Monocyte differentiation toward regulatory dendritic cells is not affected by respiratory syncytial virus-induced inflammatory mediators.

Airway epithelial cells were shown to drive the differentiation of monocytes into dendritic cells (DCs) with a suppressive phenotype. In this study, we investigated the impact of virus-induced inflammatory mediator production on the development of DCs. Monocyte differentiation into functional DCs, as reflected by the expression of CD11c, CD123, BDCA-4, and DC-SIGN and the capacity to activate T cells, was similar for respiratory syncytial virus (RSV)-infected and mock-infected BEAS-2B and A549 cells. RSV-conditioned culture media resulted in a partially mature DC phenotype, but failed to up-regulate CD80, CD83, CD86, and CCR7, and failed to release proinflammatory mediators upon Toll-like receptor (TLR) triggering. Nevertheless, these DCs were able to maintain an antiviral response by the release of Type I IFN. Collectively, these data indicate that the airway epithelium maintains an important suppressive DC phenotype under the inflammatory conditions induced by infection with RSV.

Active uptake of dendritic cell-derived exovesicles by epithelial cells induces the release of inflammatory mediators through a TNF-alpha-mediated pathway.

Dendritic cells (DCs) can release hundreds of membrane vesicles, called exovesicles, which are able to activate resting DCs and distribute antigen. Here, we examined the role of mature DC-derived exovesicles in innate and adaptive immunity, in particular their capacity to activate epithelial cells. Our analysis of exovesicle contents showed that exovesicles contain major histocompatibility complex-II, CD40, and CD83 molecules in addition to tumor necrosis factor (TNF) receptors, TNFRI and TNFRII, and are important carriers of TNF-alpha. These exovesicles are rapidly internalized by epithelial cells, inducing the release of cytokines and chemokines, but do not transfer an alloantigen-presenting capacity to epithelial cells. Part of this activation appears to involve the TNF-alpha-mediated pathway, highlighting the key role of DC-derived exovesicles, not only in adaptive immunity, but also in innate immunity by triggering innate immune responses and activating neighboring epithelial cells to release cytokines and chemokines, thereby amplifying the magnitude of the innate immune response.

Understanding the multifaceted role of inflammatory mediators in ischemic stroke.

The evolution of ischemic brain damage is strongly affected by an inflammatory reaction that involves soluble mediators, such as cytokines and chemokines, and specialized cells activated locally or recruited from the periphery. The immune system affects all phases of the ischemic cascade, from the acute intravascular reaction due to blood flow disruption, to the development of brain tissue damage, repair and regeneration. Increased endothelial expression of adhesion molecules and blood-brain barrier breakdown promotes extravasation and brain recruitment of blood-borne cells, including macrophages, neutrophils, dendritic cells and T lymphocytes, as demonstrated both in animal models and in human stroke. Nevertheless, most anti-inflammatory approaches showing promising results in experimental stroke models failed in the clinical setting. The lack of translation may reside in the redundancy of most inflammatory mediators, exerting both detrimental and beneficial functions. Thus, this review is aimed at providing a better understanding of the dualistic role played by each component of the inflammatory/immune response in relation to the spatio-temporal evolution of ischemic stroke injury.

Neuroprotection in cerebral ischemia : an effect of c-Jun N-terminal kinase inhibition on the inflammatory response

