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Am J Physiol Gastrointest Liver Physiol 294: G452-G459, 2008. First published November 21, 2007; doi:10.1152/ajpgi.00077.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages

Robert M. Rowlett,1 Carol A. Chrestensen,2 Mark Nyce,1 Mary G. Harp,1 Jared W. Pelo,1 Fabio Cominelli,1 Peter B. Ernst,1 Theresa T. Pizarro,1 Thomas W. Sturgill,2 and Mark T. Worthington3

1Department of Medicine and 2Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia; and 3Division of Digestive Diseases, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 13 February 2007 ; accepted in final form 19 November 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.

MNK kinases; p38 MAP kinase; ERK; macrophages; cytokines; Crohn's disease; Toll-like receptor

TUMOR NECROSIS FACTOR (TNF) plays a pivotal role in the regulation of normal immunity (3) as well as pathogenic inflammatory responses seen in diseases such as rheumatoid arthritis and Crohn's disease, in which anti-TNF strategies have revolutionized the treatment of patients suffering from these chronic inflammatory disorders (37). It is well known that the synthesis of biologically active TNF is regulated as several different levels and that MAP kinase pathways are critical for its production as well as that of several other proinflammatory cytokines (38). As such, interfering with MAP kinase pathways has a significant impact on downstream TNF production and its subsequent biological effects.

The MAP kinase-interacting serine/threonine kinases (MNKs) are kinases located downstream of both the p38 and ERK pathways. The two family members, MNK1 and MNK2, are substrates for activating phosphorylation by both ERK and p38 (46) and are induced by either pathway (11). MNKs are the only kinases known to phosphorylate Ser-209 of the cap-binding eIF4E protein, which acts to increase translation (16, 27, 41, 44). Both MNK1 and MNK2 are autoinhibited by the COOH terminus in the absence of p38 and p42/ERK phosphorylation (18) but are not required for normal development or viability when both mouse genes are inactivated (45). A recent report regarding MNK kinases in the regulation of TNF in response to Escherichia coli lipopolysaccharide (LPS) suggested that this family regulates the innate immune response in macrophages (2). Whether an anti-inflammatory effect of MNK inhibition is a feature unique to LPS or involves cytokines other than TNF is currently unknown. It is also unknown whether MNK inhibition is a viable strategy for the treatment of inflammatory diseases. If so, the lack of a phenotype in an unstressed MNK1/2 dual-knockout mouse suggests that these kinases could be excellent targets for anti-inflammatory drug development (45).

A well-studied agonist of TNF production in macrophages is E. coli LPS (4), which signals through the p38, JNK/SAPK, and ERK pathways to produce TNF (38). In the present study, we used RAW264.7 macrophage-like cells as well as bone marrow-derived macrophages (BMDM) from SAMP1/YitFc (SAMP) mice, a model of spontaneous Crohn's disease in which TNF is known to play an important pathogenic role (23, 29), to determine the role of MNK kinases in regulating macrophage effector cell function.


   METHODS
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 METHODS
RESULTS
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Reagents. CGP57380 was a kind gift from Hermann Gram (Arthritis and Bone Metabolism, Novartis, Basel, Switzerland) and the anti-tristetraprolin (TTP) antibody was a kind gift from Jiahuai Han (Scripps Institute, La Jolla, CA). In experiments in which CGP53870 was used, cell-culture grade DMSO in sealed ampoules (Sigma-Aldrich, no. 2650) was used both to resuspend the drug and in equal amounts without the drug in negative controls. The anti-actin antibody was obtained from ICN; the anti-eIF4E and anti-phospho-eIF4E antibodies were obtained from Cell Signaling. Anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were obtained from Upstate Bioscience and Novagen, respectively. RAW264.7 cells were obtained from the American Type Culture Collection and grown in RPMI 1640 with 10% heat-inactivated FBS. Phenol-extracted and ion exchange-purified E. coli 055:B5 LPS (no. L4524) and other laboratory chemicals were obtained from Sigma-Aldrich. Endotoxin and RNase-free sterile water was used for all solutions. Endotoxin testing was performed using the Pyrotell Limulus amebocyte lysate assay (Associates of Cape Cod).

