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Humans can taste many compounds but are able to distinguish between five basic tastes, bitter, sweet, umami, sour and salt. Bitter taste, which is mediated by 25 bitter taste receptors (T2Rs) in humans, acts as a central warning signal against the ingestion of toxic compounds. In addition to their expression in the oral cavity, T2Rs are expressed in various extra-oral tissues, suggesting that they have additional functions apart from sensing taste. The recent finding that T2Rs, upon activation with bitter tastants, cause muscle relaxation and bronchodilation of pre-contracted airway smooth muscle, has been a topic of consideration. The bronchodilatory role of T2Rs, which was three fold greater than that elicited by currently used beta-adrenergic receptor agonists, has implicated them as potential therapeutic targets for the treatment of asthma. In view of the importance of T2R function in extra-oral tissues, it is of fundamental importance to determine their physiological role in extra-oral regions like the vascular tissues, and understand how T2R signal is regulated. In this study, two representative bitter taste receptors, T2R1 and T2R4, were selected to elucidate the function and signal regulation of T2Rs. The expression of T2Rs was characterized in pulmonary artery smooth muscle cells, and studies were pursued to explore the effects of dextromethorphan (DXM) on the pulmonary artery. DXM caused vasoconstriction in pulmonary arterial cells by activating endogenous T2R1. The structure-function role of the C-terminus of T2R4 receptor was characterized by site-directed mutagenesis. A conserved KLK motif was identified in the C-terminus and many residues involved in cell surface targeting and function of T2R4 were revealed. A constitutively active mutant (CAM) was also discovered within the T2R4 C-terminus. To identify the regulatory proteins involved in T2R4 desensitization, a molecular and pharmacological approach was used. The kinases involved in this process were

One of the major challenges facing clinicians is how to effectively manage excessive host immune response to pathogenic insults resulting in sepsis. This is demonstrated by the fact that despite over half-century research efforts, sepsis and its spectrum of diseases (severe sepsis and septic shock) are still associated with poor clinical outcome. Currently, sepsis is a leading cause of death in intensive care units. The immune system protects the host against pathogens and is therefore armed with an arsenal of deadly ammunitions (including chemicals, cells and proteins) necessary for the elimination of microbes. It is therefore paramount that the immune system must develop mechanisms necessary to prevent destruction of the host it is designed to protect. A good example of such a mechanism is found in the subset of lymphocytes known as regulatory T cells (Tregs). There is unequivocal experimental evidence of the role of Tregs in the maintenance of immune homeostasis and self tolerance and aberrant Treg function has been linked with several inflammatory diseases. Since sepsis is a disease marked by a hyper-inflammatory state, I investigated the possible role of Tregs in dampening sepsis-induced excessive inflammation. Using a murine model of lipopolysaccharide (LPS) infusion and bacterial infection, I show that Tregs are essential for survival during sepsis because their depletion leads to acute death to an otherwise non-lethal dose of LPS. This enhanced susceptibility to LPS following Treg depletion was also observed using live E. coli infection. Next, I probed the mechanism by which Tregs protect against LPS challenge. I found that defective Treg function leads to exaggerated activity of two immune cells – CD4+ effector T cells and neutrophils in response to LPS, leading to severe inflammatory response. Hence, this work successfully illustrates the critical role of Tregs in regulating other immune cells and the catastrophic consequences of defective Treg function du

Thesis Abstract: Pentraxin 3 (PTX3) is a pattern recognition receptor that plays an important role in providing immunity against various pulmonary inflammatory conditions. Having known its role in fostering lung immunity, aim of my PhD thesis was to examine the role of PTX3 in asthma. In my thesis, I showed an enhanced level of PTX3 in the airway fluid of severe asthmatics and also in the airway fluid and the lungs of allergen exposed mice. To further investigate the role of PTX3, I used a murine model of ovalbumin (OVA)-induced experimental asthma and performed experiments on PTX3+/+ and PTX3-/- mice. I observed an enhanced airway inflammation and hyperactivity in OVA-exposed PTX3 Knockout (PTX3-/-) mice. In PTX3-/- mice, there was an IL-17A dominant inflammation as compared to PTX3+/+ mice upon OVA challenge. This response was accompanied with increased CD4 T cell survival and reduced IL-2 production by these cells. As Th17 phenotype of CD4 T cells is shaped by IL-6 and IL-23 producing dendritic cells, we observed increased preponderance of such DCs in the lungs and the draining lymph nodes of PTX3-/- mice as compared to their PTX3+/+ counterparts, plausibly supporting Th17/ IL-17A dominant inflammation in the former. Furthermore, there was increased infiltration of inflammatory DCs in the lungs in PTX3-/- mice upon OVA exposure. This observation was in line with increased generation of common myeloid progenitors in the bone marrow of these mice. PTX3-/- DCs showed reduced MHCII expression but increased surface expression of CD80 and CD86 along with increased ability of PTX3-/- DCs to uptake and process OVA. Since these DCs showed increase IL-6 and IL-23 production, co-culture of PTX3-/- DCs with OT II CD4 T cells resulted in greater IL-17A production as compared to those that were co-cultured with PTX3+/+ DCs. Altogether, I have observed that deletion of PTX3 resulted in enhanced IL-17 immune response plausibly through increased IL-6 and IL-23 producing DCs in OV

