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Macrophage integrin aDb2 promotes macrophage retention and accumulation within inflamed tissue, a key event in development of chronic inflammation. Recently, the P5 peptide was identified as a specific inhibitor for integrin aDb2 interaction with 2-(ω-carboxyethyl) pyrole (CEP), a ligand at inflammatory sites. This thesis aims to identify integrin aD I-domain amino acids involved in binding P5 peptide and likewise to CEP. We propose that non-conserved, basic amino acids of the integrin aDb2 I-domain are responsible for binding to P5 peptide and likewise to CEP. Eight amino acids were analyzed by generating six mutant aD I-domains: K180[A], R189[Q], K205[L], HHK223-225[NIT], K233[A], and K246[A]. Mutagenic constructs were created using PCR site-directed mutagenesis, then transformed into E.coli BL21 cells for IPTG-induced protein expression. Of the 6 mutant I-domains analyzed, amino acid K246 was critical in binding to P5 peptide and CEP through ForteBio Protein-Protein Assay, as well as to CEP by cell adhesion assay.

<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)

Salmonella is a Gram-negative intracellular pathogen that causes gastroenteritis and typhoid fever in humans. Salmonella can survive and replicate within host cells and has adapted several mechanisms to evade host immune defenses. The innate immune system plays an important role as a first-line of defense against pathogens such as Salmonella, and is mediated in part by toll-like receptors (TLRs). TLRs recognize fundamental components of pathogenic microorganisms and activation of TLRs leads to downstream signaling cascades eventually resulting in the expression of pro-inflammatory cytokines (4) and also has a role in activating adaptive immunity through presentation of antigens to lymphocytes (86). There are several lines of evidence that suggest that TLR activation may have therapeutic potential in therapies against infectious disease and several TLR agonists have been shown to protect against both bacterial and viral infection in mice (7; 8; 38; 66; 75; 84; 89; 121). To understand how TLR-agonist treatment of host cells affects Salmonella pathogenesis, RAW 264.7 murine macrophages were treated with the TLR agonists liposaccharide (LPS), poly(I:C), peptidoglycan, and CpG-ODN. Treatment of macrophages with all TLR-agonists results in increased phagocytosis of Salmonella compared to control-treated macrophages. These increases in phagocytic activity, however, do not enhance macrophage anti-microbial activity, since Salmonella infection of TLR-treated macrophages results in increased intracellular replication compared to control-treated cells. Infection with Salmonella mutants indicates that increased intracellular replication of Salmonella in TLR-treated macrophages is dependent on a functional SPI-2 type III secretion system. This also indicates that there was not a generalized defect in macrophage anti-bacterial function. These data exemplify how interactions between macrophage defense mechanisms and bacterial virulence factors can result in evasion of the innate immune response. Studying how TLR-agonist treatment affects Salmonella pathogenesis will give us a better understanding of the host-pathogen relationship and may provide insight into novel strategies to fight intracellular microorganisms. / Thesis / Master of Science (MSc)

Obesity is a disease characterized by chronic inflammation and the progressive loss in systemic insulin sensitivity. One of the more effective medications in the treatment of insulin resistance have been the thiazolidinediones (TZDs), which act through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma ). Due to the many side-effects of TZDs, our laboratory sought out a natural phytochemical, abscisic acid (ABA), with chemical similarities to TZDs. Our first study demonstrated that ABA activates PPARgamma in vitro and significantly ameliorates white adipose tissue (WAT) inflammation and glucose tolerance in db/db mice. We next further examined the effect of ABA on the phenotype of adipose tissue macrophages (ATMs). In doing so, we discovered two separate ATM populations which differed in their expression of the macrophage surface glycoprotein and maturation marker F4/80 (F4/80hi vs F4/80lo). Dietary ABA-supplementation significantly reduced F4/80hiCCR2+ ATMs and had no effect on the F4/80lo population. Utilizing a tissue-specific knockout generated through Cre-lox recombination, we were able to determine that this effect was dependent on PPARgamma in immune cells. To further characterize the differences between the ATM subsets that were affected by ABA, we performed a multi-organ assessment (i.e., WAT, skeletal muscle and liver) of the effect of diet-induced obesity on the phenotype of infiltrating macrophages and T cells into metabolic organs. Based on our new data, we formulated a model by which F4/80hiCCR2hi ATMs infiltrate WAT and ultimately induce a CD11c+ pro-inflammatory phenotype in the resident F4/80loCCR2lo subset. Ultimately, our findings provide evidence that ABA has potential as an alternative preventive intervention, expound the role of PPARgamma in immune cells and, in general, expand our knowledge concerning the immunopathogenesis of obesity-induced insulin resistance. / Ph. D.