Involvement of CD73, equilibrative nucleoside transporters and inosine in rhythm and conduction disturbances mediated by adenosine A1 and A2A receptors in the developing heart.

We previously established that exogenous adenosine (ADO) induces transient arrhythmias in the developing heart via the adenosine A1 receptor (A1AR) and downstream activation of NADPH oxidase/ERK and PLC/PKC pathways. Here, we investigated the mechanisms by which accumulation of endogenous ADO and its derived compound inosine (INO) in the interstitial compartment induce rhythm and conduction troubles. The validated model of the spontaneously beating heart obtained from 4-day-old chick embryos was used. Quantitative RT-PCR showed that enzymes involved in ADO and INO metabolism (CD39, CD73 and eADA) as well as equilibrative (ENT1, -3, -4) and concentrative (CNT3) nucleoside transporters were differentially expressed in atria, ventricle and outflow tract. Inactivation of ENTs by dipyridamole, 1) increased myocardial ADO level, 2) provoked atrial arrhythmias and atrio-ventricular blocks (AVB) in 70% of the hearts, 3) prolonged P wave and QT interval without altering contractility, and 4) increased ERK2 phosphorylation. Blockade of CD73-mediated phosphohydrolysis of AMP to ADO, MEK/ERK pathway inhibition or A1AR inhibition prevented these arrhythmias. Exposure to exogenous INO also caused atrial ectopy associated with AVB and ERK2 phosphorylation which were prevented by A1AR or A2AAR antagonists exclusively or by MEK/ERK inhibitor. Inhibition of ADA-mediated conversion of ADO to INO increased myocardial ADO and decreased INO as expected, but slightly augmented heart rate variability without provoking AVB. Thus, during cardiogenesis, disturbances of nucleosides metabolism and transport, can lead to interstitial accumulation of ADO and INO and provoke arrhythmias in an autocrine/paracrine manner through A1AR and A2AAR stimulation and ERK2 activation.

Nuclear hormone receptors and mouse skin homeostasis: implication of PPARbeta.

PPARbeta is expressed in the mouse epidermis during fetal development, and progressively disappears from the interfollicular epidermis after birth. Interestingly, its expression is strongly reactivated in the adult epidermis in conditions where keratinocyte proliferation is induced and during wound healing. Data obtained on PPARbeta heterozygous mice reveal that PPARbeta is implicated in the control of keratinocyte proliferation and is necessary for rapid healing of a skin wound.

5-HT2A and alpha1b-adrenergic receptors entirely mediate dopamine release, locomotor response and behavioural sensitization to opiates and psychostimulants.

Addictive properties of drugs of misuse are generally considered to be mediated by an increased release of dopamine (DA) in the ventral striatum. However, recent experiments indicated an implication of alpha1b-adrenergic receptors in behavioural responses to psychostimulants and opiates. We show now that DA release induced in the ventral striatum by morphine (20 mg/kg) is completely blocked by prazosin (1 mg/kg), an alpha1-adrenergic antagonist. However, morphine-induced increases in DA release in the ventral striatum were found to be similar in mice deleted for the alpha1b-adrenergic receptor (alpha1b-AR KO) and in wild-type (WT) mice, suggesting the presence of a compensatory mechanism. This acute morphine-evoked DA release was completely blocked in alpha1b-AR KO mice by SR46349B (1 mg/kg), a 5-HT2A antagonist. SR46349B also completely blocked, in alpha1b-AR KO mice, the locomotor response and the development of behavioural sensitization to morphine (20 mg/kg) and D-amphetamine (2 mg/kg). Accordingly, the concomitant blockade of 5-HT2A and alpha1b-adrenergic receptors in WT mice entirely blocked acute locomotor responses but also the development of behavioural sensitization to morphine, D-amphetamine or cocaine (10 mg/kg). We observed, nevertheless, that inhibitory effects of each antagonist on locomotor responses to morphine or D-amphetamine were more than additive (160%) in naïve WT mice but not in those sensitized to either drug. Because of these latter data and the possible compensation by 5-HT2A receptors for the genetic deletion of alpha1b-adrenergic receptors, we postulate the existence of a functional link between these receptors, which vanishes during the development of behavioural sensitization.

