Pharmacologic Inhibition of MEK–ERK Signaling Enhances Th17 Differentiation

Interleukin-17 (IL-17)–producing CD4+ T helper (Th17) cells constitute a lineage of T cells that contributes to inflammation, autoimmunity, and host defense against bacteria and fungi (1–3). It is well established that TGF-β synergizes with either IL-6 (4–7) or IL-21 (8–10) to drive Th17 differentiation, with IL-21 needed for the autocrine expansion of Th17 cells. IL-23 is thought to stabilize the Th17 phenotype, and IL-23R expression on Th17 cells is critical for their terminal commitment (11). Transcription factors required to drive Th17 development include retinoic acid-related orphan nuclear receptor (ROR)γt (12, 13), RORα (13), STAT3 (14, 15) and interferon-regulatory factor 4 (16).

Comparatively, much less is known of the cytokines and signaling pathways that inhibit Th17 differentiation. Studies of Th1 and Th2 cells indicate that they inhibit development of the other via the action of their respective lineage-specific cytokines IFN-γ and IL-4 (17). These cytokines also curtail initial Th17 development, although they fail to suppress committed Th17 cells (18, 19). IL-27, first characterized as a Th1-promoting cytokine, was found to suppress Th17 differentiation through STAT1 activation (20, 21) and/or inhibition of RORγt expression. IL-2, a growth factor for activated T and regulatory T cells, antagonizes Th17 differentiation via STAT5 (22), and IL-2 deficiency leads to increased IL-17–producing T cells infiltrating the skin in a mouse model of autoimmunity (23). IL-10 and IL-25 were also reported to inhibit Th17 responses, but the effect of these cytokines is indirect and mediated mainly through dendritic cells and macrophages. For example, IL-10 deficiency in mice led to enhanced IL-12 and IL-23 production by accessory cells and promoted Th17-associated colitis (24) whereas IL-25 deficiency resulted in high levels of IL-23 in the periphery and increased numbers of infiltrating T cells that produced IL-17, TNF-α, and IFN-γ in the CNS (25), and worsened murine experimental autoimmune encephalomyelitis (EAE).

Stimulation of T cells activates three major groups of MAPKs: ERK, JNK, and p38 (26). Of these, the ERK pathway is important for the development and positive selection of thymocytes (27). ERK signaling has been shown to regulate cytokine expression, cell proliferation, survival, and adhesion of activated peripheral T cells. Recent studies examining the role of ERK in modulating Th1/2 balance found that TLR2 agonists induced dendritic cells to produce more IL-10 and less IL-12 through enhanced ERK signaling, and this favored a Th2-biased response (28, 29). Consistent with this, ERK1^−/− mice exhibited increased Th1 polarization and were more susceptible to EAE (30, 31). However, how Th cell-intrinsic ERK signaling influences Th differentiation is relatively unknown. One report suggested that reduced ERK activity in the presence of strong TCR signaling induced early IL-4 production and Th2 generation (32), whereas another demonstrated that ERK signaling promoted Th2 differentiation by stabilizing GATA3 (33). In this study, we examined the role of ERK signaling in Th17 differentiation and provided evidence that ERK signaling negatively regulates Th17 development.

C57BL/6 and Rag^−/− mice were bred in our animal facilities and used between 6 and 8 wk of age, according to Institutional Animal Care and Use Committee regulations. Murine EL4 thymoma cells were cultured as previously reported (34).

