Regulation of translation is required for dendritic cell function and survival during activation

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. Here, we show that in response to lipopolysaccharides, protein synthesis is rapidly enhanced in DCs. This enhancement occurs via a PI3K-dependent signaling pathway and is key for DC activation. In addition, we show that later on, in a manner similar to viral or apoptotic stress, DC activation leads to the phosphorylation and proteolysis of important translation initiation factors, thus inhibiting cap-dependent translation. This inhibition correlates with major changes in the origin of the peptides presented by MHC class I and the ability of mature DCs to prevent cell death. Our observations have important implications in linking translation regulation with DC function and survival during the immune response

Ribosomal protein mRNAs are translationally-regulated during human dendritic cells activation by LPS

International audienceBACKGROUND: Dendritic cells (DCs) are the sentinels of the mammalian immune system, characterized by a complex maturation process driven by pathogen detection. Although multiple studies have described the analysis of activated DCs by transcriptional profiling, recent findings indicate that mRNAs are also regulated at the translational level. A systematic analysis of the mRNAs being translationally regulated at various stages of DC activation was performed using translational profiling, which combines sucrose gradient fractionation of polysomal-bound mRNAs with DNA microarray analysis. RESULTS: Total and polysomal-bound mRNA populations purified from immature, 4 h and 16 h LPS-stimulated human monocyte-derived DCs were analyzed on Affymetrix microarrays U133 2.0. A group of 375 transcripts was identified as translationally regulated during DC-activation. In addition to several biochemical pathways related to immunity, the most statistically relevant biological function identified among the translationally regulated mRNAs was protein biosynthesis itself. We singled-out a cluster of 11 large ribosome proteins mRNAs, which are disengaged from polysomes at late time of maturation, suggesting the existence of a negative feedback loop regulating translation in DCs and linking ribosomal proteins to immuno-modulatory function. CONCLUSION: Our observations highlight the importance of translation regulation during the immune response, and may favor the identification of novel protein networks relevant for immunity. Our study also provides information on the potential absence of correlation between gene expression and protein production for specific mRNA molecules present in DCs

Induction of GADD34 Is Necessary for dsRNA-Dependent Interferon-β Production and Participates in the Control of Chikungunya Virus Infection

Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection

Unfolded protein response et auto-immunité : focus sur GADD34 et le lupus érythémateux systémique

L'Unfolded Protein Response (UPR) est un réseau de voies de signalisation qui permet de rétablir l'homéostasie au sein du réticulum endoplasmique suite à l'accumulation de protéines mal repliées. GADD34, cofacteur inductible par le stress de la phosphatase PP1, est responsable d'une boucle de régulation négative de l'UPR ; de plus il a été montré être nécessaire pour la production de cytokines pro-inflammatoires dans un modèle murin d'infection virale. Nous avons formulé l'hypothèse que GADD34 soit une molécule clé de l'inflammation également chez l'homme, notamment dans le cas de pathologies auto-immunes avec une composante inflammatoire. Le PHRC interrégional « GADD34_LES » qui a pour objectif principal l'évaluation de l'augmentation de l'expression du gène codant pour GADD34 chez les patients lupiques comme possible biomarqueur pronostique de poussée a été mis en place par le laboratoire d'immunologie du CHUGA. Dans le cadre d'une étude ancillaire de ce PHRC portant sur 26 patients et 26 sujets sains, l'expression de GADD34 a été mesurée par PCR quantitative après séparation magnétique des lymphocytes T, lymphocytes B et monocytes. La surexpression de GADD34 principalement dans les lymphocytes B des patients lupiques par rapport aux autres populations cellulaires étudiées était statistiquement significative. Dans le cadre du PHRC, l'augmentation de l'expression de GADD34 chez les patients lupiques comme marqueur pronostique de poussée reste à évaluer. Par ailleurs, des inhibiteurs spécifiques du complexe PP1/GADD34 existent, et ils pourraient éventuellement être testés in vitro pour cibler spécifiquement les cellules productrices d'anticorps ou de cytokines pro-inflammatoires

Role of cellular responses to stress and regulation of innate immunity in dendritic cells activated by double-stranded RNA