Western blotting. RAW264.7 cells were split into 12-well plates at 1.0 x 106 cells/well and allowed to attach overnight. Protein gels, transfer blotting, and development were performed as previously described using the ECL Plus Kit (Amersham) (8), except that Ponceau S staining was used to verify the uniformity of transfers. Secondary antibodies were typically used at a 1:5,000 dilution. E. coli LPS was used at 100 ng/ml for stimulation and at 10 ng/ml for overnight incubation designed to induce tolerance. Except for a single image developed by using the STORM scanner (Amersham; fig. 4A), all Western blots were imaged with X-ray film.


Figure 4
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  		Fig. 4. Effect of MNK inhibition on tristetraprolin (TTP) expression. A: RAW264.7 cells were plated overnight, exposed to 100 ng/ml E. coli LPS for 4 h the next morning, then harvested for TTP (top) and actin (bottom) Western blots performed in response to an increasing amount of CGP57380. These images were scanned on a STORM Imager (Amersham). B: the same experiment was performed with the MNK inhibitor added at 50 mM concentration either before (–30 min, –15 min), with (0 min), or after (+15 min, +30 min) LPS stimulation. C: the effect of CGP57380 on TTP production in response to E. coli LPS is compared with the TLR9 agonist ODN1826, individually or together, by Western blot. A nucleotide with the same base composition as ODN1826 but a scrambled sequence (Cont NT) that does not stimulate TNF production is used as a TLR9 control. Results are representative of at least 2 experiments.

 
Time course of TNF mRNA production. RAW264.7 cells were split into 12-well plates at 1.0 x 106 cells/well and allowed to attach overnight. At 30 min prior to stimulation with LPS, the cells were pretreated with CGP57380 to 50 µM vs. DMSO control wells, and then LPS was added to 100 ng/ml. For the actinomycin D experiments, the cells were similarly stimulated with LPS for 1 h, except that actinomycin D was added to a 5 µM final concentration with either CGP57380 or DMSO control.

RNA methods and real-time PCR. Total cellular RNA was purified by using the Qiagen RNeasy kit and quantified spectrophotometrically, and then 2 µg total RNA were used for reverse transcription using the Invitrogen SuperScript First Strand Synthesis cDNA kit with the accompanying random primers according to the manufacturer's protocol. Real-time PCR was performed on an ABI Prism SDS 7000 in paired reactions using mouse TNF Taqman primers (Mm00443258_m1, Applied Biosystems) with 12.5 µl of Taqman Universal Mix, 1.25 µl of 20x Taqman primers, 10.25 µl of sterile water, and 1 µl of cDNA. 18S ribosomal RNA was quantified by using the Taqman primer probe set (no. 431-893E, Applied Biosystems), and the amount of TNF gene expression was normalized to 18S by the {Delta}{Delta}Ct (Ct, cycle threshold) method (1). Thermocycling conditions were: 50°C x 2 min, 95°C x 10 min, then 40 cycles of 95°C x 15 s, 60°C x 1 min. Controls without reverse transcriptase and without template RNA were included.

Animal methods. All mice were housed under specific pathogen-free conditions at the University of Virginia in autoclaved microisolator cages under positive pressure HEPA-filtered air and were given sterile food and water ad libitum. All mice were used and cared for in accordance with protocols approved by the Institutional Animal Care and Use Committee and Association for Assessment of Laboratory Animal Care guidelines.

