA, a process that was also observed to a lesser degree in control cultures as a result of spontaneous prophage induction. Furthermore, addition of the Ciona immune protein VCBP-C to static cultures of this Shewanella sp. 3313 also enhanced biofilm formation; a similar phenomenon was noted in another bacteria, a Pseudoalteromonas sp. 6751. Interestingly, both of these isolates contained inducible prophages and binding of the VCBP-C protein to these lysogenic strains was found to influence prophage induction in vitro. Colonization of the gut in vivo also correlated with differential up-regulation of VCBP-C expression in germ-free animals and a subsequent induction of prophages. This dissertation makes an important contribution to the symbiosis field by developing a new model system in which novel aspects of host-microbe interactions can be investigated. The discovery that an innate immune effector can influence bacterial biofilms and result in the induction of prophages capable of lytic infection of other co-occurring bacteria reveals a previously unrecognized intersection between secretory immune molecules and phages in shaping the microbiome. These findings establish Ciona as a relevant and tractable model for studying trans-kingdom interactions during colonization of the gut epithelium.

<p>Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.</p><p>We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of <em>E. coli</em> decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.</p><p>Type 1 fimbria-expressing <em>E. coli </em>adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of <em>E. coli</em>, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production <em>per se</em> is important.</p><p>Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.</p>

<p>Prophenoloxidase (proPO) is a key enzyme for generation of melanin and is activated by the proPO activating enzyme (ppA) to its active form, PO. The active ppA was purified and cloned from crayfish hemocytes and it is a typical serine proteinase containing a clip, a proline-rich, and a glycine-rich domain. A recombinant protein containing the clip-domain, with homology to horseshoe crab big defensin and mammalian â-defensin, had antibacterial activity <i>in vitro</i> against gram-positive bacteria.</p><p>The proPO activating system (proPO system) is triggered by lipopolysaccharides (LPS) or â-1,3-glucans. An LPS and â-1,3-glucan binding protein (LGBP) was characterized from crayfish hemocytes. The results of an LGBP antibody inhibition assay suggest that LGBP is directly involved in the proPO system.</p><p>The primary structure of a crayfish masquerade-like (mas) protein has homology to serine proteinases except for a substitution within the catalytic triad, which renders it without proteinase activity. The crayfish mas-like protein has also binding activity to various gram-negative bacteria and yeast. When the mas-like protein binds to microorganisms, it is processed by a proteolytic enzyme. The mas-like protein exhibited cell adhesion and opsonic activities suggesting that it plays a role in defense against parasites.</p>

Pre-B Cell Colony-Enhancing Factor (PBEF) is a cytokine-like molecule that functions as a nicotinamide phosphoribosyl transferase (Nampt) in a salvage pathway of NAD biosynthesis. PBEF has well-characterized activity as an extracellular inflammatory mediator and has been proposed to signal through the insulin receptor (IR). As neutrophils are key effectors of the innate immune response to infection and injury, we hypothesized that PBEF promotes pro-inflammatory function in neutrophils and that these pro-inflammatory effects may occur through interactions with the neutrophil IR or through PBEF���s enzymatic Nampt activity. Our studies focused on two important facets of neutrophil inflammatory function: their ability to generate reactive oxygen species (ROS) and undergo constitutive apoptosis. We found that, although PBEF does not activate oxidative burst on its own, it primes for ROS generation through the NADPH oxidase. PBEF promotes membrane translocation of cytosolic NADPH oxidase subunits p40phox and p47phox, but not p67phox, induces p40phox phosphorylation and activates Rac. Priming, translocation and phosphorylation are dependent on activation of p38 and ERK mitogen activated protein kinases. PBEF priming of neutrophils occurs independent of its Nampt capacity or of interactions with IR. We next investigated the effects of PBEF on neutrophil constitutive apoptosis. Our lab previously established that extracellular PBEF delays neutrophil apoptosis. Accordingly, we next investigated the mechanism through which this delay was occurring. PBEF-induced delayed apoptosis was enhanced in the presence of Nampt substrates, and NAD alone could delay apoptosis to an extent comparable to PBEF. Delayed apoptosis was blocked by a Nampt inhibitor and was lacking when a mutated PBEF deficient in Nampt activity was utilized. The cell-surface NAD glycohydrolase, CD38, can convert NAD to cyclic ADP-ribose (cADPR). Blocking CD38 activity with a blocking antibody partially reversed the delay of apoptosis induced by PBEF in conjunction with its substrates, and delayed apoptosis could be achieved by addition of the CD38 product cADPR. Finally, we found that delayed apoptosis induced by PBEF did not involve IR. These results indicate that PBEF can prime for enhanced oxidative burst and delay apoptosis in neutrophils, and that these phenomena occur through distinct mechanisms.

