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Proc Natl Acad Sci U S A. Feb 21, 2012; 109(8): 3006–3011.
Published online Feb 6, 2012. doi:  10.1073/pnas.1104491109
PMCID: PMC3286954
Immunology

Protein phosphatase 1 subunit Ppp1r15a/GADD34 regulates cytokine production in polyinosinic:polycytidylic acid-stimulated dendritic cells

Abstract

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed. In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2α was observed during DC activation. Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis. GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo. These observations have important implications in linking further pathogen detection with the integrated stress response pathways.

Keywords: type-I interferon, Toll-like receptor, protein kinase RNA activated, puromycin, unfolded protein response

Dendritic cells (DCs) are regulators of the immune response, which, upon stimulation by conserved microbial products (PAMPs), are most efficient at inducing differentiation of naive T cells through massive cytokine production and optimization of their antigen presentation capacity (1). Double-stranded RNAs (dsRNA), a hallmark of virus replication, is recognized by Toll-like receptor 3 (TLR3) (2) or cytosolic RNA DExD/H-box helicases, such as melanoma-associated gene-5 (MDA5) (3) or the DDX1, DDX21, and DHX36 (4). Detection of pI:C, a dsRNA mimic, promotes the nuclear translocation of IRF-3 and IRF-7 and antiviral type-I IFN production (5). IFN triggers, among many other antiviral genes, the dsRNA-dependent protein kinase (PKR) (6, 7). PKR activation by dsRNA promotes translation initiation factor 2 alpha (eIF2α) phosphorylation leading to protein synthesis shutoff and inhibition of viral replication (8). PKR is also necessary for normal type-I IFN secretion by DCs in response to dsRNA stimulation (9, 10).

Different stress signals trigger eIF2α phosphorylation, thus attenuating mRNA translation and promoting a specific transcriptional response known as the integrated stress response (ISR) (11). To date four eIF2α kinases have been identified, PKR, PKR-like ER kinase (PERK) (12), general control nonderepressible-2 (GCN2) (13), and heme-regulated inhibitor (HRI) (14). PERK, which is activated by an excess of unfolded proteins in the ER lumen (12), is necessary to mount part of a particular ISR, known as the unfolded protein response (UPR).

The UPR encompasses a group of signals that cope with perturbations in ER homeostasis. In addition to PERK, the UPR is initiated by additional ER sensors and associated transcription factors such as IRE1, XBP1 and ATF6 (15, 16). In addition to inhibiting global protein synthesis, eIF2α phosphorylation by PERK allows the specific translation of the transcription factor ATF4 (14). This factor promotes cell adaptation to stress by heightening ER functions through specific mRNA transcription. Immune responses and immune cell development can be profoundly affected by abnormalities in the UPR (17, 18) and TLR-dependent activation of XBP1, via the production of reactive oxygen species (ROS), is required for normal production of proinflammatory cytokines in macrophages (19).

We show here that activated DCs resist the translation arrest, normally elicited by the PKR-dependent eIF2α phosphorylation in response to cytosolic dsRNA delivery. Concomitantly, pI:C detection promotes the expression of the inducible phosphatase 1 (PP1)-cofactor, GADD34 (20). GADD34 efficiently dephosphorylates eIF2α in activated DCs, but has little impact on the specific resistance of DCs to dsRNA-triggered translation arrest. Alternatively, GADD34 is required for optimal transcription and production of the cytokines IFN-β and interleukin-6 (IL-6), suggesting that induction of the ATF4-dependent branch of the UPR in DCs is a critical signaling module of the innate response to dsRNA.

Results

DC Stimulation by pI:C Induces the ATF4 Transcription Factor.

To identify signaling pathways involved in dsRNA response, a genomewide expression analysis was performed in pI:C-stimulated mouse bone marrow-derived DCs (bmDCs) using Affymetrix Mouse Genome 430 2.0 arrays. We found that at least nine transcripts, typically expressed in different cells exposed to tunicamycin (11, 21, 22), were also induced in DCs responding to pI:C (Table S1).