BMDM were derived from the SAMP1/YitFc (SAMP) mouse strain, which was propagated from brother-sister breeding (over 20 generations) at the University of Virginia. Initial breeding mice (SAMP1/Yit) were generously provided by S. Matsumoto (Yakult Central Institute for Microbiological Research, Tokyo, Japan) and were originally derived from the AKR/J strain (Jackson Laboratories, Bar Harbor, ME). Spontaneous ileitis develops in these animals without chemical, genetic, or immunological manipulation, reaching virtually 100% penetrance by 10 wk of age. Their phenotype shows a striking resemblance to human Crohn's disease, with segmental transmural inflammation, mucosal and submucosal granulomas, altered epithelial cell morphology, and muscular and neural hypertrophy, all localized to the terminal ileum (23, 36).

Femurs and tibiae from SAMP mice with established ileitis (10 wk of age; n = 4) were isolated and cleaned, and the bones were left in 70% ethanol for 2–5 min for disinfection and washed in PBS. BMDM were produced for in vitro cell culture experiments using a standard method (7) by irrigation with PBS of bone marrow using a 0.45 µM diameter needle, vigorous pipetting to disintegrate clustered cells, and subsequent steps of serial washing and centrifugation in cold PBS. Bone marrow cells were then cultured at a density of 2 x 106 cells/ml in 100 mm petri dishes in cell culture medium consisting of RPMI-1640 (high glucose) supplemented with 1x penicillin/streptomycin, glutamine (2 mM), 2β-mercaptoethanol (50 µM) (all from Sigma-Aldrich), 200 U/ml (20 ng/ml) rmGM-CSF (Peprotech), and 10% heat-inactivated FBS (Invitrogen) at 37°C in a 5% CO2 atmosphere. On day 2, cells were washed 2x with PBS to remove nonadherent cells, and 10 ml of fresh medium were added. On days 6 and 8, half of the culture supernatant was collected and centrifuged, and the cell pellet was resuspended in 5 ml of fresh medium containing 200 U/ml rmGM-CSF and returned to the original plate. On day 10, cells were washed 2x in PBS, detached by use of 0.02% EDTA in PBS, and collected by centrifugation at 300 g for 5 min at room temperature. Resulting BMDM were split into 12-well plates, seeded at 1.0 x 106 cells/well, and allowed to attach overnight. At 30 min prior to the addition of LPS (100 ng/ml), BMDM were pretreated with either 50 µM CGP57380 or vehicle, and supernatants were collected at 0, 4, and 24 h following stimulation.

Immunological methods. The mouse Quantikine TNF ELISA kits were obtained from R&D systems and performed by using the accompanying protocol; 50,000 cells per well in 100 µl of media were used for ELISA assays in 96-well microtiter plates, including experiments performed using the Invivogen Toll-like receptor (TLR) agonist kit (San Diego). Pam3CSK4 was used at 1 µg/ml, heat-killed Listeria monocytogenes (HKLM) at 108 cells/ml, poly(I:C) at 25 µg/ml, Salmonella LPS at 1 µg/ml, flagellin at 1 µg/ml, FSL1 at 1 µg/ml, imiquimod at 10 µg/ml, ssRNA40 at 10 µg/ml, and ODN2006 at 5 µM. The Cytometric Bead Array was used to measure cytokine protein production in supernatants obtained from LPS-stimulated RAW264.7 cells and BMDM from SAMP mice and was performed per the manufacturer's protocol (BD Biosciences). The previously validated (24) ODN1826 (TLR9 agonist, TCC ATG ACG TTC CTG ACG TT) and ODN1982 (inactive, tcc agg act tct ctc agg tt) were synthesized by Integrated DNA Technologies as RNAse-free phosphothiorate oligonucleotides.


   RESULTS
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 ABSTRACT
 METHODS
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Macrophages are major effectors of innate immunity, stimulated by a broad variety of bacterial products through specific TLRs on the cell surface to produce proinflammatory cytokines, such as TNF. E. coli LPS is a potent stimulus to macrophage gene expression, especially TNF, by engaging the TLR4 membrane signaling complex (15). Andersson and Sundler (2) reported that 4 h of MNK inhibition with the compound CGP57380 reduced mouse macrophage TNF production, but whether this compound leads to sustained effects to inhibit MNK kinases is not known. We used the macrophage RAW264.7 cell line to pursue these questions. In macrophages, prolonged treatment with the MNK inhibitor CGP57380 abolishes RAW264.7 cell eIF4E phosphorylation after LPS stimulation, without changing total eIF4E levels (Fig. 1). Tolerizing doses of LPS (10 ng/ml overnight, followed by 100 ng/ml stimulation), also led to mild decreases of phospho-eIF4E at early time points (not shown).


