Philippe Pierre

Developmental regulation of MHC class II transport in mouse dendritic cells

Dendritic cells (DCs) have the unique capacity to initiate primary and secondary immune responses. They acquire antigens in peripheral tissues and migrate to lymphoid organs where they present processed peptides to T cells. DCs must therefore exist in distinct functional states, an idea that is supported by observations that they downregulate endocytosis and upregulate surface molecules of the class II major histocompatibility complex (MHC) upon maturation. Here we investigate the features of DC maturation by reconstituting the terminal differentiation of mouse DCs in vitro and in situ. We find that early DCs, corresponding to those found in peripheral tissues, exhibit a phenotype in which most class II molecules are intracellular and localized to lysosomes. Upon maturation, these cells give rise to a new intermediate phenotype in which intracellular class II molecules are found in peripheral non-lysosomal vesicles, similar to the specialized CIIV population seen in B cells. The intermediate cells then differentiate into late DCs which express almost all of their class II molecules on the plasma membrane. These variations in class II compartmentalization are accompanied by dramatic alterations in the intracellular transport of the new class II molecules and in antigen presentation. We found that although early DCs could not present antigen immediately after uptake, efficient presentation of the previously internalized antigen occurred after maturation, 24–48 hours later. By regulating class II transport and compartmentalization, DCs are able to delay antigen display, a property crucial to their role in immune surveillance.

MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control immunity. Here, we show that in response to Lipopolysaccharides (LPS), several microRNAs (miRNAs) are regulated in human monocyte-derived dendritic cells. Among these miRNAs, miR-155 is highly up-regulated during maturation. Using LNA silencing combined to microarray technology, we have identified the Toll-like receptor/interleukin-1 (TLR/IL-1) inflammatory pathway as a general target of miR-155. We further demonstrate that miR-155 directly controls the level of TAB2, an important signal transduction molecule. Our observations suggest, therefore, that in mature human DCs, miR-155 is part of a negative feedback loop, which down-modulates inflammatory cytokine production in response to microbial stimuli.

SUnSET, a nonradioactive method to monitor protein synthesis

We developed a nonradioactive fluorescence-activated cell sorting–based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.

Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling

BackgroundDendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8α in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes.ResultsWe show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively.ConclusionOur study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.

Developmental Regulation of Invariant Chain Proteolysis Controls MHC Class II Trafficking in Mouse Dendritic Cells

Dendritic cells (DCs) developmentally regulate their capacity for antigen presentation by controlling the transport and surface expression of MHC class II molecules. These events reflect a developmental regulation of invariant (Ii) chain cleavage, most likely by the cysteine protease cathepsin S. In immature DCs, inefficient Ii chain cleavage due to low cathepsin S activity leads to the transport of class II-Ii chain complexes to lysosomes, while in mature DCs, elevated cathepsin S activity results in efficient delivery of class II alphabeta dimers to the plasma membrane. Cathepsin S is not controlled transcriptionally but by a novel mechanism involving alterations in the expression and localization of an endogenous cathepsin S inhibitor cystatin C. Thus, the ratio of cystatin C to cathepsin S in developing DCs helps to determine the fate of newly synthesized MHC class II molecules.

CLIP-170 links endocytic vesicles to microtubules

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BIK1 proteins. These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.

Antigen capture, processing, and presentation by dendritic cells: recent cell biological studies.

Antigen uptake, processing and presentation by dendritic cells [DCs] have become amenable to cell biological approaches. The critical events occur in DCs that are undergoing maturation in response to inflammatory stimuli. Successful antigen presentation can be monitored directly using antibodies that are specific for particular MHC-peptide complexes. What a contrast to earlier times when it was difficult to visualize even the uptake of antigen into isolated DCs and DCs in the T cell areas of lymphoid organs! We emphasize here the efficiency of antigen capture and presentation by maturing DCs, especially for dying cells. This presentation of cellular antigens by DCs likely explains the phenomenon of cross priming in the setting of transplantation and other clinical states.

Suppression of eIF2α kinases alleviates Alzheimer's disease–related plasticity and memory deficits

Expression of long-lasting synaptic plasticity and long-term memory requires protein synthesis, which can be repressed by phosphorylation of eukaryotic initiation factor 2 α-subunit (eIF2α). Elevated phosphorylation of eIF2α has been observed in the brains of Alzheimers disease patients and Alzheimers disease model mice. Therefore, we tested whether suppressing eIF2α kinases could alleviate synaptic plasticity and memory deficits in Alzheimers disease model mice. Genetic deletion of eIF2α kinase PERK prevented enhanced phosphorylation of eIF2α and deficits in protein synthesis, synaptic plasticity and spatial memory in mice that express familial Alzheimers disease–related mutations in APP and PSEN1. Similarly, deletion of another eIF2α kinase, GCN2, prevented impairments of synaptic plasticity and defects in spatial memory exhibited by the Alzheimers disease model mice. Our findings implicate aberrant eIF2α phosphorylation as a previously unidentified molecular mechanism underlying Alzheimers disease–related synaptic pathophysioloy and memory dysfunction and suggest that PERK and GCN2 are potential therapeutic targets for treatment of individuals with Alzheimers disease.

Brucella Control of Dendritic Cell Maturation Is Dependent on the TIR-Containing Protein Btp1

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyers patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique

In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [3H]phenylalanine method when detecting synergist ablation‐induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation‐induced increase in PS when used in vivo (IV‐SUnSET). In addition, IV‐SUnSET detected food deprivation‐induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV‐SUnSET (IV‐IHC‐SUnSET). By combining IV‐IHC‐SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB‐independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV‐IHC‐SUnSET exposed the existence of fiber type‐dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.—Goodman, C. A., Mabrey, D. M., Frey, J. W., Miu, M. H., Schmidt, E. K., Pierre, P., Hornberger, T. A. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 25, 1028–1039 (2011). www.fasebj.org