RESUMESuite à un accident vasculaire cérébral (AVC) ischémique, les cellules gliales ducerveau deviennent activées, de nombreuses cellules inflammatoires pénètrent dans letissu lésé et sécrètent une grande variété de cytokines et chémokines. Aujourd'hui, ilexiste des interrogations sur les effets bénéfiques ou délétères de cette inflammation surla taille de la lésion et le pronostic neurologique.Ce projet vise à évaluer l'effet d'un peptide neuroprotecteur, D-JNKI1, inhibiteur de lavoie pro-apoptotique de signalisation intracellulaire c-Jun N-terminal kinase (JNK), surl'inflammation post-ischémique.Nous montrons d'abord que la microglie est largement activée dans toute la région lésée48 h après l'induction d'une ischémie chez la souris. Cependant, malgré l'inhibition dela mort neuronale par D-JNKI1 évaluée à 48 h, nous n'observons de modification ni del'activation de la microglie, ni de son nombre. Ensuite, nous montrons que le cerveaupeut être protégé même s'il y a une augmentation massive de la sécrétion de médiateursinflammatoires dans la circulation systémique très tôt après induction d'un AVCischémique. De plus, nous notons que la sécrétion de molécules inflammatoires dans lecerveau n'est pas différente entre les animaux traités par D-JNKI1 ou une solutionsaline, bien que nous ayons obtenu une neuroprotection significative chez les animauxtraités.En conclusion, nous montrons que l'inhibition de la voie de JNK par D-JNKI1n'influence pas directement l'inflammation post-ischémique. Ceci suggère quel'inhibition de l'inflammation n'est pas forcément nécessaire pour obtenir en hautdegré de neuroprotection du parenchyme lésé après ischémie cérébrale, et que lesmécanismes inflammatoires déclenchés lors d'une ischémie cérébrale ne sont pasforcément délétères pour la récupération du tissu endommagé.SUMMARYAfter cerebral ischemia, glial cells become activated and numerous inflammatory cellsinfiltrate the site of the lesion, secreting a large variety of cytokines and chemokines. Itis controversial whether this brain inflammation is detrimental or beneficial and how itinfluences lesion size and neurological outcome.This project was aimed at critically evaluating whether the neuroprotective peptide DJNKI,an inhibitor of the pro-apopotic c-Jun N-terminal kinase (JNK) pathway,modulates post-ischemic inflammation in animal models of stroke. Specifically, it wasasked whether JNK inhibition prevents microglial activation and the release ofinflammatory mediators.In the first part of this study, we showed that microglia was activated throughout thelesion 48 h after experimental stroke. However, the activation and accumulation ofmicroglia was not reduced by D-JNKI1, despite a significant reduction of the lesionsize. In the second part of this project, we demonstrated that neuroprotection measuredat 48 h occurs even though inflammatory mediators are released in the plasma veryearly after the onset of cerebral ischemia. Furthermore, we found that secretion ofinflammatory mediators in the brain was not different in groups treated with D-JNKI1or not, despite a significant reduction of the lesion size in the treated group.Altogether, we show that inhibition of the JNK pathway using D-JNKI1 does notinfluence directly post-stroke inflammation. Inhibition of inflammation is therefore notnecessarily required for neuroprotection after cerebral ischemia. Thus, post-strokeinflammation might not be detrimental for the tissue recovery.

Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures.

Brain inflammatory response is triggered by the activation of microglial cells and astrocytes in response to various types of CNS injury, including neurotoxic insults. Its outcome is determined by cellular interactions, inflammatory mediators, as well as trophic and/or cytotoxic signals, and depends on many additional factors such as the intensity and duration of the insult, the extent of both the primary neuronal damage and glial reactivity and the developmental stage of the brain. Depending on particular circumstances, the brain inflammatory response can promote neuroprotection, regeneration or neurodegeneration. Glial reactivity, regarded as the central phenomenon of brain inflammation, has also been used as an early marker of neurotoxicity. To study the mechanisms underlying the glial reactivity, serum-free aggregating brain cell cultures were used as an in vitro model to test the effects of conventional neurotoxicants such as organophosphate pesticides, heavy metals, excitotoxins and mycotoxins. This approach was found to be relevant and justified by the complex cell-cell interactions involved in the brain inflammatory response, the variability of the glial reactions and the multitude of mediators involved. All these variables need to be considered for the elucidation of the specific cellular and molecular reactions and their consequences caused by a given chemical insult.

The aldosterone-mineralocorticoid receptor pathway exerts anti-inflammatory effects in endotoxin-induced uveitis.

