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

Giovanna Clavarinoa,b,c,1, Nuno Cláudioa,b,c,1, Alexandre Daleta,b,c, Seigo Terawakia,b,c, Thérèse Coudercd,e, Lionel Chassona,b,c, Maurizio Ceppia,b,c, Enrico K. Schmidta,b,c, Till Wengera,b,c, Marc Lecuitd,e,f, Evelina Gattia,b,c,2, and Philippe Pierrea,b,c,2

aCentre d’Immunologie de Marseille-Luminy, Aix-Marseille University, 13288 Marseille, France; bInstitut National de la Santé et de la Recherche Médicale (INSERM), U631, 13288 Marseille, France; cCentre National de la Recherche Scientifique, Unité Mixte de Recherche 6102, 13288 Marseille, France; dMicrobes and Host Barriers Group, Institut Pasteur, 75015 Paris, France; eINSERM, Equipe avenir U604, 75015 Paris, France; and fService des Maladies Infectieuses et Tropicales, Université Paris Descartes, Hôpital Necker-Enfants malades, Assistance Publique-Hôpitaux de Paris, 75015 Paris, France

Edited by Douglas T. Fearon, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, and approved December 9, 2011 (receivedfor review March 21, 2011) In response to inflammatory stimulation, dendritic cells (DCs) have factors such as IRE1, XBP1 and ATF6 (15, 16). In addition to a remarkable pattern of differentiation that exhibits specificmech- inhibiting global protein synthesis, eIF2α phosphorylation by anisms to control the immune response. Here we show that in re- PERK allows the specific translation of the transcription factor sponse to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount ATF4 (14). This factor promotes cell adaptation to stress by aspecific integrated stress response during which the transcription heightening ER functions through specific mRNA transcription. factor ATF4 and the growth arrest and DNA damage-inducible pro- Immune responses and immune cell development can be pro- tein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are foundly affected by abnormalities in the UPR (17, 18) and TLR- expressed. In agreement with increased GADD34 levels, an exten- dependent activation of XBP1, via the production of reactive sive dephosphorylation of the translation initiation factor eIF2α was oxygen species (ROS), is required for normal production of observed during DC activation. Unexpectedly, although DCs display proinflammatory cytokines in macrophages (19). an unusual resistance to protein synthesis inhibition induced in re- We show here that activated DCs resist the translation arrest, sponse to cytosolic dsRNA, GADD34 expression did not have a major normally elicited by the PKR-dependent eIF2α phosphorylation in impact on protein synthesis. GADD34, however, was shown to be response to cytosolic dsRNA delivery. Concomitantly, pI:C de- required for normal cytokine production both in vitro and in vivo. tection promotes the expression of the inducible phosphatase 1 These observations have important implications in linking further (PP1)-cofactor, GADD34 (20). GADD34 efficiently dephosphor- pathogen detection with the integrated stress response pathways. ylates eIF2α in activated DCs, but has little impact on the specific resistance of DCs to dsRNA-triggered translation arrest. Alter- type-I interferon | Toll-like receptor | protein kinase RNA activated | natively, GADD34 is required for optimal transcription and pro- puromycin | unfolded protein response duction of the cytokines IFN-β and interleukin-6 (IL-6), suggesting that induction of the ATF4-dependent branch of the UPR in DCs endritic cells (DCs) are regulators of the immune response, is a critical signaling module of the innate response to dsRNA. Dwhich, upon stimulation by conserved microbial products (PAMPs), are most efficient at inducing differentiation of naive Results T cells through massive cytokine production and optimization of DC Stimulation by pI:C Induces the ATF4 Transcription Factor. To their antigen presentation capacity (1). Double-stranded RNAs identify signaling pathways involved in dsRNA response, a (dsRNA), a hallmark of virus replication, is recognized by Toll- genomewide expression analysis was performed in pI:C-stimu- like receptor 3 (TLR3) (2) or cytosolic RNA DExD/H-box hel- lated mouse bone marrow-derived DCs (bmDCs) using Affy- icases, such as melanoma-associated -5 (MDA5) (3) or the metrix Mouse Genome 430 2.0 arrays. We found that at least DDX1, DDX21, and DHX36 (4). Detection of pI:C, a dsRNA nine transcripts, typically expressed in different cells exposed to mimic, promotes the nuclear translocation of IRF-3 and IRF-7 tunicamycin (11, 21, 22), were also induced in DCs responding and antiviral type-I IFN production (5). IFN triggers, among to pI:C (Table S1). many other antiviral , the dsRNA-dependent protein ki- Up-regulation of these transcripts was confirmed by quanti- nase (PKR) (6, 7). PKR activation by dsRNA promotes trans- tative RT-PCR (qPCR) (Fig. 1 and Fig. S1). Among them, the lation initiation factor 2 alpha (eIF2α) phosphorylation leading key UPR transcription factors, ATF4 and CCAAT/enhancer to protein synthesis shutoff and inhibition of viral replication (8). binding protein homologous protein (CHOP) mRNAs were 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 Author contributions: G.C., N.C., E.G., and P.P. designed research; G.C., N.C., A.D., S.T., attenuating mRNA translation and promoting a specific tran- T.C., L.C., M.C., E.K.S., and T.W. performed research; G.C., N.C., A.D., M.L., E.G., and P.P. analyzed data; and G.C., N.C., E.G., and P.P. wrote the paper. scriptional response known as the integrated stress response The authors declare no conflict of interest. (ISR) (11). To date four eIF2α kinases have been identified, PKR, PKR-like ER kinase (PERK) (12), general control non- This article is a PNAS Direct Submission. derepressible-2 (GCN2) (13), and heme-regulated inhibitor Data deposition: The expression profiling by arrayGEO data have been deposited in the Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. (HRI) (14). PERK, which is activated by an excess of unfolded GSM452757). in the ER lumen (12), is necessary to mount part of 1G.C. and N.C. contributed equally to this work. a particular ISR, known as the unfolded protein response (UPR). 2To whom correspondence may be addressed. E-mail: [email protected] or gatti@ The UPR encompasses a group of signals that cope with ciml.univ-mrs.fr. perturbations in ER homeostasis. In addition to PERK, the UPR This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is initiated by additional ER sensors and associated transcription 1073/pnas.1104491109/-/DCSupplemental.

