Helicobacter pylori infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during H. pylori infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon H. pylori stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, H. pylori LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. H. pylori infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in H. pylori infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, H. pylori infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.

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.

Innate leukocytes manifest dynamic and distinct inflammatory responses upon challenges with rising dosages of pathogen associated molecular pattern molecules (PAMPs) such as lipopolysaccharide (LPS). To differentiate signal strengths, innate leukocytes may utilize distinct intra-cellular signaling circuitries modulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules in Toll-like receptor 4 (TLR4) signaling and potentially playing key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip may serve as a negative regulator of NFkB signaling pathway in cells challenged with higher dosages of LPS, it acts as a positive regulator for low-grade chronic inflammation in leukocytes programmed by subclinical low-dosages of LPS. We aim to show recent progress in our understanding of complex innate leukocyte dynamics and its relevance in the pathogenesis of resolving versus non-resolving chronic inflammatory diseases. / Ph. D. / White blood cells, or leukocytes, have a dynamic inflammatory response to rising doses of bacterial cell wall components. Lipopolysaccharide (LPS) is a ubiquitous component of gram negative bacteria that is recognized by Toll-like receptor 4 (TLR4) and can shed into the blood stream, causing low-grade non-resolving inflammation. In order to differentiate between varying signal strengths of LPS, leukocytes utilize signaling within the cell, which is often regulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules in TLR4 signaling and potentially plays key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip serves to inhibit the pro-inflammatory NFκB signaling in cells challenged with higher dosages of LPS, it acts to increase low-grade chronic inflammation in leukocytes programmed by low-dosages of LPS. In these studies we show recent progress in elucidating the mechanism for Tollip involvement in low-grade non-resolving inflammation in mouse fibroblast cells.

Pattern Recognition Receptors (PRRs) are a group of evolutionarily conserved and germline-encoded cellular receptors of the innate immune system that are responsible for recognizing and responding to the entirety of the pathogens a host encounters. The ingenuity of the innate immune system is that with a comparatively miniscule pool of receptors, these receptors are capable of responding to a diverse and large array of pathogens and damage signals. Two highly relevant subsets of PRRs include nucleotide binding domain leucine rich repeat containing (NOD-like) receptors (NLRs) and Toll-like receptors (TLRs). Both NLRs and TLRs have been implicated in several diseases, including autoimmune disorders, inflammatory conditions, and cancer. Mice lacking a specific NLR, NLRP1, are more susceptible to chemically induced colitis and colitis-associated tumorigenesis. We investigated whether the absence of NLRP1 in the gastrointestinal tract influenced the composition of the microbiome, and whether it was responsible for the predisposition of these animals to colitis-associated cancer. By carefully controlling for non-genotype influences, we found that in fact maternal and housing factors were greater predictors over genotype of gut flora composition. This study concluded with a clearer understanding of NLRP1. We next investigated the effectiveness of a novel tumor ablation therapy, termed High-Frequency Irreversible Electroporation (H-FIRE) in a murine model of triple negative breast cancer. The chosen 4T1 model closely mimics aggressive human metastatic triple negative breast cancer, and metastasizes to the same organs. After ablation of the primary mammary tumor, we saw significant improvements in disease burden and metastases, both of which were accompanied by PRR activation within the tumor microenvironment, implicating PRRs in the successful treatment outcome following H-FIRE ablation. Lastly, we generated novel CRISPR-Cas9 plasmids to genetically manipulate the Tlr4 gene of wild type C57Bl/6 mice in order to recapitulate the LPS-hyporesponsive TLR4 protein of C3H/HeJ mice. This proof-of-concept study successfully demonstrated that PRRs can be targets for gene editing purposes, and that nanoparticle delivery leads to enhanced and improved delivery. Collectively, this work attempts to better appreciate the role of PRRs in understanding, treating, and targeting cancer. / Doctor of Philosophy / The work presented here focuses on the role of the immune system in the progression of cancer. Put simply, the properly functioning immune system of a healthy individual should recognize and eliminate mutated or cancerous cells prior to the development of a tumor, thereby implying that the progression to a tumor is due to some dysfunction of the immune system. The immune system is made up of two arms: the innate and adaptive. A key difference between the innate and adaptive immune systems is that upon an infection, the adaptive response is slow and specific while the innate response is rapid and broad. Pattern Recognition Receptors (PRRs) are a group of cellular receptors of the innate immune system that are responsible for recognizing and responding to the entirety of the pathogens a host encounters. The ingenuity of the innate immune system is that with a comparatively miniscule pool of receptors, these receptors are capable of responding to a diverse and large array of pathogens. Two highly relevant PRR families are nucleotide binding domain leucine rich repeat containing (NOD-like) receptors (NLRs) and Toll-like receptors (TLRs). Both NLRs and TLRs have been implicated in several diseases, including autoimmune disorders, inflammatory conditions, and cancer. In this work, we investigated whether the absence of an NLR protein influenced the composition of the microbes that reside within the gastrointestinal tract, and whether this absence was responsible for the predisposition of these animals to colitis-associated cancer. By carefully controlling for all additional influences, we found that in our mice, the other animals with which they shared a cage were more influential on the microbes within the gut, rather than the NLR deficiency. We next investigated a novel tumor ablation therapy in an animal model of breast cancer, which closely mimics human metastatic triple negative breast cancer and metastasizes to the same organs. After treatment of the mammary tumor, we saw significant improvements in disease burden and metastases, both of which were accompanied by PRR activation. Lastly, we manipulated a TLR gene in mice to demonstrate that PRRs can be targeted for therapeutic gene editing. Collectively, this work provides evidence that PRRs are a highly useful tool for improving our understanding of cancer.