One of the major challenges facing clinicians is how to effectively manage excessive host immune response to pathogenic insults resulting in sepsis. This is demonstrated by the fact that despite over half-century research efforts, sepsis and its spectrum of diseases (severe sepsis and septic shock) are still associated with poor clinical outcome. Currently, sepsis is a leading cause of death in intensive care units. The immune system protects the host against pathogens and is therefore armed with an arsenal of deadly ammunitions (including chemicals, cells and proteins) necessary for the elimination of microbes. It is therefore paramount that the immune system must develop mechanisms necessary to prevent destruction of the host it is designed to protect. A good example of such a mechanism is found in the subset of lymphocytes known as regulatory T cells (Tregs). There is unequivocal experimental evidence of the role of Tregs in the maintenance of immune homeostasis and self tolerance and aberrant Treg function has been linked with several inflammatory diseases. Since sepsis is a disease marked by a hyper-inflammatory state, I investigated the possible role of Tregs in dampening sepsis-induced excessive inflammation. Using a murine model of lipopolysaccharide (LPS) infusion and bacterial infection, I show that Tregs are essential for survival during sepsis because their depletion leads to acute death to an otherwise non-lethal dose of LPS. This enhanced susceptibility to LPS following Treg depletion was also observed using live E. coli infection. Next, I probed the mechanism by which Tregs protect against LPS challenge. I found that defective Treg function leads to exaggerated activity of two immune cells – CD4+ effector T cells and neutrophils in response to LPS, leading to severe inflammatory response. Hence, this work successfully illustrates the critical role of Tregs in regulating other immune cells and the catastrophic consequences of defective Treg function du

Thesis Abstract: Pentraxin 3 (PTX3) is a pattern recognition receptor that plays an important role in providing immunity against various pulmonary inflammatory conditions. Having known its role in fostering lung immunity, aim of my PhD thesis was to examine the role of PTX3 in asthma. In my thesis, I showed an enhanced level of PTX3 in the airway fluid of severe asthmatics and also in the airway fluid and the lungs of allergen exposed mice. To further investigate the role of PTX3, I used a murine model of ovalbumin (OVA)-induced experimental asthma and performed experiments on PTX3+/+ and PTX3-/- mice. I observed an enhanced airway inflammation and hyperactivity in OVA-exposed PTX3 Knockout (PTX3-/-) mice. In PTX3-/- mice, there was an IL-17A dominant inflammation as compared to PTX3+/+ mice upon OVA challenge. This response was accompanied with increased CD4 T cell survival and reduced IL-2 production by these cells. As Th17 phenotype of CD4 T cells is shaped by IL-6 and IL-23 producing dendritic cells, we observed increased preponderance of such DCs in the lungs and the draining lymph nodes of PTX3-/- mice as compared to their PTX3+/+ counterparts, plausibly supporting Th17/ IL-17A dominant inflammation in the former. Furthermore, there was increased infiltration of inflammatory DCs in the lungs in PTX3-/- mice upon OVA exposure. This observation was in line with increased generation of common myeloid progenitors in the bone marrow of these mice. PTX3-/- DCs showed reduced MHCII expression but increased surface expression of CD80 and CD86 along with increased ability of PTX3-/- DCs to uptake and process OVA. Since these DCs showed increase IL-6 and IL-23 production, co-culture of PTX3-/- DCs with OT II CD4 T cells resulted in greater IL-17A production as compared to those that were co-cultured with PTX3+/+ DCs. Altogether, I have observed that deletion of PTX3 resulted in enhanced IL-17 immune response plausibly through increased IL-6 and IL-23 producing DCs in OV