3006–3011 | PNAS | February 21, 2012 | vol. 109 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1104491109 Downloaded by guest on September 30, 2021 increased by two- and eightfold, respectively (Fig. 1 A and C). repression during ER stress (22, 24, 25). In response to soluble ATF4 mRNA is normally poorly translated in unstressed cells, but pI:C, GADD34 mRNA transcription was enhanced at least 14- upon eIF2α phosphorylation, a rapid synthesis of the ATF4 protein fold (Fig. 1E), contrasting with the modest 1.5-fold up-regulation has been observed (14). In DCs, ATF4 protein levels were in- of PP1 expression (Fig. 1G) and 2-fold up-regulation of the creased upon pI:C stimulation and detected in the nuclear extracts “constitutive” regulatory subunit 15B after 8 h of stimulation, similarly to control DCs, in which a bona (CReP) mRNA (Fig. 1H) (26). Matching the transcriptional fide UPR was induced with thapsigargin (Fig. 1B and Fig. S2). analysis, and irrespectively of the mode of pI:C delivery (soluble Nuclear translocation of ATF4 normally induces CHOP ex- or lipofected), GADD34 protein levels were induced and con- pression, which can trigger apoptosis, and enhance the transcrip- tinuously increased during activation (Fig. 1F). Addition of α tion of several ATF4 target genes such as GADD34 and ERO1- proteasome inhibitor MG132 during the last 2 h of pI:C treat- (22). As expected from the array analysis, increased CHOP mRNA ment facilitated the detection of short-lived GADD34 by pre- levels were detected (Fig. 1C), however, we failed to visualize venting its degradation (Fig. 1F) (27) and confirmed GADD34 protein expression in LPS and pI:C-stimulated DCs extracts. In expression during DC maturation. contrast, CHOP was detected in control tunicamycin-treated DC as well as in LPS-stimulated mouse leukaemic macrophages eIF2α Is Dephosphorylated During DC Activation. Protein synthesis (RAW 264.7 cell line) (Fig. 1D). We tested different doses and was monitored using puromycin labeling followed by immunoblot modes of pI:C delivery to DCs and in agreement with the lack of (Fig. 2A) (28). As previously shown for LPS-activated DCs (29), CHOP expression, we found no major apoptotic death induction translation was enhanced in the first hours of pI:C stimulation in our experimental system (Fig. S3A). Thus, pI:C-activated DCs followed by a reduction at 16 h. The levels of phosphorylated eIF2α display an ATF4 transcriptional signature during which, CHOP (P-eIF2α) were gradually lost during pI:C stimulation (Fig. 2B), expression seems to be specifically down-modulated at the trans- independently of the mode of dsRNA delivery (Fig. S3B). Levels of lational level, which presumably contributes to the prevention of P-eIF2α were surprisingly high in nonstimulated DCs compared apoptosis in activated DCs (23). with mouse embryonic fibroblasts (MEFs), and no up-regulation α GADD34 Is Up-Regulated in Activated DCs. GADD34 acts together of P-eIF2 could be visualized during maturation (Fig. 2 B and C). α with PP1 to dephosphorylate eIF2α and relieves translation We further investigated whether these high levels of P-eIF2 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 sen- tinel function (29).