Figure 1
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  		Fig. 1. The MAP kinase-interacting serine/threonine kinase (MNK) kinase inhibitor blocks RAW264.7 eIF4E phosphorylation in extended culture. RAW264.7 cells were plated overnight in a 12-well tissue culture plate, then incubated in parallel tissue culture wells with either 50 µM CGP57380 (CGP, left) or an equivalent amount of control DMSO vehicle (DMSO, right). The cells were lysed at the times in hours across the top of the figure, and parallel Western blots were performed with the antibodies specific for serine 209 phosphorylated eIF4E (top), total eIF4E (middle), and actin as a loading control (bottom). Results are representative of at least 3 experiments.

 
Whether TLR bacterial pattern recognition pathways other than for LPS signal through the MNK kinases to promote TNF production is not known. Our first approach to resolve whether active MNK is required to produce TNF in RAW264.7 cells was to use E. coli LPS (TLR4 pathway) and a previously validated CpG DNA agonist, ODN1826 (24) (TLR9 pathway) vs. a scrambled nucleotide control. In both cases, CGP57380 inhibited TNF production individually as well as when both agonists were combined (Fig. 2A). To determine whether other TLR signaling pathways require MNK expression, we used a commercial TLR agonist panel on these cells to determine whether other TLR receptors would be inhibited by CGP57380 and to further establish whether the effect was dose responsive. TNF was increased in response to Salmonella LPS (TLR4), ODN2006 (TLR 9), HKLM (TLR2), FSL (TLR 6/2), and imiquimod (TLR7). In each case, the TNF production measured by ELISA was inhibited by CGP57380 in a dose-responsive fashion from 0 to 50 µmol/l (Fig. 2A). RAW264.7 cells do not express TLR5 (32); not surprisingly, we did not see TNF production in response to flagellin (TLR5, Fig. 2B), nor was there TNF production by PAM3CSK4 (TLR1/2), poly(I:C) (TLR3), and ssRNA40 (TLR8), which were also included in the kit (not shown).


Figure 2
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  		Fig. 2. TNF production by multiple Toll-like receptor (TLR) agonists requires active MNK kinases. The regulation of TNF production by MNK kinases in RAW264.7 cells was studied by using various TLR receptor agonists in 96-well plates, assayed by TNF ELISA of the media after 4-h incubations. CGP57380 (CGP) was added 30 min before stimulation with TLR agonists. A: effect of Escherichia coli LPS (TLR4) was compared with the CpG nucleotide ODN1826 (TLR9) alone and in combination, vs. a scrambled nucleotide control (Control NT) with and without CGP57380. Duplicate paired wells were assayed by TNF ELISA. Equivalent amounts of DMSO were added to the wells without CGP57380. B: whether other TLR agonists require active MNK kinases was similarly assayed by use of a commercial TLR agonist kit. Salmonella LPS (TLR4, bullet), HKLM (TLR2, {blacksquare}), imiquimod (TLR7, {circ}), ODN2006 (TLR9, {square}), FSL (TLR6/2, {blacklozenge}), and flagellin (TLR5, {lozenge} are shown) at 0, 25, and 50 µM CGP57380. The DMSO concentration was kept constant in each well. Duplicate paired wells were assayed by TNF ELISA. Not shown are PAM3CSK4 (TLR1/2), poly(I:C) (TLR3), and ssRNA40 (TLR8), which, like flagellin, did not result in significant TNF production in these cells. Results are representative of at least 2 experiments.

