Several successful pathogens evade host defenses resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory oral bone loss and systemic inflammation manifested as atherosclerosis. The pathogenic mechanisms contributing to P. gingivalis evasion of host immunity and chronic inflammation are not well defined. P. gingivalis evades host immunity at Toll-like receptor (TLR)-4 through expression of an atypical lipopolysaccharide (LPS) that contains lipid A species that exhibit TLR4 agonist or antagonist activity or fail to activate TLR4. By utilizing a series of P. gingivalis lipid A mutants we demonstrated that expression of antagonist lipid A structures resulted in weak induction of proinflammatory mediators. Moreover, expression of antagonist lipid A failed to activate the inflammasome, which correlated with increased bacterial survival in macrophages. Oral infection of atherosclerotic prone apolipoprotein E (ApoE) deficient mice with the antagonist lipid A strain resulted in vascular inflammation characterized by macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain expressing exclusively agonist lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11 dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE deficient mice infected with the agonist lipid A strain exhibited diminished vascular inflammation. Notably, the ability of P. gingivalis to induce local inflammatory oral bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. We next investigated the role of TLRs and lipid A on bacterial trafficking by the autophagic pathway. Originally characterized as a cell autonomous pathway for recycling damaged organelles and proteins, autophagy is now recognized to play a critical role in innate defense and release of the proinflammatory cytokine interleukin (IL)-1β. We demonstrated that P. gingivalis suppresses the autophagic pathway in macrophages for pathogen survival and intercepts autophagy-mediated IL-1β release. P. gingivalis-mediated suppression of autophagy was independent of lipid A expression but partially dependent on TLR2 signaling. Collectively, our results indicate that P. gingivalis evasion of innate immunity plays a role in chronic inflammation.

Les Récepteurs Toll-Like (TLR) permettent d'initier et d'orienter les réponses immunes dirigées contre les pathogènes. Ils participent également à la régulation des réponses induites, comme en témoigne leur implication dans de nombreuses maladies du système immunitaire.<br />Notre recherche a eu pour objectif d'étudier le rôle des voies de signalisation TLR en situation normale et immunopathologique. Nous avons démontré que : 1) les agonistes des TLR protègent efficacement de l'apparition du diabète spontané chez la souris NOD via l'activation et/ou le recrutement de cellules régulatrices et la production de cytokines immunorégulatrices; 2) Les cellules iNKT sont activées par l'agoniste TLR7 ; 3) La signalisation via TLR7 protège de l'asthme allergique. 4) la voie signalisation via TLR2/MyD88 a un effet majeur sur le développement de l'athérosclérose, ainsi que sur la production de chimiokines et cytokines ; 5) la voie de signalisation MyD88 est impliquée dans le développement et la fonction des lymphocytes iNKT. En conclusion, notre travail met en évidence les propriétés immunomodulatrices des voies de signalisation TLR.

Neutrophils are implicated in many inflammatory lung disorders. However, the mechanisms regulating neutrophil migration in acute lung inflammation are incompletely understood. Although, integrin β2 mediates neutrophil migration in lungs in response to many stimuli such as E. coli, integrin involved in <i>S. pneumoniae</i> induced neutrophil migration is not known. Therefore, the role of integrin αvβ3 in neutrophil recruitment was tested. First, it was found that the number of neutrophils expressing the integrin subunits αv and β3 is reduced or remains in lung inflammation induced by E. coli or <i>S. pneumoniae</i>, respectively. Next, the role of integrin αvβ3 using β3 knockout mice (β3-/-) and function blocking antibodies was addressed. Neutrophil recruitment did not vary between wild type and β3-/- mice. Although β3 antibodies reduced neutrophil recruitment, similar effect was observed with isotype antibodies. Therefore, one can conclude that integrin αvβ3 is not critical for neutrophil recruitment in <i>S. pneumoniae</i> induced pneumonia. <p>Apart from integrins, TLR4 also regulate neutrophil migration. Because, the pattern of TLR4 expression at various times of lung inflammation is not known, TLR4 expression during different phases of lung inflammation in a rat model of LPS-induced inflammation was studied. TLR4 expression in the septum increased and decreased at 6h and 12-36h of inflammation, respectively. Since these correlate with the time of increase and decline of neutrophil recruitment, the findings support previously observed requirement for TLR4 in neutrophil recruitment. <p>Neutrophils recruited into the lungs regulate the inflammatory process by controlling subsequent monocyte/macrophage recruitment. The mechanisms involved and the pattern of monocyte/macrophage recruitment in lungs are not completely understood. Therefore, the possible involvement of monocyte chemoattractant protein (MCP)-1, which is a premier chemokine in monocyte/macrophage migration and produced by neutrophils and other cells was tested. This was addressed by quantification of monocytes/macrophages at various times and using neutrophil depletion experiments in LPS-induced lung inflammation in rats. It was found that monocytes/macrophages migrate very early and before neutrophils in addition to their migration in the late phase of acute lung inflammation. Neutrophil depletion abrogated both early as well as the late monocyte/macrophage recruitment without altering the expression of MCP-1. Therefore, possibly other chemokines and not MCP-1 are involved in neutrophil dependent monocyte/macrophage recruitment. <p>To conclude, the experiments further the understanding on acute lung inflammation by ruling-out the involvement of integrin αvβ3 and MCP-1 in β2-independent neutrophil migration and neutrophil dependent monocyte/macrophage recruitment, respectively. Further studies are essential to find the integrins and chemokines operating in the above situations. Equally important will be to understand the functional significance of early recruited monocytes/macrophages in the lung.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