A C splenic β-actin iDC MEF CD11c+ P-eIF2α eIF2α

D dmso guana dmso sal pI:C(h) 0 8 20 0 8 20 0 8 0 8

P-eIF2α puromycin eIF2α

co chx 0 4 8 16 24 E WT GADD34ΔC/ΔC pI:C (h) PKR P-eIF2α pI:C (h) B eIF2α 0 1 4 8 16 24 β-actin P-eIF2α (h) 0 8 20 20 4 0 8 20 20 4 eIF2α lipo sol Th lipo sol Th pI:C pI:C

Fig. 2. eIF2α dephosphorylation is controlled by GADD34 during pI:C-in- Fig. 1. ATF4 and GADD34 are induced during DC activation with pI:C. duced DC maturation. (A) Protein synthesis was quantified in protein Experiments were performed in bmDCs stimulated with 10 μg/mL of pI:C or, extracts of pI:C-activated DCs using puromycin labeling followed by immu- when indicated, in RAW macrophages with 100 ng of LPS. mRNA levels of noblot. Cells not treated with puromycin (co) or cycloheximide (chx) were ATF4 (A), CHOP (C), GADD34 (E), PP1 (G), and CReP (H) were measured by used as controls. (B) Cell extracts were blotted for P-eIF2α and total eIF2α; qPCR. (B) ATF4 protein levels were detected by immunoblot in nuclear P-eIF2α decreases with time after pI:C stimulation. (C) Steady-state levels of extracts. Histone deacetylase 1 (HDAC1) is shown as loading control. Protein P-eIF2α nonstimulated bmDC (iDC), MEF, and CD11c+ splenocytes. (D and E) levels of CHOP (D) and GADD34 (F) were detected by immunoblot in total Immature and pI:C-stimulated DCs were treated with GADD34 inhibitors cell lysates. pI:C was added as soluble or lipofected (pI:C-lip). When in- guanabenz and salubrinal. Both drugs were added at the same time as pI:C dicated, MG132 was used to inhibit proteasomal degradation of GADD34 and for a period of 8 h to nonstimulated cells. Protein extracts were blotted Δ Δ and was added for 2 h before harvesting the cells. ER stress-inducing drugs for P-eIF2α and eIF2α.(E) P-eIF2α levels of WT and GADD34 C/ C DCs stim- thapsigargin (th) or tunicamycin (tun) serve as positive control treatments ulated with lipofected (lipo) or soluble pI:C (sol). Nonstimulated cells were for the expression of ATF4, CHOP, and GADD34. Data represented in A, C, E, treated with lipofectamine only. PKR blot is shown as control of DC activa- IMMUNOLOGY G, and H are mean ± SD of three independent experiments. tion. Thapsigargin (th) was used as positive control for P-eIF2α.