 
The MNK kinases are required for phosphorylation of hnRNPA1 (12). This phosphorylation allows hnRNPA1 to enter the stress granule (SG), cytoplasmic particles where unstable mRNAs like TNF are kept undegraded in response to harsh physiological conditions (i.e., arsenite exposure), but not translated. hnRNPA1 has been shown to regulate T cell-derived TNF expression via MNK phosphorylation (6). To explore the mechanism of the MNK effect, RAW264.7 cells were treated with CGP57380 or the DMSO control for 30 min before LPS stimulation, and RNA samples were collected from 0 to 4 h thereafter. The TNF mRNA was quantified by Taqman real-time RT-PCR and normalized to 18S ribosomal RNA. There was a striking inhibition of TNF mRNA production in response to LPS in the CGP57380-treated macrophages compared with the DMSO controls (Fig. 3A). To determine whether changes in mRNA stability were also affected by MNK inhibition, we stimulated these cells with E. coli LPS for 1 h to produce a pulse of TNF mRNA and then added the transcription inhibitor actinomycin D simultaneously with either the MNK inhibitor or the DMSO control. Early time points were used to avoid apoptotic responses seen with prolonged actinomycin D stimulation (13, 21). The real-time RT-PCR results (Fig. 3B) of this treatment show that, even though TNF mRNA is generally unstable, there is a subset of the mRNA (~10%) that remains undegraded after actinomycin D treatment in the mock-treated cells. The TNF mRNA in CGP57380-inhibited cells continues to be degraded at subsequent time points, suggesting that the MNK pathway assists in preventing a component of macrophage TNF mRNA decay. Thus, similar to the role of MNK in T-cell SGs, inhibition of MNK in macrophages allows uninhibited mRNA degradation.


Figure 3
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  		Fig. 3. MNK inhibition affects macrophage TNF mRNA kinetics. A: RAW264.7 cells were incubated overnight in 6-well dishes, with 50 µM CGP57380 or DMSO vehicle added to the media 30 min before stimulation with 100 ng/ml E. coli LPS. Duplicate RNA samples from each dish were reverse transcribed for TNF Taqman real-time PCR, normalized to simultaneous 18S VIC/TAMRA detection by the {Delta}{Delta}Ct (Ct, cycle threshold) method. B: parallel wells of RAW264.7 cells stimulated for 1 h with 100 ng/ml E. coli LPS; then, to arrest transcription, 5 µM actinomycin D was added with either 50 µM CGP57380 or DMSO control. Duplicate RNA samples from each well were purified and analyzed for TNF mRNA by 18S-normalized real-time PCR as noted in A. Error bars represent standard error. In both panels, where error bars are not seen, they are smaller than the graphic. {blacksquare}, CGP57380-treated cells; bullet, mock DMSO controls. Results are representative of at least 3 experiments.

 
Because of the enhanced degradation of TNF mRNA in response to CGP57380 treatment, we were curious whether MNK inhibition increased the abundance of the TTP protein, which both promotes degradation of TNF mRNA by binding to AU-rich elements in the TNF 3'-untranslated region and is a component of SGs (20, 47). The phenotype of the TTP knockout mouse is one of TNF overexpression, with resultant arthritis, dermatitis, glomerulonephritis, and ds-DNA antibodies (40). TTP can cause both 3'-exonuclease- and micro-RNA-based degradation (19, 25), and increased TTP induced by MNK inhibition would suggest a mechanism for the reduced TNF mRNA production. In T cells, MNK inhibition regulates TNF expression via the 3''-untranslated region of the TNF mRNA (6) and TTP is active in the T-cell regulation of TNF (34). A prior report suggested no effect of CGP57380 on TTP production (5), but those experiments used concentrations that we predict were too low (0.1, 1, and 10 µM) to show significant inhibition of TNF production by interpolation of our graph using TLR agonists (Fig. 2B) and at an earlier time point (2 h after LPS). Since TTP expression has been generally concordant with TNF, the prior conditions may not be relevant for TTP regulation by MNKs. RAW264.7 cells were stimulated for 4 h with 100 ng/ml E. coli LPS in the presence of 0, 25, and 50 µmol/l CGP57380, with the results shown in Fig. 4A. Rather than being upregulated by CGP57380, TTP protein production was suppressed by MNK inhibition. Thus TTP expression is regulated by the MNK kinases in a coordinate fashion with TNF, one of its major targets. This effect can be seen after the inflammatory response has started; the suppression of TTP protein can be seen 30 min after LPS stimulation (Fig. 4B). As shown in Fig. 4C, TTP is also stimulated by the CpG nucleotide agonist ODN1826, but not a scrambled control nucleotide (ODN1982, "Cont NT"). The induction of TTP by either route (TLR4 vs. TLR9) is blocked by CGP57380.