Up-regulation of these transcripts was confirmed by quantitative RT-PCR (qPCR) (Fig. 1 and Fig. S1). Among them, the key UPR transcription factors, ATF4 and CCAAT/enhancer binding protein homologous protein (CHOP) mRNAs were increased by two- and eightfold, respectively (Fig. 1 A and C). ATF4 mRNA is normally poorly translated in unstressed cells, but upon eIF2α phosphorylation, a rapid synthesis of the ATF4 protein has been observed (14). In DCs, ATF4 protein levels were increased upon pI:C stimulation and detected in the nuclear extracts after 8 h of stimulation, similarly to control DCs, in which a bona fide UPR was induced with thapsigargin (Fig. 1B and Fig. S2).

Fig. 1.
ATF4 and GADD34 are induced during DC activation with pI:C. Experiments were performed in bmDCs stimulated with 10 μg/mL of pI:C or, when indicated, in RAW macrophages with 100 ng of LPS. mRNA levels of ATF4 (A), CHOP (C), GADD34 (E), PP1 (G), ...

Nuclear translocation of ATF4 normally induces CHOP expression, which can trigger apoptosis, and enhance the transcription of several ATF4 target genes such as GADD34 and ERO1-α (22). As expected from the array analysis, increased CHOP mRNA levels were detected (Fig. 1C), however, we failed to visualize protein expression in LPS and pI:C-stimulated DCs extracts. In contrast, CHOP was detected in control tunicamycin-treated DC as well as in LPS-stimulated mouse leukaemic macrophages (RAW 264.7 cell line) (Fig. 1D). We tested different doses and modes of pI:C delivery to DCs and in agreement with the lack of CHOP expression, we found no major apoptotic death induction in our experimental system (Fig. S3A). Thus, pI:C-activated DCs display an ATF4 transcriptional signature during which, CHOP expression seems to be specifically down-modulated at the translational level, which presumably contributes to the prevention of apoptosis in activated DCs (23).

GADD34 Is Up-Regulated in Activated DCs.

GADD34 acts together with PP1 to dephosphorylate eIF2α and relieves translation repression during ER stress (22, 24, 25). In response to soluble pI:C, GADD34 mRNA transcription was enhanced at least 14-fold (Fig. 1E), contrasting with the modest 1.5-fold up-regulation of PP1 expression (Fig. 1G) and 2-fold up-regulation of the “constitutive” protein phosphatase 1 regulatory subunit 15B (CReP) mRNA (Fig. 1H) (26). Matching the transcriptional analysis, and irrespectively of the mode of pI:C delivery (soluble or lipofected), GADD34 protein levels were induced and continuously increased during activation (Fig. 1F). Addition of proteasome inhibitor MG132 during the last 2 h of pI:C treatment facilitated the detection of short-lived GADD34 by preventing its degradation (Fig. 1F) (27) and confirmed GADD34 expression during DC maturation.

eIF2α Is Dephosphorylated During DC Activation.

Protein synthesis was monitored using puromycin labeling followed by immunoblot (Fig. 2A) (28). As previously shown for LPS-activated DCs (29), translation was enhanced in the first hours of pI:C stimulation followed by a reduction at 16 h. The levels of phosphorylated eIF2α (P-eIF2α) were gradually lost during pI:C stimulation (Fig. 2B), independently of the mode of dsRNA delivery (Fig. S3B). Levels of P-eIF2α were surprisingly high in nonstimulated DCs compared with mouse embryonic fibroblasts (MEFs), and no up-regulation of P-eIF2α could be visualized during maturation (Fig. 2 B and C). We further investigated whether these high levels of P-eIF2α were physiologically relevant by analyzing purified mouse spleen CD11c+ DCs. Explanted CD11c+ DCs displayed even higher P-eIF2α levels than nonstimulated bmDCs. This observation was further confirmed by immunohistochemistry of spleen sections, in which high P-eIF2α was mostly detected in resting CD11c+ DCs and not in neighboring CD3+ T cells in situ (Fig. S4). Spleen DCs and nonstimulated bmDCs, therefore, display naturally high levels of P-eIF2α, which might explain their low level of translation in vitro and in vivo and might be important for exerting their sentinel function (29).

