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Activation of p38 MAPK in CD4 T cells controls IL-17 production and autoimmune encephalomyelitis

Rajkumar Noubade, Dimitry N. Krementsov, Roxana del Rio, Tina Thornton, Viswas Nagaleekar, Naresha Saligrama, Anthony Spitzack, Karen Spach, Guadalupe Sabio, Roger J. Davis, Mercedes Rincon, Cory Teuscher

Abstract

Although several transcription factors have been shown to be critical for the induction and maintenance of IL-17 expression by CD4 Th cells, less is known about the role of nontranscriptional mechanisms. Here we show that the p38 MAPK signaling pathway is essential for in vitro and in vivo IL-17 production by regulating IL-17 synthesis in CD4 T cells through the activation of the eukaryotic translation initiation factor 4E/MAPK-interacting kinase (eIF-4E/MNK) pathway. We also show that p38 MAPK activation is required for the development and progression of both chronic and relapsing-remitting forms of experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis. Furthermore, we show that regulation of p38 MAPK activity specifically in T cells is sufficient to modulate EAE severity. Thus, mechanisms other than the regulation of gene expression also contribute to Th17 cell effector functions and, potentially, to the pathogenesis of other Th17 cell–mediated diseases.

Introduction

CD4 Th lymphocytes are central in regulating host immune responses as well as inflammatory and autoimmune diseases.1 Once activated, these cells expand and differentiate into different Th subsets with distinct cytokine profiles and effector functions. Th1 cells are characterized by IFNγ production and mediate cellular immunity. Th2 cells are characterized by production of IL-4, IL-5, and IL-13 and are critical in humoral immunity and allergic responses. IL-17 production is characteristic of a third subset called Th17 cells.2,3 IL-17 is a proinflammatory cytokine critical for host defense but is also implicated in the pathogenesis of multiple autoimmune diseases. Elevated levels of IL-17 have been observed in patients with multiple sclerosis (MS), rheumatoid arthritis (RA), inflammatory bowel disease, psoriasis, and asthma.2,3 The pathogenic role of IL-17 is demonstrated by the fact that mice deficient in IL-17 and IL-17R are less susceptible to several autoimmune diseases including collagen-induced arthritis and experimental allergic encephalomyelitis (EAE), the autoimmune model of MS.2,3 Further, blockade of IL-17 signaling results in substantial reduction in EAE severity and reverses the progression of active EAE.24

The differentiation of naive CD4 T cells, on TCR activation, into Th17 cells is dependent on several cytokines including IL-6 and TGFβ, which leads to up-regulation of the Th17 master transcriptional regulators Rorc and Rora (retinoic acid receptor-related orphan receptor γ and α, respectively).2,3 Induction of these transcription factors is Stat3-dependent, as deletion of Stat3 in T cells abrogates Th17 differentiation.2,3 Runx1 (runt-related transcription factor 1), a member of the Runx family of transcription factors, regulates IL-17 production and Th17 differentiation by inducing Rorc expression and by binding to promoter and enhancer regions of Il17 along with Rorc. Irf4 (interleukin regulatory factor 4), considered to be a Th2 transcription factor,5 has also been shown to play an important role in optimal differentiation of Th17 cells. T cells lacking Irf4 exhibit reduced Rorc expression and impaired Th17 differentiation.2,3 Batf (basic leucine zipper transcription factor, ATF-like), an AP-1 transcription factor, is also critical for differentiation of Th17 cells, because T cells lacking Batf fail to induce Rorc and Th17 differentiation.6,7 Thus, most studies have focused on the gene transcription of the Il17 gene as the major regulatory mechanism of Th17 cell function.

p38 MAPK is activated by phosphorylation primarily by the upstream MAPK kinases MKK3 and MKK6,8,9 although an alternative, T cell–specific, p38 MAPK activation pathway downstream of the TCR has been recently described.10 The p38 MAPK pathway has been involved in mediating cell death and/or survival in response to stress-inducing stimuli, but it also plays a central regulatory role in the production of several cytokines, including TNFα, IL-6, and IFNγ among others.8,9 Although p38 MAPK regulates the activity of specific transcription factors (eg, ATF2) it can also regulate cytokine production by affecting mRNA stability or translation. Recent studies suggest that pharmacologic inhibition of this pathway can affect IL-17 production by CD4 T cells.1114

We show here that activation of p38 MAPK signaling in CD4 T cells plays a pivotal role in Th17 cell function by regulating IL-17 production at the translational level through indirect activation of the eIF-4E (eukaryotic translation initiation factor 4E) by MAPK-interacting kinase (MNK), one of the p38 MAPK targets. Moreover, we also show that in vivo regulation of p38 MAPK activity specifically in T cells is sufficient to alter IL-17 production and EAE severity. Importantly, inhibition of p38 MAPK not only prevents the development of chronic EAE, but it also prevents disease relapse in relapsing-remitting EAE.

Methods

Mice

C57BL/6J (B6) and B10.BR-H2k H2-T18/SgSnJ (B10.BR) mice were purchased from The Jackson Laboratory. MKK6(Glu) transgenic,15 dn-p38 transgenic,16 and MKK3−/−MKK6+/−17 mice have been described. SJL/JCrHsd (SJL) mice were purchased from Harlan Laboratories. The experimental procedures used in this study were approved by the Animal Care and Use Committee of the University of Vermont.

