Walleye (Sander vitreus M.) are valuable in commercial and recreational fisheries and are affected by bacterial, fungal and viral disease. Pattern recognition receptors (PRRs) are germline-encoded and constitutively expressed and bind non-self or altered-self for immune recognition. Walleye were hypothesised to have circulating PRRs that were capable of binding diverse pathogens. These PRRs were hypothesised to increase with infection, be distributed in immunologically relevant tissues and to be strain and age specific. PRR binding was measured by affinity chromatography, plasma binding assays,SDS-PAGE, Western blots, ELISA, PCR, and immunohistochemistry. ELISA and affinity chromatography assays were developed in rainbow trout (Oncorhynchus mykiss) with known PRRs. Trout ladderlectin was confirmed as a PRR binding viral hemorrhagic septicemia virus (VHSV). These techniques were adapted to walleye using Flavobacterium columnare, chitin, VHSV and Sepharose resin. A 22 kDa protein bound to F. columnare, a 17 kDa protein bound to chitin and a 34 kDa protein bound to VHSV were identified as similar to bass apolipoprotein, carp C3 and rainbow trout intelectin, respectively. PCR and 3'-RACE-PCR were used to generate nucleotide sequence to confirm identity of walleye apolipoprotein and mannose-binding lectin (MBL)-like protein from the intelectin-like sequence. Two rabbit polyclonal antibodies were raised to 34 and 67 kDa MBL amino acid sequences and used to verify MBL-like protein as a PRR for VHSV. Healthy walleye MBL-like protein plasma concentration was 7.5 ng/ml. Significant differences were found between geographically distant strains of walleye. An ELISA demonstrated that MBL-like protein had significant differences in binding affinity between multiple strains of VHSV and different viruses found in Ontario. MBL-like protein plasma levels increased with initial infection of naïve fish with waterborne and IP VHSV (107 pfu) but did not change with IP reinfection. Previous infection with VHSV significantly decreased walleye mortality. IHC of walleye shows MBL-like protein is distributed in epithelial surfaces, primarily skin, oropharynx, gill, gastrointestinal system, renal nephrons, connective tissue of gonads and plasma. There was no qualitative difference in MBL-like protein tissue distribution in healthy and VHSV-infected walleye. This is the first evidence for fish lectins binding viruses.

Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that mediate host-responses to pathogens. In mammals, TLR ligands promote cellular activation and the production of cytokines. Several TLR ligands have been employed prophylactically for the control of bacterial or viral diseases in the mouse model. However, the TLR-mediated responses in chickens have not been well described. Importantly, the utility of TLR agonists for the control of viral pathogens, such as avian influenza virus (AIV), has not been fully explored in chickens. To this end, the studies described in this thesis characterized the kinetics of in vivo responses in chickens to the TLR4 ligand lipopolysaccharide (LPS) and the TLR21 ligand CpG ODN. It was demonstrated that both of these ligands induced the up-regulation of several immune system genes in the spleen, including those associated with pro-inflammatory and antiviral responses, as well antigen presentation. By harnessing the immunostimulatory properties of TLR ligands, it was also demonstrated that the prophylactic administration of either poly I:C (a TLR3 ligand), LPS or CpG ODN may confer immunity to a low pathogenic avian influenza virus, as determined by a reduction in both oropharyngeal and cloacal virus shedding in infected birds. Furthermore, transcriptional analysis of genes in the spleen and lungs identified interleukin (IL)-8, interferon (IFN)-α and IFN-γ as correlates of immunity. In conclusion, TLR ligands may modulate several aspects of the chicken immune system to induce an anti-viral state, thereby conferring immunity to AIV.

No description available.

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.