Clavarino et al. PNAS | February 21, 2012 | vol. 109 | no. 8 | 3007 Downloaded by guest on September 30, 2021 Because P-eIF2α intensity was inversely correlated with GADD34 expression was not affected by PKR deletion (Fig. 3C), − − GADD34 expression in activated cells, we tested the function of correlating with the absence of abundant P-eIF2α in PKR / -acti- PP1-GADD34 and -CReP complexes by inhibiting their activity vated cells (Fig. 3A). Recently, PKR and its NOX2-dependent ac- with the pharmacological inhibitors salubrinal and guanabenz tivation have been implicated in amplifying part of the UPR and (30, 31). In the presence of both drugs, P-eIF2α levels were linking it to inflammation (19, 35, 36). To gain further insights into enhanced by pI:C treatment (Fig. 2D). The same result was the signaling pathways controlling GADD34 expression during DC Δ Δ found when using DCs inactivated for GADD34 (GADD34 C/ C) activation by pI:C, we monitored GADD34 expression in DCs (25), in which P-eIF2α was considerably augmented upon acti- derived from different knockout mice. Conversely to what was −/− vation compared with WT cells (Fig. 2E and quantification in observed in NOX2 macrophages (35), NOX2 deletion did not Fig. S3C). Thus, GADD34 and PP1 dephosphorylate eIF2α and interfere with GADD34 induction upon DC activation (Fig. 3D). counteract efficiently the activation of eIF2α kinases in pI:C- However, we found that toll/interleukin 1 receptor domain-con- stimulated DCs. taining adapter inducing IFN-β (TRIF) was absolutely required to induce GADD34 expression in response to soluble and lipofected − − PKR Is Up-Regulated and Phosphorylates eIF2α in pI:C-Activated DCs. pI:C. GADD34 remained undetectable in TRIF / DCs (Fig. 3E), PKR acts as a signal transducer in the proinflammatory response even using longer kinetics of activation (Fig. S5A), whereas its ex- − − to different PAMPs through TLR signaling and direct activation by pression was found normal in activated MDA5 / cells (Fig. 3F). cytosolic dsRNA. PKR activation results in eIF2α phosphoryla- Thus, the cytosolic helicase MDA5 is dispensable for GADD34- tion and protein synthesis arrest (7, 32). As expected, PKR, being inducible expression by dsRNA even upon lipofection delivery. type I IFN-inducible, was strongly up-regulated upon pI:C stimu- When we investigated ATF4 expression, we also found that its −/− lation (Figs. 2E and 3 A and B). In nonactivated PKR DCs, synthesis was attenuated upon TRIF inactivation, albeit not com- P-eIF2α levels were close to normal, indicating that eIF2α kinases pletely abolished (Fig. S2). This result further suggests that at least other than PKR are responsible for the high degree of eIF2α two signaling pathways are working in parallel upon pI:C detection phosphorylation in nonstimulated cells (Fig. 3A). Importantly, and that GADD34 and ATF4 expression are mostly TRIF de- −/− eIF2α phosphorylation was nearly abolished in activated PKR pendent in pI:C-activated DCs. DCs, indicating that PKR is mostly responsible for this activity after pI:C stimulation (Fig. 3A). Cytosolic dsRNA delivery by Cytosolic Delivery of pI:C Does Not Inhibit Protein Synthesis in DCs. lipofection also decreased P-eIF2α levels in maturing cells, being As cytosolic accumulation of dsRNA normally induces a PKR- − − more evident in PKR / cells (Fig. 3B). Independently of the mode dependent translational arrest (32), protein synthesis was mon- of pI:C delivery, PKR activity is therefore efficiently counteracted itored in DCs and fibroblasts exposed to soluble or lipofected by GADD34 induction. This observation contrasts with previous pI:C. pI:C lipofection of MEFs efficiently caused a PKR-depen- work on MEFs exposed to cytosolic pI:C or Sindbis virus (33). dent translation arrest within 8 h (Fig. 4A). The translation arrest Moreover, although PKR was shown to promote apoptosis in LPS- observed in WT MEFs suggests that soluble dsRNA can effi- stimulated macrophages (34), we found that procaspase 3 cleavage ciently access the cytosol of these cells and interact with PKR. was reduced in response to pI:C, again coinciding with eIF2α de- In the case of DCs, and as anticipated from the low levels of phosphorylation and contrasting with the UPR-inducing drug, P-eIF2α induced by pI:C lipofection (Fig. 3B and Fig. S3B), thapsigargin, which enhanced both caspase-3 cleavage and eIF2α translation was not inhibited even after 8 h of exposure to phosphorylation in DCs (Fig. S3B). equivalent levels of cytosolic pI:C (Fig. 4A and Fig. S5B). We