Induction and evaluation of EAE

EAE was induced in female C57BL/6J mice as described previously.18 Mice were injected subcutaneously with an emulsion containing 200 μg of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK; Peptide Core Facility, University of Vermont, Burlington, VT) and complete Freund adjuvant (CFA; Sigma-Aldrich) supplemented with 200 μg of Mycobacterium tuberculosis H37RA (Difco Laboratories) in the posterior right and left flank; 1 week later, all mice were similarly injected at 2 sites on the right and left flank anterior of the initial injection sites (2× MOG35-55 + CFA). Mice received 5 mg/kg/d SB203580 dihydrochloride (Tocris) by IP injection in a total volume of 200 μL or an equal volume of carrier everyday from the day of immunization.

EAE was induced in B10.BR, MKK6-Tg, and dn-p38-Tg mice by immunizing with a single injection of 200 μg of MOG97-114 (TCFFRDHSYQEEAAVELK) or PLP180-209 (SKTSASIGSLCADARMYGVL) in CFA. Immediately thereafter, each animal received 200 ng of PTX (List Biologic Laboratories) by IV injection. Mice were scored daily starting at day 10 after injection as previously described.18 Clinical quantitative trait variables were generated as previously described.19

EAE was induced in female SJL mice using the 2 × PLP139-151 + CFA protocol. After the initial episode of clinical signs and after 2 days of remission (defined as a score of 0 for a minimum of 2 days), daily injections of carrier or SB203580 were commenced. Because the relapsing-remitting disease was somewhat asynchronous between individual mice, clinical scores from individual mice were synchronized by the initiation of the first disease phase and/or of the relapse for data presentation and analysis.

Cell preparation and culture conditions

Total CD4 T cells were isolated from spleen and lymph nodes as previously described,18 by negative selection for CD8-, MHC class II–, NK1.1- and CD11b-positive cells using magnetic beads from QIAGEN. For FACS sorting, negatively selected CD4 T cells were stained with anti-TCRβ-allophycocyanin and anti-CD4–Texas Red (Invitrogen), and sorted using a FACSAria cell sorting system (BD Biosciences). Th17 CD4 T cells were generated by activating total CD4 T cells (1 × 106 cells/mL) in RPMI containing 10% FBS (Hyclone), with plate bound anti-CD3 (5 μg/mL) and soluble anti-CD28 (1 μg/mL) mAbs from BD Pharmingen in the presence of 1 ng/mL TGFβ (PeproTech Inc), 30 ng/mL IL-6 (R&D Systems), 10 μg/mL anti-IFNγ, and 10 μg/mL anti–IL-4 mAbs. For the FACS-sorted cells, the Th17 conditions included activating cells with plate-bound anti-CD3 (5 μg/mL) and soluble anti-CD28 (1 μg/mL) mAbs in the presence of 1 ng/mL TGFβ and 100 ng/mL IL-6 and no neutralizing mAbs. Depending on the experiment, cells were treated the p38 MAPK inhibitors SB203580 (Calbiochem) or BIRB796 (Axon Medchem) or the MNK inhibitor CGP57380 (Sigma-Aldrich). Cells were incubated at 37°C and 5% CO2 for the desired lengths of time as described in the figure legends.

Cytokine quantification

For the detection of cytokines in the cell-culture supernatants, ELISAs were performed as described previously,18 using the primary mAbs: anti-IFNγ, anti–IL-2, and anti–IL-17A and their corresponding biotinylated mAbs (BD Pharmingen). Other ELISA reagents included: HRP-conjugated avidin D (Vector Laboratories) and TMB microwell peroxidase substrate and stop solution (Kirkegaard & Perry Laboratories). rIFNγ, rIL-17A, and rIL-2 (R&D Systems) were used as standards.

For cytokine analysis of ex vivo–stimulated splenocytes from mice immunized with 2× MOG35-55 + CFA, spleen and draining LN (DLN) were harvested on day 10 or day 21 after immunization, single-cell suspensions were prepared at 1 × 106 cells/mL in RPMI medium, and stimulated with 50 μg/mL MOG35-55 in the presence or absence of 5μM SB203580. Cell-culture supernatants were collected at 72 hours and cytokine levels were measured by ELISA.

Proliferation assays

Mice were immunized with 2× MOG35-55 + CFA, and spleen and DLN cells were collected on day 10 after immunization. Single-cell suspensions of spleen and DLN were prepared at 2.5 × 105 cells/well in RPMI medium and stimulated in a 96-well plate with different concentrations of MOG35-55 in the presence or absence of 5μM SB203580 for 72 hours. Proliferation was determined by [3H]-thymidine incorporation during the last 18 hours of culture.

Flow cytometry

For intracellular cytokine staining, either FACS-sorted or total CD4 T cells polarized to Th17 cells in the absence or the presence of 5μM SB203580 were stimulated with 5 ng/mL PMA, 250 ng/mL ionomycin, and 2μM monensin (Sigma-Aldrich) for the last 4 hours of culture. Cells harvested at the end of the incubation were first stained with LIVE/DEAD fixable stain (Invitrogen) and anti-CD4–Texas Red. Cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with buffer containing 0.2% saponin and stained with anti–IL-17A–PE (BD Pharmingen) and anti-IFNγ–Alexa 647 (BD Pharmingen). Cells were collected using a LSR II cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc).

Quantitative real-time PCR

Total RNA was extracted from CD4 T cells using RNeasy RNA isolation reagent (QIAGEN) as recommended by the manufacturer. The generated cDNA was used in quantitative real-time PCR using the assay-on-demand (AOD) TaqMan probe and primers for IL-17A and β2-microglobulin (Applied Biosystems). β2-microglobulin was used as a reference gene and relative mRNA levels were calculated using the comparative CT method.