LPS stimulation of macrophages releases a program of proinflammatory cytokines, including IL-6, IL-10, IL-12, and TNF. To understand how the MNK kinases affect the innate immune response, we stimulated RAW264.7 cells with LPS for 4 h and performed a mouse multiplexed bead immunoassay (Cytometric Bead Array) that measures the proinflammatory cytokines IL12p70, monocyte chemoattractant protein (MCP)-1, TNF, IL-6, and IFN-{gamma} and the anti-inflammatory cytokine IL-10, comparing mock and CGP57380-treated cells. The results, shown in Fig. 5, reveal a striking suppression by MNK inhibition for the proinflammatory cytokines TNF (Fig. 5A) and IL-6 (Fig. 5B), but an increased production of IL-10 (Fig. 5C), suggesting a change from a pro- to an anti-inflammatory cytokine response with inhibition of the MNK pathway in macrophages. IFN-{gamma}, MCP-1, and IL-12p70 were not appreciably produced by these cells (not shown).


Figure 5
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  		Fig. 5. MNK inhibition changes the cytokine profile of RAW264.7 cells. RAW264.7 cells were incubated with either CGP57380 vs. DMSO control and stimulated with 100 ng/ml E. coli LPS for 4 h. Cytometric bead array assays of paired aliquots of media for TNF (A), IL-6 (B), and IL-10 (C) are shown. Error bars are the standard error of the mean. Results are representative of at least 2 experiments.

 
We were interested whether the effects observed in the RAW264.7 cell line could also be obtained by using primary cultured macrophages isolated from a murine model of human disease. SAMP1/YitFc (SAMP) mice spontaneously develop discontinuous, ileal-specific lesions with 100% penetrance by 10 wk of age, which closely resembles human Crohn's disease (23, 36). Histologically, these mice show segmental transmural inflammation, mucosal and submucosal granulomas, altered epithelial cell morphology, and, in a small percentage of mice, perianal disease (36). Importantly, the disease in SAMP mice is responsive to several biologics that are currently used to treat the human condition, including anti-TNF antibodies; in fact, TNF is known to play an important role in the pathogenesis of ileitis in these mice (23, 29). We used BMDM macrophages from SAMP mice that were pretreated with either CGP57380 or vehicle control (DMSO) and stimulated with 100 ng/ml LPS. Supernatants were collected after 0, 4, and 24 h and analyzed of cytokine production via a Cytometric Bead Array. CGP57380 was capable of significantly reducing the amount of TNF produced by SAMP BMDM by more than 84 and 82%, at 4 and 24 h, respectively (P < 0.02 vs. control for both time points) (Fig. 6A). CGP57380 also potently inhibited the production of IL-6 by 93% at 4 h (P < 0.03) and 72.7% at 24 h (Fig. 6B), as well as MCP-1 by 68.7 and 86.9% at 4 and 24 h (P < 0.04 and P < 0.05), respectively (Fig. 6C), in BMDM from SAMP mice. Interestingly, MNK inhibition did not prevent the production of IL-10 from these cells (Fig. 6D). Thus MNK signaling in primary BMDM from Crohn's disease-like SAMP mice is associated with a proinflammatory cytokine response but does not inhibit production of the anti-inflammatory cytokine, IL-10.


