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Infect Immun. 2010 June; 78(6): 2868–2876.
Published online 2010 March 29. doi:  10.1128/IAI.00018-10
PMCID: PMC2876557

Mitogen-Activated Protein Kinase Phosphatase 2 Regulates the Inflammatory Response in Sepsis[down-pointing small open triangle]

Abstract

Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2−/− mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2−/− mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2−/− BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production.

Severe sepsis has a significant impact on public health, with an estimated incidence of nearly 800,000 cases per year, resulting in over 200,000 deaths at an annual cost of over $17 billion (2). The pathophysiology of sepsis involves a dysregulation of the inflammatory response, leading to an imbalance between pro- and anti-inflammatory mediators (1, 4, 15, 21). In the setting of sepsis, this imbalance is a result of complex interactions of signal transduction pathways, triggered by host exposure to microbe-associated molecular patterns (MAMPs), such as lipopolysaccharide (LPS). These MAMPs bind Toll-like receptors (TLR) on the cell surface and utilize a variety of pathways to propagate signals to the nucleus, triggering gene expression responses.

Among the most active signal transduction pathways involved in the immune response are the mitogen-activated protein kinase (MAPK) pathways. Three major families of the MAPK pathway exist in mammalian species: c-Jun NH2-terminal kinases (JNK), the extracellular signal-regulated protein kinase (ERK), and the p38 mitogen-activated protein kinase (p38). These MAPKs are activated by dual phosphorylation on tyrosine and threonine residues through a conserved cascade of upstream kinases, termed MAPK kinases (MKK) and MAPK kinase kinases (MKKK). The activation of the terminal kinases results in the nuclear translocation and promoter binding of transcription factors resulting in the gene expression of numerous mediators involved in the inflammatory response (12).

The kinase-mediated phosphorylation involved in the MAPK pathways is balanced by the presence of a dephosphorylating system comprised of phosphatases to create a dichotomous regulatory process (17). The observation that the MAPKs are phosphorylated on tyrosine and threonine residues makes them unique targets for the dual-specific phosphatases (DUSPs), which specifically dephosphorylate these residues (18). The best-studied dual-specific phosphatase, MAP kinase phosphatase-1 (MKP-1), has been shown to be a negative regulator of the innate immune response (10, 14, 23, 29). MKP-2 is another DUSP, which is closely related to MKP-1 (13, 19). MKP-2 is a 42-kDa inducible phosphatase known to be upregulated in response to growth factors, oncogenes, phorbol 12-myristate 13-acetate (PMA), oxidative stress, and UV light as well as LPS (5, 11, 13, 16, 19, 26-28); however, the role of MKP-2 in regulating the innate immune response has not been elucidated. We hypothesized that MKP-2 would play a complementary and redundant role to MKP-1 in negatively regulating the MAPK pathways following infectious stimuli. To test this hypothesis, we utilized the MKP-2−/− mouse by using both in vivo and in vitro models of sepsis. Surprisingly, our results show a positive regulatory role for MKP-2 in that in its absence, a significant downregulation of inflammation and a survival advantage occur in response to LPS and polymicrobial peritonitis.

MATERIALS AND METHODS

Mice.

MKP-2−/− mice were provided by Jeffery Molkentin (Cincinnati Children's Hospital Research Foundation, Cincinnati, OH). The mutation was generated in embryonic stem cells from the C57BL/6 mouse strain (Jackson Laboratories, Bar Harbor, ME), and these mice have been described previously (22). Homozygous MKP-2−/− mice were generated by breeding heterozygous mice. To confirm homozygous null mice, the genotype was confirmed by first obtaining tail DNA by using the Extract-N-Amp tissue PCR kit (Sigma-Aldrich, St. Louis, MO) per the manufacturer's protocol, and PCR was performed on this tail DNA. PCR primers used to confirm the genotype were as follows: wild-type (WT) forward primer, 5′-CATCGAGTACATCGGTAGG-3′; WT reverse primer, 5′-GGGAAGTCACATGGCAGAG-3′; MKP-2−/− forward primer, 5′-CTCTATGGCTTCTGAGGCG-3′; and MKP-2−/− reverse primer, 5′-GGGAAGTCACATGGCAGAG-3′.

Endotoxin sepsis model.