A B

C D

E

F

− − 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)orfor8h(B). Protein extracts were blotted for PKR, P-eIF2α (A and B) and GADD34 (C). Sodium arsenite (as) was used as − − a positive control for P-eIF2α induction. (D) GADD34 immunoblot was performed on lysate of NOX2 / bmDCs activated with either pI:C or LPS for 8 and 24 h. (E) WT and TRIF−/− bmDCs were stimulated with pI:C as indicated and tested for the presence of GADD34 in protein extracts. (F) WT and MDA5−/− bmDCs were stimulated with lipofected pI:C and immunoblotted for GADD34. Treatment with MG132 (2 h) was used to facilitate GADD34 visualization.

3008 | www.pnas.org/cgi/doi/10.1073/pnas.1104491109 Clavarino et al. Downloaded by guest on September 30, 2021 A MEFs DCs WT PKR-/- WT PKR-/- β-actin

puromycin

co chx mock lip sol co chx mock lip sol co chxmock lip sol co chx mock lip sol pI:C pI:C pI:C pI:C + puromycin WT GADD34ΔC/ΔC B WT GADD34ΔC/ΔC C β-actin

puromycin

puromycin

P-eIF2α P-S6 eIF2α S6 β-actin chx 0 4 8 16 24 chx 0 4 8 16 24 chx 0 0.5 1 3 6 0 0.5 1 3 6 pI:C pI:C thapsigargin (h) + puromycin + puromycin

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 (lip) for 8 h. Controls: no puromycin (co), chx, and Δ Δ lipofectamine-treated (mock) cells. (B) WT and GADD34 C/ C DCs were lipofected with pI:C for the indicated time and translation measured by antipuromycin Δ Δ immunoblot. (C) bmDCs from WT and GADD34 C/ C mice were challenged with thapsigargin for the indicated time. Protein translation, P-eIF2α, and total eIF2α were detected by immunoblotting.

