During steady state? Are Dendritic Cells arrested in translation?

RODRIGUEZ RODRIGUES C

Paris

Simposio; Labex DC Bio; 2013

CNRS-INSERM

Dendritic cells (DCs) share specific functional properties that distinguish them as a pivotal link between innate and adaptive immunity. They represent the most important family of professional antigen-presenting cells specialized in capturing a broad spectrum of antigens, processing and efficiently present to naive T cells, a process that leads to two distinct outcomes: induction of tolerance or the generation of a protective immune response. As the initial description of dendritic cells (DCs) by Steinman and Cohn, an increasing number of DC subpopulations has been phenotypically classified and functionally designated due of their enormous plasticity and ability to adapt to the influences of the microenvironment. Diverse stresses such as nutrient deprivation, radiation, oxidative stress or viral infection can promote translation initiation factor 2 alpha (eIF2á) phosphorylation leading to global protein synthesis shutoff and activating the expression of the transcription factor ATF4, and its downstream targets, including the growth arrest and DNA-damage-inducible protein 34 (GADD34), a phosphatase 1 cofactor that functions as a negative-feedback regulator of eIF2á phosphorylation. Surprisingly, previous data of our team show unusually high levels of P- eIF2á in nonstimulated DCs compared with mouse embryonic fibroblasts (MEFs), and no up-regulation of P- eIF2á could be visualized during maduration. Moreover, the levels of P-eIF2á in purified mouse spleen CD11c+ DCs were higher than nonstimilated bmDCs. To our knowledge this level of P- eIF2á has never been documented before and must indicate a particular immunological status of resting DCs. From these data, we formulate several questions, What is the function of have high levels of P-eIF2a in splenic DCs?, Which signaling pathway controls eIF2á phosphorylation in splenic DCs?.  Is it involved in the development of the DCs? or this protein synthesis inhibition affects  a particular DCs subpopulation ? To address these questions, we will use a combination of cell biology and immunological techniques, as well as different transgenic mice strains to perform a precise dissection of the state of eIF2á phosphorylation in DCs subpopulations both in vivo and in vitro. We will correlate the state of eIF2-a phosphorylation in DCs with their protein synthesis using Sunset, a technique designed by our laboratory. Our primary interest will be to define, which signaling pathway controls eIF2á phosphorylation in inactive tissue resident cells and microbe-activated DCs. Second, we will establish, whether eIF2á phosphorylation regulation is required for DC development and function, by investigating their capacity to present antigens, prime T cells and produce cytokines. DCs deficient for different eIF2á kinases (e.g. PKR, PERK, GCN2) or expressing a non-phosphorylable eIF2-A/A form (eIF2á S51A) will be used to analyze DC populations and behavior. PKR -/- cells will be of particular interest since this eIF2á kinase has been shown to be also activated downstream of different TLRs. Mice deficient for GADD34 will be used to explore the biochemical and immunological consequences for animals and cells in which eIF2á remains overly phosphorylated upon stimulation. All together this strategy should allow us to understand the importance of DCs eIF2á phosphorylation.