Fig. 2.
eIF2α dephosphorylation is controlled by GADD34 during pI:C-induced DC maturation. (A) Protein synthesis was quantified in protein extracts of pI:C-activated DCs using puromycin labeling followed by immunoblot. Cells not treated with puromycin ...

Because P-eIF2α intensity was inversely correlated with GADD34 expression in activated cells, we tested the function of PP1-GADD34 and -CReP complexes by inhibiting their activity with the pharmacological inhibitors salubrinal and guanabenz (30, 31). In the presence of both drugs, P-eIF2α levels were enhanced by pI:C treatment (Fig. 2D). The same result was found when using DCs inactivated for GADD34 (GADD34ΔC/ΔC) (25), in which P-eIF2α was considerably augmented upon activation compared with WT cells (Fig. 2E and quantification in Fig. S3C). Thus, GADD34 and PP1 dephosphorylate eIF2α and counteract efficiently the activation of eIF2α kinases in pI:C-stimulated DCs.

PKR Is Up-Regulated and Phosphorylates eIF2α in pI:C-Activated DCs.

PKR acts as a signal transducer in the proinflammatory response to different PAMPs through TLR signaling and direct activation by cytosolic dsRNA. PKR activation results in eIF2α phosphorylation and protein synthesis arrest (7, 32). As expected, PKR, being type I IFN-inducible, was strongly up-regulated upon pI:C stimulation (Figs. 2E and 3 A and B). In nonactivated PKR−/− DCs, P-eIF2α levels were close to normal, indicating that eIF2α kinases other than PKR are responsible for the high degree of eIF2α phosphorylation in nonstimulated cells (Fig. 3A). Importantly, eIF2α phosphorylation was nearly abolished in activated PKR−/− DCs, indicating that PKR is mostly responsible for this activity after pI:C stimulation (Fig. 3A). Cytosolic dsRNA delivery by lipofection also decreased P-eIF2α levels in maturing cells, being more evident in PKR−/− cells (Fig. 3B). Independently of the mode of pI:C delivery, PKR activity is therefore efficiently counteracted by GADD34 induction. This observation contrasts with previous work on MEFs exposed to cytosolic pI:C or Sindbis virus (33). Moreover, although PKR was shown to promote apoptosis in LPS-stimulated macrophages (34), we found that procaspase 3 cleavage was reduced in response to pI:C, again coinciding with eIF2α dephosphorylation and contrasting with the UPR-inducing drug, thapsigargin, which enhanced both caspase-3 cleavage and eIF2α phosphorylation in DCs (Fig. S3B).

Fig. 3.
PKR phosphorylates eIF2α in pI:C-activated DCs. WT and PKR−/− DCs were stimulated with pI:C alone (sol) or in combination with lipofectamine (lip) for the indicated time points (A and C) or for 8 h (B). Protein extracts were blotted ...

GADD34 expression was not affected by PKR deletion (Fig. 3C), correlating with the absence of abundant P-eIF2α in PKR−/−-activated cells (Fig. 3A). Recently, PKR and its NOX2-dependent activation have been implicated in amplifying part of the UPR and linking it to inflammation (19, 35, 36). To gain further insights into the signaling pathways controlling GADD34 expression during DC activation by pI:C, we monitored GADD34 expression in DCs derived from different knockout mice. Conversely to what was observed in NOX2−/− macrophages (35), NOX2 deletion did not interfere with GADD34 induction upon DC activation (Fig. 3D). However, we found that toll/interleukin 1 receptor domain-containing adapter inducing IFN-β (TRIF) was absolutely required to induce GADD34 expression in response to soluble and lipofected pI:C. GADD34 remained undetectable in TRIF−/− DCs (Fig. 3E), even using longer kinetics of activation (Fig. S5A), whereas its expression was found normal in activated MDA5−/− cells (Fig. 3F). Thus, the cytosolic helicase MDA5 is dispensable for GADD34-inducible expression by dsRNA even upon lipofection delivery. When we investigated ATF4 expression, we also found that its synthesis was attenuated upon TRIF inactivation, albeit not completely abolished (Fig. S2). This result further suggests that at least two signaling pathways are working in parallel upon pI:C detection and that GADD34 and ATF4 expression are mostly TRIF dependent in pI:C-activated DCs.