Les cellules dendritiques (DCs) sont, parmi les cellules présentatrices d antigène, les plus efficaces dans l initiation des réponses immunitaires, induisant la différentiation des lymphocytes T naïfs. La régulation de la synthèse protéique pendant la maturation des DCs induite par la détection de motifs pathogéniques a été montrée par notre groupe avoir un rôle essentiel pour leur fonction et survivance. Nous avons développé une nouvelle technologie pour mesurer l intensité de la traduction, basée sur l incorporation de puromycine dans les chaînes polypeptidiques néosynthétisées et sa détection par des anticorps monoclonaux spécifiques. En utilisant cette méthode, nous avons montré que les DCs, suite à la détection du poly I:C, un analogue synthétique des ARNs double-brins, présentent une résistance à l inhibition de la traduction, contrairement à d autres types cellulaires tels que les fibroblastes. De plus, les DCs activées montrent une résistance générale à différents types de réponse au stress cellulaire, notamment la formation de granules de stress et l arrêt traductionnel suite à la carence en tryptophane. De façon intéressante, nous avons trouvé que les DCs activées par le poly I:C présentent un profil de transcrits similaire à ce qui est produit au cours d une unfolded protein response . Parmi les gènes induits, GADD34, cofacteur de la phosphatase PP1, a été identifié comme la molécule responsable de la régulation des réponses au stress dans les DCs, à travers le changement de l équilibre biochimique vers la déphosphorylation de la sous-unité du facteur d initiation de la traduction eIF2. L importance de cette régulation pour la fonction des DCs est démontrée par l altération de la production de IFN- et l activation de la caspase 3 suite à l inhibition de l activité de GADD34/PP1. Nous proposons que ce mécanisme de régulation puisse permettre aux DCs d exercer leurs fonctions spécifiques dans la réponse immunitaire, en conditions dans lesquelles un arrêt traductionnel pourrait empêcher leur activité.Among antigen presenting cells, dendritic cells (DCs) are the most efficient at initiating antigen-specific responses, inducing differentiation of naive T cells. Regulation of protein synthesis during DC maturation induced by the detection of pathogen-associated molecular patterns has been shown by our group to have a crucial role for their function and survival. We developed a new technology to measure the intensity of translation, based on the incorporation of puromycin in newly synthesized polypeptide chains and its subsequent detection by specific monoclonal antibodies. By using this method, we show that DCs, following the detection of poly I:C (a synthetic double-stranded RNA analogue) resist to the inhibition of translation normally observed in other cell types such as fibroblasts. Moreover, activated DCs show a general inhibition of several types of response to cellular stress, such as stress granule formation and translational arrest due to tryptophan starvation. Interestingly, we found that poly I:C-activated DCs display a transcription profile similar to that induced during an unfolded protein response . Among the upregulated genes, we identified GADD34, a phosphatase 1 cofactor, as the responsible for the regulation of stress responses in DCs, by shifting the biochemical equilibrium toward the dephosphorylation of the subunit of the translation initiation factor eIF2. The importance of this regulation for DC function is demonstrated by the alteration of IFN- production and the induction of caspase-3 cleavage upon inhibition of PP1/GADD34 activity. We propose that this mechanism could allow DCs to exert their specific functions in the immune response, in conditions in which a translational arrest might impair their activity.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Antibodies targeting circulating protective molecules in lupus nephritis: Interest as serological biomarkers

International audienceLupus nephritis (LN) is one of the most frequent and severe manifestations of systemic lupus erythematosus (SLE), considered as the major predictor of poor prognosis. An early diagnosis of LN is a real challenge in the management of SLE and has an important implication in guiding treatments. In clinical practice, conventional parameters still lack sensitivity and specificity for detecting ongoing disease activity in lupus kidneys and early relapse of nephritis. LN is characterized by glomerular kidney injury, essentially due to deposition of immune complexes involving autoantibodies against cellular components and circulating proteins. One of the possible mechanisms of induction of autoantibodies in SLE is a defect in apoptotic cells clearance and subsequent release of intracellular autoantigens. Autoantibodies against soluble protective molecules involved in the uptake of dying cells, including complement proteins and pentraxins, have been described. In this review, we present the main autoantibodies found in LN, with a focus on the antibodies against these protective molecules. We also discuss their pathogenic role and conclude with their potential interest as serological biomarkers in LN

The Immunopathology of Complement Proteins and Innate Immunity in Autoimmune Disease

International audienceThe complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions

Are p53 inhibitors potentially useful therapeutics?

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The endosomal proteome of macrophage and dendritic cells.

International audienceThe essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types