New insights into the regulatory mechanisms of the NALP3 inflammasome

Résumé : Les vertébrés ont recours au système immunitaire inné et adaptatif pour combattre les pathogènes. La découverte des récepteurs Toll, il y a dix ans, a fortement augmenté l'intérêt porté à l'immunité innée. Depuis lors, des récepteurs intracellulaires tels que les membres de la famille RIG-like helicase (RLHs) et NOD-like receptor (NLRs) ont été décrits pour leur rôle dans la détection des pathogènes. L'interleukine-1 beta (IL-1β) est une cytokine pro-inflammatoire qui est synthétisée sous forme de précurseur, la proIL-1β. La proIL-1β requiert d'être clivée par la caspase-1 pour devenir active. La caspase-1 est elle-même activée par un complexe appelé inflammasome qui peut être formé par divers membres de la famille NLR. Plusieurs inflammasomes ont été décrits tels que le NALP3 inflammasome ou l'IPAF inflammasome. Dans cette étude nous avons identifié la co-chaperone SGT1 et la chaperone HSP90 comme partenaires d'interaction de NALP3. Ces deux protéines sont bien connues chez les plantes pour leurs rôles dans la régulation des gènes de résistance (gène R) qui sont structurellement apparentés à la famille NLR. Nous avons pu montrer que SGT1 et HSP90 jouent un rôle similaire dans la régulation de NALP3 et des protéines R. En effet, nous avons démontré que les deux protéines sont nécessaires pour l'activité du NALP3 inflammasome. De plus, la HSP90 est également requise pour la stabilité de NALP3. En se basant sur ces observations, nous avons proposé un modèle dans lequel SGT1 et HSP90 maintiennent NALP3 inactif mais prêt à percevoir un ligand activateur qui initierait la cascade inflammatoire. Nous avons également montré une interaction entre SGT1 et HSP90 avec plusieurs NLRs. Cette observation suggère qu'un mécanisme similaire pourrait être impliqué dans la régulation des membres de la famille des NLRs. Ces dernières années, plusieurs PAMPs mais également des DAMPs ont été identifiés comme activateurs du NALP3 inflammasome. Dans la seconde partie de cette étude, nous avons identifié la réponse au stress du réticulum endoplasmique (RE) comme nouvel activateur du NALP3 inflammasome. Cette réponse est initiée lors de l'accumulation dans le réticulum endoplasmique de protéines ayant une mauvaise conformation ce qui conduit, en autre, à l'arrêt de la synthèse de nouvelles protéines ainsi qu'une augmentation de la dégradation des protéines. Les mécanismes par lesquels la réponse du réticulum endoplasmique induit l'activation du NALP3 inflammasome doivent encore être déterminés. Summary : Vertebrates rely on the adaptive and the innate immune systems to fight pathogens. Awarness of the importance of the innate system increased with the identification of Toll-like receptors a decade ago. Since then, intracellular receptors such as the RIG-like helicase (RLH) and the NOD-like receptor (NLR) families have been described for their role in the recognition of microbes. Interleukin- 1ß (IL-1ß) is a key mediator of inflammation. This proinflammatory cytokine is synthesised as an inactive precursor that requires processing by caspase-1 to become active. Caspase-1 is, itself, activated in a complex termed the inflammasome that can be formed by members of the NLR family. Various inflammasome complexes have been described such as the IPAF and the NALP3 inflammasome. In this study, we have identified the co-chaperone SGT1 and the chaperone HSP90 as interacting partners of NALP3. SGT1 and HSP90 are both known for their role in the activity of plant resistance proteins (R proteins) which are structurally related to the NLR family. We have shown that HSP90 and SGT1 play a similar role in the regulation of NALP3 and in the regulation of plant R proteins. Indeed, we demonstrated that both HSP90 and SGT1 are essential for the activity of the NALP3 inflammasome complex. In addition, HSP90 is required for the stability of NALP3. Based on these observations, we have proposed a model in which SGT1 and HSP90 maintain NALP3 in an inactive but signaling-competent state, ready to receive an activating ligand that induces the inflammatory cascade. An interaction between several NLR members, SGTI and HSP90 was also shown, suggesting that similar mechanisms could be involved in the regulation of other NLRs. Several pathogen-associated molecular patterns (PAMPs) but also danger associated molecular patterns (DAMPs) have been identified as NALP3 activators. In the second part of this study, we have identified the ER stress response as a new NALP3 activator. The ER stress response is activated upon the accumulation of unfolded protein in the endoplasmic reticulum and results in a block in protein synthesis and increased protein degradation. The mechanisms of ER stress-mediated NALP3 activation remain to be determined.