Cytosolic Delivery of pI:C Does Not Inhibit Protein Synthesis in DCs.

As cytosolic accumulation of dsRNA normally induces a PKR-dependent translational arrest (32), protein synthesis was monitored in DCs and fibroblasts exposed to soluble or lipofected pI:C. pI:C lipofection of MEFs efficiently caused a PKR-dependent translation arrest within 8 h (Fig. 4A). The translation arrest observed in WT MEFs suggests that soluble dsRNA can efficiently access the cytosol of these cells and interact with PKR. In the case of DCs, and as anticipated from the low levels of P-eIF2α induced by pI:C lipofection (Fig. 3B and Fig. S3B), translation was not inhibited even after 8 h of exposure to equivalent levels of cytosolic pI:C (Fig. 4A and Fig. S5B). We also observed by confocal microscopy the efficient access of soluble pI:C in the DC cytosol (Fig. S5C), likely to induce concomitantly the different dsRNA-sensing pathways (e.g., TLR3 and MDA5), as suggested by the levels of IFN-β production observed in TRIF-inactivated cells upon soluble pI:C stimulation in vitro (Fig. S5D) (4) and in vivo (37). On the basis of these observations and for greater consistency in our experiments, we decided to use only lipofected pI:C, which is more likely to be relevant physiologically (38).

Fig. 4.
DCs are protected from translation inhibition induced by cytosolic pI:C detection. (A) Protein synthesis was monitored by immune detection of puromycin incorporation in WT and PKR−/− MEFs and DCs treated with soluble pI:C (sol) or lipofected ...

When GADD34ΔC/ΔC DCs were exposed to cytosolic pI:C, their translation levels remained similar to those of WT cells (Fig. 4B). GADD34ΔC/ΔC DCs were clearly activated as shown by the pattern of S6 ribosomal protein phosphorylation (P-S6) normally associated with TRIF-dependent mTOR activation and protein synthesis enhancement (29). Activated DCs are therefore able to resist PKR-dependent translational arrest and rely on the induction of GADD34 to shift the biochemical equilibrium toward eIF2α dephosphorylation. GADD34, however, is not important to establish or maintain DC resistance to dsRNA-induced translation arrest (Fig. 4B). In contrast, GADD34 was absolutely required for eIF2α dephosphorylation and translation recovery upon thapsigargin treatment (Fig. 4C). Thus, GADD34 seems to play a specific role in the innate response toward dsRNA independently of its previously characterized function in the UPR.

Cytokine Production Is Affected by GADD34 Inactivation.

GADD34 impact on translational initiation being limited, we examined the phenotype of activated GADD34ΔC/ΔC DCs. Lipofected pI:C-driven maturation of GADD34ΔC/ΔC CD11c+ cells was found normal, judging by the increase of surface MHC II and CD86 levels (Fig. S6). In contrast with these results, a reduction in IFN-β and IL-6 levels was detected in the cell culture supernatants of GADD34ΔC/ΔC DCs (Fig. 5A). To define whether GADD34 deficiency impacted cytokine production at the transcriptional or translational level, we measured the mRNA expression of IFN-β and IL-6 transcripts and observed that, in the absence of functional GADD34, the levels of these transcripts were reduced by half after 8 h of pI:C stimulation (Fig. 5B). GADD34 activity, therefore, enhances the transcription of different cytokines downstream of the TRIF adapter.

Fig. 5.
GADD34 promotes normal secretion of IFN-β and IL-6 in response to pI:C. (A) Culture supernatants of WT and GADD34ΔC/ΔC bmDCs in which pI:C was delivered in the cytoplasm by lipofection were analyzed by ELISA for IFN-β and ...

GADD34 Regulates Levels of IFN-β in Vivo.