CD4+CD25- T cells were isolated from lymph nodes and spleens of mice using CD4+ T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) with biotinylated anti-CD25 (7D4) Ab added. Purity of cells was routinely >95%, as assessed by FACS using anti-CD4 (RM4-5), anti-CD8 (53-6.7) and anti-CD25 (7D4) mAb (BD Pharmingen, San Diego, CA). Cells were plated at 1 × 10⁶/ml in 48-well, flat-bottom plates and stimulated with 1 μg/ml plate-bound anti-TCRβ (H57-597) and 2 μg/ml soluble anti-CD28 (37.51) mAbs (eBioscience, San Diego, CA). To differentiate cells, human TGF-β1 (1 ng/ml), IL-6 (20 ng/ml), or both were added to cultures. Where indicated, chemical inhibitors, U0126 (Cell Signaling Technology, Beverly, MA), SB203580, and SP600125 (Calbiochem, San Diego, CA) were added at 5 μM unless stated otherwise. Cells were collected 72 h later and restimulated with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 0.5 μg/ml ionomycin (Sigma-Aldrich) in the presence of 1 μl GolgiPlug Protein Transport Inhibitor (containing brefeldin A, BD Pharmingen) per milliliter of culture at 37°C for 4–6 h. Cells were then fixed, permeabilized with Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit (BD Pharmingen), and stained with anti–IFN-γ (XMG1.2), anti–IL-17A (eBioTC11-18H10.1), anti–IL-17F (eBio18F10), anti–IL-10 (JES5-16E3), anti–IL-2 (JES6-5H4), or anti–TNF-α (MP6-XT22) mAbs (all from eBioscience). Cells were then visualized and analyzed on a FACSCalibur (BD Biosciences, San Jose, CA) using CellQuest Pro software (BD Biosciences, San Diego, CA).

Total RNA was isolated using TRIzol and treated with DNase I (Invitrogen, Carlsbad, CA) to eliminate genomic DNA. CDNA was prepared using SuperScript III Reverse Transcriptase (Invitrogen) with oligo(dT)_12–18 as primer. The primers used for real-time PCR were: IL-17A, 5′-TCC AGA AGG CCC TCA GAC TA-3′ and 5′-AGC ATC TTC TCG ACC CTG AA-3′; IL-17F, 5′-CAA AAC CAG GGC ATT TCT GT-3′ and 5′-ATG GTG CTG TCT TCC TGA CC-3′; IL-21, 5′-TCA TCA TTG ACC TCG TGG CCC-3′ and 5′-ATC GTA CTT CTC CAC TTG CAA TCC C-3′; IL-22, 5′-TCA TCG GGG AGA AAC TGT TC-3′ and 5′-GCT GAT GTG ACA GGA GCT GA-3′; IL-23R, 5′-AGG CTT TTC GGA ACC TCA T-3′ and 5′-GTA GGC TTC CCA GTG TTC CA-3′; IFN-γ, 5′-GGA TGC ATT CAT GAG TAT TGC-3′ and 5′-CCT TTT CCG CTT CCT GAG G-3′; IL-4, 5′-CGA AGA ACA CCA CAG AGA GTG AGC T-3′ and 5′-GAC TCA TTC ATG GTG CAG CTT ATC G-3′; T-bet, 5′-CAA CAA CCC CTT TGC CAA AG-3′ and 5′-TCC CCC AAG CAG TTG ACA GT-3′; GATA-3, 5′-GAA GGC ATC CAG ACC CGA AAC-3′ and 5′-ACC CAT GGC GGT GAC CAT GC-3′; RORγt, 5′-TAC CTT GGC CAA AAC AGA GG-3′ and 5′-ATG CCT GGT TTC CTC AAA A-3′; β-actin, 5′-GAT CTG GCA CCA CAC CTT CT-3′ and 5′-ACC AGA GGC ATA CAG GGA CA-3′. Each reaction (50°C, 2 min; 95°C, 10 s; 60°C, 30 s; 72°C, 30 s, 40 cycles) was performed using SYBR Green PCR Master Mix in an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). All experiments were performed at least twice, and mRNA levels of gene transcripts were normalized to those of β-actin.

Purified CD4+CD25- T cells (1 × 10⁶) activated with anti-TCRβ and anti-CD28 mAbs were incubated with TGF-β1, IL-6, or both for 30 min, 3 h, or 24 h. U0126, anti–IL-2 mAb (10 ng/ml; S4B6, BD Pharmingen) or hIL-2 (100 U; PeproTech, Rocky Hill, NJ) was also added to parallel cultures. Cells were fixed for 10 min at 37°C with 2% (w/v) paraformaldehyde and permeabilized for 30 min on ice with 90% (v/v) methanol and stained with Abs to phosphorylated STAT3 (pY705; 4/P-STAT3), STAT5 (pY694; 47), p38 MAPK (pT180/pY182; 36/p38), JNK (pT183/pY185; polyclonal), or ERK1/2 (pT202/pY204; 20a).

