Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor (GEF) that converts inactive eIF2 GDP-bound binary complexes into active eIF2 GTP-bound complexes that can bind initiator t-RNA molecules and ribosomes to begin translation. eIF2B is functionally divided into two subcomplexes: the catalytic core comprised of eIF2B epsilon and eIF2B gamma, and the regulatory core comprised of eIF2B alpha, eIF2B beta and eIF2B delta. While the catalytic subunits are responsible for exerting GEF activity, the regulatory subunits recognize eIF2 and respond to eIF2 alpha phosphorylation. Cellular stress, such as virus infection, inhibits host protein synthesis by activating specific kinases that are capable of phosphorylating the alpha subunit of eIF2, which can then sequester eIF2B to stall guanine nucleotide exchange by a currently unresolved mechanism. Importantly, we demonstrate that loss of eIF2B alpha or expression of a variant of the human eIF2B alpha subunit harboring a single point mutation (T41A) is sufficient to neutralize the consequences of eIF2 alpha phosphorylation, and render primary MEFs significantly more susceptible to vesicular stomatitis virus infection. To extend this analysis, we further exhibit the vital function of eIF2B alpha in protein synthesis through phenotypic studies in yeast. Here, we report that this subunit can sufficiently substitute for its yeast counterpart, GCN3, and reproduce similar growth phenotypes under normal and amino acid deprived conditions. In addition, the human eIF2B alpha-T41A variant was unable derepress GCN4 translation in response to an inhibitor of amino acid biosynthesis in yeast, an activity that requires sensitivity to phosphorylation of the yeast eIF2 alpha homolog, SUI2. Previously, we have demonstrated that vesicular stomatitis virus can infect and replicate to high levels in tumor cells. Moreover, these cells appear to contain defects in eIF2 alpha-mediated translational control, plausibly due to disregulation of eIF2B activity, which overcomes the inhibitory effects of eIF2 alpha phosphorylation. Our data suggest a role for eIF2B, specifically eIF2B alpha, in suppression of translation following virus infection, and imply that this complex may contribute to oncogenic transformation. These results emphasize the importance of eIF2B alpha in mediating eIF2 kinase translation inhibitory activity and may provide insight into the complex nature of viral oncolysis and cellular transformation.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In press. Paper 4: Manuscript.<strong></strong></p>

All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the Drosophila hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as Galleria mellonella. The Drosophila clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in Drosophila coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, in vivo when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as Galleria mellonella and Ephestia kuehniella, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

Probiotics can influence intestinal responses and mucosal immunity either directly or indirectly through transient modulation of the endogenous microenvironment or the immune system. During space travel, astronauts experience various physiological stresses including putting them at risk for infections or inappropriate immune responses. Macrophages and monocytes are a key cell type involved in innate immunity. The effects of dairy milk or soy milk base fermented with S. thermophilus ST5 in combination with either B. longum R0175 or L. helveticus R0052 on the cell line U937 and all-trans retinoic acid differentiated U937 were examined under normal gravity and simulated microgravity conditions, in order to screen for effects on monocytes and macrophages. Soy and milk ferments demonstrated the ability to modulate certain aspects of the innate immune system, both in normal gravity and in simulated microgravity. These probiotics affected U937 cells differently depending on differentiation stage (monocyte or macrophage) and whether or not the cells were tested in regular gravity or in simulated microgravity conditions. These results provide insight into effects on this aspect of innate immunity and may provide guidelines to potential in vivo administration. / UOIT

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

The over-expression of Down syndrome cell adhesion molecules (DSCAMs) is partially responsible for the mental retardation associated with Down syndrome. Previous work in our lab showed that a DSCAM homolog in C. elegans, Y32G9A.8, is expressed at all developmental stages and appears to be crucial for survival. In an effort to map the expression pattern, I used the Genome Sciences Centre’s primer design program (http://elegans.bcgsc.bc.ca/gfp_primers/) to design a GFP promoter fusion product that was used to monitor gene expression. The results indicate that Y32G9A.8 is expressed in the animal’s gut, suggesting that it may function in the worm’s innate immune response. I also designed a primer set to amplify the Y32G9A.8 transcript. RT-PCR of the entire Y32G9A.8 coding region resulted in a single product; there appears to be no alternative splicing. Although this gene shows homology to other N-CAMS, results indicate that this gene may function in the innate immune system of C. elegans.

