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RIG-I-mediated Activation of p38 MAPK Is Essential for Viral Induction of Interferon and Activation of Dendritic Cells

2009; Elsevier BV; Volume: 284; Issue: 16 Linguagem: Inglês

10.1074/jbc.m807272200

ISSN

1083-351X

Autores

Susie Sommer Mikkelsen, Søren B. Jensen, Srikanth Chiliveru, Jesper Melchjorsen, Ilkka Julkunen, Matthias Gaestel, J. Simon C. Arthur, Richard A. Flavell, Sankar Ghosh, Søren R. Paludan,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

The innate immune system provides an initial defense system against microbial infections and contributes to the development of adaptive immune response. Type I interferons play a pivotal role for the first line of defense against virus infections, and dendritic cells (DCs) are important sensors of pathogens responsible for priming of adaptive immune responses in lymphoid organs. Here we have investigated the role and mechanisms of activation of the MAPK pathway in innate immune responses induced by Sendai virus, a negative sense single-stranded RNA virus. Both p38 and JNK were activated in fibroblasts and DCs after infection with Sendai virus in a manner dependent on virus replication and RIG-I. Virus replication was also required for stimulation of interferon production in both cell types and interleukin-12 production in DCs. Blocking of p38 MAPK activation by the specific inhibitor SB202190 abolished the expression of these cytokines. p38 MAPK exerted its function independent of the MAPK-activated protein kinases MK2, MNK, and MSK1/2. We also observed that TRAF2 and TAK1 were essential for RIG-I-mediated activation of p38 MAPK. Interestingly, the kinase activity of p38 MAPK was required for its own phosphorylation, which was kinetically associated with TAB1 interaction. By contrast, the canonical p38 upstream kinase MKK3 was not involved in the p38-dependent response. Thus, activation of p38 MAPK by RIG-I proceeds via a TRAF2-TAK1-dependent pathway, where the enzymatic activity of the kinase plays an essential role. The p38 MAPK in turn stimulates important processes in the innate antiviral response. The innate immune system provides an initial defense system against microbial infections and contributes to the development of adaptive immune response. Type I interferons play a pivotal role for the first line of defense against virus infections, and dendritic cells (DCs) are important sensors of pathogens responsible for priming of adaptive immune responses in lymphoid organs. Here we have investigated the role and mechanisms of activation of the MAPK pathway in innate immune responses induced by Sendai virus, a negative sense single-stranded RNA virus. Both p38 and JNK were activated in fibroblasts and DCs after infection with Sendai virus in a manner dependent on virus replication and RIG-I. Virus replication was also required for stimulation of interferon production in both cell types and interleukin-12 production in DCs. Blocking of p38 MAPK activation by the specific inhibitor SB202190 abolished the expression of these cytokines. p38 MAPK exerted its function independent of the MAPK-activated protein kinases MK2, MNK, and MSK1/2. We also observed that TRAF2 and TAK1 were essential for RIG-I-mediated activation of p38 MAPK. Interestingly, the kinase activity of p38 MAPK was required for its own phosphorylation, which was kinetically associated with TAB1 interaction. By contrast, the canonical p38 upstream kinase MKK3 was not involved in the p38-dependent response. Thus, activation of p38 MAPK by RIG-I proceeds via a TRAF2-TAK1-dependent pathway, where the enzymatic activity of the kinase plays an essential role. The p38 MAPK in turn stimulates important processes in the innate antiviral response. Activation of immune responses against microbial pathogens depends on the recognition of pathogen-associated molecular patterns (PAMPs) 5The abbreviations used are: PAMP, pathogen-associated molecular patterns; PRR, pattern recognition receptor; IFN, interferon; DC, dendritic cell; IL, interleukin; NF, nuclear factor; IRF, IFN regulatory factor; MAPK, mitogen-activated protein kinase; MK, MAPK-activated protein kinase; MNK, MAPK-interacting kinases; MSK, mitogen- and stress-activated protein kinase; MEF, mouse embryo fibroblast; BM, bone marrow; SeV, Sendai virus; PE, phycoerythrin; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; RLR, RIG-like receptor. by pattern recognition receptors (PRRs), which trigger a first line of defense against the infection and also promote development of antigen-specific adaptive immune response. 