A síntese noturna de melatonina pela glândula pineal é inibida por padrões moleculares associados à patógenos ou à danos, como por exemplo lipopolissacarídeo (LPS) ou peptídeo &#946;-amilóide. A interação destas moléculas com receptores toll 4 (TLR4) ativa o eixo imune-pineal, favorecendo a migração de leucócitos para o local da injúria. Heparan sulfato (HS) é um glicosaminoglicano da matrix extracelular que por dano tecidual, inflamação generalizada ou migração de células tumorais, liberam dissacarídeos que podem ligar a TLR4, levando a formação de uma resposta inflamatória. Avaliamos se HS poderia prejudicar a atividade da melatonina. HS é capaz de inibir a síntese noturna de melatonina, através da supressão da expressão gênica e do conteúdo enzimático de acetilserotonina O-metiltransferase (ASMT). Este efeito é modulado pela interação de HS com TLR4, mas não envolve a via de translocação nuclear de NF-&#954;B. Estes dados sugerem que um aumento de moléculas de HS na matriz da glândula pineal é traduzido a todo o organismo por uma redução no pico noturno de melatonina. / The nocturnal synthesis of melatonin by the pineal gland is inhibited by pathogen or damage-associated molecular patterns, such as lipopolysaccharide (LPS) and &#946;-amyloid peptide. The interaction of these molecules with toll like receptors 4 (TLR4) activates the immune-pineal axis, favoring the migration of leukocytes for the site of lesion. Heparan sulfate (HS), a glycosaminoglycan of the extracellular matrix, that in case of tissue injury, generalized inflammation or migration of tumor cells, releases disaccharide, which can bind to TLR4 triggering an inflammatory response. Here we evaluated if HS could impair nocturnal melatonin activity. HS is capable of inhibit the melatonin synthesis by the suppression of the gene expression and enzymatic content of acetylserotonin O-methyltransferase (ASMT). This effect is modulated by the interaction of HS with TLR4, but does not involve the NF-&#954;B nuclear translocation pathway. This data suggest that the increase in HS in pineal gland matrix is translated to the whole organism by a reduction in the nocturnal melatonin peak.