Age- and sex-matched C57BL/6 WT and MKP-2−/− mice were challenged with 30 mg/kg LPS from Escherichia coli O55:B5 (Sigma-Aldridge, St. Louis, MO) or 0.9% saline via intraperitoneal (i.p.) injection. Concentrations of LPS were calculated and diluted with 0.9% NaCl, so all mice received a total of 1 ml (approximately 40 ml/kg) of i.p. fluid. Mice were examined every 12 h for a total of 5 days.

Additional WT and MKP-2−/− mice that received i.p. injection of 30 mg/kg of LPS were sacrificed at the time points stated below. For serum sample collections, the mice were anesthetized with ketamine, midline abdominal incisions were made, and blood samples were obtained by venipuncture of the inferior vena cava. Blood samples were transferred to BD Microtainer SST tubes (BD, Franklin Lakes, NJ) and centrifuged at 10,000 rpm for 5 min. Serum samples were transferred to microcentrifuge tubes and stored at −80°C.

Cecal ligation and puncture model.

Anesthesia of age- and sex-matched WT and MKP-2−/− mice was induced with isoflurane and maintained via a self-scavenging anesthesia machine by the use of a nose cone (flow, 2.25 liters/min, with a fraction of inspired O2 of 0.7). The abdomen was prepped with alcohol, and a 1-cm incision was made in the skin and peritoneum. The cecum was identified, and a 3-0 silk suture was used to ligate the cecum at its proximal aspect without occlusion of the intestinal lumen. A 21-gauge needle was used to make two complete (through-and-through) punctures of the cecum. Fecal material was expressed to ensure the patency of both punctures. The peritoneum and skin were sutured closed. Mice were resuscitated with 1 ml of warmed, normal saline injected subcutaneously and placed in a warming bed until ambulatory and fully recovered from anesthesia. Sham animals received the same treatment as the experimental animals, but without ligation or puncture of the cecum. Mice were examined every 12 h for a total of 7 days.

While we were aware of the role of antibiotics and fluid administration in affecting survival from cecal ligation and puncture (CLP), antibiotics were not provided because the specific objective of these experiments was to determine the impact of the presence of polymicrobial organisms in MKP-2−/− mice.

All animal care and procedures were conducted under the guidelines and policies of the University of Michigan's Unit for Laboratory and Animal Medicine in compliance with the University Committee on the Use and Care of Animals.

Isolation of bone marrow-derived macrophages (BMDMs).

Bone marrow cells were harvested and cultured in RPMI supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, glutamine, and 30% L929 cell supernatant as previously reported (25). Briefly, following harvest, cells were allowed to grow for 7 days, at which time they were replated in RPMI supplemented with 10% FBS, penicillin-streptomycin, and glutamine to a density of 4 × 106 cells per well in six-well plates. Cells were allowed to adhere for 4 to 6 h. The media were changed to RPMI supplemented with 0.5% FBS, penicillin-streptomycin, and glutamine, and the cells were incubated overnight. The following morning, cells were stimulated with ultrapure LPS (100 ng/ml), lipoteichoic acid (LTA) (10 μg/ml) or poly(I:C) (1 μg/ml) (InvivoGen, San Diego, CA) for the times indicated below.

Cytokine concentrations.

Serum cytokine (interleukin-1β [IL-1β], IL-6, IL-10, IL-12, gamma interferon [IFN-γ], KC, MCP-1, and tumor necrosis factor alpha [TNF-α]) concentrations were determined using the Bio-Rad multiplex suspension array system (Bio-Rad, Hercules, CA) per the manufacturer's instructions. Immunoreactive IL-10 and TNF-α concentrations from cell culture supernatants were also determined using a commercially available mouse IL-10 or TNF-α enzyme-linked immunosorbent assay (ELISA) kit (BioSource International, Camarillo, CA). All procedures were performed in triplicate and according to the manufacturer's protocol.

Immunoblotting.