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

Postoperative Cognitive Dysfunction (POCD) and Inflammatory Markers in Elderly Patients

Introduction: Particularly in elderly patients, the brain responds to a systemic inflammatory response with an increased production of inflammatory mediators. This has hypothetically been linked to the development of postoperative cognitive dysfunction (POCD). Methods: We investigated 31 patients aged >65 yrs undergoing elective major surgery under standardized general anaesthesia (thiopental, sevoflurane, fentanyl, atracurium). Cognitive function was measured preoperatively and 7 days postoperatively using the extended version of the Consortium to Establish a Registry for Alzheimer's Disease - Neuropsychological Assessment Battery (CERAD-NAB, validated German version) for which we developed a diagnostic cut-off in healthy elderly volunteers. Systemic C-reactive protein (CRP) and interleukin 6 (IL-6) were measured preoperatively, 2 days postoperatively, and 7 days postoperatively. Values for CRP, IL-6, operative characteristics and hospital length of stay in patients with POCD and without POCD were compared using the Mann- Whitney U test and are shown as median [range]. Results: Fourteen patients (45%) developed POCD. Values for CRP were not statistically different in patients with POCD and without POCD but tended to be higher in patients with POCD 2 days postoperatively. Patients with POCD had significantly higher IL-6 values on postoperative days 2 and 7 (table 1). These patients also had a significantly longer duration of anaesthesia (305 [195-620] vs.190 [150-560] min, p = 0.034), larger intraoperative blood loss (425 [0-1600] vs. 100 [0-1500] ml, p = 0.018) and longer hospital stays (15 [8-45] vs. 8 [4-40] days, p = 0.008). Table 1 POCD (n = 14) No POCD (n = 17) p value CRP (mg/dl) preop. 4.0 [1.0-245] 4.2 [0.3-36.2] 0.6 2 days postop. 223 [20-318] 98 [4.5-384] 0.07 7 days postop. 58 [15-147] 44 [11-148] 0.2 IL-6 (U/ml) preop. 2[2-28.1] 2 [2-7.3] 0.8 2 days postop. 56 [17-315] 20 [2-123] 0.009 7 days postop. 9[2-77] 4 [2-16] 0.03 Interpretation: In this small group of patients, high IL-6 values postoperatively were associated with POCD supporting a role for systemic inflammation in the development of POCD. In patients with POCD, duration of anaesthesia was significantly longer, and intraoperative blood losses were larger. These risk factors will need to be confirmed in a larger group of patients. The difference in length of stay may be indicative of postoperative complications, which have been linked to POCD earlier.

Immune regulation and function of the NOD-like receptors NLRC5 and NLRP3

AbstractThe vertebrate immune system is composed of the innate and the adaptive branches. Innate immune cells represent the first line of defense and detect pathogens through pattern recognition receptors (PRRs), detecting evolutionary conserved pathogen- and danger- associated molecular patterns. Engagement of these receptors initiates the inflammatory response, but also instructs antigen-specific adaptive immune cells. NOD-like receptors (NLRs) are an important group of PRRs, leading to the production of inflammatory mediators and favoring antigen presentation to Τ lymphocytes through the regulation of major histocompatibility complex (MHC) molecules.In this work we focused our attention on selected NOD-like receptors (NLRs) and their role at the interface between innate and adaptive immunity. First, we describe a new regulatory mechanism controlling IL-1 production. Our results indicate that type I interferons (IFNs) block NLRP1 and NLRP3 inflammasome activity and interfere with LPS-driven proIL-Ια and -β induction. As type I IFNs are produced upon viral infections, these anti-inflammatory effects of type I IFN could be relevant in the context of superinfections, but could also help explaining the efficacy of IFN-β in multiple sclerosis treatment.The second project addresses the role of a novel NLR family member, called NLRC5. The function of this NLR is still matter of debate, as it has been proposed as both an inhibitor and an activator of different inflammatory pathways. We found that the expression of this protein is restricted to immune cells and is positively regulated by IFNs. We generated Nlrc5-deficient mice and found that this NLR plays an essential role in Τ, NKT and, NK lymphocytes, in which it drives the expression of MHC class I molecules. Accordingly, we could show that CD8+ Τ cell-mediated killing of target lymphocytes lacking NLRC5 is strongly impaired. Moreover, NLRC5 expression was found to be low in many lymphoid- derived tumor cell lines, a mechanism that could be exploited by tumors to escape immunosurveillance.Finally, we found NLRC5 to be involved in the production of IL-10 by CD4+ Τ cells, as Nlrc5- deficient Τ lymphocytes produced less of this cytokine upon TCR triggering. In line with these observations, Mrc5-deficient CD4+ Τ cells expanded more than control cells when transferred into lymphopenic hosts and led to a more rapid appearance of colitis symptoms. Therefore, our work gives novel insights on the function of NLRC5 by using knockout mice, and strongly supports the idea that NLRs direct not only innate, but also adaptive immune responses.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).

