Education of Murine NK Cells Requires Both cis and trans Recognition of MHC Class I Molecules.

Although NK cells use invariant receptors to identify diseased cells, they nevertheless adapt to their environment, including the presence of certain MHC class I (MHC-I) molecules. This NK cell education, which is mediated by inhibitory receptors specific for MHC-I molecules, changes the responsiveness of activating NK cell receptors (licensing) and modifies the repertoire of MHC-I receptors used by NK cells. The fact that certain MHC-I receptors have the unusual capacity to recognize MHC-I molecules expressed by other cells (trans) and by the NK cell itself (cis) has raised the question regarding possible contributions of the two types of interactions to NK cell education. Although the analysis of an MHC-I receptor variant suggested a role for cis interaction for NK cell licensing, adoptive NK cell transfer experiments supported a key role for trans recognition. To reconcile some of these findings, we have analyzed the impact of cell type-specific deletion of an MHC-I molecule and of a novel MHC-I receptor variant on the education of murine NK cells when these mature under steady-state conditions in vivo. We find that MHC-I expression by NK cells (cis) and by T cells (trans), and MHC-I recognition in cis and in trans, are both needed for NK cell licensing. Unexpectedly, modifications of the MHC-I receptor repertoire are chiefly dependent on cis binding, which provides additional support for an essential role for this unconventional type of interaction for NK cell education. These data suggest that two separate functions of MHC-I receptors are needed to adapt NK cells to self-MHC-I

Etude du rôle des LT non conventionnels pendant la réponse anti-infectieuse à un pathogène intracellulaire (Modèle Brucella suis)

Les LTg9d2 et les cellules iNKT sont des lymphocytes non conventionnels impliqués dans la réponse immunitaire. Ils interviennent rapidement durant la phase innée et régulent la phase adaptative. Nous avons étudié l'activité anti-infectieuse de ces deux populations lymphocytaires dans un modèle d'infection bactérien de macrophages humains. Brucella suis est une bactérie pathogène responsable de la fièvre de Malte qui infecte et se multiplie dans les macrophages. Les LTg9d2 inhibent le développement de Brucella dans les macrophages par une combinaison de divers mécanismes. Nous avons montré que les LTg9d2 sécrètent des peptides antimicrobiens (LL-37) qui inhibent le développement de Brucella. Nous avons également étudié le rôle du récepteur NKG2D et montré son implication dans l'activité anti-infectieuse des LTg9d2. Concernant les cellules NKT, nous avons tout d'abord étudié les mécanismes qui régulent leur activation puis nous nous sommes intéressés à leur activité anti-infectieuse. L'activation des cellules iNKT par l'IL-2 déclenche un profil signalétique particulier conduisant à la production de cytokines pro- et anti-inflammatoires. Ces cellules sont également capables d'inhiber le développement intramacrophagique de Brucella. Divers mécanismes comme la production d'IFN-g, le déclenchement de l'apoptose via le recrutement de la voie du Fas et la libération de granules lytiques sont impliqués dans cette activité anti-infectieuse. Nos résultats suggèrent que les LT g9d2 et les cellules iNKT jouent un rôle important dans la réponse immunitaire anti-infectieuse aussi bien en tant que cellules régulatrices (sécrétion de cytokines) qu'effectrices (activité cytotoxique)g9d2 T cells and iNKT cells are nonconventional lymphocytes early involving in the innate phase of immune response and regulate the adaptive phase. We study their involvement during bacterial infection of macrophages. Brucella suis is the bacteria responsible of brucellosis infecting macrophages and surviving inside. g9d2 T cells inhibit intramacrophagic development of B. suis by diverse mechanisms. We demonstrated that g9d2 T cells secrete antimicrobial peptides (LL-37) which decrease Brucella's survival. We also demonstrated that NKG2D receptor is involved in anti-infectious effect of g9gd T cells. Regarding NKT cells, we first studied their activation mechanisms and then their anti-infectious activity. IL-2 activation of iNKT cells triggers recruitment of several specific intracellular signalling pathways leading to pro- and anti-inflammatory cytokine production. iNKT cells are also able to decrease intramacrophagic development of B. suis. Apoptose induced by Fas pathway and by release of lytic granules as well as production of IFN-g involve in anti-infectious effect of iNKT cells. In conclusion, our results suggest that g9d2 T cells and iNKT cells play an important role during anti-infectious immune response as regulator cells by cytokine production and as effector cells by cytotoxic activityMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

IL-2 triggers specific signaling pathways in human NKT cells leading to the production of pro- and anti-inflammatory cytokines.

International audienceNKT cells belong to a conserved T lymphocyte subgroup that has been implicated in the regulation of various immune responses, including responses to viruses, bacteria, and parasites. They express a semi-invariant TCR that recognizes glycolipids presented by the nonpolymorphic MHC class I-like molecule CD1d, and upon activation, they produce various pro- and anti-inflammatory cytokines. Recent studies have shed light on the nature of glycolipids and the environmental signals that may influence the production of cytokines by NKT cells and thus, modulate the immune response. To better understand the regulation mechanisms of NKT cells, we explored their behavior following activation by IL-2 and investigated the signaling pathways and biological responses triggered. We demonstrated that IL-2 activates not only STAT3 and -5 and the PI-3K and ERK-2 pathways as in all IL-2 responder cells but also STAT4 as in NK cells and the p38 MAPK pathway as in alphabeta T cells. We also showed that STAT6 is activated by IL-2 in NKT cells. Moreover, IL-2 induces the production of IFN-gamma and IL-4. The ability of IL-2 to induce pro- and anti-inflammatory cytokine production, in addition to proliferation, could open new therapeutic approaches for use in combination with molecules that activate NKT cells through TCR activation

In Vitro and In Vivo Analysis of the Gram-Negative Bacteria-Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells.