Le système du complément est une cascade de défense immunitaire complexe et étroitement régulée, conduisant à des dommages tissulaires lorsqu’il est suractivé. L’hème, un motif moléculaire de danger dérivant de l’hémolyse, est capable d’activer le complément dans le sérum et à la surface des cellules endothéliales (CE) in vitro, suggérant un rationel pour examiner l’impact de l’activation du complément dans les maladies hémolytiques. L’objectif de ce projet était d’étudier si et comment l’hémolyse intravasculaire active le complément in vivo, et de comprendre les mécanismes sous-jacent conduisant à l’acquisition d’un phénotype activateur du complément par les CE afin d’identifier de nouvelles cibles thérapeutiques. Nous avons détecté des dépôts de complément, de C3 et de C5b-9, dans des reins de patients souffrants de nephropathie drépanocytaire ainsi que dans un modèle murin de drépanocytose. Nous avons mis en place un modèle murin d’hémolyse intravasculaire massive, déclenchée par la phénylhydrazine (PHZ), et caractérisé l’atteinte rénale. Nous avons détecté des dépôts de C3 au niveau des reins de ces souris. Cet effet a été inhibé par l’administration préventive du scavenger naturel de l’hème, l’hémopexine (Hx), et reproduit par des injections d’hème libre, démontrant une activation hème-dépendante in vivo. Les microvésicules d’érythrocytes (MVs) drépanocytaires représentent une source naturelle d’hème, de par leur concentration en hème trois fois supérieure à celle observée chez les donneurs sains. Nous avons démontré que les MVs drépanocytaires activent le complément dans le sérum et sur les CE, de manière en partie hème-dépendante. Ces résultats révèlent le rôle activateur de l’hème sur le complément dans les maladies hémolytiques. De plus, nous avons démontré que l’interaction de l’hème avec TLR4 peut en partie expliquer les dépôts de C3 sur l’endothélium in vivo et les CE in vitro. L’utilisation d’un inhibiteur de TLR4, le TAK-242, a réduit de 50% les dépôts de complément sur les CE, confirmé par une réduction des dépôts sur l’endothélium vasculaire chez des souris TLR4-/- traitées par PHZ ou hème. De plus, nous avons montré que ces dépôts hème/TLR4 dépendants sont liés à l’expression rapide de P-sélectine, qui recrute C3b et C3(H2O) à la membrane des CE, révélé par l’analyse des interactions protéiques en temps réel et l’utilisation d’un anticorps bloquant anti-P-sélectine. Ensemble, ce projet démontre que l’hème et les MVs sont les produits dérivés de l’hémolyse responsables de l’activation du complément. Au niveau cellulaire, l’induction par l’hème d’un phénotype activateur du complément des CE dépend de l’axe TLR4/P-sélectine, induisant des dépôts de C3 à la surface cellulaire. Ainsi, ces études soulignent les bénéfices potentiels de l’Hx et du TAK-242 contre l’activation du complément dans des pathologies associées à une hémolyse. / Complement system is a complex and tightly regulated innate immune defensive cascade, which can promote tissue damage, when overactivated. Hemolysis-derived danger associated molecular pattern heme is able to activate complement in serum and on endothelial cells (EC) in vitro, providing a rational for scrutinizing the impact of complement activation in hemolytic diseases. The objectives of this work were to study whether and how intravascular hemolysis induces complement activation in vivo, and to understand the underlying mechanism that leads to the acquisition of a complement activating phenotype of the endothelium in order to identify novel therapeutic strategies. We found complement deposits, including C3 activation fragments and C5b-9, within kidneys of patients with sickle cell disease (SCD) nephropathy (a prototypical hemolytic disease) as well as in a mouse model of SCD. We set up and characterized the renal injury of a mouse model of massive intravascular hemolysis, triggered by injection of phenylhydrazine (PHZ). We revealed C3 deposition within kidneys of the PHZ-treated animals. It was prevented by heme scavenging with hemopexin (Hx) and reproduced by injections of free heme, thus demonstrating the importance of heme for the complement activation in vivo. SCD erythrocytes microvesicles (MVs), are a pathologically relevant source of labile heme, since they carry three times more heme on their surface compare to MVs from healthy donors. We demonstrated that MVs, generated from SCD erythrocytes, activate complement in human serum and on EC surface, in part on a heme-dependent manner. These data highlight the importance of heme as a complement activator in hemolytic diseases. Further, we found that the C3 activation fragments deposits on endothelium in vivo and on EC in vitro can be in part explained by interaction of heme with TLR4. Indeed, the use of a specific inhibitor of TLR4, TAK-242, reduced about 50% the complement deposits on EC surface and such deposits on vascular endothelium in PHZ- or heme-injected mice were attenuated TLR4-/- mice. Moreover, we found that heme/TLR4-dependent complement deposition was mediated by the rapid expression of P-selectin, which in turn, recruited C3b and C3(H2O) on the EC surface, as evidenced by real time protein interaction analyses and using of blocking antibodies. Together our results demonstrated that heme and erythrocytes MVs are the hemolysis-derived products which promoted complement activation. At cellular level, heme induced complement-activating phenotype of EC by triggering TLR4/P-selectin axis and resulting in C3 activation fragments on cell surface. Together, these studies underline the potential benefits of Hx and TAK-242 against complement activation in pathologies related to hemolysis.