Cell lysates and Western blot analysis

Whole-cell lysates were prepared from 1-5 × 106 cells in Triton lysis buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes as described previously.18 Primary Abs used for Western blot analysis included anti-phospho–STAT3 (Tyr705), anti-phospho–STAT3 (Ser727), anti-STAT3, anti-phospho–eIF-4E (Ser209; Cell Signaling Technologies), and anti-actin (Santa Cruz Biotechnology). Anti–rabbit-HRP, anti–mouse-HRP (Jackson ImmunoResearch Laboratories), and anti–goat-HRP (Santa Cruz Biotechnology) were used as secondary Abs.

CNS-infiltrating mononuclear cell isolation

Animals were perfused with saline and brains and spinal cords removed. A single-cell suspension was obtained and passed through a 70-μm strainer. Mononuclear cells were obtained by Percoll gradient (37%/70%) centrifugation and collected from the interphase. Cells were washed and stimulated for 4 hours with PMA/ionomycin in the presence of brefeldin A (Golgi Plug; BD Biosciences). Cell were labeled with LIVE/DEAD UV-Blue dye (Invitrogen) followed by surface staining (CD45 from Invitrogen and CD4, CD8 and TCRβ from BD Biosciences). Afterward, cells were fixed, permeabilized, and stained for intracellular IL-17A (BD Biosciences) and IFNγ (Invitrogen).

RNA-immunoprecipitation assay

Purified CD4 T cells were cultured under Th17 differentiation conditions (see “Cell preparation and culture conditions”). On day 3, cells were harvested and RNA-immunoprecipitation assays (RIPs) were preformed as per the manufacturer's instructions using the RIP-Assay kit (MBL International). Western blot analysis was used to determine the efficiency of the immunoprecipitation using the Exacta Cruz E homologous Western blot/immunoprecipitation reagent (Santa Cruz Biotechnology). RNA isolated from the input lysate, isotype control immunoprecipitate, and the anti-eIF-4E immunoprecipitate was reverse transcribed and the levels of c-myc and Il17 mRNA were determined using the AOD probes (Applied Biosystems). The relative levels of each mRNA were calculated using the standard Δ-Δ CT formula substituting the level of the target mRNA in the input lysate for the endogenous control.

Statistical analysis

The statistical analyses, as indicated in the figure legends, were performed using GraphPad Prism 4 software (GraphPad Software Inc) or SPSS (IBM Corp).

Results

Activation of p38 MAPK in CD4 T cells is essential for IL-17 production

Recent studies using pharmacologic inhibitors of the p38 MAPK pathway suggested a regulatory effect of this pathway on Th17 cell differentiation.1114 We investigated whether p38 MAPK could have a direct effect on the production of IL-17 by CD4 T cells. Thus, CD4 T cells were activated with anti-CD3 and anti-CD28 mAbs together with TGFβ plus IL-6 in the presence of varying concentrations of SB203580, a specific inhibitor of p38 MAPK α and β,20 or BIRB796, another pharmacologic compound that inhibits of p38 MAPKγ in addition to α and β.21,22 There was a clear dose-dependent inhibition of IL-17 production by SB203580 (Figure 1A). The reduction of IL-17 levels was not due to SB203580 toxicity because neither cell viability (supplemental Figure 1A-B, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) nor IL-2 production (supplemental Figure 1C) were affected. As with SB203580, the presence of either BIRB796 (Figure 1B) or SB239063 (supplemental Figure 2), 2 additional p38 MAPK inhibitors, caused a significant reduction in IL-17 production. Because these pharmacologic compounds could have effects other than those on p38 MAPK activity, to further demonstrate the role of p38 MAPK in regulating IL-17 production by Th17 cells, we used cells from genetically manipulated mice. p38 MAPK is activated primarily by phosphorylation mediated by 2 upstream MAPK kinases (MKKs), MKK3 (mitogen-activated protein kinase kinase 3) and MKK6 (mitogen-activated protein kinase kinase 6).23 Simultaneous disruption of both MKK3 and MKK6 genes (MKK3−/−MKK6−/− mice) causes embryonic lethality.17 However, mice that are MKK3−/−MKK6+/− are viable.17 We have previously shown that activation of p38 MAPK in MKK3−/−MKK6+/− mice is strongly reduced in thymocytes, although not abrogated.24 Analysis of phospho-p38 MAPK in effector CD4 T cells (Th17 or Th1) from these mice also showed a marked reduction in the activation of p38 MAPK (supplemental Figure 3). We therefore examined cytokine production in MKK3−/−MKK6+/− Th17 cells. CD4 T cells from wild-type (WT) and MKK3−/−MKK6+/− mice were in vitro–differentiated into Th17 cells. MKK3−/−MKK6+/− Th17 cells produced significantly less IL-17 compared with WT cells (Figure 1C). We also examined IL-17 production by CD4 T cells from transgenic mice expressing a dominant-negative (dn) mutant allele of p38 MAPK (dn-p38-Tg) where the activity of endogenous p38 MAPK is highly reduced specifically in T cells.15 Similar to CD4 T cells from MKK3−/−MKK6+/− mice, Th17 cells from dn-p38-Tg mice produced significantly less IL-17 compared with WT Th17 cells (Figure 1D). Expression of a constitutively active mutant allele of MKK6 in T cell–specific transgenic mice (MKK6-Tg) leads to activation of p38 MAPK in CD4 T cells even before stimulation15 (supplemental Figure 4). We therefore tested whether the presence of activated p38 MAPK in CD4 T cells from these mice could further enhance IL-17 production. In vitro–differentiated Th17 cells from MKK6-Tg mice produced significantly greater amounts of IL-17 compared with those from WT mice (Figure 1E). Taken together, the data from these genetic studies demonstrate that the activation of p38 MAPK intrinsic to CD4 T cells is critical for IL-17 production. In addition to the activation of p38 MAPK by the canonical MKK pathway, an alternative pathway of activation of p38 MAPK through phosphorylation on Tyr323 by Zap70 has been identified in T cells.10 Although our results indicate that the activation of p38 MAPK by the canonical MKK3/6 pathway plays a major role in IL-17 production, the alternative pathway also appears to contribute to Th17 differentiation.25