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A third important PRR-activated signaling pathway is the mitogen-activated protein kinase (MAPK) pathway, which have multiple functions, including cell cycle control, cell survival, and regulation of transcription, mRNA stability, and translation (22Roux P.P. Blenis J. Microbiol. Mol. Biol. Rev... 2004; 68: 320-344Google Scholar). These functions are mediated via the activation of MAPKs (p38, JNK, and ERK), which act by phosphorylating specific target proteins directly or via the activation of downstream MAPK-activated protein kinases (MKs) such as MAPK-interacting kinases (MNKs), mitogen- and stress-activated protein kinases (MSKs), MK2, and MK3 (22Roux P.P. Blenis J. Microbiol. Mol. Biol. Rev... 2004; 68: 320-344Google Scholar, 23Ueda T. Watanabe-Fukunaga R. Fukuyama H. Nagata S. Fukunaga R. Mol. Cell Biol... 2004; 24: 6539-6549Google Scholar, 24Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol... 1999; 1: 94-97Google Scholar-25Gaestel M. Nat. Rev. Mol. Cell Biol... 2006; 7: 120-130Google Scholar). The RIG-I signaling pathway is dependent on the adaptor protein MAVS/IPS-1/CARDIF/VISA (26Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell.. 2005; 122: 669-682Google Scholar, 27Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol... 2005; 6: 981-988Google Scholar, 28Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Nature.. 2005; 437: 1167-1172Google Scholar-29Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell.. 2005; 19: 727-740Google Scholar), which is located in the outer mitochondrial membrane (26Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell.. 2005; 122: 669-682Google Scholar), where the intracellular signaling is initiated (28Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Nature.. 2005; 437: 1167-1172Google Scholar). To induce type I IFN response, MAVS signals to IRF-3, through a pathway regulated by TRAF3 (30Oganesyan G. Saha S.K. Guo B. He J.Q. Shahangian A. Zarnegar B. Perry A. Cheng G. Nature.. 2005; 439: 211Google Scholar, 31Saha S.K. Pietras E.M. He J.Q. Kang J.R. Liu S.Y. Oganesyan G. Shahangian A. Zarnegar B. Shiba T.L. Wang Y. Cheng G. EMBO J... 2006; 25: 3257-3263Google Scholar). RIG-I also stimulates the NF-κB pathway, and recent work has shown that RIG-I signaling shares significant similarity with the signaling pathway downstream of the TNF receptor. For instance, both receptors rely on TRADD, RIP1, and FADD to activate NF-κB (27Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol... 2005; 6: 981-988Google Scholar, 32Michallet M.C. Meylan E. Ermolaeva M.A. Vazquez J. Rebsamen M. Curran J. Poeck H. Bscheider M. Hartmann G. Konig M. Kalinke U. Pasparakis M. Tschopp J. Immunity.. 2008; 28: 651-661Google Scholar, 33Balachandran S. Thomas E. Barber G.N. Nature.. 2004; 432: 401-405Google Scholar-34Takahashi K. Kawai T. Kumar H. Sato S. Yonehara S. Akira S. J. Immunol... 2006; 176: 4520-4524Google Scholar). However, many of the details in the regulation of signaling by TNF-α and RIG-I to NF-κB need to be clarified. Similarly, the mechanism of how RIG-I activates the p38 MAPK is not well characterized. In the present work, we show that the activation of the RIG-I pathway leads to activation of p38 MAPK, via a process that is dependent on TRAF2, TAK1, and p38 kinase activity. The activity of p38 in turn is essential for viral induction of type I IFNs as well as activation of conventional DCs. Cells and Virus—Mouse embryonic fibroblasts (MEFs) derived from C57BL/6, Rig-I+/-, Rig-I-/- (S. Akira, Osaka Japan), Traf2-/-, Traf6+/-, Traf6-/- (T. Mak, Toronto, Canada), and Tak1-/- mice (14Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. Yamaguchi O. Otsu K. Tsujimura T. Koh C.S. 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The plates were left overnight in 5% CO2 at 37 °C to allow the cells to attach before further experiments were carried out. For isolation of total cellular RNA and Luminex experiments, cells were plated in 6-well tissue culture plates in a concentration of 5 × 106 cells/well in 2 ml of medium. The murine dendritic cell line BC-1 was maintained in Iscove's modified Dulbecco's medium containing 10% fetal calf serum, 200 IU/ml penicillin, 100 μg/ml streptomycin, 600 mg/ml l-glutamine, 50 μm 2-mercaptoethanol, 30% NIH/3T3 conditioned medium, and 10 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (R&D Systems). For experiments, cells were plated in 96-well tissue culture plates in a concentration of 1 × 105 cells/well. The plates were left overnight in 5% CO2 at 37 °C to allow the cells to attach before the experiments. For isolation of total cellular RNA and Luminex experiments, cells were plated in 6-well tissue culture plates in a concentration of 3 × 106 cells/well in 2 ml of medium. Bone marrow (BM)-derived DCs were prepared as described elsewhere (37Petersen M.S. Toldbod H.E. Holtz S. Hokland M. Bolund L. Agger R. Scand. J. Immunol... 