Our work provides an additional layer of analysis to the DC activation process. Although transcriptional regulation is necessary to acquire new functions, the regulation of gene expression at the level of translation provides the cell with the plasticity that is needed to respond to rapid changes in its environment. This has been shown recently in naive T helper-2 cells, which enter a stress response by blocking translation upon T cell receptor stimulation and retaining this state until restimulation (Scheu et al., 2006
). Our results demonstrate that DC activation is divided in two distinct phases. A phase of protein synthesis increase during the first 4 h of DC maturation is followed by a phase of down-regulation involving cap-mediated translation inhibition. We have also provided examples of the importance of these two phases in DC function, illustrating how basic cellular processes such as regulation of mRNA translation can control cell differentiation and function.
Detection of LPS by TLR4 activates PI3K through the recruitment of the MyD88 and TRIF adaptors, leading to AKT and mTOR activation and finally ending by S6 protein phosphorylation by S6K1. This signal transduction pathway is also activated by growth factors in replicating cells. TLR signaling is therefore likely to control the cellular metabolism, in addition to its stimulating role on immunity. It is a puzzling observation because mDCs are not dividing and therefore do not need a high rate of protein synthesis to sustain proliferation. Putatively, a high rate of translation is required for achieving the massive cellular changes observed upon DC maturation such as MHC and costimulatory molecules up-regulation, as well as cytokine production. Interestingly, our observation that IL-12 secretion is prevented during PI3K inhibition by LY contrasts with previous results obtained by Fukao et al. (Fukao et al., 2002
), in which DC treatment with wortmannin was found to enhance IL-12 production. We attribute this discrepancy to the use of wortmannin, which, at the concentration used, is less efficient than LY to inhibit PI3K in DCs (as shown in ). Partial inhibition of the PI3K pathway could lead to potential compensation mechanisms able to promote IL-12 production.
The abrupt increase in protein synthesis is also likely to enhance markedly the proportion of DRiPs available for processing. In this context, DALIS probably represent an adapted stress response to these radical changes in protein synthesis and to the massive accumulation of DRiPs in such a short time lapse. DALIS are dependent on the signaling pathway inducing the translational boost, comprising the PI3K/AKT/mTOR signaling axis, which is key to achieve functional DC maturation. DALIS formation and disappearance follow the enhancement and inhibition of cap-mediated translation during DC maturation. By accumulating DRiPs, DALIS provide probably a mean to control the cytotoxic effects of these misfolded proteins, as well as their availability for degradation. Surprisingly, although the rate of surface arrival of peptide-loaded MHC class I does not fluctuate with maturation progression, the type of antigen presented changes dramatically. From fully dependent on newly synthesized antigens at early time of maturation, in agreement with the DRiPs hypothesis, MHC class I presentation switches to a pool of preexisting antigens at late stages of maturation. Thus, these antigens could be provided by decaying DALIS or alternatively they could be of exogenous origin and mostly cross-presented. In absence of a mean to control DALIS formation efficiently without affecting DC maturation, the elucidation of their exact function will remain unanswered. However, we could demonstrate that translation regulation affects the pool of antigen available for MHC class I presentation and that MHC class I restricted presentation is radically different in cells stimulated with LPS for different times.
A reduction in cap-dependent translation and a shift toward translation of certain IRES-containing mRNAs was observed after 8 h of maturation. Remarkably, this process is similar to the stress-induced attenuation of protein synthesis required for cell survival under harsh conditions. In addition to a mild phosphorylation of eIF2α, we observed a good correlation between eIF4GI and DAP5 proteolysis and the intensity of their translation during maturation. Moreover, protease inhibitors preventing eIF4GI and DAP5 cleavage had a strong inhibitory effect on the production of these molecules in a manner similar to protein synthesis inhibitors, thus definitely linking proteolysis and translation regulation. We could show that the protease(s) responsible for the cleavage of eIF4GI are different from the major known apoptotic caspases, such as caspase-3, which is responsible for translation inhibition during apoptosis. Based on MG132 effects and on a recent report (Baugh and Pilipenko, 2004
), we further propose that proteasome activity could be responsible, at least in part, for the degradation of the translation initiation factors and, thus, exert a control on translation in mDCs. Interestingly, MG132 treatment is known to affect viral IRES activity (Baugh and Pilipenko, 2004
) as well as to induce translation reprogramming of myoblasts (Cowan and Morley, 2004
). The effect observed with the broad range inhibitor of caspases Z-VAD-FMK could be due either by inhibition of an additional unidentified protease or by inhibition of the caspase-like activity of the proteasome itself because a survey of eIF4GI primary sequence for sites potentially targeted by the proteasome caspase-like activity (Kisselev et al., 2003
) indicates the presence of several consensus sites in areas matching eIF4GI cleavage map.
Cell death appears to be a normal response to counter-balance the activation of the proliferation machinery (including protein synthesis enhancement). The establishment of anti-apoptotic conditions during DC maturation seems dependent, at least in part, on the switch in the quality of translation induced by proteolytic cleavage of eIF4GI and DAP5. LPS detection by DCs is therefore integrated in a binary response containing a growth factor-like and a stress-like phase. This response is adapted to the need for rapid changes in the physiology of DCs after pathogen detection. Marked up-regulation of protein synthesis probably favors apoptosis through the accumulation of misfolded proteins. DCs therefore take advantage of the second phase to enhance their survival rate and augment their capacity to interact with other cells during their migration. Whether this phase is directly triggered by LPS or indirectly by an autocrine cytokine loop remains to be evaluated. IL-6 could cause some of these changes because it has been shown to favor some IRES-mediated translation (Yamagiwa et al., 2004