Host defense against pathogenic organisms represents one of the most important and highly-conserved biological processes across the evolutionary timescale. The ability to detect, engulf and destroy particulates for either nutrition or host defense is conserved from single-celled protists to complex multicellular organisms. A central component of host defense is the recognition of invariant, conserved patterns on pathogenic organisms through the use of pattern recognition receptors (PRRs), such as macrophage receptor with collagenous structure (MARCO). MARCO modulates binding and phagocytosis of unopsonized microorganisms and particulates, tethers ligands to signalling complexes and enhances cellular adhesion. Current literature suggests these functions are mediated via the C-terminal scavenger receptor cysteine rich (SRCR) domain. The relative importance of this domain remains unclear, as other, closely-related scavenger receptors function independently of the SRCR domain via a shared lysine-rich motif. In chapter 3.1, we discovered and cloned a naturally-occurring transcript variant of MARCO which lacks the SRCR domain, termed MARCOII. We demonstrated that the SRCR domain is required for ligand binding and internalization and that MARCOII can form heteromeric complexes with MARCO and reduce receptor function. Furthermore, the SRCR domain enhanced TLR2/CD14-mediated pro-inflammatory responses to Streptococcus pneumoniae. Finally, it was demonstrated that the SRCR domain modulates MARCO-mediated cellular adhesion. In chapter 3.2, we used comparative phylogenetics to identify human specific mutations and residues undergoing positive selection in human MARCO. We demonstrated that humans possess a unique phenylalanine residue at position 282 that is polymorphic, with some humans encoding an ancestral serine residue. We also demonstrated that glutamine at position 452 is found in Denisovans, Neanderthals, and extant humans, but all other non-primate, terrestrial, and aquatic mammals possess an aspartic acid residue. We cloned the ancestral residues and loss-of-function mutations and demonstrated that these residues enhance ligand association and phagocytosis of Escherichia coli. / Thesis / Doctor of Philosophy (PhD) / Some of the most ancient mechanisms of host defense rely on invariant recognition of pathogens through the use of pattern recognition receptors, such as the macrophage receptor with collagenous structure (MARCO). MARCO plays an integral role to allow for specialized subsets of white blood cells to bind pathogens, activate signalling complexes and to bring pathogens inside the white blood cell for destruction. Current literature suggests the C-terminal Scavenger Receptor Cysteine Rich (SRCR) domain of MARCO is required for these processes. This remains under scrutiny, as other closely-related receptors have been shown to operate independently of the SRCR domain. Herein, we utilized a variant of MARCO which lacks the SRCR domain and patterns of evolution to confirm both that the SRCR domain is critical for receptor function and to discover novel sites within the human MARCO protein that play indirect, but important roles in receptor function.

Gene therapy can potentially treat a wide range of diseases ranging from inherited diseases to cancer. The successful use of nucleic acids to treat genetic diseases is limited by rapid capture and degradation of the nanoparticle by Kupffer cells in the liver. Scavenger receptors on the cell surface, capture both viral and non-viral nanoparticles leading to reduced efficacy. PEG-peptides were found to inhibit scavenger receptors on the surface of Kupffer cells by forming albumin nanoparticles when intravenously dosed. This work explores the development of potent, low-molecular weight PEG-peptide inhibitors. In order to study the in vivo activity of the nanoparticle, an in vivo assay was developed to directly assess the potency of inhibition. High molecular weight polylysine peptides (33.5 kDa) inhibited liver uptake with an IC50 of 18 μM. Incorporation of four leucine residues, to improve albumin binding, allowed for a decrease in PEG molecular weight and number of lysine residues, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys (7.4 kDa) that inhibited scavenger receptors with an IC50 = 20 μM. Further decrease in the PEG molecular weight resulted in the discovery of PEG2kDa- Cys-Tyr- (Leu-Lys4)3-Leu-Lys (4.4 kDa) with potency of 3 μM. The increase in potency could be attributed to a decrease in the zeta potential of the albumin nanoparticle resulting in more efficient scavenger receptor mediated uptake. Co- administration of PEG2kDa- Cys-Tyr-(Leu-Lys4)3-Leu-Lys with a stable PEGylated polyacridine DNA polyplex resulted in inhibition of rapid polyplex uptake by the liver with an IC50 = 11 μM. Other properties including spatial distribution of leucine, hydrophobicity and peptide length were also explored to determine their effect on liver uptake. Hydrophobic peptides resulted in the formation of micelles which were inactive as scavenger receptor inhibitors and exhibited increased liver uptake upon dose escalation. Reduction in the peptide length resulted in peptides that were not captured by the liver. Inhibition scavenger receptors has the potential to improve the efficacy of viral and non-viral nanoparticles. The findings of this work provide a framework for the development of PEG-peptide inhibitors capable of blocking live uptake of viral and non-viral nanoparticles.