Bovine Spongiform Encephalopathy (BSE) belongs to a group of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) which affect many species. From 1986 more than 184,000 cattle in the UK have been confirmed to be infected with this disease, and in Canada total losses to the economy reached $6 billion. This study examines the gene expression in three major innate immunity components: complement system, toll-like receptors, interleukins, and selected proteins of their signaling pathways. Quantitative real time polymerase chain reaction analyses were performed on caudal medulla samples to identify differentially expressed genes between non-exposed and orally challenged animals. In general, immune genes were down-regulated in comparison to non-challenged animals during first 12 months of disease with a tendency to be up-regulated at terminal stage of BSE. The results from this study provide a basis for further research on the mechanisms modifying immune responses and altering progression of the disease. / Animal Science

Mastitis, or mammary gland inflammation, causes the greatest loss in profit for dairy producers. Mastitis susceptibility differs among cows due to environmental, physiological, and genetic factors. Prior research identified a genetic marker in a chemokine receptor, CXCR1, associated with mastitis susceptibility and decreased neutrophil migration. Current research seeks to identify reasons behind mastitis susceptibility by validating this model through in vivo challenge with Streptococcus uberis and studying specific mechanisms causing impaired neutrophil migration. Holstein cows with GG (n=19), GC (n=28), and CC (n=20) genotypes at CXCR1+777 were challenged intramammarily with S. uberis strain UT888. After challenge 68% of quarters from GG genotype, 74% from CC genotype and only 47% from GC genotype cows had ≥10 colony forming units/ml S. uberis for at least two sampling time points (P<0.05). However, among infected cows, number of S. uberis, somatic cell count, rectal temperature, milk scores and mammary scores were comparable among genotypes throughout infection. These findings suggest that cows with GC genotypes may be more resistant to S. uberis mastitis, but have similar responses if infected. To better understand the mechanisms associated with disease resistance, migration patterns in neutrophils from cows with different CXCR1+777 genotypes were evaluated. Neutrophils from cows with GG (n=11) and CC (n=11) genotypes were isolated and stimulated with zymosan activated sera (ZAS). Cells were fixed and stained for F-actin and evaluated for F-actin content, distribution, and cell morphology. Neutrophils from CC cows had significantly lower average F-actin polymerization than GG cows v (P=0.05). Directed migration of neutrophils from GG (n=10) and CC (n=10) genotypes was imaged and tracking data was analyzed for individual cells. Cells from GG genotype traveled further on an X axis and had higher X/Y movement towards IL8 compared to CC genotype, meaning they moved more directly towards IL8. Our findings suggest lower F-actin polymerization in combination with lower ability to directly move towards IL8 could impair neutrophil response to infection in cows with a CC genotype and may contribute to increased mastitis susceptibility. Finding what makes certain cows more susceptible to mastitis could lead to strategies aimed at improved prevention and treatment of mastitis.

This thesis focuses on enteroviral effects on human pancreatic islets. Most knowledge of viral effects on host cells relies on studies of immortalized cell lines or animal models. The islets represent a fundamentally different and less well studied cellular host. Also, enterovirus has been implicated in the etiology of type 1 diabetes (T1D). We show that when enterovirus replicates in human islets it activates innate immunity genes and induces secretion of the chemokines MCP-1 and IP-10. An important difference in activation of innate immunity by replicating EV and synthetic dsRNA is suggested, since the chemokine secretion induced by EV infection but not by dsRNA is reduced by female sex hormone. We also demonstrate a direct antiviral effect of nicotinamide, and even though this substance failed to prevent T1D in a large-scale study, this finding could have implications for the treatment/prevention of virus- and/or immune-mediated disease. We also had access to human pancreata from two organ donors with recent onset T1D and several donors with T1D-related autoantibodies, which gave us the opportunity to study ongoing pathogenic processes at and before the onset of T1D. Despite this, we could neither confirm nor reject the hypothesis that EV is involved in T1D development. Several observations, such as ultrastructural remodeling of the beta cell, activation of innate immunity, and immunopositivity to EV capsid protein 1, supported an ongoing virus infection, but direct evidence is still lacking. An interesting finding in the donors with recent onset T1D was that the islets were positively stained for insulin, but did not secrete insulin in response to glucose-stimulation. A similar effect was observed in EV-infected islets in vitro; EV destroyed islet function and insulin gene expression, but the islets still stained positive for insulin. This may be indicative of that a functional block in addition to beta cell destruction is involved in T1D pathogenesis. In conclusion, these studies of EV in isolated human islets in vitro support that this virus can cause T1D in vivo, but future studies will have to show if and how frequently this happens.

Prophenoloxidase (proPO) is a key enzyme for generation of melanin and is activated by the proPO activating enzyme (ppA) to its active form, PO. The active ppA was purified and cloned from crayfish hemocytes and it is a typical serine proteinase containing a clip, a proline-rich, and a glycine-rich domain. A recombinant protein containing the clip-domain, with homology to horseshoe crab big defensin and mammalian â-defensin, had antibacterial activity in vitro against gram-positive bacteria. The proPO activating system (proPO system) is triggered by lipopolysaccharides (LPS) or â-1,3-glucans. An LPS and â-1,3-glucan binding protein (LGBP) was characterized from crayfish hemocytes. The results of an LGBP antibody inhibition assay suggest that LGBP is directly involved in the proPO system. The primary structure of a crayfish masquerade-like (mas) protein has homology to serine proteinases except for a substitution within the catalytic triad, which renders it without proteinase activity. The crayfish mas-like protein has also binding activity to various gram-negative bacteria and yeast. When the mas-like protein binds to microorganisms, it is processed by a proteolytic enzyme. The mas-like protein exhibited cell adhesion and opsonic activities suggesting that it plays a role in defense against parasites.