2000; 51: 586-594Google Scholar). Briefly, C57BL/6, MK2-/-, MSK1/2-/-, and MKK3-/- mice were sacrificed by cervical dislocation, and femur and tibia were removed. Bone marrow cells were flushed from the bone shafts with PBS, pipetted to break up cell aggregates, centrifuged, washed, counted, and put into culture in Petri dishes (Falcon; BD Biosciences). The cells were cultured at 2 × 105 cells/ml in culture medium containing 40 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (R&D systems) in 5% CO2 at 37 °C. Fresh medium containing 40 ng/ml granulocyte-macrophage colony-stimulating factor was added after 3 days, and on day 5, half of the medium was replaced with fresh medium. After 7 days of culture, non-adherent cells were harvested for flow cytometry or infection with infectious or UV-inactivated Sendai virus (SeV). The cells were 80% CD11c-positive, as determined by flow cytometry (data not shown). For isolation of cell culture supernatant, cells were seeded at a density of 1 × 105 cells/well in a 96-well tissue culture plate and left overnight in an atmosphere of 5% CO2 at 37 °C to allow the cells to settle, after which they were infected with virus and/or chemical inhibitors/activators as indicated. For total RNA isolation and Luminex experiments, cells were plated on 6-well tissue culture plates in a concentration of 3 × 106 cells/well. The plates were left for 2 h in 37 °C in a humidified atmosphere with 5% CO2 before further treatment. SeV (strain Cantell) was grown in 11-day-old embryonated hen eggs, as previously described (38Pirhonen J. Sareneva T. Kurimoto M. Julkunen I. Matikainen S. J. Immunol... 1999; 162: 7322-7329Google Scholar). The infectivity titer of the virus in DCs was 4 × 109 plaque-forming units/ml (39Osterlund P. Veckman V. Siren J. Klucher K.M. Hiscott J. Matikainen S. Julkunen I. J. Virol... 2005; 79: 9608-9617Google Scholar). UV inactivation of the virus was performed by exposing the virus to UV light for 5 min unless otherwise indicated. The uninfected hen egg allantoic fluid did not stimulate proinflammatory cytokine expression in DCs, and the virus preparation did not contain lipopolysaccharide (data not shown). Reagents—For inhibition of specific molecules in the MAPK pathways, the following compounds were used: SB202190 (p38 inhibitor) (Sigma), PD169316 (p38 inhibitor) (Sigma), BIRB 796 (p38 inhibitor) (Axon Medchem), Sp600125 (JNK inhibitor) (Sigma), and U0126 (MEK-1 inhibitor) (Sigma). CGP057380 (MNK inhibitor) was kindly provided by Dr. Hermann Gram (Novartis Pharma AG, Basel, Switzerland). Flow Cytometry—Cells were counted and resuspended in RPMI 1640 supplemented with 10% fetal calf serum at a concentration of 1 × 107 cells/ml. Cells were incubated in a 96-well plate on ice in the dark for 40 min with combinations of the fluorescein isothiocyanate- and PE-conjugated antibodies at a concentration of 0.5 mg/ml. Antibodies used (all from BD Pharmingen) were as follows: fluorescein isothiocyanate-conjugated hamster anti-mouse monoclonal CD11c, fluorescein isothiocyanate-conjugated CD11b, PE-conjugated anti-mouse monoclonal CD8α (Ly-2), PE-conjugated anti-mouse CD86 and CD40, PE-conjugated anti-mouse I-A (major histocompatibility complex I), and PE-conjugated anti-mouse H-2K (major histocompatibility complex II). As isotype control antibodies, we used fluorescein isothiocyanate-conjugated hamster IgG1, λ1, PE-conjugated monoclonal rat IgG2a, and PE-conjugated monoclonal mouse IgG2a. The labeled cells were fixed in 1% paraformaldehyde diluted in PBS for 15 min, and fixed cells were kept in PBS at 4 °C until they were analyzed. Acquisition and analysis were performed with a flow cytometer (Coulter FS500). The data were stored in list mode files. A total of 30,000 cells were analyzed in each experiment by using a single laser system with a wavelength of 488 nm. Compensation was determined before the acquisition of data. Cytokine Determinations—To measure cytokine production, culture supernatants were harvested after 12-24 h of infection. Cytokine levels in the supernatants were determined by ELISA. 