also observed by confocal microscopy the efficient access of half after 8 h of pI:C stimulation (Fig. 5B). GADD34 activity, soluble pI:C in the DC cytosol (Fig. S5C), likely to induce con- therefore, enhances the transcription of different cytokines comitantly the different dsRNA-sensing pathways (e.g., TLR3 downstream of the TRIF adapter. and MDA5), as suggested by the levels of IFN-β production observed in TRIF-inactivated cells upon soluble pI:C stimulation GADD34 Regulates Levels of IFN-β in Vivo. To evaluate the con- in vitro (Fig. S5D) (4) and in vivo (37). On the basis of these sequences of GADD34 deletion at the whole animal level, we observations and for greater consistency in our experiments, we injected i.v. the Friend leukemia virus B sensitive mouse strain decided to use only lipofected pI:C, which is more likely to be (FVB) mice with pI:C complexed with the liposomal transfection relevant physiologically (38). reagent DOTAP. We could show that under these conditions, Δ Δ When GADD34 C/ C DCs were exposed to cytosolic pI:C, their GADD34 is induced rapidly in CD11c+ splenocytes (Fig. S7). We translation levels remained similar to those of WT cells (Fig. 4B). next analyzed IFN-β blood levels at 3 and 6 h postinjection. In FVB Δ Δ GADD34 C/ C DCs were clearly activated as shown by the pattern WT animals, production of IFN-β peaked at 3 h postinjection, of S6 ribosomal protein phosphorylation (P-S6) normally associ- whereas after 6 h the levels returned to basal levels (Fig. 5C). As ated with TRIF-dependent mTOR activation and protein syn- predicted from the in vitro data and compared with WT mice, Δ Δ thesis enhancement (29). Activated DCs are therefore able to a marked reduction of IFN-β was observed in GADD34 C/ C mice resist PKR-dependent translational arrest and rely on the in- 3 h postinjection (Fig. 5C). We tested next the relevance of duction of GADD34 to shift the biochemical equilibrium toward GADD34 during viral infection by monitoring type-I IFN pro- eIF2α dephosphorylation. GADD34, however, is not important to duction in Chikungunya virus (CHIKV)-infected 12-d-old mice. establish or maintain DC resistance to dsRNA-induced translation We turned to this small RNA enveloped virus, because it is known arrest (Fig. 4B). In contrast, GADD34 was absolutely required for to be a strong inducer of type-I IFN in vivo (39), a response key for eIF2α dephosphorylation and translation recovery upon thapsi- mouse neonates to control the infection (40). At 72 h post- Δ Δ gargin treatment (Fig. 4C). Thus, GADD34 seems to play a spe- inoculation, GADD34 C/ C pups displayed significantly less IFN-β cific role in the innate response toward dsRNA independently of in the serum and in the joints than their WT littermates, whereas as Δ Δ its previously characterized function in the UPR. expected, virus titers were increased in GADD34 C/ C organs (Fig. 5D). GADD34 is therefore an immunologically relevant Cytokine Production Is Affected by GADD34 Inactivation. GADD34 dsRNA-inducible factor that participates in the optimization of impact on translational initiation being limited, we examined the cytokine production in response to pI:C and viral infection. Δ Δ phenotype of activated GADD34 C/ C DCs. Lipofected pI:C- Δ Δ driven maturation of GADD34 C/ C CD11c+ cells was found Discussion normal, judging by the increase of surface MHC II and CD86 DCs treated with pI:C display an ATF4 expression signature levels (Fig. S6). In contrast with these results, a reduction in IFN- sharing common features with an ISR, including GADD34 in- β and IL-6 levels was detected in the cell culture supernatants duction. Interestingly, GADD34 induction has also been singled Δ Δ of GADD34 C/ C DCs (Fig. 5A). To define whether GADD34 out in a transcriptome analysis of Listeria monocytogenes-infected deficiency impacted cytokine production at the transcriptional or macrophages (41) and during corona virus infection of fibroblasts translational level, we measured the mRNA expression of IFN-β (42), suggesting its association with pathogen detection. In vitro,

and IL-6 transcripts and observed that, in the absence of func- the penetration of dsRNA in all cellular compartments can be IMMUNOLOGY tional GADD34, the levels of these transcripts were reduced by directly detected through TLR3, DExD/H-box helicases, and PKR