Figure 1

p38 MAPK regulates IL-17 production by in vitro–generated Th17 cells. FACS-sorted CD4 T cells from WT B6 mice in vitro differentiated into Th17 cells in the presence of different concentrations of SB203580 (A) or BIRB796 (B) for 72 hours. IL-17 production by Th17-polarized FACS-sorted CD4 T cells from WT B6 or MKK3−/−MKK6+/− mice (C) or total CD4 T cells from WT B10.BR and dn-p38-Tg (D) or MKK6 Tg (E) differentiated into Th17 cells. IL-17 levels in the supernatants were assessed by ELISA. The significance of differences observed in panels A and B was determined by linear regression analysis (A, P < .0001; B, P = .0001). The significance of the differences observed in panels C-E was determined using the Student t test (*** ≤ 0.001).

p38 MAPK regulates IL-17 production by Th17 cells at the translational level

p38 MAPK can regulate cytokine gene expression by direct phosphorylation and activation of transcription factors.26 The transcription factor STAT3 has been shown to be critical for the development of Th17 cells.27 Tyrosine phosphorylation of STAT3 (Tyr705) by JAK kinases is required for its nuclear translocation.28 However, phosphorylation of STAT3 at Ser727 is required for maximal transcriptional activity.29 Both ERK and p38 MAPKs are involved in the phosphorylation of STAT3 at Ser727 in mouse epidermal cells.30 We therefore investigated the effect of p38 MAPK inhibition on STAT3 phosphorylation in CD4 T cells cultured under Th17-polarizing conditions. Western blot analysis showed that phosphorylation of STAT3 at Ser727 or Tyr705 was not affected by the addition of SB203580 (Figure 2A). Thus, p38 MAPK does not appear to contribute to STAT3 activation in Th17 cells. In addition, we examined the effect of p38 MAPK inhibition on the expression of the transcription factor Rorc, the Th17-master regulator, that is induced in CD4 T cells during activation under Th17 conditions and is required to maintain the Th17 phenotype.3 No significant difference in Rorc mRNA levels was observed with SB203580 treatment (Figure 2B).

Figure 2

p38 MAPK controls IL-17 production at the posttranscriptional level. (A) FACS-sorted CD4 T cells from WT B6 mice were differentiated into Th17 cells in the absence or presence of SB203580 (5μM) for the indicated periods of time. The phosphorylation of STAT3 at Ser727 (P-Ser) or at Tyr705 (P-Tyr) was examined by Western blot analysis. Total STAT3 (Total) is also shown. FACS-sorted CD4 T cells from WT mice were activated under Th17 conditions in the absence (control) or presence of SB203580 for 48h. Relative Rorc (B) and Il17 (C) mRNA levels were examined by quantitative real-time PCR using β2-microglobulin as the endogenous control. (D) Relative Il17 mRNA levels in FACS-sorted CD4 T cells from WT B6 and MKK3−/−MKK6+/− mice activated under Th17 conditions for 48h. (E) Intracellular staining for IL-17 and IFNγ in WT total CD4 T cells differentiated into Th17 cells in the absence (control) or presence of SB203580 for 72 hours.

p38 MAPK can also regulate cytokine expression independently of transcription by promoting mRNA stability.26 We further examined the effect of p38 MAPK inhibition on Il17 mRNA levels in CD4 T cells activated under Th17 conditions. However, despite a strong reduction in IL-17 production as determined by ELISA (Figure 1A), Il17 mRNA levels were not different between SB203580-treated Th17 cells and nontreated cells (Figure 2C). Similarly, the levels of Il17 mRNA in MKK3−/−MKK6+/− Th17 cells were comparable with those detected in WT Th17 cells (Figure 2D), despite the impaired IL-17 production detected by ELISA. To examine whether the reduced levels of IL-17 determined by ELISA could be because of a decrease in IL-17 protein synthesis, we examined intracellular IL-17. CD4 T cells were activated under Th17 conditions in the absence or presence of SB203580, stained for IL-17 and analyzed by flow cytometry. Consistent with the ELISA results, the frequency of CD4 T cells capable of synthesizing IL-17 was substantially lower in the presence of SB203580 (Figure 2E). Thus, p38 MAPK regulates IL-17 synthesis, but not gene expression.

