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, 284 (16), 10774-82

RIG-I-mediated Activation of p38 MAPK Is Essential for Viral Induction of Interferon and Activation of Dendritic Cells: Dependence on TRAF2 and TAK1

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RIG-I-mediated Activation of p38 MAPK Is Essential for Viral Induction of Interferon and Activation of Dendritic Cells: Dependence on TRAF2 and TAK1

Susie S Mikkelsen et al. J Biol Chem.

Abstract

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.

Figures

FIGURE 1.
FIGURE 1.
SeV-mediated activation of p38 MAPK is dependent on viral replication and RIG-I. MEFs (A and B) or BC-1 DCs (C and D) were infected with live or UV-inactivated SeV at an MOI of 1. At the indicated time points after infection, cells were collected, cell extracts were prepared, and the phosphorylation levels of p38 and JNK were measured using Luminex technology. For comparison, p38 samples were also analyzed by Western blotting (C, inset). E and F, Rig-I+/- and Rig-I-/- MEFs were infected with SeV for 5 h, at which point cell extracts were prepared. Phosphorylation levels of p38 and IκBα were measured using Luminex technology. The data are shown as means of duplicate cultures ± S.D. UT, untreated cells.
FIGURE 2.
FIGURE 2.
Viral replication is required for induction of type I IFN production in fibroblasts and full activation of dendritic cells. MEFs (A), BM-DCs (B), and BC-1 DCs (C-E) were infected with live or UV-inactivated SeV at MOI 1. Cell culture supernatants were collected 18 h after infection, and cytokine levels were determined. In C, the cells received infectious virus (MOI 1) or a similar dose of virus that had been UV-treated for the indicated times (1-7 min). The data are shown as means of triplicates ± S.D. UT, untreated cells.
FIGURE 3.
FIGURE 3.
MAPK p38 activity is essential for viral induction of type I IFNs in fibroblasts and full activation of dendritic cells. BC-1 DCs (A), MEFs (B), and BM-DCs (C) were pretreated with p38 MAPK inhibitors 30 min prior to infection with SeV (MOI 1). Cell culture supernatants were collected 12 h after infection, and cytokine levels were determined. Unless indicated otherwise, the concentrations used were as follows: SB202190, 30 μm; PD169316, 10 μm; BIRB 796, 10 μm. The data are shown as means of triplicates ± S.D. UT, untreated cells.
FIGURE 4.
FIGURE 4.
SeV-induced DC activation through p38 is exerted at the posttranscriptional level and is independent of MNK1, MK2, and MSK1/2. A and B, BC-1 DCs were pretreated with SB202190 (30 μm) 30 min prior to infection with SeV (MOI 1). Supernatants and total RNA were harvested 12 and 5 h, after infection, respectively, and analyzed for the presence of IL-12p40 protein (A) and IL-12p40 mRNA (B). C, BC-1 DCs were seeded and pretreated with the MNK1 inhibitor CGP57380 30 min before infection with SeV at MOI 1. Cell culture supernatants were collected 12 h after infection, and IL-12p40 levels were determined by ELISA. D and E, BM-DCs were generated from C57BL/6, MK2-/-, and MSK1/2-/- mice and plated for 2 h in tissue culture plates before treatment as described above. At 12 h after SeV infection, cell culture supernatants were collected, and IL-12p40 protein production was determined by ELISA. The data are shown as means of triplicates ± S.D. UT, untreated cells.
FIGURE 5.
FIGURE 5.
RIG-I-mediated activation of the MAPK and NF-κB pathways is dependent on TRAF2 and TAK1, which are essential for virus-induced type I IFN expression. Wild type, Tak1-/-, Traf2-/-, Traf6+/-, and Traf6-/- MEFs were seeded in triplicates and infected with SeV at MOI 1. 5 (A-D) and 6 (E-J) h postchallenge, cell extracts and total RNA, respectively, were harvested for measurement of the phosphorylation status of p38 (A and B) and IκBα (C and D) as well as mRNA expression of IFN-β (E-G) and ISG56 (H-J). The data are shown as means of duplicate cultures ± S.D. ND, not done. UT, untreated cells.
FIGURE 6.
FIGURE 6.
Viral activation of p38 MAPK requires its own kinase activity and leads to association of p38 MAPK with TAB1. A and B, BC1 DCs were pretreated with 30 μm SB202190 30 min before infection with SeV at MOI 1. 4 h later, cells were collected, cell extracts were prepared, and the amounts of phosphorylated p38 and IκBα were determined by Luminex. C, BM-DCs were generated from C57BL/6 and MKK3-/- mice and plated for 2 h in tissue culture plates before treatment as described above. At 12 h after SeV infection, cell culture supernatants were collected, and IL-12p40 protein production was determined by ELISA. D, BC-1 DCs were infected with SeV (MOI 1) for the indicated time intervals, and cell extracts were prepared and subjected to immunoprecipitation with anti-p38α antibodies. The immunoprecipitates (IP) were separated on SDS-PAGE and immunoblotted (WB) with anti-TAB1 antibodies. UT, untreated cells.

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