Scavenger receptors are a large family of structurally diverse molecules that have been implicated in a range of biological functions. In this work, a newly identified multi-domain scavenger receptor-serine protease IrSRP-1 from the tick Ixodes ricinus is characterized. IrSRP-1 is related to the serine protease Sp22D from the mosquito Anopheles gambiae. IrSRP-1 is expressed mainly in the tick gut but also in hemocytes, Malpighian tubules, tracheas and ovaries of fully fed females. This was confirmed with Western blots and immunohistological labeling with antibodies raised against recombinantly expressed IrSRP-1 trypsine-like domain. According to acquired qRT-PCR profiles relative expression of IrSRP-1 is strongly up-regulated during female feeding and remains unchanged in ticks experimentally injected with various microbes. Functional characterization by RNA interference revealed that lowering IrSRP1 expression leads to a higher mortality rate during tick female feeding.

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spα, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by β-glucan but not by mannan. The K d of the rshCD5/(1→3)-β-D-glucan phosphate interaction is 3.7 ± 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1→3)-β-D- glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.

Recognition of non-self, pathogen-associated molecular patterns is a central component of host immune response to pathogens like viruses. Intracellular detection of viral nucleic acids leads to the production of type I interferons (IFN-I) and subsequent establishment of an antiviral state in infected and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN-I responses in infected cells, however, viral nucleic acids released from dying cells can stimulate IFN-I production in surrounding or distal uninfected cells. This thesis examines the mechanisms by which cells recognize extracellular viral nucleic acids and the subsequent downstream antiviral signaling. Class A scavenger receptors (SR-As) internalize extracellular viral double-stranded RNA (dsRNA) to mediate IFN-I responses, but little is known about extracellular viral DNA. We observed that extracellular DNA is recognized and internalized by SR-As in a manner like extracellular dsRNA. Furthermore, we established that SR-A1 is sufficient in mediating extracellular dsRNA-induced cellular responses and other nucleic acid receptors like SR-J1 and DEC-205 are dispensable. Finally, a direct interaction of RNA and DNA species was demonstrated with the coiled-coil collagenous domain of SR-A1, but not the scavenger receptor cysteine rich domain of SR-A6.We elaborated the role of SR-A1 by identifying the cellular processes activated through SR-A1 following uptake of extracellular dsRNA. Cytosolic sensors are essential in mediating an antiviral response to the endocytosed dsRNA, but the mechanism of endoplasmic release and cytoplasmic entry of dsRNA remains an enigma. We demonstrated that the lack of a dsRNA-channel, SIDT2, impaired the ability of the cells to mediate an antiviral response to extracellular dsRNA. Understanding host responses to extracellular viral nucleic acids will enable the development of novel vaccines and antiviral therapeutics against RNA and DNA viruses that efficiently counteract these responses in infected cells. / Thesis / Doctor of Philosophy (PhD) / Viral infections remain a threat to global health as new diseases continue to emerge. To develop effective vaccines and antivirals to combat viruses and alleviate human disease require a deeper understanding of virus-host interactions. Host cells identify virus-associated molecules to detect viruses and eliminate them whereas, viruses employ tactics to prevent the activation of the immune system. However, virus-induced cell lysis releases viral molecules that can stimulate immune responses in neighbouring uninfected cells. This thesis examines the mechanism by which cells respond to extracellular viral nucleic acids. We showed that a protein present at the cell surface called ‘class A scavenger receptor 1’ is sufficient to internalize extracellular viral nucleic acids, leading to immune responses. The response is impaired when a channel protein, SIDT2, is absent in the cells. Further work is necessary to understand how this knowledge can be harnessed to develop vaccines and antiviral therapeutics.