p38 MAPK regulates IL-17 production via activation of the MNK/eIF-4E pathway

In addition to regulating gene expression, p38 MAPK has also been shown to regulate cytokine production at the translational level through direct phosphorylation and activation of MNKs.3134 Activated MNKs promote initiation of translation through phosphorylation and activation of eIF-4E, a translation initiation factor that binds the 5′ cap structure of cytoplasmic mRNA and assists in cap-dependent translation of specific mRNAs.35 Because little is known about the regulation of eIF-4E in CD4 T cells, we investigated the effect of p38 MAPK on MNK-mediated phosphorylation of eIF-4E in Th17 cells. eIF-4E was highly phosphorylated at Ser209 the target residue of MNK, during Th17 differentiation and inhibition of p38 MAPK by SB203580 significantly reduced this phosphorylation (Figure 3A). Similar results were obtained with BIRB796 (Figure 3B). Phosphorylation of eIF-4E was also highly compromised in Th17 cells from MKK3−/−MKK6+/− mice (Figure 3C) and dn-p38-Tg mice (supplemental Figure 5) compared with its phosphorylation status in WT Th17 cells. Furthermore, in correlation with the increased p38 MAPK activation and increased IL-17 production, increased levels of phosphorylated eIF-4E were also present in CD4 T cells from MKK6-Tg mice activated under Th17 conditions (Figure 3D). Higher phospho-eIF-4E levels could also be detected in MKK6-Tg CD4 T cells even before activation (Figure 3E), in correlation with the constitutive activation of p38 MAPK in the these cells15 (supplemental Figure 3). Together, these results indicate that activation of p38 MAPK during Th17 differentiation leads to MNK-mediated phosphorylation of eIF-4E.

Figure 3

p38 MAPK regulates IL-17 via eIF-4E. (A) CD4 T cells from WT B6 mice were activated under Th17 conditions in the absence (−) or presence (+) of SB203580 (SB) for the indicated periods of time and phosphorylation of eIF-4E at Ser209 (P-eIF-4E) was examined by Western blot analysis. Actin is shown as a loading control throughout. (B) B6 CD4 T cells were activated as in panel A in the presence or absence of BIRB796 (BIRB) for 48 hours, and phosphorylation of eIF-4E at Ser209 was examined by Western blot analysis. (C) CD4 T cells from WT B6 and MKK3−/−MKK6+/− (KO) mice were activated as in panel A and phosphorylation of eIF-4E was examined by Western blot analysis. (D-E) Phosphorylation of eIF-4E in CD4 T cells from WT B10.BR and MKK6-Tg mice before activation (E) or 24 and 48 hours on activation (D) as described in panel A was examined by Western blot analysis. A longer exposure was used for detection of P-eIF-4E in panel E. (F) Relative levels of Il17, c-myc, and Il2 mRNA present in the immunoprecipitates obtained with an anti-eIF-4E Ab or a control IgG using whole-cell lysates from CD4 T cells activated under Th17 conditions for 3 days. Analyses were performed by quantitative real-time PCR and mRNA values for each gene are relative to the levels detected in the control IgG immunoprecipitates. The presence of eIF-4E in the input lysate and the immunoprecipitates was determined by Western blot analysis (right panel). (G) CD4 T cells from WT B6 mice were activated under Th17 conditions in the absence (−) or the presence (+) of the MNK inhibitor CGP57380 (CGP) for the indicated periods of time. Phospho-eIF-4E was examined by Western blot analysis. (H) CD4 T cells from WT B6 mice were differentiated into Th17 cells in the presence of the indicated amounts of CGP57380, and the levels of IL-17 in the culture supernatants after 72 hours were quantified by ELISA. (I) Relative Il17 mRNA levels in CD4 T cells activated as in panel H were assessed after 48 hours by quantitative real-time PCR using β2-microglobulin as the endogenous control. (J) IL-2 production in CD4 T cells activated as in panel H was quantified by ELISA. The significance of differences observed in panels H through J was determined by linear regression analysis (H, P < .0001; I, P = .23; J, P = .27).

eIF-4E has been shown to bind specific mRNAs, for example, c-myc,36,37 and promote initiation of their translation. We therefore investigated whether eIF-4E regulated Il17 mRNA translation by examining the binding of eIF-4E to IL-17 mRNA using immunoprecipitation of mRNA-eIF-4E complexes in Th17 cells. Th17 cell extracts were used for immunoprecipitation with an anti-eIF-4E or an isotype control Abs, and the presence of Il17 mRNA in the immunoprecipicates was examined by quantitative real-time PCR. High levels of Il17 mRNA were present in the anti-eIF-4E immunoprecipitate relative to the levels found in the isotype control immunoprecipitate (Figure 3F). Similarly, high mRNA levels for c-myc, a known eIF-4E target, were present in the anti–eIF-4E immunoprecipitate (Figure 3F). In contrast, no difference between the levels of Il2 mRNA in the anti–eIF-4E and isotype immunoprecipiates was detected (Figure 3F). These results indicate that Il17 mRNA is another selective target for eIF-4E.

Because regulation of eIF-4E by p38 MAPK is indirectly mediated through MNKs, which are the only known kinases to phosphorylate eIF-4E,32 we examined whether inhibition of MNK interferes with eIF-4E phosphorylation in Th17 cells. CD4 T cells from WT mice were activated under Th17 conditions in the absence or the presence of CGP57380, a MNK inhibitor.38 Inhibition of MNK by CGP57380 blocked phosphorylation of eIF-4E in Th17 cells (Figure 3G). We therefore examined the contribution of the p38 MAPK/MNK pathway to the regulation of IL-17 production. CD4 T cells were differentiated into Th17 cells in the presence of different concentrations of the MNK inhibitor, and IL-17 production was quantified by ELISA. Inhibition of MNK significantly reduced IL-17 production in a dose-dependent manner (Figure 3H), without affecting Il17 mRNA levels (Figure 3I). In contrast to IL-17, IL-2 production was not affected by the MNK inhibitor (Figure 3J), showing that this compound did not cause a global inhibition of cytokine translation. Together, these results indicate that MNK selectively contributes to IL-17 protein synthesis in Th17 cells and that the MNK/eIF-4E pathway likely mediates the effect of p38 MAPK on IL-17 production.

p38 MAPK controls IL-17 production by Th17 cells in vivo

Th17 cells have been shown to contribute to several autoimmune inflammatory diseases, including EAE, the autoimmune model of MS. EAE and MS are organ-specific autoimmune diseases where myelin-reactive CD4 T cells infiltrate the CNS and orchestrate an inflammatory cascade primarily through the secretion of proinflammatory cytokines, including IL-17.39,40 Therefore, we examined if p38 MAPK regulates IL-17 production in vivo using the EAE model.