Figure 6
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  		Fig. 6. Effect of MNK inhibition in bone marrow-derived macrophages (BMDM) from SAMP model of Crohn's-like ileitis. BMDM from 10-wk-old SAMP mice with established ileitis (n = 4) were isolated using a standard protocol and assayed in paired cultures, pretreating for 30 min with either CGP57380 (50 µM) or vehicle control (DMSO) prior to stimulation with E. coli LPS (100 ng/ml). Supernatants were collected after 0, 4, and 24 h and cytokine protein production measured by cytometric bead array for TNF (A), IL-6 (B), MCP-1 (C), and IL-10 (D). Results are expressed as total cytokine protein concentration ± standard error of the mean. *Statistical significance, calculated by paired Student's t-test; differences were considered significant when P ≤ 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
MNK1 and 2 mediate signals from either the mitogenic (ERK) or stress (p38) MAP kinase pathways (18). They lack the DFG amino acid motif typical for other protein kinases that coordinates a Mg2+ ion and is otherwise critical for activation of {gamma}-ATP residing in the ATP binding pocket. Instead, the central phenylalanine in a DFD motif sterically prevents ATP binding unless a conformational change occurs because of p38 or ERK phosphorylation at their respective sites in the autoinhibitory loop (18). In normal tissues, the MNK1 kinase activity is inducible in response to LPS or insulin stimulation, whereas MNK2 activity is considered constitutive (45). In breast cancer cells, both phosphorylation and activity of the MNKs are constitutive, and this activity correlates with increased HER2 overexpression and is required for optimal colony formation in soft agar, both in vitro correlates of tumorigenicity (9).

Both MNK1 and MNK2, which are 78% homologous in their catalytic domains, bind to eIF4G and phosphorylate eIF4E at Ser209, changing the affinity of the eIF4E for the mRNA cap (42). Ser209 phosphorylation of eIF4E also promotes the nucleocytoplasmic transport of certain transforming mRNAs, like cyclin D1, through a defined motif in the 3'-untranslated region of its mRNA (10, 43). We do not know which MNK isoform (or both) is responsible for our results in this manuscript. In one report, in human embryonal kidney 293 cells, the IC50 for human MNK1 was reported to be ~3 µM, with complete inhibition of eIF4E phosphorylation at ~10 µM (22), whereas in another report maximal inhibition of eIF4E phosphorylation and TNF production from human Jurkat T-cells was 40 µM (6). Our findings are similar to those of Andersson and Sundler (2), who saw maximal inhibition of eIF4E and LPS-induced TNF production in murine RAW264.7 cells above 40 µM CGP57380.

The fact that multiple TLR pathways that cause macrophage TNF production are inhibited by CGP57380 in a dose-responsive manner and that multiple innate, proinflammatory cytokines are affected by MNK inhibition reveals the central role these kinases play in inflammation. The changes in TNF mRNA level and pattern of mRNA degradation were not related to the TNF regulatory protein TTP, since TTP protein production is also inhibited by MNK inhibition. A recent publication suggests that the formation of SGs is prevented when MNK is inhibited (12). SGs are subcellular organelles where untranscribed mRNAs can safely reside undegraded until the stress is removed. That observation is concordant with the ~10% of LPS-induced TNF mRNA that was stable after actinomycin D treatment without CGP57380 that was degraded when the inhibitor was added to the culture.

The finding that TTP expression requires MNK kinase activation is similar to previous reports that p38 is required for TTP expression. In that case, TTP is a direct target for p38 phosphorylation (8) and p38 promotes TTP degradation via the proteasome (5, 28). In our hands, recombinant TTP was not a substrate for activated, recombinant MNK1 kinase, unlike an eIF4E control (not shown). A previous report suggested that TTP was not affected by MNK inhibition by CGP57380 (5); however, that report used significantly lower (0.1, 1, and 10 µM) concentrations and assayed at an earlier time point (2 h) after LPS stimulation compared with the 4-h time point we used. Consistent with the requirement for a higher concentration to suppress TTP production, from our TLR agonist studies in Fig. 2B, we would not expect doses at those lower concentrations to have significant effects on TNF production.