No description available.

<p> The Scavenger Receptor, Class B, Type I (SR-BI) is an integral membrane protein whose expression in the liver is critical to reverse cholesterol transport by mediating the selective uptake of HDL-derived cholesterol. SR-BI is expressed in a variety of tissues including bone marrow derived macrophages and foam cells in atherosclerotic lesions. We have explored the effect of eliminating SR-BI in leukocytes on advanced stages of atherosclerotic plaque development in apoE KO mice. We observed statistically significant cardiomegaly as a result of the elimination of SR-BI in bone marrow derived cells compared to controls (P=0.02). We report that the elimination of SR-BI in bone marrow derived cells in apoE KO mice induced to undergo atherosclerosis by feeding a high fat diet for four weeks leads to no significant difference in cross-sectional atherosclerotic plaque area at the aortic root (4.9±0.9x10^4 μm^2 when SR-BI-/- apoE-/- --> apoE-/- [n=9] and 5.5±0.9x10^4 μm^2 when SR-BI +/+ apoE-/- --> apoE -/- [n=12], P=0.68) or plaque volume through the aortic sinus (1.8±0.3x 10^7 μm^3 when SR-BI-/- apoE-/- --> apoE-/- [n=9] and 1.9±0.3x10^7 μm^3 when SR-BI +/+ apoE-/- --> apoE -/- [n=12], P=0.69). We demonstrate that macrophage SR-BI protein expression can be decreased by cholesterol associated with lipoproteins. Furthermore, we report that in Raw 264.7 macrophage-like cells the expression of SR-BI can also decrease in response to glucosamine treatment. The expression of SR-BI is decreased significantly in cells overexpressing SR-BI (1d1A[mSR-BI] cells [P=0.003]) due to treatment with glucosamine with increased protein mobility. We support this finding by demonstrating that this difference may be the result of altered glycosylation.</p> / Thesis / Master of Science (MSc)