EL4 cells were plated, transfected with empty or caMEK2 expressing vector, stimulated with PMA and ionomycin, and harvested 16 h later for RNA extraction as described (34). Real-time RT-PCR analyses of il-17a, il-17f, and il-22 transcripts were then performed as described above.

Rag^−/− mice were injected i.p. with PBS alone or with 5 × 10⁵ syngeneic Th17 cells differentiated in the presence or not of U0126. Mice were observed for signs of colitis and weighed weekly. Any mice showing clinical symptoms of severe disease and became moribund were sacrificed following Institutional Animal Care and Use Committee regulations. The weight losses of such mice were not included in the generation of curves depicted in Fig. 6A. Colons and mesenteric lymph nodes were removed from mice 4–8 or 12–16 wk after cell transfer. Colons were fixed in 10% neutral-buffered formalin, and 6-μm paraffin-embedded sections were cut and stained with H&E. Th cells were prepared from mesenteric lymph nodes to assess transcript and cytokine expression.

Differences in numerical values between samples were compared by Student t test using GraphPad Prism 5 (GraphPad, San Diego, CA). Values of p ≤ 0.05 were regarded as statistically significant.

To determine whether and which MAPKs play a role in Th17 differentiation, we assessed by flow cytometry the level of phosphorylation and activation of ERK, JNK, and p38 in in vitro differentiated Th17 cells. We found that although TGF-β or IL-6 alone attenuated ERK phosphorylation in Th cells compared with unpolarized (no cytokine) cells, the combination of TGF-β and IL-6 produced a synergistic suppressive effect on phospho-ERK levels in Th cells (Fig. 1). In contrast, down-modulation of p38 and JNK phosphorylation was less drastic, and the effect was primarily mediated by TGF-β. Thus, our data suggest that Th17-differentiated Th cells have greatly reduced phosphorylation and activation of ERK.

Because Th17 cells have reduced ERK phosphorylation compared with control cells (Fig. 1), we hypothesized that ERK activity might have to be modulated for optimal Th17 differentiation. To test this, we differentiated Th17 cells in the presence or absence of inhibitors of ERK (U0126), JNK (SP600125), and p38 (SB203580) and assayed their expression of IL-17A, IFN-γ, and IL-10 by intracellular cytokine staining (Fig. 2A). Interestingly, we found a greater fraction of IL-17A–expressing cells in the ERK-inhibited culture (45%) compared with the untreated (18%), JNK-inhibited (14%), or p38-inhibited (13%) cultures. Alternatively, the fractions of IFN-γ–expressing cells were largely comparable across various treatments, whereas there were reductions in IL-10–expressing cells in JNK- or p38-inhibited cultures. In contrast, the fraction of Foxp3-expressing cells was slightly reduced in the ERK-inhibited culture (14%) compared with the untreated (17%), JNK-inhibited (23%), or p38-inhibited (23%) cultures. Consistent with previous data (35), activated Th cells cultured in the absence of cytokines upregulated Foxp3 expression in the presence of ERK inhibitor (3.9% versus 0.8%).

Although the generation of IL-17A–expressing cells was enhanced when ERK signaling was curtailed in the Th17-polarized cultures, ERK-inhibited Th cells per se were not predisposed to become IL-17A–expressing cells. When differentiated in the absence of cytokines, Th cells treated or not with U0126 or other MAPK inhibitors did not express high levels of IL-17A. Interestingly, the expression of effector cytokines such as IL-17A, IL-17F, and TNF-α was greatly enhanced with increasing doses of U0126 treatment of Th17 cells (Fig. 2B), suggesting that the strength of ERK signaling quantitatively influences the Th17 effector phenotype.

The inhibition of MAPKs, particularly ERK, in cells differentiated under Th1-polarizing conditions did not appear to perturb the percentage of IFN-γ-expressing cells to the same extent as IL-17A–expressing cells (Fig. 2C). This finding suggests that the modulation of Th17 differentiation by ERK signaling is likely to be lineage-specific. Collectively, our data indicate that dampening ERK signaling increases Th17 differentiation.