One of the fundamental questions in mucosal immunology is how the intestine maintains tolerance to food antigens and commensal flora, and yet it is capable of mounting immune responses to pathogens. Peyers patches (PP) are lymphoid aggregates that are found in the small intestine and are the primary sites where adaptive immune responses are initiated in the intestine. An understanding of how PP cells regulate innate immune responses may provide information on how immune responses are regulated in the intestine. The toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) which provide a sensory mechanism for the detection of infectious threats. TLR9 recognizes bacterial DNA or synthetic CpG oligodeoxynucleotides (ODN). Cells that express TLR9 when stimulated with CpG ODN proliferate and produce Th1-like pro-inflammatory cytokines and upregulate co-stimulatory molecules. Because the intestine is constantly exposed to bacterial DNA from commensal flora, immune cells from the gut must have evolved mechanisms to modulate responses to TLR9 stimulation to prevent responses to harmless bacteria. Our hypothesis is that innate immune responses to the TLR9 agonist CpG ODN in Peyers patches (PP) are attenuated compared to other tissues such as blood or lymph nodes. This is due to local regulatory mechanisms unique to the intestinal microenvironment.<p> We conducted a number of experiments to test this hypothesis. We initially assessed the immunostimulatory activity of three available classes of CpG ODN in lymph nodes (LN), peripheral blood mononuclear cells (PBMC) and PP since this had not been done in ruminants. We found that CpG ODN induced strong IFNá, IFN-gamma, IL-12, lymphocyte proliferation and NK-like activity in LN and PBMC. In contrast, these responses were significantly less in PP stimulated with CpG ODN. We wondered whether the reduced responses of PP cells to CpG ODN were unique to the TLR9 agonist. For this reason we tested responses of cells from these tissues to poly (I:C), LPS, and single-stranded RNA, which are agonists for TLR3, TLR4, and TLR7/8 respectively. Additionally, we tested combinations of TLRs since others have reported that multiple TLR agonists may induce synergistic responses. All TLR agonists or their combinations either failed to induce detectable responses or the responses were significantly less in PP compared to other tissues. Thus we concluded that PP cells responses to TLR stimulation were attenuated. In all tissues tested, there were no synergistic responses (IFN-alpha, IFN-gamma and lymphocyte proliferation) following stimulation with combinations of agonists. However, there was inhibition of PBMC responses when TLR7/8 agonists were combined with CpG ODN (TLR9 agonist). Importantly, TLR7/8 agonists reduced the CpG-induced proliferative responses in purified blood B cells. Interestingly, ovine B cells constitutively expressed TLR7/8 and TLR9 mRNA, suggesting the potential for cross-talk between the receptors.<p> Interestingly, cell from all isolated tissues [ileal PP (IPP), jejunal PP (JPP), mesenteric LN (mLN) and PBMC] expressed similar levels of TLR9 mRNA, suggesting that the reduced responsiveness to CpG ODN stimulation in PP was not due to a lack of TLR9 expression.<p> Surprisingly, we observed that PP cells spontaneously secreted significant amounts of the immunoregulatory cytokine IL-10. Furthermore, we confirmed that CD21+ B cells were the source of the IL-10. We then examined the role of IL-10 in regulating IFN and IL-12 responses in PP. Neutralization of IL-10 resulted in a significant increase in the numbers of CpG-induced IFNá-secreting cells detected and in IFN-gamma and IL-12 production by PP cells (both follicular and interfollicular lymphocytes). Similarly, depletion of the CD21+ B cells resulted in significant increases in IFNá, IFN-gamma and IL-12 responses. These observations support the conclusion that IL-10-secreting PP CD21+ B cells suppress innate immune responses in PP. Further characterization by flow cytometry revealed that these cells were CD1b-CD5-CD11c-CD72+CD21+ IgM+ B cells. We have proposed that these IL-10-secreting PP CD21+ B cells are a novel subset of regulatory B cells (Bregs).<p> Finally, we examined the capacity of IL-10 secreting B cells (Bregs) to respond to CpG ODN. To achieve this, we compared CD21+ B cells from blood and JPP. Unlike blood CD21+ B cells, CD21+ B cells from JPP proliferated poorly in response to CpG ODN. Moreover, PP CD21+ B cells, unlike blood CD21+ B cells, do not secrete IgM or IL-12 in response to CpG stimulation, although both PP and blood CD21+ B cells express similar level of TLR9 mRNA. Neutralization of IL-10 did not enhance CpG-induced proliferative responses in PP CD21+ B cells. Thus IL-10 does not play a direct role in the hyporesponsiveness of PP CD21+ B cells to CpG ODN. To further explore the mechanism by which PP Bregs fail to respond to CpG ODN stimulation, we used a kinome analysis to determine whether the TLR9 pathway was functional in PP Bregs compared to blood CD21+ B cells. We observed that peptides representing critical adaptor molecules downstream of TLR9 such as IRAK1, TAK1, Casp8, p-38 MAPK, JNK, FOS, IKKá, NF-KB-p65 were not phosphorylated in JPP CD21+ B cells following CpG ODN stimulation. However, in blood CD21+ B cells stimulated with CpG ODN, the same peptides on the array were all highly phosphorylated leading to a functional TLR9 signaling pathway. Thus PP Bregs have evolved mechanisms by which the TLR9 signaling pathway is not activated following exposure to the TLR9 agonist, CpG ODN.<p> In conclusion, we clearly demonstrated that TLR9-induced responses in cells from PP are significantly attenuated. This is a consequence of PP CD21+ B cells (Bregs) that spontaneously secrete IL-10, which in turn conditions an anti-inflammatory environment in this tissue leading to poor cytokine responses to the TLR9 agonist, CpG ODN. Additionally, we show that Bregs are unresponsiveness to TLR9 stimulation. This unresponsiveness is due to regulatory mechanisms in Bregs leading to a dysfunctional TLR9 signaling pathway. These may represent strategies by which PP dampen innate responses to pathogen-associated molecular patterns (PAMPs) in intestinal immune tissues to maintain intestinal immune homeostasis. These conclusions are consistent with our hypothesis that TLR responses in PP cells are attenuated, and this is due to B cell-mediated regulatory mechanisms that are unique to the intestinal microenvironment.