VEGF and TNF up-regulate, NSAID down-regulate SOX18 protein level in HUVEC

Angiogenesis, the process of generating new blood vessels, is essential to embryonic development, organ formation, tissue regeneration and remodeling, reproduction and wound healing. Also, it plays an important role in many pathological conditions, including chronic inflammation and cancer. Angiogenesis is regulated by a complex interplay of growth factors, inflammatory mediators, adhesion molecules, morphogens and guidance molecules. Transcription factor SOX18 is transiently expressed in nascent endothelial cells during embryonic development and postnatal angiogenesis, but little is known about signaling pathways controlling its expression. The aim of this study was to investigate whether pro-angiogenic molecules and pharmacological inhibitors of angiogenesis modulate SOX18 expression in endothelial cells. Therefore, we treated human umbilical vein endothelial cells (HUVEC) with angiogenic factors, extracellular matrix proteins, inflammatory cytokines and nonsteroidal anti-inflammatory drugs (NSAID) and monitored SOX18 expression. We have observed that the angiogenic factor VEGF and the inflammatory cytokine TNF increase, while the NSAID ibuprofen and NS398 decrease the SOX18 protein level. These results for the first time demonstrate that SOX18 expression is modulated by factors and drugs known to positively or negatively regulate angiogenesis. This opens the possibility of pharmacological manipulation of SOX18 gene expression in endothelial cells to stimulate or inhibit angiogenesis.

Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice.

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders.

Developments in the scientific and clinical understanding of gout.

Gout is the most common form of inflammatory arthritis in the elderly. In the last two decades, both hyperuricemia and gout have increased markedly and similar trends in the epidemiology of the metabolic syndrome have been observed. Recent studies provide new insights into the transporters that handle uric acid in the kidney as well as possible links between these transporters, hyperuricemia, and hypertension. The treatment of established hyperuricemia has also seen new developments. Febuxostat and PEG-uricase are two novel treatments that have been evaluated and shown to be highly effective in the management of hyperuricemia, thus enlarging the therapeutic options available to lower uric acid levels. Monosodium urate (MSU) crystals are potent inducers of inflammation. Within the joint, they trigger a local inflammatory reaction, neutrophil recruitment, and the production of pro-inflammatory cytokines as well as other inflammatory mediators. Experimentally, the uptake of MSU crystals by monocytes involves interactions with components of the innate immune system, namely Toll-like receptor (TLR)-2, TLR-4, and CD14. Intracellularly, MSU crystals activate multiple processes that lead to the formation of the NALP-3 (NACHT, LRR, and pyrin domain-containing-3) inflammasome complex that in turn processes pro-interleukin (IL)-1 to yield mature IL-1 beta, which is then secreted. The inflammatory effects of MSU are IL-1-dependent and can be blocked by IL-1 inhibitors. These advances in the understanding of hyperuricemia and gout provide new therapeutic targets for the future.

Do glial cells control pain?

Management of chronic pain is a real challenge, and current treatments focusing on blocking neurotransmission in the pain pathway have only resulted in limited success. Activation of glia cells has been widely implicated in neuroinflammation in the central nervous system, leading to neruodegeneration in many disease conditions such as Alzheimer's and multiple sclerosis. The inflammatory mediators released by activated glial cells, such as tumor necrosis factor-α and interleukin-1β can not only cause neurodegeneration in these disease conditions, but also cause abnormal pain by acting on spinal cord dorsal horn neurons in injury conditions. Pain can also be potentiated by growth factors such as BDNF and bFGF that are produced by glia to protect neurons. Thus, glia cells can powerfully control pain when they are activated to produce various pain mediators. We will review accumulating evidence supporting an important role of microglia cells in the spinal cord for pain control under injury conditions (e.g. nerve injury). We will also discuss possible signaling mechanisms in particular MAP kinase pathways that are critical for glia control of pain. Investigating signaling mechanisms in microglia may lead to more effective management of devastating chronic pain.