Other than increased IL-17A–expressing cells in the ERK-inhibited Th17 differentiation culture, we also found that ERK-inhibited Th17 cells expressed abnormally higher levels of il-17a and il-17f transcripts compared with cells that were untreated or treated with inhibitors against JNK and p38 (Fig. 3A). Moreover, we observed significant overexpression of il-21, il-22, and il-23r transcripts in ERK-inhibited Th17 cells. Contrarily, there was no obvious difference in the levels of rorγt and foxp3 between untreated and ERK-inhibited Th17 cells. Moreover, the transcript levels of Th1-associated ifn-γ and t-bet were comparable between untreated and ERK-inhibited Th17 cells, although levels of Th2-associated il-4 and gata-3 were reduced in ERK-inhibited cells compared with untreated cells. In complementary experiments, we ectopically expressed a constitutively active form of MEK2 (caMEK2), which is upstream of ERK signaling, in EL4 cells, a cell line that upregulated IL-17A expression upon stimulation with PMA and ionomycin overnight (data not shown). Overexpression of caMEK2 in EL4 cells dampened PMA and ionomycin-mediated induction of il-17a, il-17f, and il-22 transcript levels in a dose-dependent manner (Fig. 3B). Hence, ERK signaling affects Th17-associated cytokine expression at the transcriptional level.

Because IL-2 had been reported to limit Th17 differentiation (22), we asked whether ERK signaling mediates the inhibitory effect of this cytokine. To address this, we differentiated Th17 cells in the presence of anti–IL-2 Ab, recombinant human IL-2 (hIL-2), ERK-inhibitor U0126, or combination of hIL-2 and U0126. Consistent with previous data (22), the addition of anti–IL-2 Ab to Th cells cultured under Th17-polarizing conditions enhanced the generation of Th17 cells (increase of 17.8–36.6% IL-17A–expressing cells; Fig. 4A), whereas the addition of exogenous IL-2 to the culture suppressed the differentiation of these cells (decrease of 17.8–8.2% IL-17A–expressing cells). Interestingly, when hIL-2 and U0126 were concurrently added to the culture, the inhibitory effect of IL-2 on Th17 differentiation was largely abolished (modest decrease of 34.5–32.0% IL-17A–expressing cells), suggesting that ERK signaling plays an important role in the inhibitory effect of IL-2 in Th17 differentiation. To further ascertain this finding, we examined the level of phospho-ERK in Th cells differentiated with TGF-β and IL-6 in the presence of hIL-2 or anti–IL-2 Ab. We found that under Th17-polarizing conditions, ERK phosphorylation was further diminished in Th cells treated with anti–IL-2 Ab for 30 min or 3 h compared with untreated cells (Fig. 4B). Conversely, the addition of hIL-2 to Th cells augmented the phosphorylation of ERK at the same time points examined. Thus, IL-2 likely induces ERK signaling to inhibit Th17 differentiation.

Because ERK signaling is important for il-2 transcription in activated T cells (36, 37) and our data indicated that IL-2 activates ERK signaling, there appears to be an ERK–IL-2 autoregulatory loop that constrains Th17 differentiation. Consistent with this, we noted that ERK but not JNK or p38 inhibition led to profound reduction in IL-2 expression in cells differentiated for 24 h under Th17-polarizing conditions (Fig. 4C), compared with cells differentiated in the absence of cytokines (data not shown). Because IL-2–IL-2R signaling is known to activate JAK3-STAT5 axis, we also observed changes in STAT5 phosphorylation that were similar to those of ERK phosphorylation, at 30 min and 3 h of culture (Fig. 4B). In contrast, STAT3 phosphorylation was comparable between Th cells cultured under different treatment conditions, suggesting that IL-2 represses Th17 differentiation independently of STAT3. These data suggest the existence of an autoregulatory circuit involving IL-2 and ERK signaling that negatively regulates Th17 differentiation.