Following LPS stimulation (100 ng/ml), BMDMs were harvested and lysed using radioimmunoprecipitation assay (RIPA) buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS, with 10 μl Halt protease inhibitor cocktail (Pierce, Rockford, IL) added for each 1 ml of buffer. Samples were separated using SDS-PAGE and transferred to the nitrocellulose. Nitrocellulose blots were probed with antibodies against MKP-2, MKP-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), ERK, phospho-ERK, p38, phospho-p38, JNK (Cell Signaling, Danvers, MA), phospho-JNK (Promega, Madison, WI), or GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Abcam, Cambridge, MA) as indicated in the results section. Following incubation with primary antibody, blots were probed with anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP; ECL, Little Chalfont, Buckinghamshire, United Kingdom). Blots were developed using an Immobilon Western chemiluminescent HRP kit (Millipore, Billerica, MA). Blots were imaged using a Gel-Doc XR system (Bio-Rad, Hercules, CA), and densitometry measurements were obtained using Quality One 1-D analysis software v4.5 (Bio-Rad, Hercules, CA).

qRT-PCR.

RNA was isolated from BMDMs by using the RNeasy mini kit (Qiagen, Valencia, CA) following TLR agonist exposure at the times indicated below. Quantitative reverse transcription-PCR (qRT-PCR) was performed on the samples by using TaqMan gene expression probes for MKP-2 (Mm00723761_m1; Applied Biosystems, Foster City, CA) following cDNA production using the high-capacity cDNA reverse transcriptase kit (Applied Biosystems, Foster City, CA).

siRNA transfection.

WT and MKP-2−/− BMDMs were transfected with On-Targetplus SmartPool MKP-1 siRNA (siMKP-1) (catalog number L-040753-00) or On-Targetplus SmartPool control small interfering RNA (siRNA; catalog number D-001810-10) from Dharmacon (Lafayette, CO) via electroporation using the mouse macrophage Nucleofector kit (Lonza, Basel, Switzerland) for BMDMs from C57BL/6 mice. Briefly, BMDMs were isolated as described above and differentiated for 7 days. Cells were harvested, and 1 × 106 cells were resuspended in 100 μl of Nucleofector solution. A concentration of 40 nM siMKP-1 or control siRNA was added to the cell suspension, and the cells underwent electroporation per the manufacturer's program, using the Nucleofector device (Lonza, Basel, Switzerland). Cell suspensions were then added to 2 ml of RPMI media with 20% FBS and plated onto one well of a six-well plate. Following overnight incubation, cells were stimulated with ultrapure LPS (100 ng/ml) for 2 h. Control cells were exposed to all conditions except siRNA.

Of note, transfection efficiency was determined using 2 μg pmaxGFP (Lonza, Basel, Switzerland) in place of siRNA. Cells were then viewed using fluorescence microscopy, and transfection efficiency was calculated by dividing the number of cells containing green fluorescent protein (GFP) by the total number of cells in the field. Transfection efficiency ranged from 40 to 50% for each of the individual experiments (not shown).

Statistical analysis.

Results are reported as means ± standard errors of the mean (SEM). Log rank test (Mantel-Cox) was performed to determine statistical significance for survival. Statistical significance for parametric data was determined using an unpaired t test for experiments comprising two groups and a one-way analysis of variance (ANOVA) for experiments comprising three or more groups. Statistical tests were conducted using GraphPad Prism 5.01 for Windows (GraphPad Software, Inc., La Jolla, CA).

RESULTS

MKP-2 null mice have improved survival following endotoxin challenge and cecal ligation and puncture.

To investigate the MKP-2's regulation of the innate immune response triggered by TLR4 activation, we examined the effects of i.p. endotoxin injection in MKP-2−/− mice and compared this response to age- and sex-matched WT mice. As prior studies employing FVB/n and C57BL/6 strains of mice had shown a 50% lethal dose (LD50) for mortality at 5 days of 30 mg/kg LPS delivered i.p., MKP-2−/− mice were challenged with 30 mg/kg LPS i.p. MKP-2−/− mice (mortality rate of 20%; n = 29) showed a 55% improved survival rate (P < 0.01) compared to that of WT mice (mortality rate of 65%; n = 29) at 5 days (Fig. (Fig.1A).1A). Furthermore, the majority of WT mice (59%) died between 24 and 48 h, with a median survival time of 36 h, while the majority (69%) of the MKP-2 mice was still alive at 5 days. No deaths were noted in control mice in either group that were injected with equal volumes of 0.9% saline (data not graphed).