Clavarino et al. PNAS | February 21, 2012 | vol. 109 | no. 8 | 3009 Downloaded by guest on September 30, 2021 (43), which can also be rapidly activated by TLR ligation to pro- in DCs, the ATF4-dependent pathway is activated upon dsRNA mote p38 and NF-κB signaling (32). This eIF2α kinase is necessary detection. Interestingly nonactivated DCs possess abnormally high to achieve functional DC maturation (9); however, its activation levels of P-eIF2α, which could, like XBP1 transcription, reflect should normally lead to translation inhibition through eIF2α a specific activation of the UPR during their differentiation and phosphorylation. Surprisingly, and in contrast to fibroblasts, DCs explain their low mRNA translation activity (29). GADD34 in- are not affected by dsRNA-induced translation inhibition. This duction by pI:C in DCs requires the adapter TRIF and, contrary to specificity could allow DCs to prioritize the signal transduction XBP1, is not linked to NOX2 activity. The fact that pI:C enters pathways governing their innate immunity function over the efficiently the cell cytosol and the recent discovery of the TRIF- pathways normally protecting cellular integrity from viral in- dependent DExD/H-box helicases DDX1, DDX21, and DHX36 fection. When fully activated, these pathways would lead to protein (4), suggests that according to the mode of dsRNA delivery, TRIF translational arrest and/or apoptosis, through CHOP production could integrate different signals initiated by TLR3 and/or these and caspase cleavage, thus impairing DC function at a crucial time helicases. The total absence of GADD34 induction by cytosolic pI: − − of the immune response (29). The lack of translation inhibition CinTRIF / DCs suggests that this pathway, leading to GADD34 together with the absence of CHOP production, could allow DCs expression, works in parallel with the induction of the MDA-5 to carry their function even when infected with viruses as already sensing pathway to produce cytokines such as IFN-β efficiently. evoked for human monocyte-derived DCs submitted to influenza Our observations also imply the existence of alternative sig- virus infection (44). naling pathways, which allow ATF4 and GADD34 translation in GADD34 expression has primarily been shown to operate as the absence of increased P-eIF2α levels (48). Potentially, the a negative feedback loop during the UPR and allows for trans- high levels of P-eIF2α present in iDCs are sufficient to promote lation recovery through eIF2α dephosphorylation. We have their synthesis during the early stages of DC activation. CHOP shown that in DCs, GADD34 is required to prevent thapsigargin- mRNA transcription was also found to be up-regulated in DCs, induced protein synthesis arrest but GADD34 inactivation does but no translation product could be detected, suggesting that not impact significantly on protein synthesis in response to CHOP synthesis is also tightly regulated in this cell type, as an- dsRNA delivery. Activated DCs are therefore atypical cells in ticipated from the potent protective effect mediated by TLR which eIF2α phosphorylation levels do not fully correlate with agonists injection in mice subjected to systemic ER stress (49). protein synthesis intensity and confirms that specific translation dsRNA is probably an extreme example of microbial-associated regulation pathways operate in during microbe detection, as ob- stressor because it can also induce PKR through direct recogni- served during the proteasome-dependent cleavage of eIF4GI and tion in the cytosol. Interestingly, although GADD34 controls DAP5 translation factors at late stages of their activation (29). eIF2α dephosphorylation, its inactivation does not overly impact DCs have been reported to express high levels of XBP1, a translational control. However, like for XBP1 deficiency, we could transcription factor essential for ER homeostasis during UPR (15, show that IFN-β and IL-6 transcriptions were decreased in Δ Δ 45, 46) and necessary for normal DC development (17, 47). In GADD34 C/ C DCs in vitro and in deficient mouse in vivo. In macrophages, TLR4 and TLR2 stimulation activates the ER-stress absence of an obvious effect of GADD34 deletion on protein sensor kinase IRE1α and its downstream target, XBP1, without synthesis, this observation suggests that GADD34/PP1 is required inducing other UPR branches (19). Our observations suggest that to dephosphorylate/activate a key factor in the signal transduction

A 50 WT 300 WT Δ Δ ΔC/ΔC 40 GADD34 C/ C 250 GADD34 200 30

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IL-6 (pg/ml) IL-6 IFN- β 10 50 0 0 pI:C (h) pI:C (h) B 0 4 8 0 4 8