Because IL-2 has been reported to limit Th17 differentiation via STAT5 (22), we examined whether STAT5 has a role in mediating the inhibitory effect of ERK signaling on Th17 differentiation. We cultured Th cells under Th17-polarizing conditions in the presence of U0126 and assessed STAT5 phosphorylation at 30 min, 3h, and 24 h. Although there was no detectable difference in p-STAT5 levels between ERK-inhibited and noninhibited cells after 30 min of culture, we found appreciable attenuation of STAT5 phosphorylation at 3 h, which was mostly reversed at 24 h (Fig. 5). In contrast, there were no obvious changes in the levels of STAT3 phosphorylation at all time points between treated and nontreated cells. Hence, our results indicate that ERK inhibition attenuates STAT5 activity during the early stage of Th17 differentiation and suggest that ERK signaling restricts Th17 differentiation in part through inhibition of STAT5 activity.

Because ERK-inhibited Th17 cells have enhanced expression of various cytokine transcripts (Fig. 3), we next examined the significance of this finding in vivo using a T cell transfer model of colitis. We transferred 5 × 10⁵ Th17 cells differentiated in the presence or absence of U0126 as well as Th cells activated in the absence of cytokines (no cytokine) or without cytokines, but with U0126 (no cytokine + U0126) as controls into Rag^−/− mice, and assessed their development of colitis by monitoring weight loss as a percentage of initial weight. Whereas unpolarized Th cells, whether treated with U0126 or not, elicited colitic disease with similar kinetics and severity in mice receiving these cells, ERK-inhibited Th17 cells (TGF-β + IL-6 + U0126) induced more rapid and severe colitis than did their untreated counterparts (TGF-β + IL-6). This finding is evident from the early and drastic decrease in the weight of mice receiving the former compared with the latter cells (Fig. 6A, phase i). The severe weight loss also correlated with the exacerbated pathologic findings seen in the distal colons of these mice, which showed massive leukocyte infiltration and goblet cell disruption (Fig. 6B, phase i). In comparison, the pathologic findings seen in the distal colons of mice that received the untreated cells were considerably milder, as they exhibited only early signs of disease at this stage. Because Rag^−/− mice typically develop fulminant disease only after 8–12 wk (50–80 d) postinjection of cells in this model, our data suggest that the pronounced levels of effector cytokines, such as IL-17A, IL-17F, and TNF-α induced by ERK inhibition in Th17 cells (Fig. 2B), likely contributed to the accelerated progression and worsening of disease. Consistent with this, we found that Th cells isolated ex vivo from the mesenteric lymph nodes of mice initially injected with ERK-inhibited Th17 cells expressed a greater fraction (2.6%) of IL-17A–expressing cells compared with those (1.0%) from mice that received untreated Th17 cells (Fig. 6C, i). Strikingly, this severe colitic phase was transient, as the majority of mice that received the ERK-inhibited Th17 cells recovered with time and gradually gained weight (Fig. 6A, ii). Disease resolution was also evident from the restoration of normal colonic histology in these mice (Fig. 6B, ii). In contrast, mice receiving untreated Th17 cells developed colitis much later and manifested pathologic signs during the time when most mice that initially received the ERK-inhibited cells had become free of disease. This finding correlated with a smaller fraction (3.2%) of IFN-γ–expressing Th1 cells from mice injected with ERK-inhibited cells compared with (10.3%) mice injected with untreated cells (Fig. 6C, ii). Moreover, although IL-22 expression was comparable between ERK-inhibited and untreated cells during phase i, the former cells expressed IL-22 more highly during phase ii when mice receiving such cells recovered from disease (Fig. 6D, ii), which is consistent with a protective role of IL-22 in intestinal inflammation (38, 39). Thus, modulation of ERK signaling in Th17 cells appeared to influence the ratio of IL-17A–expressing Th17 and IFN-γ–expressing Th1 cells and thus has a significant effect on their effector function in vivo.

We showed in this study that ERK signaling inhibits Th17 generation and affects effector function in vivo. We further provided insight into how IL-2 and ERK likely cooperate in an autoregulatory loop to constrain Th17 differentiation. In addition, our data suggest that the suppressive effect of ERK on Th17 differentiation occurred in part via STAT5 but not STAT3 activation.

