BACKGROUND A subarachnoid hemorrhage (SAH) is a bleed into the subarachnoid space surrounding the brain. This disease affects roughly 30,000 Americans each year and approximately one in six affected individuals die at the time of the ictal event. Individuals that do survive suffer from many complications including delayed cerebral vasospasm (DCV), cerebral edema, fever, and increased intracranial pressure (ICP) amongst others. These patients often suffer from brain damage due to neuronal apoptosis as a consequence of excess neuroinflammation. Microglia, the resident macrophage of the central nervous system, and Toll-like Receptor-4 (TLR4), a pro-inflammatory transmembrane receptor, have both been shown to play a role in the neuroinflammation seen in SAH. RBC components have been shown to activate microglial TLR4, and this event is suggested to trigger downstream mechanisms leading to neuronal apoptosis. The presented research takes a closer look at the role of microglial TLR4 in early neuronal apoptosis seen in an SAH model. METHODS All mice used were 10- to 12-week-old males on a C57BL/6 background: TLR4−/−, MyD88−/−, TRIF−/− and wild type (WT). To induce an SAH, a total of 60 ul of arterial blood from a donor WT mouse was injected for over 30 seconds into another mouse. For in vitro experiments, either primary microglia (PMG) or murine microglial BV2 cells were used. Microglia were separated from murine neuronal HT22 cells by 3um cell culture inserts or transwells, before being stimulated with lipopolysaccharide (LPS), red blood cells (RBCs), or RBC components including hemin (structurally similar to heme) and hemoglobin. In vivo samples were studied using either immunohistochemistry (IHC) or Fluorescence Activated Cell Sorting (FACS), and in vitro cells were studied using IHC and Light Microscopy. Neuronal cell death was measured using TUNEL and/or FloroJade C (FJC) assays. Cognitive function after SAH was measured using the Barnes Maze protocol. RESULTS In a 24-hour time course, more death occurred in the HT22 cells associated with BV2s treated with RBCs for 12-hour and 24-hour incubation time points as compared to 1-hour and 3-hour time points. Similar results were seen in the WT PMGs, as HT22 apoptosis increased in the RBC treated WT groups as the incubation time points increased. The WT PMG and MyD88−/− RBC treated PMGs showed significant death as compared to a WT untreated control (p<0.05) using a FJC assay, and both showed more death in a TUNEL assay as compared to an untreated control. WT mice treated with whole blood and hemoglobin had significantly more apoptosis as compared with a normal saline (NS)-treated control mouse (p<0.05). WT PMGs treated with whole blood and hemoglobin had more apoptosis as compared with an untreated control. MyD88-/- treated with RBC, hemoglobin, and hemin had more HT22 cell death compared with other genotypes treated with the same component. For the Barnes Maze, TLR4−/− mice performed significantly less total errors than WT mice on POD5 and 6 (p<0.01), and took significantly less time to reach the goal chamber on POD4, POD5 (p<0.05), and POD6 (p<0.01). CONCLUSION Our experimental results suggest that a microglial TLR4-dependent, MyD88-independent pathway is involved in neuronal apoptosis very early in an SAH model via RBC and hemoglobin activation, and that neuronal cell apoptosis due to TLR4 expression may be related to SAH-related cognitive and behavioral deficits. Our results suggest that TRIF may be the intracellular adaptor that is involved in this mechanism, but further experiments are needed to confirm this.

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Many bacterial species exist in nature as part of highly structured multicellular communities known as biofilms. Amyloids, proteins with a conserved β-sheet quarternary structure, show high resistance to many chemical and enzymatic processes including proteinase K and SDS treatments and are produced as essential adhesins during biofilm formation. Curli fibers expressed by Enterobacteriaceae family members including E. coli and S. Typhimurium are the most studied amyloids to date. Curli-like fibers are also produced by members of the predominant phyla found in the host gastrointestinal microbiota in environmental biofilms. Curli fibers are the predominant microbial-associated molecular pattern (MAMP) on enteric bacteria recognized by the Toll-like receptor (TLR) 2/1-heterodimer complex. Interestingly, the TLR2/1 complex has been implicated as a key player in modulating gastrointestinal homeostasis. The focus of the current studies centered on the innate immune recognition of curli fibers by cells of the gastrointestinal tract and how that contributes to gastrointestinal homeostasis. In the first phase of our studies, utilizing intestinal epithelial cells polarized on semi-permeable tissue culture inserts (Transwells®), we observed that the recognition of curli fibers on Salmonella enterica serovar Typhimurium by intestinal epithelial cells led to the augmentation of the intestinal epithelial barrier in a PI3K-dependent manner. We also observed that bacterial translocation of S. Typhimurium from the apical side to the basolateral side of the Transwell system was limited when curli fibers were present. Furthermore, infection of mice with S. Typhimurium showed that translocation of bacteria from the intestinal lumen into the cecal tissue and mesenteric lymph nodes was limited in C57BL/6 mice as compared to TLR2 knockout mice. In the second phase of our studies, we sought to further investigate the effect that curli fibers exert on gastrointestinal homeostasis through the induction of immunomodulatory cytokines such as Interleukin 10 (IL10) from subepithelial lamina propria cells. IL10 has been shown to contribute to the maintenance of the intestinal epithelial barrier and IL10-deficient mice develop lethal colitis within the first 2-3 months of life. 6-8 week-old female C57BL/6 and TLR2-/- mice were given 5mg/kg of curli fibers via intraperitoneal injection. Subsequent RT-PCR analysis of the small intestine showed a significant expression of Il10 in C57BL/6 that was absent in TLR2-/- mice. Interestingly, no changes in Ifnγ or Tgfβ mRNA were observed in these mice. This response was gut-specific, as Il10 was not detected at all in the spleen. Furthermore, in a chemically-induced colitis model, we observed that the administration of curli fibers to 8-week old Balb/c mice ameliorated disease severity as compared to colitic mice that received mock treatments. Interestingly, Il10 was also induced in the colons of colitic mice that received curli and which were euthanized 6 days after colitis was induced. Our results suggest that curli fibers induce IL10 production via a TLR2-dependent manner to dampen inflammation in the gastrointestinal tract. Overall, our results partially describe a novel role for curli amyloid fibers produced by commensal bacteria in modulating gastrointestinal health and homeostasis. We propose that the induction of immunomodulatory cytokine such as IL10 by amyloid fibers is an important mechanism utilized by commensal bacterial to confer beneficial effects that benefit both the host and microbe. We also propose curli fibers as a potential alternative in the treatment of inflammatory bowel disease. / Microbiology and Immunology

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)