To evaluate the autoreactive effector CD4 response in the target organ, we examined the frequency of encephalitogenic Th1 and Th17 cells in the CNS during the chronic phase of the disease in mice treated or not with SB203580. C57BL/6J mice immunized for the induction of EAE18 were treated daily with either SB203580 or carrier for 35 days, and mononuclear cells from the CNS were isolated and examined by intracellular staining. The number and frequency of CD45+ CNS-infiltrating cells were comparable between SB203580 or carrier-treated mice (data not shown). The frequency of CD4 T cells was also not affected by SB203580 treatment (Figure 4A left panel), but the frequency of IL-17–producing CD4 T cells was significantly decreased, while that of IFNγ-producing cells did not change (Figure 4A right panel). To examine the peripheral T cell response, cells from spleen and DLN from carrier- and SB203580-treated MOG35-55-immunized mice were harvested on day 21 and restimulated with MOG35-55 ex vivo. We found that IL-17, but not IFNγ production by cells from SB203580-treated mice was reduced compared with cells from carrier-treated mice (Figure 4B). To confirm that p38 MAPK regulates IL-17 production by neuroantigen-specific effector Th17 cells, spleen and DLN cells from immunized mice were restimulated ex vivo with MOG35-55 in the absence or presence of SB203580. Inhibition of p38 MAPK in vitro inhibited IL-17 production by Ag-specific T cells (Figure 4C). The decrease in IL-17 production was not because of an impaired activation of the Ag-specific cells, because proliferation of these cells was not affected (Figure 4D). Thus, p38 MAPK is essential for the optimal production of IL-17 by autoreactive Th17 cells in an autoimmune disease model.

Figure 4

p38 MAPK controls IL-17 production by Th17 cells in vivo. (A) Mononuclear cells were isolated from the CNS of 2× MOG35-55-CFA immunized mice on day 35 after immunization, stimulated with PMA/ionomycin for 4 hours in the presence of brefeldin A, stained, and analyzed by flow cytometry. (Left panel) Cells were gated on CD45 and the frequency of CD4+TCRβ+ cells was determined. (Right panels) Cells were gated on CD4 and TCRβ and the frequency of IL-17+ and IFNγ+ cells was determined. (B) Spleen and draining lymph node (DLN) cells from 2× MOG35-55-CFA immunized mice treated with either carrier (n = 7) or the SB203580 (n = 8) were harvested on day 21 after immunization and stimulated with MOG35-55 (50 μg/mL) for 72 hours and IL-17 and IFNγ in the supernatants was quantified by ELISA. (C) Spleen and DLN cells from 2× MOG35-55-CFA immunized mice (n = 10) were harvested on day 10 after immunization and stimulated with MOG35-55 in the presence or absence of SB203580 (SB; 5μM) for 72 hours. IL-17 production was quantified by ELISA. (D) Cells obtained as in panel C were stimulated with the indicated amounts of MOG35-55 in the presence or the absence of SB203580 and proliferation was assessed by [3H]-thymidine incorporation. The significance of differences observed in panels A through C was determined using the Student t test (* ≤ 0.05; *** ≤ 0.001), and by 2-way ANOVA for panel D (SB, P = .31; MOG35-55, P = .003; and interaction, P = .95).

p38 MAPK blockade suppresses EAE development

We also assessed whether decreased IL-17 production by Th17 in vivo also correlated with reduced EAE severity. Using the C57BL/6J 2 × MOG35-55-CFA model,18 we found that immunized mice receiving carrier exhibited a normal course of EAE (Figure 5A, Table 1), while mice that received daily injections of the p38 MAPK inhibitor SB20358020 were resistant to EAE (Figure 5A, Table 1). We therefore investigated whether the effect of p38 MAPK inhibition was because of the requirement of p38 MAPK during the induction phase of the disease (priming) or if continuous inhibition of p38 MAPK was necessary. Mice were immunized with 2 × MOG35-55-CFA and treated with either SB203580 or carrier for 30 days, at which point SB203580 treatment was stopped. Within 2-3 days of discontinuing SB203580 treatment immunized mice exhibited clinical signs of EAE, which by day 6-7 were equivalent in severity to those seen in carrier-treated mice (Figure 5B, Table 2). Readministration of SB203580 to these mice 10 days after treatment was terminated led to an overall reduction and stabilization of their disease severity, which was not seen in carrier-treated controls (Figure 5B, Table 2). Together, these results indicate that p38 MAPK activation is essential for the initiation and the progression of the disease, and that continuous inhibition of this pathway is required for disease suppression.