Because ERK signaling activates AP-1 and the Ets family of transcription factors, it is possible that some of these factors mediated the Th17-inhibitory effects of ERK. Indeed, Ets-1^−/− Th cells, when differentiated under Th17-polarizing conditions, expressed higher levels of IL-17 compared with wild-type cells (40). Similar to what was observed with Ets-1^−/− cells, inhibition of ERK in developing Th17 cells also rendered them resistant to IL-2 suppression that was not due to defective RORγt induction. However, unlike Ets-1 deficiency, which preserved STAT5 activation, ERK inhibition dampened STAT5 phosphorylation (Fig. 5). This could be caused by an indirect effect of ERK inhibition on IL-2 production, impairment of which may lead to defective STAT5 phosphorylation. Intriguingly, Th17 polarizing conditions appeared to sensitize IL-2 expression in Th cells selectively to control by ERK signaling (Fig. 4C), because IL-2 expression in Th cells activated under nonpolarizing conditions is activated strongly by JNK and ERK and weakly by p38 (41 and data not shown).

The Th17-produced cytokine IL-22 is known to exacerbate pathology in some disease contexts, such as in a mouse model of psoriasis-like skin inflammation (42) and IL-23-induced dermal inflammation and acanthosis (43). One recent study showed that IL-22 could protect against the aberrant immune response accompanying inflammatory bowel disease (38), whereas another demonstrated that IL-22 could ameliorate established colitis (39). Based on these data, we speculate that, despite the increased colitogenic potential of Th17 cells differentiated in the absence of ERK signaling, elevated IL-22 expression following in vitro culture (Fig. 3B) and ex vivo when isolated from lymph nodes draining the colons of Rag^−/− mice previously injected with these cells (Fig. 6D) may have contributed to the overt disappearance of disease at a later time in mice that received such cells, compared with those that received cells with intact ERK signaling. It is also well established that the immunosuppressive cytokine IL-10 has a protective function in T cell-mediated colitis, because IL-10-deficient mice developed spontaneous colitis (44) and IL-10 is essential to control intestinal inflammation (45). In addition, Foxp3+ regulatory T cells have been shown to cure colitis (46). However, we did not observe any significant difference in the fraction of IL-10–expressing Th cells in the presence or absence of the ERK inhibitor under Th17 polarizing conditions, although there was a modest decrease in Foxp3 expression when ERK was inhibited (Figs. 2A, 3A). We observed that acute but transient disease in mice that received ERK-inhibited Th17 cells was associated with a greater fraction of IL-17A–expressing Th cells isolated ex vivo from these mice compared with cells from mice that received untreated Th17 cells (Fig. 6C, i), but later recovery from disease was not correlated with a reciprocally lower fraction of IL-17A-expressing cells (Fig. 6C, ii). Because previous studies have determined an important role for IFN-γ–expressing Th1 cells in disease pathogenesis in this Th cell-transfer model of colitis (47), disappearance of disease in mice that received ERK-inhibited Th17 cells could be caused by a lower fraction of IFN-γ–expressing Th cells isolated from such mice during phase ii (Fig. 6C, ii). More in-depth studies are required to ascertain how ERK-inhibited Th17 cells elicit a disease pattern divergent from that evoked by untreated Th17 cells.

Our present studies do not permit a clear functional demarcation between the roles of ERK1 and ERK2 in regulating Th17 differentiation. Further studies using Th cells singly or multiply deficient in various ERK isoforms should help to illuminate the cell-intrinsic contribution of ERK in shaping Th17 differentiation. Although ERK1^−/− mice manifested increased Th1 polarization and were more susceptible to EAE, the induction of which is currently regarded to be Th17-dependent, such a phenotype was a result of ERK1 deficiency in dendritic cells that skewed the ensuing Th response (30, 31). Our study provides, for the first time, evidence of a cell-intrinsic and differential role for ERK vis-à-vis JNK and p38 MAPK in modulating Th17 differentiation.

We thank staff of the animal facilities for care and maintenance of mice, the Histology Unit for preparation and staining of mouse colons, and members of the laboratory for helpful discussions.

Disclosures The authors have no financial conflict of interest.