FIG. 1.
Improved survival in MKP-2−/− mice following LPS and CLP models of sepsis. (A) Age- and sex-matched WT (n = 29) and MKP-2−/− (n = 29) mice received i.p. injections of LPS (30 mg/kg). The percentage of alive ...

While endotoxin challenge is an important in vivo model that assists in elucidating the complex host inflammatory response to activation of TLR4, it does not assess the ability of the innate immune system to contain and/or eradicate viable, pathogenic organisms. Given our observation of improved survival to endotoxin challenge in the MKP-2 null mice, we suspected that a dampened host immune response in the absence of MKP-2 may be detrimental to pathogen clearance. Therefore, to examine this possibility, we used a cecal ligation and puncture (CLP) model to assess the impact of MKP-2's regulation of the innate immune response to polymicrobial peritonitis. Following CLP, MKP-2−/− mice (mortality rate of 25%; n = 20) showed a 44% improved survival rate (P < 0.01) compared to that of WT mice (mortality rate of 66%; n = 20) at 7 days following CLP (Fig. (Fig.1B).1B). No deaths were observed in sham animals from either group (data not shown). These data indicate a survival benefit for mice lacking MKP-2 in both a sterile and viable pathogen LPS-induced inflammatory process.

MKP-2−/− mice have decreased serum cytokine levels following endotoxin challenge.

Since the survival benefits were similar in both our endotoxin and CLP models, we chose to further investigate the regulatory role of MKP-2 on cytokine production by using the endotoxin model, thus eliminating any confounding inflammatory response to the surgical procedure. Consistent with the improved survival observed in this mouse model of i.p. endotoxin challenge, a significantly attenuated inflammatory cytokine response was observed in the MKP-2−/− mice compared to that in WT mice (Fig. (Fig.2).2). Serum levels of IL-1β, IL-6, and TNF-α were significantly decreased in the MKP-2−/− mice at 8 h (P values of <0.01, <0.05 and <0.05, respectively) after i.p. LPS injection compared to those in WT mice. TNF-α levels were also significantly reduced in the MKP-2−/− mice (P < 0.01) compared to those in WT mice 24 h after LPS injection. This result did not appear to result from an augmented anti-inflammatory response, as IL-10 production was also attenuated at 24 h in the MKP-2−/− mice (P < 0.05) compared to that in WT mice (Fig. (Fig.2).2). In addition to these time points, attenuated proinflammatory cytokine production was also noted at earlier times (2 and 4 h after LPS injection) for several cytokines, including significant reductions in MIP-1α and MCP-1 and trends toward reduced TNF-α and IL-1β (data not shown). This regulatory effect by MKP-2 appeared to involve specific cytokines, as there was no change observed in IFN-γ levels. These data indicate overall decreased expression of key inflammatory cytokines in response to LPS in the MKP-2−/− mice associated with improved survival.

FIG. 2.
Attenuated serum cytokine levels in the absence of MKP-2. Multiplex cytokine array on serum samples from mice following i.p. injections of LPS (30 mg/kg) at the times indicated (at 8 h, n = 10 for WT and n = 10 for MKP-2−/− ...

MKP-2 influences activation of ERK, JNK, and p38.

As MKP-2 had been shown to modulate MAPK activation and since macrophages and monocytes are key sources of cytokine production during the LPS-induced inflammatory response, we hypothesized that macrophages isolated from MKP-2−/− mice would have attenuated cytokine production in response to LPS. To test this hypothesis, we utilized BMDMs from MKP-2−/− and WT mice to investigate the effect of the absence of MKP-2 on MAPK signaling and cytokine production. Consistent with the serum cytokine levels noted in the in vivo studies, macrophages from MKP-2−/− mice produced significantly less TNF-α and IL-10 over time (Fig. 3A and B). The greatest difference in the production levels of TNF-α was noted at 2 h after LPS stimulation, with a 4-fold decrease in production from MKP-2−/− BMDMs (P < 0.01) compared to that from WT BMDMs (Fig. (Fig.3A).3A). The production of the anti-inflammatory cytokine IL-10 was also attenuated in MKP-2−/− BMDMs, albeit at 8 h (P < 0.01) after LPS exposure (Fig. (Fig.3B3B).