Histone deacetylase inhibitors impair innate immune responses

SUMMARYThe innate immune system plays a central role in host defenses against invading pathogens. Innate immune cells sense the presence of pathogens through pattern recognition receptors that trigger intracellular signaling, leading to the production of pro-inflammatory mediators like cytokines, which shape innate and adaptive immune responses. Both by excess and by default inflammation may be detrimental to the host. Indeed, severe sepsis and septic shock are lethal complications of infections characterized by a dysregulated inflammatory response.In recent years, members of the superfamily of histone deacetylases have been the focus of great interest. In mammals, histone deacetylases are broadly classified into two main subfamilies comprising histone deacetylases 1-11 (HDAC1-11) and sirtuins 1-7 (SIRT1-7). These enzymes influence gene expression by deacetylating histones and numerous non-histone proteins. Histone deacetylases have been involved in the development of oncologic, metabolic, cardiovascular, neurodegenerative and autoimmune diseases. Pharmacological modulators of histone deacetylase activity, principally inhibitors, have been developed for the treatment of cancer and metabolic diseases. When we initiated this project, several studies suggested that inhibitors of HDAC 1-11 have anti-inflammatory activity. Yet, their influence on innate immune responses was largely uncharacterized. The present study was initiated to fill in this gap.In the first part of this work, we report the first comprehensive study of the effects of HDAC 1- 11 inhibitors on innate immune responses in vitro and in vivo. Strikingly, expression studies revealed that HDAC1-11 inhibitors act essentially as negative regulators of basal and microbial product- induced expression of critical immune receptors and antimicrobial products by mouse and human innate immune cells like macrophages and dendritic cells. Furthermore, we describe a new molecular mechanism whereby HDAC1-11 inhibitors repress pro-inflammatory cytokine expression through the induction of the expression and the activity of the transcriptional repressor Μί-2β. HDAC1-11 inhibitors also impair the potential of macrophages to engulf and kill bacteria. Finally, mice treated with an HDAC inhibitor are more susceptible to non-severe bacterial and fungal infection, but are protected against toxic and septic shock. Altogether these data support the concept that HDAC 1-11 inhibitors have potent anti-inflammatory and immunomodulatory activities in vitro and in vivo.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a central role in innate immune responses, cell proliferation and oncogenesis. In the second part of this manuscript, we demonstrate that HDAC1-11 inhibitors inhibit MIF expression in vitro and in vivo and describe a novel molecular mechanism accounting for these effects. We propose that inhibition of MIF expression by HDAC 1-11 inhibitors may contribute to the antitumorigenic and anti-inflammatory effects of these drugs.NAD+ is an essential cofactor of sirtuins activity and one of the major sources of energy within the cells. Therefore, sirtuins link deacetylation to NAD+ metabolism and energy status. In the last part of this thesis, we report preliminary results indicating that a pharmacological inhibitor of SIRT1-2 drastically decreases pro-inflammatory cytokine production (RNA and protein) and interferes with MAP kinase intracellular signal transduction pathway in macrophages. Moreover, administration of the SIRT1-2 inhibitor protects mice from lethal endotoxic shock and septic shock.Overall, our studies demonstrate that inhibitors of HDAC1-11 and sirtuins are powerful anti-inflammatory molecules. Given their profound negative impact on the host antimicrobial defence response, these inhibitors might increase the susceptibility to opportunistic infections, especially in immunocompromised cancer patients. Yet, these inhibitors might be useful to control the inflammatory response in severely ill septic patients or in patients suffering from chronic inflammatory diseases.