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Made available in DSpace on 2014-07-29T15:07:28Z (GMT). No. of bitstreams: 1 Adriano Queiroz de Mesquita -30-07-2010.pdf: 3684854 bytes, checksum: bc87bf213d6700cac487c2b4580b87a1 (MD5) Previous issue date: 2010-07-30 / A mastite tem sido considerada, mundialmente, a doença de maior impacto nos rebanhos leiteiros, devido à elevada prevalência e aos prejuízos econômicos que determina. As desordens decorrentes da mastite por agente etiológico de origem bacteriana são complexas, dependentes do microrganismo envolvido, e desencadeiam inúmeros processos de reconhecimento. As estruturas moleculares dos microrganismos são conhecidas como padrões moleculares associados aos patógenos (PAMPS) e os receptores nas células do hospedeiro como receptores de reconhecimento de padrões (PRR). O presente trabalho foi desenvolvido com o objetivo de identificar a presença de polimorfismos de nucleotídeo único no gene codificador do TLR4 em vacas leiteiras da raça holandesa em uma propriedade leiteira em Goiás, avaliando a relação dos alelos identificados, com a ocorrência de mastite subclínica e contagem celular somática. Foram coletadas 150 amostras de leite individual de vacas para identificação de microrganismos, contagem celular somática e composição centesimal, e 150 amostras de sangue para genotipagem em uma propriedade rural do Estado de Goiás. A discriminação alélica foi realizada por meio da técnica de PCR em tempo real, baseada em 4 SNPs de referência no gene codificador do TLR4 depositados no NCBI (rs8193046, rs8193047, rs8193060 e rs29017188). Os resultados obtidos revelam maior frequência de microrganismos Gram negativos na propriedade de estudo (52,47%) e que, animais identificados com os genótipos AACCCC, GGTCGG e GACCGC são os mais indicados para seleção assistida por marcadores moleculares. / Mastitis has been considered, worldwide, the disease of greatest impact in dairy herds because of the high prevalence and the economic losses that determines. The disorders caused by mastitis causative agent of bacterial origin are complex, depending on the microrganism involved, and trigger numerous processes of recognition. The molecular structures of microrganisms are known as Pathogen- Associated Molecular Patterns (PAMPs) and the receptors on host cells as pattern recognition receptors (PRR). This study was developed with the aim of identifying the presence of single nucleotide polymorphisms in TLR4 in Holstein dairy cows on a dairy farm in Goiás, evaluating the relationship between identified alleles, occurrence of subclinical mastitis and somatic cell count. 150 milk samples from individual cows were collected for identification of microrganisms, somatic cell count and composition, and 150 blood samples for genotyping on a farm in the State of Goiás. The allelic discrimination was performed by Real-time PCR, based on four reference SNPs in TLR4 gene from NCBI (rs8193046, rs8193047, rs8193060 and rs29017188). The results showed higher frequency of Gram negative microrganisms (52.47%) and that animals with the genotypes AACCCC, GGTCGG GACCGC are best suited for marker-assisted selection.

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