FIG. 3.
MKP-2 regulates cytokine production in BMDMs. ELISAs for immunoreactive TNF-α (A) and IL-10 (B) performed on MKP-2−/− and WT BMDM media following stimulation with LPS (100 ng/ml) for the times indicated. Data are shown as mean ...

As production of these cytokines is at least in part dependent on MAPK activation, we hypothesized that the pathways were modulated in the absence of MKP-2. We tested this hypothesis of MKP-2's regulation of the MAPK signaling by first determining the kinetics of MKP-2 induction in BMDMs following LPS stimulation. qRT-PCR for MKP-2 in WT BMDMs demonstrated a significant increase in MKP-2 transcription over the first 2 h of stimulation, with a maximum increase in mRNA occurring at 1 h (Fig. (Fig.4A).4A). Western blots of LPS-stimulated WT BMDM lysates probed for MKP-2 showed maximal protein production occurring between 1 and 2 h (Fig. (Fig.4B).4B). As expected, neither mRNA nor protein of MKP-2 was detected in the MKP-2−/− BMDM following LPS stimulation (data not shown). Of note, MKP-2 induction also occurred in response to other TLR agonists—LTA (TLR2 agonist; 10 μg/ml) and poly(I:C) (TLR3 agonist; 1 μg/ml)—although induction was approximately 100-fold less than induction with LPS and peak induction occurred at 2 h following stimulation (Fig. 4C and D).

FIG. 4.
MKP-2 is induced by TLR ligands. qRT-PCR (A) and Western blot analysis (B) results showing mRNA and protein induction of MKP-2 in WT BMDMs following stimulation with LPS (100 ng/ml) for the times indicated. (C and D) qRT-PCR data indicating an increase ...

We next performed Western blot analyses of both MKP-2−/− and WT BMDMs to determine the regulatory effect of MKP-2 on the terminal kinases in the MAPK pathway. Consistent with a known targeting of the ERK pathway by MKP-2 (8, 11, 19), phosphorylation of ERK was increased in MKP-2−/− BMDMs compared to that at the same time points in WT BMDMs (Fig. (Fig.5).5). Under similar experimental conditions, phosphorylation of JNK and p38 was decreased in MKP-2−/− BMDMs (Fig. (Fig.5)5) compared to that in WT BMDMs. These data indicated that the absence of MKP-2 critically altered the phosphorylation state of ERK, JNK, and p38 in response to LPS, and we next sought to identify the link between this regulation and the effect on cytokine production.

FIG. 5.
MKP-2 regulates activation of ERK, JNK, and p38. BMDMs from WT and MKP-2−/− mice were stimulated with LPS (100 ng/ml) for the times indicated. (A) Western blots of cell lysates probed with antibodies to the phosphorylated forms of JNK, ...

MKP-1 induction is increased in the absence of MKP-2.

As stated above, MKP-1 has been shown to be a negative regulator of the inflammatory response (10, 14, 23, 29), and interestingly, ERK activation results in decreased MKP-1 protein degradation (6, 9). Based on this knowledge, we suspected that expression of MKP-2 via regulation of ERK activation could affect MKP-1 induction. Given our observations, we hypothesized that in the absence of MKP-2 and subsequent augmented ERK activation, MKP-1 expression would be significantly increased, resulting in reduced cytokine production. Consistent with this hypothesis, we observed an increase in MKP-1 in MKP-2−/− BMDMs compared with that in WT BMDMs following LPS stimulation (Fig. (Fig.6A).6A). These data suggest a role for MKP-1 in the regulatory mechanism of MKP-2.

FIG. 6.
Increased levels of MKP-1 in MKP-2−/− BMDMs attenuate cytokine production. (A) Western blots of WT and MKP-2−/− BMDM whole-cell lysates probed with antibodies to MKP-1 indicate increased levels of MKP-1 in MKP-2−/− ...