Severe malaria infections cause almost 1 million deaths annually, mostly among non-immune African children. The pathogenesis of severe malaria is poorly understood. It is increasingly appreciated that while host innate immune responses such as inflammation and phagocytosis are critical for control of parasite replication, they can become dysregulated and contribute to severe disease. The goals of this work were: (1) to characterize inflammatory responses to malaria by defining their relationship to phagocytosis and identifying novel molecular mediators, and (2) to evaluate the utility of biomarkers of inflammation and other host responses for predicting outcome in severe malaria infection. Using an in vitro model of the malaria-macrophage interaction, inflammatory and phagocytic responses to Plasmodium falciparum were found to be partially coupled. Activation of Toll-like receptors (TLRs) by purified parasite components increased internalization of parasitized erythrocytes, but uptake of parasitized erythrocytes did not require TLRs, nor did it trigger cytokine production via TLRs or other receptors. Two candidate molecules – Triggering receptor expressed on myeloid cells-1 (TREM-1) and Chitinase-3 like-1 (CHI3L1) – did not appear to critically modulate inflammation to malaria in vitro or in murine models. However, exogenous TREM-1 activation enhanced the pro- inflammatory nature of the response to P. falciparum, with potential implications for malarial-bacterial co-infection. CHI3L1-deficient mice showed a trend towards earlier death in experimental cerebral malaria, suggesting that CHI3L1 may protect against severe malaria; however, further investigation in more informative models is required. Admission levels of plasma TREM-1, CHI3L1, and other biomarkers of inflammation and endothelial activation were increased in Ugandan children with severe malaria. Simple combinations of these biomarkers predicted mortality among severe malaria patients with high accuracy, warranting larger validation studies. Taken together, these findings identify host responses as putative targets for adjunctive therapies, and suggest the utility of host biomarker combinations as prognostic tests for severe malaria.