Figure 5

p38 MAPK blockade ameliorates EAE. (A) Clinical course of EAE in B6 mice immunized with 2× MOG35-55-CFA treated with either carrier or SB203580. (B) Clinical course of EAE in B6 mice immunized with 2× MOG35-55-CFA treated with either carrier or SB203580 starting on day 0 (first arrow), treatment terminated on day 31 (arrow with cross), and retreated from day 41 through day 60 (second arrow). (C) Clinical course of EAE in B6 mice immunized with 2× MOG35-55-CFA and randomly selected for treatment with either carrier or SB203580 on reaching a clinical score ≥ 1. (D) Remitting-relapsing EAE course in SJL mice immunized with 2× PLP135-151-CFA. After the initial episode of EAE and after 2 days of remission defined as a score of 0 for a minimum of 2 days, mice were treated with either carrier or SB203580 (arrow). The significance of the differences between the clinical courses of disease in panels A and B (day 0-day 40), and panel C was calculated by regression analysis and best-fit curves are shown. In pane D, 2-way ANOVA followed by Bonferroni posthoc comparisons was used to determine significant differences in disease severity for individual days (*P = .01, **P = .001).

View this table:
Table 1

Inhibition of p38 MAPK activity prevents EAE

View this table:
Table 2

Termination of p38 MAPK inhibitor SB203580 treatment leads to EAE in immunized mice

We further examined whether SB203580 can be used as a disease-modifying therapy (DMT), which when administered on initial diagnosis can halt disease progression. C57BL/6J mice were immunized with 2× MOG35-55-CFA and monitored daily for the onset of clinical signs. On exhibiting a clinical score ≥ 1 the mice were randomly treated either with SB203580 or carrier. Compared with carrier-treated controls, the course of clinical disease in SB203580-treated mice was significantly less severe (Figure 5C, supplemental Table 1). In SB203580-treated mice the disease severity was stabilized at a clinical score of ∼ 1 during the chronic phase of the disease. Thus, although administration of a pharmacologic inhibitor of p38 MAPK on disease onset does not cure the disease, it nevertheless is capable of halting disease progression.

Among the different forms of MS, the majority of patients exhibit remitting-relapsing disease. To determine whether p38 MAPK blockers administered during remission can prevent disease relapse, we used the well-established remitting-relapsing model of EAE in SJL mice, where relapses are characterized by the presence of predominantly Th17 cells in the CNS.41 SJL mice were immunized using the 2× proteolipid protein peptide 139-151 (PLP139-151)–CFA protocol. Onset of clinical signs occurred around day 15, and remission by day 20 (Figure 5D). On day 2 of remission daily injections of SB203580 or carrier were initiated. Mice receiving the carrier exhibited a severe disease relapse, whereas SB203580-treated mice showed minimal clinical signs (Figure 5D). Together, these results demonstrate that p38 MAPK plays a key role in regulating the Th17-mediated pathogenesis of EAE, and suggest that inhibition of this pathway may be effective in treating both chronic and remitting-relapsing autoimmune disease of the CNS.

p38 MAPK activity in T cells controls EAE susceptibility and severity

The above studies show that in vivo treatment with the p38 MAPK inhibitor SB203580 can prevent the development and/or progression of clinical signs of EAE, and that this effect is associated with impaired IL-17 production by Th17 cells. Our in vitro studies also show that p38 MAPK activation in CD4 T cells is required for maximal IL-17 production. To address the contribution of the p38 MAPK pathway specifically in T cells to EAE susceptibility, we used dn-p38-Tg mice where p38 MAPK activity is markedly and selectively inhibited in T cells. Because the dn-p38-Tg mice are on the B10.BR background (H2K haplotype), the mice were immunized for the induction of EAE using the class II H2K-restricted MOG97-114 peptide.42 We found that WT B10.BR mice immunized with MOG97-114 developed classic EAE, and that the course of the clinical disease was comparable with that seen in C57BL/6J mice (Figure 6A). Interestingly, the course of clinical disease in the dn-p38-Tg mice was significantly less severe (Figure 6A, supplemental Table 2), indicating that inactivation of p38 MAPK selectively in T cells is sufficient to alter disease severity.

Figure 6

Modulation of p38 MAPK activity specifically in T cells influences EAE susceptibility and severity. (A) Clinical course of EAE in WT B10.BR and dn-p38-Tg mice immunized with 1× MOG97-114-CFA + PTX. (B) Clinical course of EAE in WT and MKK6-Tg mice immunized with 1× MOG97-114-CFA + PTX. (A-B) Significance of the differences in the clinical course of disease was determined by regression analysis and the best-fit curves are shown. (C-D) EAE was induced in WT (n = 29) and MKK6-Tg (n = 8) mice by immunization with 1× PLP190-209-CFA + PTX. The incidence (C) and the cumulative disease score (CDS; D) are shown. The significance of the differences was determined using the independent samples t test within SPSS (Levene test for equality of variances and t test for equality of means; ***≤ 0.001).

The results from our in vitro studies indicated that constitutive activation of p38 MAPK in CD4 T cells from MKK6-Tg mice leads to increased production of IL-17 (Figure 1E). We therefore examined the development of EAE in MKK6-Tg mice. As expected, the course of clinical EAE in these mice was significantly more severe than that of WT mice (Figure 6B, supplemental Table 3). The role of the p38 MAPK pathway was also examined in a second model of EAE elicited by immunization with class II H2k-restricted PLP180-209. WT B10.BR mice were completely resistant to PLP180-209–induced EAE, whereas 80% of MKK6-Tg mice developed clinical signs of disease (Figure 6C-D, supplemental Table 4). These results demonstrate that augmented p38 MAPK activity in T cells can impart susceptibility to mice that are otherwise resistant to EAE. Taken together, these results demonstrate that activation of p38 MAPK intrinsically in T cells is required and sufficient for the development of EAE.