To evaluate the consequences of GADD34 deletion at the whole animal level, we injected i.v. the Friend leukemia virus B sensitive mouse strain (FVB) mice with pI:C complexed with the liposomal transfection reagent DOTAP. We could show that under these conditions, GADD34 is induced rapidly in CD11c+ splenocytes (Fig. S7). We next analyzed IFN-β blood levels at 3 and 6 h postinjection. In FVB WT animals, production of IFN-β peaked at 3 h postinjection, whereas after 6 h the levels returned to basal levels (Fig. 5C). As predicted from the in vitro data and compared with WT mice, a marked reduction of IFN-β was observed in GADD34ΔC/ΔC mice 3 h postinjection (Fig. 5C). We tested next the relevance of GADD34 during viral infection by monitoring type-I IFN production in Chikungunya virus (CHIKV)-infected 12-d-old mice. We turned to this small RNA enveloped virus, because it is known to be a strong inducer of type-I IFN in vivo (39), a response key for mouse neonates to control the infection (40). At 72 h postinoculation, GADD34ΔC/ΔC pups displayed significantly less IFN-β in the serum and in the joints than their WT littermates, whereas as expected, virus titers were increased in GADD34ΔC/ΔC organs (Fig. 5D). GADD34 is therefore an immunologically relevant dsRNA-inducible factor that participates in the optimization of cytokine production in response to pI:C and viral infection.

Discussion

DCs treated with pI:C display an ATF4 expression signature sharing common features with an ISR, including GADD34 induction. Interestingly, GADD34 induction has also been singled out in a transcriptome analysis of Listeria monocytogenes-infected macrophages (41) and during corona virus infection of fibroblasts (42), suggesting its association with pathogen detection. In vitro, the penetration of dsRNA in all cellular compartments can be directly detected through TLR3, DExD/H-box helicases, and PKR (43), which can also be rapidly activated by TLR ligation to promote p38 and NF-κB signaling (32). This eIF2α kinase is necessary to achieve functional DC maturation (9); however, its activation should normally lead to translation inhibition through eIF2α phosphorylation. Surprisingly, and in contrast to fibroblasts, DCs are not affected by dsRNA-induced translation inhibition. This specificity could allow DCs to prioritize the signal transduction pathways governing their innate immunity function over the pathways normally protecting cellular integrity from viral infection. When fully activated, these pathways would lead to protein translational arrest and/or apoptosis, through CHOP production and caspase cleavage, thus impairing DC function at a crucial time of the immune response (29). The lack of translation inhibition together with the absence of CHOP production, could allow DCs to carry their function even when infected with viruses as already evoked for human monocyte-derived DCs submitted to influenza virus infection (44).

GADD34 expression has primarily been shown to operate as a negative feedback loop during the UPR and allows for translation recovery through eIF2α dephosphorylation. We have shown that in DCs, GADD34 is required to prevent thapsigargin-induced protein synthesis arrest but GADD34 inactivation does not impact significantly on protein synthesis in response to dsRNA delivery. Activated DCs are therefore atypical cells in which eIF2α phosphorylation levels do not fully correlate with protein synthesis intensity and confirms that specific translation regulation pathways operate in during microbe detection, as observed during the proteasome-dependent cleavage of eIF4GI and DAP5 translation factors at late stages of their activation (29).

DCs have been reported to express high levels of XBP1, a transcription factor essential for ER homeostasis during UPR (15, 45, 46) and necessary for normal DC development (17, 47). In macrophages, TLR4 and TLR2 stimulation activates the ER-stress sensor kinase IRE1α and its downstream target, XBP1, without inducing other UPR branches (19). Our observations suggest that in DCs, the ATF4-dependent pathway is activated upon dsRNA detection. Interestingly nonactivated DCs possess abnormally high levels of P-eIF2α, which could, like XBP1 transcription, reflect a specific activation of the UPR during their differentiation and explain their low mRNA translation activity (29). GADD34 induction by pI:C in DCs requires the adapter TRIF and, contrary to XBP1, is not linked to NOX2 activity. The fact that pI:C enters efficiently the cell cytosol and the recent discovery of the TRIF-dependent DExD/H-box helicases DDX1, DDX21, and DHX36 (4), suggests that according to the mode of dsRNA delivery, TRIF could integrate different signals initiated by TLR3 and/or these helicases. The total absence of GADD34 induction by cytosolic pI:C in TRIF−/− DCs suggests that this pathway, leading to GADD34 expression, works in parallel with the induction of the MDA-5 sensing pathway to produce cytokines such as IFN-β efficiently.