International audienceMucosal-associated invariant T (MAIT) cells recognize microbial compounds presented by the MHC-related 1 (MR1) protein. Although riboflavin precursor derivatives from Gram-positive bacteria have been characterized, some level of ligand heterogeneity has been suggested through the analysis of the MAIT cell TCR repertoire in humans and differential reactivity of human MAIT cell clones according to the bacteria. In this study, using Gram-negative bacteria mutated for the riboflavin biosynthetic pathway, we show a strict correlation between the ability to synthesize the 5-amino-ribityl-uracil riboflavin precursor and to activate polyclonal and quasi-monoclonal mouse MAIT cells. To our knowledge, we show for the first time that the semipurified bacterial fraction and the synthetic ligand activate murine MAIT cells in vitro and in vivo. We describe new MR1 ligands that do not activate MAIT cells but compete with bacterial and synthetic compounds activating MAIT cells, providing the capacity to modulate MAIT cell activation. Through competition experiments, we show that the most active synthetic MAIT cell ligand displays the same functional avidity for MR1 as does the microbial compound. Altogether, these results show that most, if not all, MAIT cell ligands found in Escherichia coli are related to the riboflavin biosynthetic pathway and display very limited heterogeneity

The new species Brucella microti replicates in macrophages and causes death in murine models of infection

Background. The recent isolation of Brucella microti from the common vole, the red fox, and the soil raises the possibility of an eventual reemergence of brucellosis in Europe. In this work, the pathogenic potential of this new Brucella species in both in vitro and in vivo models of infection was analyzed. Methods. The ability of B. microti (as compared to that of the closely related species Brucella suis) to replicate in human macrophages and in human and murine macrophage-like cells was determined. The behavior of B. microti and B. suis was evaluated in vivo in murine models of infection with Balb/c, CD1, and C57BL/6 mice. Results.B. microti showed an enhanced capacity for intramacrophagic replication compared with that of B. suis. Surprisingly, and in contrast to other species of Brucella, 105 colony-forming units of B. microti killed 82% of Balb/c mice within 7 days. Infection of spleen and liver with B. microti peaked at day 3, compared with B. suis infection, which peaked at day 7. Sublethal doses of B. microti induced good protection against a subsequent challenge with lethal doses. Conclusions. In experimental cellular and murine infections, B. microti exhibited a high pathogenic potential, compared with other Brucella species

MAIT cells detect and efficiently lyse bacterially-infected epithelial cells.

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells

Spheroplexes: Hybrid PLGA-cationic lipid nanoparticles, for in vitro and oral delivery of siRNA

International audienceVectorized small interfering RNAs (siRNAs) are widely used to induce gene silencing. Among the delivery systems used, lipid-based particles are the most effective. Our objective was the development of novel lipid-polymer hybrid nanoparticles, from lipoplexes (complexes of cationic lipid and siRNAs), and poly (lactic-co-glycolic acid) (PLGA), using a simple modified nanoprecipitation method. Due to their morphology, we called these hybrid nanoparticles Spheroplexes. We elucidated their structure using several physico-chemical techniques and showed that they are composed of a hydrophobic PLGA matrix, surrounded by a lipid envelope adopting a lamellar structure, in which the siRNA is complexed, and they retain surface characteristics identical to the starting nanoparticles, i.e. lipoplexes siRNA. We analyzed the composition of the particle population and determined the final percentage of spheroplexes within this population, 80 to 85% depending on the preparation conditions, using fluorescent markers and the ability of flow cytometry to detect nanometric particles (approximately 200 nm). Finally, we showed that spheroplexes are very stable particles and more efficient than siRNA lipoplexes for the delivery of siRNA to cultured cells. We administered spheroplexes contain siRNAs targeting TNF-α to mice with ulcerative colitis induced by dextran sulfate and our results indicate a disease regression effect with a response probably mediated by their uptake by macrophages / monocytes at the level of lamina propria of the colon. The efficacy of decreased level of TNF-α in vivo seemed to be an association of spheroplexes polymer-lipid composition and the specific siRNA. These results demonstrate that spheroplexes are a promising hybrid nanoparticle for the oral delivery of siRNA to the colon

Mucosal-associated invariant T cell alterations in obese and type 2 diabetic patients

Obesity and type 2 diabetes (T2D) are associated with low-grade inflammation, activation of immune cells, and alterations of the gut microbiota. Mucosal-associated invariant T (MAIT) cells, which are innate-like T cells that recognize bacterial ligands, are present in blood and enriched in mucosal and inflamed tissues. Here, we analyzed MAIT cells in the blood and adipose tissues of patients with T2D and/or severe obesity. We determined that circulating MAIT cell frequency was dramatically decreased in both patient groups, and this population was even undetectable in some obese patients. Moreover, in both patient groups, circulating MAIT cells displayed an activated phenotype that was associated with elevated Th1 and Th17 cytokine production. In obese patients, MAIT cells were more abundant in adipose tissue than in the blood and exhibited a striking IL-17 profile. Bariatric surgery in obese patients not only improved their metabolic parameters but also increased circulating MAIT cell frequency at 3 months after surgery. Similarly, cytokine production by blood MAIT cells was strongly decreased after surgery. This study reveals profound MAIT cell abnormalities in patients harboring metabolic disorders, suggesting their potential role in these pathologies