Background During the first stages of development human infants are either fed human milk or human milk substitutes (infant formulas). The composition of infant formulas and human milk differ drastically, including a difference in protein constituents and bacterial load. Due to the high global frequency of infant formula use, the humanization of infant formulas to better reflect the complex nature of human milk is warranted. To better understand the role of human milk components, the fate and function of a key bacterial sensor in human milk, soluble CD14, was determined. Additionally, the microbiome of human milk was analyzed from a metagenomic standpoint in an attempt to determine which types of bacteria are present in human milk and what their potential biological function might be. Results In rodent models, ingested sCD14 persisted in the gastrointestinal tract and was transferred intact into the blood stream. Once transferred to the blood, ingested sCD14 retained its ability to recognize lipopolysaccharide and initiate an immune response in pups. This transfer of sCD14 across the epithelial barrier was also observed in human cells in vitro, where it appears to be dependent on Toll-like receptor 4. Using Illumina sequencing and the MG-RAST pipeline, the human milk metagenome of ten mothers was sequenced. DNA from human milk aligned to over 360 prokaryotic genera, and contained 30,128 open reading frames assigned to various functional categories. The DNA from human milk was also found to harbor immune-modulatory DNA motifs that may play a significant role in immune development of the infant. Conclusions Given the complex nature of human milk in comparison to its bovine or plant based substitutes, the results presented in this thesis warrant future modification of infant formulas to include non-nutritive bioactive components. Current human milk components not yet present in infant formulas include the diverse microbiome of human milk, the immune-modulatory DNAs which those microbes harbor, and bioactive human proteins such as sCD14.

Surfactant protein D (SP-D) is an important innate immune collectin involved in uptake and clearance of microbes and allergens in the lungs. SP-D has been shown to ameliorate allergic asthma reactions in mice; however, the mechanisms for this are not fully understood. We investigated the role of SP-D in the uptake and clearance of the model allergen ovalbumin (OVA) by macrophages. We discovered that SP-D does not bind OVA but binds fractions with contaminating proteins; ovomucin and ovomacroglobulin. We extended these findings to show that SP-D binds human alpha-2-macroglobulin (A2M) in its cleaved or intact state, in a concentration-, calcium-, and carbohydrate-dependent manner. A2M increases the innate immune potential of SP-D by increasing its ability to agglutinate the bacteria Escherichia coli and Bacillus subtilis. We found that SP-D does not increase the uptake of OVA by murine macrophage cell lines, or by alveolar macrophages in vivo in BALB/cJ mice.

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.

Surfactant protein D (SP-D) is an important innate immune collectin involved in uptake and clearance of microbes and allergens in the lungs. SP-D has been shown to ameliorate allergic asthma reactions in mice; however, the mechanisms for this are not fully understood. We investigated the role of SP-D in the uptake and clearance of the model allergen ovalbumin (OVA) by macrophages. We discovered that SP-D does not bind OVA but binds fractions with contaminating proteins; ovomucin and ovomacroglobulin. We extended these findings to show that SP-D binds human alpha-2-macroglobulin (A2M) in its cleaved or intact state, in a concentration-, calcium-, and carbohydrate-dependent manner. A2M increases the innate immune potential of SP-D by increasing its ability to agglutinate the bacteria Escherichia coli and Bacillus subtilis. We found that SP-D does not increase the uptake of OVA by murine macrophage cell lines, or by alveolar macrophages in vivo in BALB/cJ mice.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Inflammatory Bowel Diseases (IBD) result from the nexus of a genetic predisposition, dysregulated immunologic insult against commensal microflora, and an environmental trigger. The intestinal epithelium is a single cell layer that separates a highly active mucosal immune system from a large antigenic load in the intestinal lumen. Innate immune recognition combined with a highly regulated adaptive immune response maintains this tolerance. The intestinal epithelium in collusion with antigen presenting cells primarily modulates this activity. In this thesis, we show that, in response to DNA isolated from bacteria, innate toll like receptor 9 (TLR9) activation in intestinal epithelial cells modulates both arms of the immune system, to regulate intestinal homeostasis, and through this mechanism, Bifidobacteria breve DNA exerts its anti-inflammatory function. / Experimental Medicine