Our observations also imply the existence of alternative signaling pathways, which allow ATF4 and GADD34 translation in the absence of increased P-eIF2α levels (48). Potentially, the high levels of P-eIF2α present in iDCs are sufficient to promote their synthesis during the early stages of DC activation. CHOP mRNA transcription was also found to be up-regulated in DCs, but no translation product could be detected, suggesting that CHOP synthesis is also tightly regulated in this cell type, as anticipated from the potent protective effect mediated by TLR agonists injection in mice subjected to systemic ER stress (49).

dsRNA is probably an extreme example of microbial-associated stressor because it can also induce PKR through direct recognition in the cytosol. Interestingly, although GADD34 controls eIF2α dephosphorylation, its inactivation does not overly impact translational control. However, like for XBP1 deficiency, we could show that IFN-β and IL-6 transcriptions were decreased in GADD34ΔC/ΔC DCs in vitro and in deficient mouse in vivo. In absence of an obvious effect of GADD34 deletion on protein synthesis, this observation suggests that GADD34/PP1 is required to dephosphorylate/activate a key factor in the signal transduction pathway downstream of TRIF. Recently, PP1-mediated deactivation of IkB kinase (IKK) in response to TNF-α, has been linked to GADD34 recruitment by the CUE domain-containing 2 protein (CUEDC2) (50). CUEDC2 silencing in macrophages led to increased transcription of inflammatory cytokine IL-6, by decreasing putative GADD34-dependent dephosphorylation of IKK. We could not recapitulate this finding in DCs, because IL-6 mRNA was less induced in activated GADD34ΔC/ΔC cells. A search for additional GADD34/PP1 targets will allow better understanding of the regulation pathways controlled by this specific pathogen-induced stress response. Activation of ATF4 and GADD34 should therefore be considered as an integral part of the innate immunity signaling cascades downstream of dsRNA sensors.

Experimental Procedures

Mouse bmDCs were treated with pI:C for different time periods. An Affymetrix Mouse Genome MOE 430 2.0 gene microarray chip was used to identify genes involved in dsRNA response of pI:C-activated DCs. Potential targets were confirmed by qPCR. Protein extracts of DCs from WT or different gene-targeted mice were analyzed by immunoblot for the presence of GADD34, P-eIF2α, or to study protein translation through puromycin incorporation (28). In vivo injections of pI:C were performed as shown before (3). Cytokine levels were measured by ELISA. Detailed experimental procedures can be found in SI Experimental Procedures.

Supplementary Material

Supporting Information:

Acknowledgments

We thank L. Alexopoulou for the TRIF−/− mice; M. Colonna for MDA5−/− mouse bone marrow; C. Reis e Sousa for PKR−/− mouse bone marrow, PKR−/− MEFs, and for sharing unpublished results; L. Wrabetz for the GADD34ΔC/ΔC mice; and S. Meresse for the NOX2−/− mice. We also thank the Plateforme d'Imagerie Commune du Site de Luminy (PICsL) Ministère de l'Education Nationale et de la Recherche (MENRT) Fundação para a Ciência e Tecnologia (FCT) for expert technical assistance. This work is supported by grants (to P.P.) from La Ligue Nationale Contre le Cancer, the Agence Nationale pour la Recherche 07-MIME-005 “DC-TRANS,” and the Agence Nationale de Recherches sur le SIDA. G.C. is supported by fellowships from la Fondation pour la Recherche Médicale. N.C. is supported by Fundação para a Ciência e Tecnologia Grant SFRH/BD/40112/2007 (Portugal).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The expression profiling by arrayGEO data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSM452757).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1104491109/-/DCSupplemental.

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