If increased expression of MKP-1 was responsible for the observed reduction in MAPK activation and, thus, cytokine production in the MKP-2−/− cells, knockdown of MKP-1 in this model should reverse this effect. Thus, we utilized RNA interference directed at MKP-1 to determine if the increase in MKP-1 suppressed cytokine production in the MKP-2−/− BMDMs. Consistent with our hypothesis, we detected a significant increase (~2-fold; P < 0.01) in TNF-α production from LPS-stimulated MKP-2−/− BMDMs transfected with siMKP-1 compared to that from MKP-2−/− BMDMs transfected with nontargeting control siRNA (Fig. (Fig.6B).6B). A similar 2-fold increase in TNF-α (P < 0.05) production was also detected in LPS-stimulated WT BMDMs transfected with siMKP-1 compared to that in WT BMDMs transfected with nontargeting control siRNA (Fig. (Fig.6C).6C). However, absolute TNF-α production remained increased in WT BMDMs compared to that in MKP-2−/− BMDMs. Taken together, these data suggest a strong mechanistic link between MKP-2 and regulation of MKP-1 expression in the modulation of the MAPK activation and cytokine production following LPS stimulation.

DISCUSSION

We aimed to study the role of MKP-2 in regulating the inflammatory response in sepsis by utilizing the MKP-2 null mouse. Initially, we anticipated that MKP-2 would play a similar and perhaps redundant role to MKP-1 in negatively regulating cytokine production in response to a canonical inflammatory trigger (e.g., LPS). Contrary to our initial hypothesis, we observed that mice lacking the DUSP MKP-2 exhibited a decreased inflammatory response following LPS stimulation. Furthermore, MKP-2−/− mice were conferred a survival benefit not only in this “sterile” in vivo model but also in the polymicrobial CLP model of sepsis (Fig. (Fig.1B).1B). This survival benefit correlated with significantly reduced production of pro- and anti-inflammatory cytokines (Fig. (Fig.2).2). These findings are intriguing, as it suggests that the functional role of MKP-2 in regulating the host's immune response can involve modulating the cytokine response, but preliminarily, without affecting the ability to contain and/or eradicate a viable pathogen. Ongoing studies are examining the effect of MKP-2 in regulating components of this immune response that are beyond the scope of these current studies, including antigen presentation, phagocytosis, and oxidative burst. Immune responsive cells in the form of isolated BMDMs also exhibited decreased production of archetypal pro- and anti-inflammatory cytokines TNF-α and IL-10 in in vitro studies. Mechanistically, this regulation of cytokine production by MKP-2 was associated with altered phosphorylation of ERK, JNK, and p38 as well as an increased production of the negative regulatory DUSP, MKP-1. LPS stimulation of MKP-2−/− BMDMs increased phosphorylation of ERK and decreased phosphorylation of both JNK and p38 compared to that of BMDMs derived from WT mice (Fig. (Fig.5).5). Additionally, MKP-1 was increased in MKP-2−/− BMDMs starting at 30 min following LPS stimulation (Fig. (Fig.6).6). This increase in MKP-1, which is a negative regulator of both pro- and anti-inflammatory cytokine production via JNK and p38 regulation (10, 14, 20, 23, 29) likely explains the decreased cytokine production in the MKP-2−/− cells compared to WT cells. As further evidence of this mechanistic link between MKP-2 and MKP-1 expression, we also found that silencing MKP-1 in the MKP-2 null BMDMs reversed this phenotype, as we observed increased production of TNF-α from MKP-1-silenced MKP-2−/− BMDMs (Fig. (Fig.6B).6B). These data suggest that increased expression of MKP-1 driven by augmented ERK activation in the absence of MKP-2 was involved in the attenuated cytokine production.

Given these observations, we believe that our data demonstrate a critical mechanism of cross talk between MKP-2 and MKP-1 that is mediated through ERK activation. This is highly plausible given prior findings that ERK activation is important for maximal induction of MKP-1 (24) and stabilization of the MKP-1 protein (6, 9). Furthermore, this link likely explains our findings that all three terminal MAPKs were altered in the absence of MKP-2. This was initially not expected, as several studies have shown that MKP-2 has much a higher specificity for ERK than for either JNK or p38 (8, 11, 13, 19). Thus, it is not surprising that in the absence of MKP-2, we observed increased activation of ERK; however, given that we also observed increased expression of MKP-1, a known regulator of JNK and p38, it is likely that MKP-1 is responsible for the significant reduction in phosphorylation of JNK and p38 observed in the LPS-stimulated MKP-2 null BMDMs. We therefore propose a mechanism of MKP-2 regulation of cytokine production in which, following LPS binding to TLR4, the three arms of the MAPK pathway are simultaneously activated (Fig. (Fig.7).7). JNK and p38 are directly involved in the induction and production of proinflammatory cytokines, while ERK activation leads to induction and stabilization of MKP-1, which serves as a negative regulator of proinflammatory cytokine production by inhibiting the action of JNK and p38 (10, 14, 20, 23, 29). Subsequently, MKP-2 is induced to deactivate ERK and thus destabilize MKP-1, resetting the cellular mechanism for cytokine production.