Discussion

In this study, we show that the p38 MAPK pathway plays an important role in the regulation of Th17 cell function. It is well established that the Rorc/Rora, STAT3, and other transcription factors (Irf4, Runx1, and Batf) are essential for Il17 expression.2,3,7 However, it is becoming increasingly evident that, in addition to transcriptional regulation, the production of several cytokines is subject to other regulatory mechanisms such as mRNA stability and translation of existing mRNAs.31,32,43 Here we show that regulation of IL-17 production also involves an alternative mechanism independent of gene expression, and that p38 MAPK is a key component of this pathway. Regulation of translation by p38 MAPK through its indirect effect on eIF-4E has been described for specific cytokines, such as TNFα.31,32 Our data suggest that indirect phosphorylation of eIF-4E through MNKs is also the mechanism by which p38 MAPK regulates IL-17 production by Th17 cells. To our knowledge this is the first time that a transcription-independent regulatory mechanism has been implicated in the regulation of IL-17 production.

Previous studies have shown that p38 MAPK controls IFNγ production by Th1 cells in vitro, but its contribution to Th1 differentiation in vivo is less clear.9,44 In the EAE model we failed to observe a reduction in IFNγ, despite the inhibition of IL-17 production. Recent studies have identified IL-1 as a selective and potent enhancer of Th17 activity, because Th17 cells express higher levels of IL-1R compared with Th1 cells.11,44,45 Activation of p38 MAPK by IL-1 in CD4 T cells in vitro has recently been shown to contribute to the effects of IL-1 on Th17 differentiation.11,12 Thus, it is possible that the effect of SB203580 on IL-17 production in EAE is because of an impaired signaling downstream of IL-1R and thus has little consequence on IFNγ production by Th1 cells.

We also show in this study that the activation of p38 MAPK is critical for the development of EAE, because inhibition of the p38 MAPK pathway with selective small-molecule inhibitors can prevent the development of clinical disease when administered prophylactically. Moreover, it can also prevent the progression of clinical signs if treatment is initiated at their onset and, more importantly, during disease remission. Current approved DMT for MS have limited efficacies and in some instances untoward toxicities.46 Importantly, advances in our understanding of the complex immunopathogenesis of MS afford the possibility of more personalized treatment based on targeting multiple pathways.46 Examples include strategies for neuroprotection, neurorepair and remyelination, altering the entry of inflammatory cells into the CNS, induction of regulatory cells, and suppression of pathogenic Th effector functions. Inhibitors of p38 MAPK have been evaluated in several clinical trials for treating inflammatory and autoimmune diseases, including RA.26 However, no studies have evaluated the therapeutic potential of inhibition of the p38 MAPK pathway in MS. According to our data, it is possible that pharmacologic inhibition of the p38 MAPK pathway may be useful as an additional DMT for MS.

In agreement with our results, Lu et al reported that treatment of mice with SB203580 reduced susceptibility to EAE in association with decreased IL-17 production by CD4 T cells.11 However, it is unclear whether this is a direct effect of inhibition of intrinsic p38 MAPK activity in CD4 T cells, or an indirect effect via other cell types. The p38 MAPK pathway is implicated in RA and other autoimmune diseases mostly for its role in regulating macrophage responses.26 However, using genetically manipulated mice, we show here that modulation of the p38 MAPK pathway in T cells alone is sufficient to modify EAE susceptibility and severity. It is possible that activation of p38 MAPK in T cells also contributes to the pathogenesis of other inflammatory T cell–dependent autoimmune diseases, such as autoimmune diabetes, where inhibition of p38 MAPK can ameliorate disease.47,48

Although Th17 cells are a major contributor to EAE pathogenesis, it remains a debate whether other Th17-derived cytokines, in addition to IL-17, contribute to the disease.49,50 It is therefore possible that the protective effect of p38 MAPK inhibition may occur because of alterations in the production of additional effector cytokines/chemokines produced by Th17 cells. Nonetheless, our data demonstrate that the p38 MAPK pathway may be a novel target for the therapeutic intervention and treatment of MS by selectively regulating the encephalitogenic activity of Th17 cells. Future studies will focus on identifying additional pathogenic factors produced by Th17 cells that may be regulated by the p38 MAPK pathway, potentially providing additional T cell–specific druggable targets for MS therapies.

Authorship

Contribution: R.N., V.N., M.R., and C.T. conceived the study; R.N., D.N.K., R.d.R., T.T., V.N., N.S., A.S., and K.S. performed or assisted with experiments; R.N., D.N.K., R.d.R., and C.T. analyzed data; G.S. and R.J.D. contributed novel reagents; and R.N., D.N.K., R.d.R., M.R., and C.T. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Dr Cory Teuscher, Immunobiology Program, C331 Given Medical Bldg, University of Vermont, Burlington, VT 05405; e-mail: c.teuscher{at}uvm.edu; or Dr Mercedes Rincon, Immunobiology Program, C331 Given Medical Bldg, University of Vermont, Burlington, VT 05405; e-mail: mrincon{at}uvm.edu.

Acknowledgments

The authors thank Dr Sean Diehl and other members of the Teuscher laboratory for helpful discussions, the staff at the University of Vermont DNA Sequencing Facility for assistance with quantitative real-time PCR, and Colette Charland for cell sorting.

This work was supported by National Institute of Health grants AI041747, NS036526, and NS060901 (C.T.), and AI054154 (M.R.).

National Institutes of Health

Footnotes

  • The online version of this article contains a data supplement.

  • The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

  • Submitted February 15, 2011.
  • Accepted July 6, 2011.

References

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