Bacterial flagellin elicits innate immune defenses and cardiac dysfunction in vivo

Résumé Les agents pathogènes responsables d'infection entraînent chez l'hôte deux types de réponses immunes, la première, non spécifique, dite immunité innée, la seconde, spécifique à l'agent concerné, dite immunité adaptative. L'immunité innée, qui représente la première ligne de défense contre les pathogènes, est liée à la reconnaissance par les cellules de l'hôte de structures moléculaires propres aux micro-organismes (« Pathogen-Associated Molecular Patterns », PAMPs), grâce à des récepteurs membranaires et cytoplasmiques (« Pattern Recognition Receptors », PRRs) identifiant de manière spécifique ces motifs moléculaires. Les récepteurs membranaires impliqués dans ce processus sont dénommés toll-like récepteurs, ou TLRS. Lorsqu'ils sont activés par leur ligand spécifique, ces récepteurs activent des voies de signalisation intracellulaires initiant la réponse inflammatoire non spécifique et visant à éradiquer l'agent pathogène. Les deux voies de signalisation impliquées dans ce processus sont la voie des « Mitogen-Activated Protein Kinases » (MAPKs) et celle du « Nuclear Factor kappaB » (NF-κB), dont l'activation entraîne in fine l'expression de protéines de l'inflammation dénommées cytokines, ainsi que certaines enzymes produisant divers autres médiateurs inflammatoires. Dans certaines situations, cette réponse immune peut être amplifiée de manière inadéquate, entraînant chez l'hôte une réaction inflammatoire systémique exagérée, appelée sepsis. Le sepsis peut se compliquer de dysfonctions d'organes multiples (sepsis sévère), et dans sa forme la plus grave, d'un collapsus cardiovasculaire, définissant le choc septique. La défaillance circulatoire du choc septique touche les vaisseaux sanguins d'une part, le coeur d'autre part, réalisant un tableau de «dysfonction cardiaque septique », dont on connaît mal les mécanismes pathogéniques. Les bactéries à Gram négatif peuvent déclencher de tels phénomènes, notamment en libérant de l'endotoxine, qui active les voies de l'immunité innée par son interaction avec un toll récepteur, le TLR4. Outre l'endotoxine, la plupart des bactéries à Gram négatif relâchent également dans leur environnement une protéine, la flagelline, qui est le constituant majeur du flagelle bactérien, organelle assurant la mobilité de ces micro-organismes. Des données récentes ont indiqué que la flagelline active, dans certaines cellules, les voies de l'immunité innée en se liant au récepteur TLRS. On ne connaît toutefois pas les conséquences de l'interaction flagelline-TLRS sur le développement de l'inflammation et des dysfonctions d'organes au cours du sepsis. Nous avons par conséquent élaboré le présent travail en formulant l'hypothèse que la flagelline pourrait déclencher une telle inflammation et représenter ainsi un médiateur potentiel de la dysfonction d'organes au cours du sepsis à Gram négatif, en nous intéressant plus particulièrement àl'inflammation et à la dysfonction cardiaque. Dans la première partie de ce travail, nous avons étudié les effets de la flagelline sur l'activation du NF-κB et des MAPKs, et sur l'expression de cytokines inflammatoires au niveau du myocarde in vitro (cardiomyocytes en culture) et in vivo (injection de flagelline recombinante à des souris). Nous avons observé tout d'abord que le récepteur TLRS est fortement exprimé au niveau du myocarde. Nous avons ensuite démontré que la flagelline active la voie du NF-κB et des MAP kinases (p38 et JNK), stimule la production de cytokines et de chemokines inflammatoires in vitro et in vivo, et entraîne l'activation de polynucléaires neutrophiles dans le tissu cardiaque in vivo. Finalement, au plan fonctionnel, nous avons pu montrer que la flagelline entraîne une dilatation et une réduction aiguë de la contractilité du ventricule gauche chez la souris, reproduisant les caractéristiques de la dysfonction cardiaque septique. Dans la deuxième partie, nous avons déterminé la distribution du récepteur TLRS dans les autres organes majeurs de la souris (poumon, foie, intestin et rein}, et avons caractérisé dans ces organes l'effet de la flagelline sur l'activation du NF-κB et des MAPKs, l'expression de cytokines, et l'induction de l'apoptose. Nous avons démontré que le TLRS est exprimé de façon constitutive dans ces organes, et que l'injection de flagelline y déclenche les cascades de l'immunité innée et de processus apoptotiques. Finalement, nous avons également déterminé que la flagelline entraîne une augmentation significative de multiples cytokines dans le plasma une à six heures après son injection. En résumé, nos données démontrent que la flagelline bactérienne (a) entraîne une inflammation et une dysfonction importantes du myocarde et (b) active de manière très significative les mécanismes d'immunité innée dans les principaux organes et entraîne une réponse