96-well tissue culture plates were coated overnight at 4 °C with 100 μl of anti-IL-12 p40 (6 μg/ml; Pharmingen) capture antibodies diluted in coating buffer (15 mm NaHCO3, 35 mm Na2CO3, 0.2% sodium azide, pH 9.6). The cells were washed three times with washing buffer (PBS plus 0.05% Tween), blocked for 3 h with blocking buffer (PBS, 5% sucrose, 0.05% sodium azide, 1% bovine serum albumin), after which 100 μl of serial dilutions of IL-12 (rmIL12-p40; Pharmingen) standard or cell supernatants were added in duplicates or triplicates. The plates were left overnight at 4 °C. The plates were washed three times with washing buffer, and 100 μl of anti-IL-12 p40/p70 (Pharmingen) detection antibodies diluted in a reagent diluent (TBS, 0.05% Tween, 0.1% bovine serum albumin) was added and left to incubate for 2-3 h at room temperature. The plates were washed three times, incubated for 20 min with Streptavidin-horseradish peroxidase (R&D Systems) at room temperature, washed again three times, and developed with 100 μl of substrate reagent (Substrate Reagent Pack; R&D Systems) at room temperature for 2-5 min followed by the addition of 2 n H2SO4 to stop the reaction. Cytokine concentration was measured by an automated ELISA reader. For measurement of IL-12p70 and CXCL10, we used Luminex kits purchased from Bio-Rad and BIOSOURCE, respectively. Briefly, the filter plate was washed with assay buffer, and 50 μl of freshly vortexed antibody-conjugated beads was added to each well. The plate was washed with assay buffer, and samples and standards were added. After a brief shake (30 s at 1,100 rpm), the plate was incubated at room temperature in the dark for 2 h with light shaking (300 rpm). After one wash step, 25 μl of the detection antibody was added to each well, and the plate was shaken and incubated as above. Subsequently, the plate was washed and incubated for 30 min with 50 μl of a streptavidin-PE solution with shaking (30 s at 1,100 rpm, 10 min at 300 rpm). Finally, the plate was washed, and 125 μl of assay buffer was added to each well, and the plate was shaken for 10 s at 1,100 rpm. Measurement was carried out immediately on the Bio-Plex reader. IFN-α/β Bioassay—IFN-α/β bioactivity was measured by an L929 cell-based bioassay. L929 cells (2 × 104 cells/well in 100 μl) grown in minimum Eagle's medium supplemented with 5% fetal calf serum and antibiotics were incubated overnight at 37 °C in successive 2-fold dilutions of samples or murine IFN-α/β as a standard. Subsequently, vesicular stomatitis virus (VSV/V10) was added to the wells, and the cells were incubated for 2-3 days. The dilution giving 50% protection was defined as 1 unit/ml of IFN-α/β. To ensure that the bioassay was useful for measurement of IFN activity in samples containing p38 MAPK inhibitors, we performed control experiments, where samples spiked with IFN in the presence or absence of the inhibitors were subjected to IFN measurement by bioassay. We found that the concentrations of p38 MAPK inhibitor used in this study did not affect the result of the bioassay (data not shown). Quantitative Reverse Transcription-PCR—Total RNA was extracted with the High Pure RNA isolation kit (Roche Applied Science) according to the recommendations of the manufacturer. For cDNA generation, RNA was subjected to reverse transcription with oligo(dT) as primer and Expand reverse transcriptase (both from Roche Applied Science). The cDNA was amplified by PCR using the following primers: IFN-β, 5′-CACTGGGTGGAATGAGACTAT-3′ (forward) and 5′-GACATCTCCCACGTCAATC-3′ (reverse); ISG56, 5′-GCCAGGAGGTTGTGCATC-3′ (forward) and 5′-ACCATGGGAGAGAATGCTGAT-3′ (reverse); IL12-p40, 5′-ACGTGAACCGTCCGGAGTAA-3′ (forward) and 5′-CCACAAAGGAGGCGAGACTCT-3′ (reverse); β-actin, 5′-TAGCACCATGAAGATCAAGAT-3′ (forward) and 5′-CCGATCCACACAGAGTACTT-3′ (reverse). The products were measured by the use of SYBR Green I (from Qiagen). All primers were obtained from DNA Technology (Aarhus, Denmark). Detection of Phosphoproteins—For detection of the phosphorylation status of IκBα, p38, and JNK, we used Luminex technology. Briefly, the filter plate was washed with assay buffer, and 50 μl of freshly vortexed antibody-conjugated beads were added to each well. The plate was washed with assay buffer, and samples and standards were added. After a brief shake (30 s at 1,100 rpm), the plate was incubated at 4 °C overnight in the dark with light shaking (300 rpm). After one wash step, 25 μl of the detection antibody was added to each well, and the plate was shaken and incubated as above. Subsequently, the plate was washed and incubated for 30 min with 50 μl of a streptavidin-PE solution with shaking (30 s at 1,100 rpm, 10 min at 300 rpm). Finally, the