Red blood cells (RBCs) are vital for oxygen delivery to tissues and constitute the vast majority of all cells in blood. After leaving the red bone marrow as mature cells, RBCs have a lifespan of approximately 120 days before they are removed from the circulation by macrophages, mainly in the spleen and liver. RBC transfusion is a common therapy in modern healthcare. Major surgery, numerous cancer treatments and other, often lifesaving, interventions would be unthinkable without available blood supply. For this reason, hospitals store donated RBCs in blood banks. The metabolic and structural changes that occur during prolonged storage of RBCs (the storage lesion) have been studied in detail in vitro and include oxidative stress, a reduction in glycolysis, increased membrane rigidity and shedding of microparticles from the RBC membrane. Stored RBCs share several features of senescent RBCs, but also with RBCs undergoing an apoptotic-like process called eryptosis. A consequence of the storage lesion is the fact that as much as 25% of stored RBCs could be rapidly removed from the circulation within 24 hours after transfusion. The mechanisms behind this rapid macrophage-mediated recognition and removal of stored RBCs, and its immunological consequences, remain largely unknown. Therefore, the aims of this thesis were to investigate if cryopreserved human RBCs induced an inflammatory response following autologous transfusion into healthy volunteers, and to further understand the mechanisms behind macrophage recognition of stored RBCs in vitro and in vivo. Autologous transfusion of two units of cryopreserved RBCs into healthy human recipients was found to be associated with an increased extravascular RBC elimination already at 2 hours after transfusion. However, there were no signs of an increased production of any of the investigated pro-inflammatory cytokines, indicating that an increase in the destruction of RBCs per se did not induce an inflammatory response. Eryptosis is a form of induced RBC death associated with an increased cytoplasmic Ca2+ uptake. We found that a subset of human RBCs increased their Ca2+ permeability during prolonged storage at +4°C. Using a murine model, to further understand how RBCs with an increased Ca2+ permeability were eliminated by phagocytic cells in the spleen, it was found that such RBCs were taken up by marginal zone macrophages and dendritic cells (DCs) in a manner distinct from that of naturally senescent RBCs. The DC population particularly efficient in this process expressed CD207 and are known for their ability to promote immunological tolerance. Eryptotic cell uptake was not regulated by the phagocytosis-inhibitory protein CD47 on the RBCs. To investigate how RBCs damaged during liquid storage are recognized and taken up by macrophages, a model to store and transfuse murine RBCs was developed. This storage model generated murine RBCs with several characteristics similar to that of stored human RBCs (i.e. loss of ATP, formation of RBC microparticles and rapid clearance of up to 35% of the RBCs during the first 24 h after transfusion). In vitro phagocytosis of human as well as murine stored RBCs was serum dependent and could be inhibited by blocking class A scavenger receptors using fucoidan or dextran sulphate. In conclusion, the findings of this thesis contribute to further understanding how changes inflicted to RBCs during storage direct the fate of these cells in their interaction with cells of the immune system after transfusion. The observation of an increased Ca2+ permeability of stored RBCs, and the possible recognition of such cells by tolerance-promoting DCs, in combination with the findings that class A scavenger receptors and serum factors may mediate recognition of stored RBCs, may result in novel new directions of research within the field of transfusion medicine.

In recent years, studies have revealed a central role for the intestine in regulation of lipid homeostasis and development of insulin resistance and type-2 diabetes. The function of intestinal Scavenger Receptor Class-B type-I remains unknown, however it is believed to play a role in dietary lipid uptake. Recently, our laboratory demonstrated a correlation between intestinal SR-BI expression and chylomicron secretion. We hypothesized that intestinal SR-BI is involved in chylomicron secretion and contributes to chylomicron oversecretion in insulin resistance. I first characterized chylomicron production in healthy and insulin resistant Syrian golden hamsters. Inhibition of SR-BI resulted in reduced postprandial chylomicron accumulation in plasma, and resistance to diet-induced hyperlipidemia and weight-gain. Lower postprandial triglyceride levels were also observed in SR-BI-/- mice. In summary, these data demonstrate a key role for intestinal SR-BI in chylomicron secretion and control of lipid homeostasis, implicating intestinal SR-BI in chylomicron overproduction in insulin resistant states.

In recent years, studies have revealed a central role for the intestine in regulation of lipid homeostasis and development of insulin resistance and type-2 diabetes. The function of intestinal Scavenger Receptor Class-B type-I remains unknown, however it is believed to play a role in dietary lipid uptake. Recently, our laboratory demonstrated a correlation between intestinal SR-BI expression and chylomicron secretion. We hypothesized that intestinal SR-BI is involved in chylomicron secretion and contributes to chylomicron oversecretion in insulin resistance. I first characterized chylomicron production in healthy and insulin resistant Syrian golden hamsters. Inhibition of SR-BI resulted in reduced postprandial chylomicron accumulation in plasma, and resistance to diet-induced hyperlipidemia and weight-gain. Lower postprandial triglyceride levels were also observed in SR-BI-/- mice. In summary, these data demonstrate a key role for intestinal SR-BI in chylomicron secretion and control of lipid homeostasis, implicating intestinal SR-BI in chylomicron overproduction in insulin resistant states.