Neutrophils are implicated in many inflammatory lung disorders. However, the mechanisms regulating neutrophil migration in acute lung inflammation are incompletely understood. Although, integrin β2 mediates neutrophil migration in lungs in response to many stimuli such as E. coli, integrin involved in <i>S. pneumoniae</i> induced neutrophil migration is not known. Therefore, the role of integrin αvβ3 in neutrophil recruitment was tested. First, it was found that the number of neutrophils expressing the integrin subunits αv and β3 is reduced or remains in lung inflammation induced by E. coli or <i>S. pneumoniae</i>, respectively. Next, the role of integrin αvβ3 using β3 knockout mice (β3-/-) and function blocking antibodies was addressed. Neutrophil recruitment did not vary between wild type and β3-/- mice. Although β3 antibodies reduced neutrophil recruitment, similar effect was observed with isotype antibodies. Therefore, one can conclude that integrin αvβ3 is not critical for neutrophil recruitment in <i>S. pneumoniae</i> induced pneumonia. <p>Apart from integrins, TLR4 also regulate neutrophil migration. Because, the pattern of TLR4 expression at various times of lung inflammation is not known, TLR4 expression during different phases of lung inflammation in a rat model of LPS-induced inflammation was studied. TLR4 expression in the septum increased and decreased at 6h and 12-36h of inflammation, respectively. Since these correlate with the time of increase and decline of neutrophil recruitment, the findings support previously observed requirement for TLR4 in neutrophil recruitment. <p>Neutrophils recruited into the lungs regulate the inflammatory process by controlling subsequent monocyte/macrophage recruitment. The mechanisms involved and the pattern of monocyte/macrophage recruitment in lungs are not completely understood. Therefore, the possible involvement of monocyte chemoattractant protein (MCP)-1, which is a premier chemokine in monocyte/macrophage migration and produced by neutrophils and other cells was tested. This was addressed by quantification of monocytes/macrophages at various times and using neutrophil depletion experiments in LPS-induced lung inflammation in rats. It was found that monocytes/macrophages migrate very early and before neutrophils in addition to their migration in the late phase of acute lung inflammation. Neutrophil depletion abrogated both early as well as the late monocyte/macrophage recruitment without altering the expression of MCP-1. Therefore, possibly other chemokines and not MCP-1 are involved in neutrophil dependent monocyte/macrophage recruitment. <p>To conclude, the experiments further the understanding on acute lung inflammation by ruling-out the involvement of integrin αvβ3 and MCP-1 in β2-independent neutrophil migration and neutrophil dependent monocyte/macrophage recruitment, respectively. Further studies are essential to find the integrins and chemokines operating in the above situations. Equally important will be to understand the functional significance of early recruited monocytes/macrophages in the lung.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

The main goal of this thesis was to study cancer immunotherapy based on combination of ligands of TLR and phagocytic receptor agonists. This study is focused on looking for proper and save TLR stimulation and specific anchoring of phagocytic ligands.

The main goal of this thesis was to optimize the current therapeutic approach using TLR agonists and anchored agonists of phagocytic receptor to treating cancer. The study is focused on searching a suitable agonist of TLR as the replacement of LPS.

This thesis is focused on analysis of immune processes during cancer immunotherapy which is based on synergistic combination of TLR agonists and phagocytosis stimulating ligands anchored into the tumour cell membrane. This immunotherapy was tested in murine melanoma B16-F10 and murine pancreatic adenocarcinoma Panc02 models. The aims of this thesis were to analyse the tumor infiltration, elucidate the role of innate immunity in this immunotherapy, and study the possibility of strengthening immunotherapeutic effect using anti-CD40 and anti-CTLA-4.