Macrophage migration inhibitory factor: a counter-regulator of glucocorticoid action and critical mediator of septic shock.

Recent studies have led to the discovery of a mediator that acts as an endogenous counter-regulator of glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, this protein was found to have the sequence of macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described. Macrophages and T cells release MIF in response both to various inflammatory stimuli and upon incubation with low concentrations of glucocorticoids. The glucocorticoid-induced secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once secreted, MIF "overrides" the anti-inflammatory and immunosuppressive effects of steroids on macrophage and T-cell cytokine production. The physiological role of MIF thus appears to be to counter-balance steroid inhibition of the inflammatory response. Anti-MIF antibodies fully protect animals from experimentally induced gram-negative or gram-positive septic shock, an effect that may be the result of the increased anti-inflammatory effects of glucocorticoids after neutralization of endogenous MIF. Anti-MIF therapeutic strategies are presently under development and may prove to be a means to modulate cytokine production in septic shock as well as in other inflammatory disease states.

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 widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice.

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms

Characterization of two receptors for TRAIL.

Two receptors for TRAIL, designated TRAIL-R2 and TRAIL-R3, have been identified. Both are members of the tumor necrosis factor receptor family. TRAIL-R2 is structurally similar to the death-domain-containing receptor TRAIL-R1 (DR-4), and is capable of inducing apoptosis. In contrast, TRAIL-R3 does not promote cell death. TRAIL-R3 is highly glycosylated and is membrane bound via a putative phosphatidylinositol anchor. The extended structure of TRAIL-R3 is due to the presence of multiple threonine-, alanine-, proline- and glutamine-rich repeats (TAPE repeats). TRAIL-R2 shows a broad tissue distribution, whereas the expression of TRAIL-R3 is restricted to peripheral blood lymphocytes (PBLs) and skeletal muscle. All three TRAIL receptors bind TRAIL with similar affinity, suggesting a complex regulation of TRAIL-mediated signals.

T cell clones from Schistosoma haematobium infected and exposed individuals lacking distinct cytokine profiles for Th1/Th2 polarisation

T cell clones were derived from peripheral blood mononuclear cells of Schistosoma haematobium infected and uninfected individuals living in an endemic area. The clones were stimulated with S. haematobium worm and egg antigens and purified protein derivative. Attempts were made to classify the T cell clones according to production of the cytokines IL-4, IL-5 and IFN-gamma. All the T cell clones derived were observed to produce cytokines used as markers for the classification of Th1/Th2 subsets. However, the 'signature' cytokines marking each subset were produced at different levels. The classification depended on the dominating cytokine type, which was having either Th0/1 or Th0/2 subsets. The results indicated that no distinct cytokine profiles for polarisation of Th1/Th2 subsets were detected in these S. haematobium infected humans. The balance in the profiles of cytokines marking each subset were related to infection and re-infection status after treatment with praziquantel. In the present study, as judged by the changes in infection status with time, the T cell responses appeared to be less stable and more dynamic, suggesting that small quantitative changes in the balance of the cytokines response could result in either susceptibility or resistant to S. haematobium infection.