plate was washed, 125 μl of assay buffer was added to each well, and the plate was shaken for 10 s at 1,100 rpm and read immediately on the Bio-Plex machine. Immunoprecipitation and Western Blotting—For co-immunoprecipitations, cells were washed twice in phosphate-buffered saline and lysed in 850 μl of lysis buffer (50 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm EDTA, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40 containing 1 mm phenylmethylsulfonyl fluoride, 0.01% (v/v) aprotinin, and 1 mm sodium orthovanadate). For immunoprecipitation, rabbit polyclonal anti-p38α (Abcam, Cambridge, UK) was precoupled to protein A-Sepharose beads overnight at 4 °C. The beads were then washed twice in lysis buffer and incubated with 1.5 mg of cell lysate/sample overnight at 4 °C. The immune complexes were washed three times in lysis buffer, boiled, and analyzed by standard SDS-PAGE and Western blotting techniques using rabbit polyclonal anti-p38α (as above) and rabbit polyclonal anti-TAB1 antibodies (Cell Signaling Technology, Danvers, MA) for blotting. SeV Replication Stimulates Activation of MAPKs in a RIG-I-dependent Manner—To examine the ability of SeV to stimulate activation of MAPKs, we first studied MEFs, in which we observed that the virus strongly activated p38 and to a lesser extent also JNK (Fig. 1, A and B). For both kinases, the activation was dependent on the ability of the virus to replicate, since UV-irradiated virus was not able to activate these kinases. The MAPKs ERK1/2 were not activated by SeV (data not shown). To study the mechanism of DC activation during viral infection, we used a murine DC cell line, BC-1 (40Yanagawa Y. Iijima N. Iwabuchi K. Onoe K. J. Leukocyte Biol... 2002; 71: 125-132Google Scholar), which was grown in parallel with BM-DCs. BC-1 cells resemble myeloid DCs, as assessed by their high expression of CD11b and CD11c and low expression of CD8α (Fig. S1). The cells express low levels of CD40 and major histocompatibility complex class I and II and intermediary levels of CD86, thus displaying a semimature phenotype. When looking for MAPK activation in response to SeV infection in BC-1 cells, we observed the same pattern of activation as in MEFs, although the kinetics of activation was somewhat faster in DCs (Fig. 1, C and D). To study whether virus-induced activation of MAPKs was dependent on RIG-I, we compared the ability of Rig-I+/- and Rig-I-/- MEFs to activate p38 MAPK during SeV infection. As shown in Fig. 1E, SeV-mediated activation of p38 was fully dependent on RIG-I, hence resembling the activation of the NF-κB pathway (Fig. 1F). In BC-1 cells, the cellular response to SeV infection was not inhibited by chloroquine, which was capable of inhibiting TLR9 (Toll-like receptor 9) signaling (Fig. S2), thus showing that the response was independent of endosomal Toll-like receptors and hence indicating that the RIG-I pathway was responsible for activation of this cell type in response to SeV infection. SeV Replication and p38 MAPK Activation Are Essential for Stimulation of Type I IFN Production and Activation of DCs—To examine whether the secretion of cytokines in response to virus infection required viral replication and was dependent on MAPKs, we stimulated MEFs, BC-1, and BM-DCs with infectious or UV-inactivated virus for subsequent examination of cytokine production. As shown in Fig. 2A, MEFs responded to SeV infection with a robust type I IFN response, which was fully dependent on viral replication. In DCs (BM-DCs or BC-1 cells), the production of IL-12p40 was also observed to be dependent on the replication of the virus (Fig. 2, B and C). Similar findings were made when examining expression of the bioactive IL-12 p70 heterodimer (Fig. 2D). In BC-1 cells, the type I IFN response was also abolished in cells stimulated with UV-inactivated virus (Fig. 2E). To investigate the involvement of the different MAPK pathways in induction of IFN production and maturation of DCs, we treated the cells with SB202190, SP600125, or U0126, which inhibit p38, JNK, and the ERK activators MEK1/2, respectively, and subsequently infected them with SeV. Supernatants were harvested for measurement of relevant cytokines. As seen in Fig. 3, A-C, treatment of MEF, BC-1 DC, and BM-DC cells with SB202190 inhibited virus-induced production of IFN-α/β and IL-12. By contrast, inhibition of the JNK and ERK pathways did not affect SeV-induced IL-12 production in BC-1 cells (Fig. S3). Importantly, SeV-induced expression of CXCL10, which is regulated throug

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