Several lines of evidence support the concept that cytokines are important mediators of intestinal inflammation and a broad array of cytokines have been implicated in the pathogenesis in inflammatory bowel disease (IBD). In fact, successful strategies that have been recently developed (and are currently in development) to treat IBD are based on intervention of cytokine pathways, with the end goal of designing therapeutics with greater specificity and decreased toxicity for these patients (33). MNK inhibition may represent an example of such a strategy. We have shown that MNK inhibition reduces proinflammatory cytokines known to be important in mucosal innate immune responses and chronic intestinal inflammation, including TNF, IL-6, and MCP-1. In fact, TNF plays a critical role in the pathogenesis of Crohn's disease as inhibition of TNF using monoclonal antibody therapy dramatically improves disease activity in this patient population (39). Similarly, in the SAMP mouse strain, a single injection of a chimeric anti-murine TNF antibody markedly suppresses the ileal-specific disease in which the mechanism of action involves homeostatic regulation of intestinal mucosal cell apoptosis resulting in a net decrease of chronic gut inflammation (29). In regard to IL-6, high serum levels in quiescent Crohn's patients are predictive of clinical relapse (26), and an anti-IL-6 monoclonal antibody has recently been shown to hold promise in treating Crohn's disease (17). The ileitis characteristic of SAMP mice improves, in fact, when signaling through the STAT3 transcription factor is blocked, thereby reducing IL-6 production, and it is worsened by exogenous IL-6 (31). MCP-1 is increased in the intestinal mucosal of IBD patients (30, 35), and the degree of intestinal inflammation in Crohn's disease is associated with MCP-1 tissue levels; furthermore, there is evidence for different MCP-1 genotypes of a functional single nucleotide polymorphism (G/A) located in the distal regulatory region of the MCP-1 gene and an association with different disease behavior in Crohn's disease (14). Finally, the release of IL-10 from SAMP BMDM after LPS stimulation and pretreatment with CGP57380 is exciting, suggesting that an anti-inflammatory response is not prevented by MNK inhibition. Therefore, our results would suggest that MNK inhibition promotes the overall dampening of inflammation mediated by macrophages by reducing proinflammatory cytokine production while permitting responses mediated by anti-inflammatory cytokines, such as IL-10.

In summary, we demonstrate that MNK inhibitors may represent promising therapeutic agents in inflammatory diseases in which bacterial products and innate, proinflammatory cytokines play a critical role in their pathogenesis. Initial studies of the MNK 1/2 double knockout mouse appeared to have no obvious phenotype (45), suggesting that pharmacological inhibitors of these kinases might have great potential in treating inflammatory diseases like Crohn's disease in which TNF and IL-6 have known pathogenic roles and in which the anti-inflammatory effects of IL-10 may limit disease severity. Although our studies show that MNK inhibition can potently inhibit TLR responses and innate, proinflammatory cytokine production in macrophages, further studies will be required to see whether these effects can be achieved in whole animal models of inflammation.


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This work was supported by National Institutes of Health (NIH) Grant DK-060720 and a Crohn's and Colitis Foundation of America Senior Research Award (to M. T. W.), NIH GM-62890 (to T. W. Sturgill), NIH DK-057880 and DK-056762 (to T. T. Pizarro), United States Army Medical Research and Materiel Command Grant under DAMD17-03-1-0555 (to C. A. Chrestensen), and the Immunology and Cell Isolation Cores of the University of Virginia Digestive Health Research Center (DK-67629).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. T. Worthington, 4940 Eastern Ave., Rm. A501, Johns Hopkins Bayview Medical Center, Baltimore, MD 21224-2780 (e-mail: mworthi3{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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