Peroxisome proliferator-activated receptors beta/delta: emerging roles for a previously neglected third family member.

PURPOSE OF REVIEW: Peroxisome proliferator-activated receptors alpha, beta/delta and gamma are members of the nuclear receptor superfamily. They mediate the effects of fatty acids and their derivatives at the transcriptional level, and are considered to be lipid sensors that participate in the regulation of energy homeostasis. Compared with the alpha and gamma peroxisome proliferator-activated receptor isotypes, peroxisome proliferator-activated receptor beta functions have long remained an enigma. In this review, we focus on emerging knowledge about peroxisome proliferator-activated receptor beta activation and roles. RECENT FINDINGS: We review recent data that suggest key roles in basic cell functions, such as proliferation, differentiation and survival, and in embryonic development and lipid metabolism in peripheral tissues. SUMMARY: The newly unveiled roles of peroxisome proliferator-activated receptor beta in important basic cell functions certainly justify a further exploration of its potential as a therapeutic target in pathologies such as metabolic syndrome X or skin diseases.

Implicacions diagnòstiques del strem-1 (soluble triggering receptor expressed on myeloid cells) en malalts amb síndrom del distress respiratori agut (sdra) i patologia abdominal. estudi preliminar

Els pacients amb patologia abdominal aguda poden desenvolupar una insuficiència respiratòria secundària a la resposta inflamatòria generada o a una pneumònia nosocomial. El mesurament del “soluble *triggering receptor expressed on myeloid cells 1” (sTREM-1) en líquid alveolar, pleural, sinovial o cefalorraquidi ha demostrat la seva utilitat en el diagnòstic d'infecció. Es va determinar el sTREM-1 alveolar i peritoneal en pacients amb quadre abdominal agut més síndrom de distress respiratori agut (SDRA). Es va concloure que el sTREM-1 és útil per diagnosticar infeccions abdominals i pulmonars en aquests pacients i que la relació sTREM-1 alveolar/peritoneal podria ser útil per determinar el focus infecciós.

High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors.

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.

Pathological Reorganization of NMDA Receptors Subunits and Postsynaptic Protein PSD-95 Distribution in Alzheimer's Disease.

In Alzheimer's disease (AD), synaptic alterations play a major role and are often correlated with cognitive changes. In order to better understand synaptic modifications, we compared alterations in NMDA receptors and postsynaptic protein PSD-95 expression in the entorhinal cortex (EC) and frontal cortex (FC; area 9) of AD and control brains. We combined immunohistochemical and image analysis methods to quantify on consecutive sections the distribution of PSD-95 and NMDA receptors GluN1, GluN2A and GluN2B in EC and FC from 25 AD and control cases. The density of stained receptors was analyzed using multivariate statistical methods to assess the effect of neurodegeneration. In both regions, the number of neuronal profiles immunostained for GluN1 receptors subunit and PSD-95 protein was significantly increased in AD compared to controls (3-6 fold), while the number of neuronal profiles stained for GluN2A and GluN2B receptors subunits was on the contrary decreased (3-4 fold). The increase in marked neuronal profiles was more prominent in a cortical band corresponding to layers 3 to 5 with large pyramidal cells. Neurons positive for GluN1 or PSD-95 staining were often found in the same localization on consecutive sections and they were also reactive for the anti-tau antibody AD2, indicating a neurodegenerative process. Differences in the density of immunoreactive puncta representing neuropile were not statistically significant. Altogether these data indicate that GluN1 and PSD-95 accumulate in the neuronal perikarya, but this is not the case for GluN2A and GluN2B, while the neuropile compartment is less subject to modifications. Thus, important variations in the pattern of distribution of the NMDA receptors subunits and PSD-95 represent a marker in AD and by impairing the neuronal network, contribute to functional deterioration.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.