inflammatoire systémique. Par conséquent, la flagelline peut représenter un médiateur puissant de l'inflammation et de la dysfonction d'organes, notamment du coeur, au cours du choc septique déclenché par les bactéries à Gram négatif. Summary Pathogenic microorganisms trigger two kinds of immune responses in the host. The first one is immediate and non-specific and is termed innate immunity, whereas the second one, specifically targeted at the invading agent, is termed adaptative immunity. Innate immunity, which represents the first line of defense against invading pathogens, confers the host the ability to recognize molecular structures common to many microbial pathogens, ("Pathogen-Associated Molecular Patterns", PAMPs), through cytosolic or membrane-associated receptors ("Pattern Recognition Receptors", PRRs), the latter being represented by a family of receptors termed "toll-like receptors or TLRs". Once activated by the binding of their specific ligand, these receptors activate intracellular signaling pathways, which initiate the non-specific inflammatory response aimed at eradicating the pathogens. The two pathways implicated in this process are the mitogen-activated protein kinases (MAPK) and the nuclear factor kappa B (NF-κB) signaling pathways, whose activation elicit in fine the expression of inflammatory proteins termed cytokines, as well as various enzymes producing a wealth of additional inflammatory mediators. In some circumstances, the innate immune response can become amplified and dysregulated, triggering an overwhelming systemic inflammatory response in the host, identified as sepsis. Sepsis can be associated with multiple organ dysfunction (severe sepsis), and in its most severe form, with cardiovascular collapse, defming septic shock. The cardiovascular failure associated with septic shock affects blood vessels as well as the heart, resulting in a particular form of acute heart failure termed "septic cardiac dysfunction ", whose pathogenic mechanisms remain partly undefined. Gram-negative bacteria can initiate such phenomena, notably by releasing lipopolysaccharide (LPS), which activates innate immune signaling by interacting with its specific toll receptor, the TLR4. Besides LPS, most Gram-negative bacteria also release flagellin into their environment, which is the main structural protein of the bacterial flagellum, an appendage extending from the outer bacterial membrane, responsible for the motility of the microorganism. Recent data indicated that flagellin activate immune responses upon binding to its receptor, TLRS, in various cell types. However, the role of flagellin/TLRS interaction in the development of inflammation and organ dysfunction during sepsis is not known. Therefore, we designed the present work to address the hypothesis that flagellin might trigger such inflammatory responses and thus represent a potential mediator of organ dysfunction during Gram-negative sepsis, with a particular emphasis on cardiac inflammation and contractile dysfunction. In the first part of this work, we investigated the effects of flagellin on NF-κB and MAPK activation and the generation of pro-inflammatory mediators within the heart in vitro (cultured cardiomyocytes) and in vivo (injection of recombinant flagellin into mice). We first observed that TLRS protein is strongly expressed by the myocardium. We then demonstrated that flagellin activates NF-κB and MAP kinases (p38 and JNK), upregulates the transcription of pro-inflammatory cytokines and chemokines in vitro and in vivo, and stimulates the activation of polymorphonuclear neutrophils within the heart in vivo. Finally, we demonstrated that flagellin triggers acute cardiac dilation, and a significant reduction of left ventricular contractility, mimicking characteristics of clinical septic cardiac dysfunction. In the second part, we determined the TLRS distribution in other mice major organs (lung, liver, gut and kidney) and we characterized in these organs the effects of flagellin on NF-κB and MAPK activation, on the expression of pro-inflammatory çytokines, and on the induction of apoptosis. We demonstrated that TLRS protein is constitutively expressed and that flagellin activates prototypical innate immune responses and pro-apoptotic pathways in all these organs. Finally, we also observed that flagellin induces a significant increase of multiple cytokines in the plasma from 1 to 6 hours after its intravenous administration. Altogether, these data provide evidence that bacterial flagellin (a) triggers an important inflammatory response and an acute dysfunction of the myocardium, and (b) significantly activates the mechanisms of innate immunity in most major organs and elicits a systemic inflammatory response. In consequence, flagellin may represent a potent mediator of inflammation and multiple organ failure, notably cardiac dysfunction, during Gram-negative septic shock.