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C D WT 4 Δ Δ p=0,01 80 WT GADD34 C/ C GADD34ΔC/ΔC 3 60 p=0,11 p=0,04 Virus titers (/g of tissue):

40 2 (pg/ml) WT GADD34 ΔC/ΔC

20 (ng/ml or ng/g) 1 Muscle 7,3x104 5,0x105

IFN- β Joint 5,2x105 2,5x106 0 IFN- β 0 0 3 6 serum muscle joint pI:C (h)

Δ Δ Fig. 5. GADD34 promotes normal secretion of IFN-β and IL-6 in response to pI:C. (A) Culture supernatants of WT and GADD34 C/ C bmDCs in which pI:C was delivered in the cytoplasm by lipofection were analyzed by ELISA for IFN-β and IL-6. (B) IFN-β and IL-6 mRNA expression levels were measured by qPCR. One of four independent experiments with similar results is shown for each panel. (C and D) GADD34 is relevant for normal IFN-β protein levels in vivo. IFN-β levels were measured in the serum of mice that were i.v. injected with 20 μg/mL of pI:C for 3 or 6 h. pI:C was delivered conjugated with DOTAP; controls were performed injecting PBS with DOTAP. (D) IFN-β dosage in tissues and serum of mice inoculated with 106 pfu of CHIKV via the ID route. Fifteen-day-old mice were analyzed 72 h after inoculation. Each data point represents the arithmetic mean ±SD for at least four mice. Average virus titers in the different organs are indicated.

3010 | www.pnas.org/cgi/doi/10.1073/pnas.1104491109 Clavarino et al. Downloaded by guest on September 30, 2021 pathway downstream of TRIF. Recently, PP1-mediated de- identify genes involved in dsRNA response of pI:C-activated DCs. Potential activation of IkB kinase (IKK) in response to TNF-α, has been targets were confirmed by qPCR. Protein extracts of DCs from WT or different gene-targeted mice were analyzed by immunoblot for the presence of linked to GADD34 recruitment by the CUE domain-containing 2 α protein (CUEDC2) (50). CUEDC2 silencing in macrophages led GADD34, P-eIF2 , or to study protein translation through puromycin in- fl corporation (28). In vivo injections of pI:C were performed as shown before to increased transcription of in ammatory cytokine IL-6, by de- (3). Cytokine levels were measured by ELISA. Detailed experimental proce- creasing putative GADD34-dependent dephosphorylation of IKK. dures can be found in SI Experimental Procedures. We could not recapitulate this finding in DCs, because IL-6 mRNA ΔC/ΔC − − was less induced in activated GADD34 cells. A search for ACKNOWLEDGMENTS. We thank L. Alexopoulou for the TRIF / mice; M. Colonna for MDA5−/− mouse bone marrow; C. Reis e Sousa for PKR−/− additional GADD34/PP1 targets will allow better understanding − − mouse bone marrow, PKR / MEFs, and for sharing unpublished results; of the regulation pathways controlled by this specific pathogen- Δ Δ − − L. Wrabetz for the GADD34 C/ C mice; and S. Meresse for the NOX2 / mice. induced stress response. Activation of ATF4 and GADD34 We also thank the Plateforme d’Imagerie Commune du Site de Luminy should therefore be considered as an integral part of the innate (PICsL) Ministère de l’Education Nationale et de la Recherche (MENRT) Fun- immunity signaling cascades downstream of dsRNA sensors. dação 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 Can- Experimental Procedures cer, 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 fellow- Mouse bmDCs were treated with pI:C for different time periods. An Affy- ships from la Fondation pour la Recherche Médicale. N.C. is supported by metrix Mouse Genome MOE 430 2.0 gene microarray chip was used to Fundação para a Ciência e Tecnologia Grant SFRH/BD/40112/2007 (Portugal).

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