In humans, taste is one of the five senses, and helps in the recognition of nutritionally important and potentially harmful substances. It triggers innate behaviour to accept or reject food. Humans can sense five basic tastes, which are sweet, umami, bitter, salt and sour. The receptors that mediate bitter, sweet and umami tastes belong to the G protein-coupled receptor (GPCR) superfamily. A group of three receptors sense sweet and umami tastes, whereas bitter taste is sensed by 25 bitter taste receptors (referred as T2Rs). T2Rs are activated by structurally diverse natural and synthetic bitter compounds. Many common pharmaceutical compounds are bitter in taste and these are effective ligands for T2Rs. Recent finding of T2Rs in extra-oral tissues suggests these receptors are also involved in various physiological and pathophysiological processes. To understand the structure and function of these receptors, studies directed at elucidating their mechanisms of activation, and identification of novel ligands including bitter blockers (antagonists and inverse agonists), are required. To obtain mechanistic insights into the role of the highly conserved, and receptor specific residues, two bitter taste receptors (T2R1 and T2R4) were targeted. In this study, a combination of molecular, biochemical and pharmacological approaches were used to identify the amino acids and motifs, important for T2Rs to switch from inactive to active state. A hydrogen-bonding network between transmembrane (TM) helices 1-2-7 was identified as important for T2R activation. Alanine-scan mutagenesis of intracellular loops (ICLs) 2 and 3 identified T2R regions important for G protein binding, and receptor activation. A pharmacological method was developed, to screen potential bitter blockers for T2Rs. Using this method, three novel bitter blockers, which include two natural antagonists and one synthetic inverse agonist for T2R4, were discovered. The role of expression tags in enhancing T2R4 expressio

In humans, taste is one of the five senses, and helps in the recognition of nutritionally important and potentially harmful substances. It triggers innate behaviour to accept or reject food. Humans can sense five basic tastes, which are sweet, umami, bitter, salt and sour. The receptors that mediate bitter, sweet and umami tastes belong to the G protein-coupled receptor (GPCR) superfamily. A group of three receptors sense sweet and umami tastes, whereas bitter taste is sensed by 25 bitter taste receptors (referred as T2Rs). T2Rs are activated by structurally diverse natural and synthetic bitter compounds. Many common pharmaceutical compounds are bitter in taste and these are effective ligands for T2Rs. Recent finding of T2Rs in extra-oral tissues suggests these receptors are also involved in various physiological and pathophysiological processes. To understand the structure and function of these receptors, studies directed at elucidating their mechanisms of activation, and identification of novel ligands including bitter blockers (antagonists and inverse agonists), are required. To obtain mechanistic insights into the role of the highly conserved, and receptor specific residues, two bitter taste receptors (T2R1 and T2R4) were targeted. In this study, a combination of molecular, biochemical and pharmacological approaches were used to identify the amino acids and motifs, important for T2Rs to switch from inactive to active state. A hydrogen-bonding network between transmembrane (TM) helices 1-2-7 was identified as important for T2R activation. Alanine-scan mutagenesis of intracellular loops (ICLs) 2 and 3 identified T2R regions important for G protein binding, and receptor activation. A pharmacological method was developed, to screen potential bitter blockers for T2Rs. Using this method, three novel bitter blockers, which include two natural antagonists and one synthetic inverse agonist for T2R4, were discovered. The role of expression tags in enhancing T2R4 expressio

There is increasing evidence suggesting the contribution of bacterial infections in atherothrombotic conditions. Studies have demonstrated that bacteria residing within the oral cavity activate platelets once they enter circulation. S. sanguis 2017-78 is capable of stimulating platelet aggregation in a thromboxane-dependent manner. In the present study, the signaling events associated with S. sanguis have been studied further. S. sanguis 2017-78 caused the phosphorylation of p38 MAP kinase and subsequently cPLA2. The p38 MAP kinase inhibitor, SB203580 inhibited S. sanguis 2017-78-induced platelet aggregation as well as the phosphorylation of both p38 MAP kinase and cPLA2. These data are consistent with cPLA2 as a physiological target of p38. A second component of the study examined the effects of aspirin, a known inhibitor of cyclooxygenase, on these signalling pathways.

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, their molecular targets in humans were poorly characterized. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium were measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2.

There is increasing evidence suggesting the contribution of bacterial infections in atherothrombotic conditions. Studies have demonstrated that bacteria residing within the oral cavity activate platelets once they enter circulation. S. sanguis 2017-78 is capable of stimulating platelet aggregation in a thromboxane-dependent manner. In the present study, the signaling events associated with S. sanguis have been studied further. S. sanguis 2017-78 caused the phosphorylation of p38 MAP kinase and subsequently cPLA2. The p38 MAP kinase inhibitor, SB203580 inhibited S. sanguis 2017-78-induced platelet aggregation as well as the phosphorylation of both p38 MAP kinase and cPLA2. These data are consistent with cPLA2 as a physiological target of p38. A second component of the study examined the effects of aspirin, a known inhibitor of cyclooxygenase, on these signalling pathways.

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, their molecular targets in humans were poorly characterized. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium were measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2.