FIG. 7.
Proposed regulatory mechanism of MKP-2 impacting MKP-1 levels via ERK deactivation. Following LPS binding to TLR4, the three MAPK pathways are activated. Activation of ERK results in the induction and stabilization of MKP-1. The activation of JNK and ...

Since MKPs remove phosphates on activated MAPKs, this putative role of a phosphatase as a positive regulator of the inflammatory response is surprising; however, such a role has been demonstrated for a related MKP, PAC-1 (16). Similar to our results, Jeffrey et al. showed decreased cytokine production by PAC-1−/− macrophages in response to LPS. MKP-2 is present in a variety of tissues (19) whereas PAC-1 is limited to immune cells (16). It is plausible that the redundancy of the positive regulation on the inflammatory response for these two MKPs is necessary in immune cells, but the ubiquitous nature of MKP-2 may provide a more global positive regulation. The complex nature of regulation of the MAPK pathway by the DUSPs is not completely understood but may impact the regulation of scaffolding and cellular trafficking (7) as well as the flexibility of the cellular response to amount and type of stimuli (3). Although our data support a positive regulatory role for MKP-2 involving ERK-mediated cross talk with MKP-1, further studies are under way to define the precise interactions involved between MKP-2, ERK, and MKP-1. We are additionally pursuing the effects of other TLR agonists on MKP-2 induction as well as the effects of MKP-2 overexpression in order to gain more insight into the regulatory role of MKP-2. Finally, we acknowledge that the altered cytokine production may be the result of preconditioned or altered phenotypic macrophages. Further investigation is needed to understand the impact MKP-2 has on macrophage development as well as involvement of other regulators besides MKP-1 that are responsible for the altered cytokine response.

An additional limit to the current studies is the complex nature of cytokine production in vivo in response to LPS. Although we used BMDMs to investigate the regulatory role of MKP-2 in cytokine production, the exact role of macrophages in the improved survival noted in our MKP-2−/− mice is unknown. Further studies are under way investigating both potential alterations in the hematopoietic differentiation and impact on the function of various tissue macrophages (e.g., lung, spleen, and peritoneum), as well as lymphocytes in the MKP-2−/− mice and its impact on modifying the response to LPS. These investigations will strengthen our understanding of the regulatory role of MKP-2 in both hematopoietic differentiation and inflammatory/immune response.

In summary, our studies demonstrated a survival benefit associated with decreases in both pro- and anti-inflammatory cytokines in the absence of MKP-2 and an increase in the phosphorylation of ERK, while there is a decrease in phosphorylation of JNK and p38 concomitant with an increase in MKP-1 induction. These data suggest a cross talk mechanism (Fig. (Fig.7)7) by which MKP-1 is involved in the global regulation of cytokine production by MKP-2.

Acknowledgments

This work was supported by National Institutes of Health grants K12GM076344 (T.T.C.) and RO1GM66839 (T.P.S.). We thank the Pediatric Critical Care Scientist Development Program (PCCSDP) for supporting the work of T.T.C.

We are grateful to Ann Marie Levine for her careful reading of the manuscript.

Notes

Editor: F. C. Fang

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 March 2010.

REFERENCES

1. Abraham, E., and M. Singer. 2007. Mechanisms of sepsis-induced organ dysfunction. Crit. Care Med. 35:2408-2416. [PubMed]
2. Angus, D. C., W. T. Linde-Zwirble, J. Lidicker, G. Clermont, J. Carcillo, and M. R. Pinsky. 2001. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 29:1303-1310. [PubMed]
3. Bhalla, U. S., P. T. Ram, and R. Iyengar. 2002. MAP kinase phosphatase as a locus of flexibility in a mitogen-activated protein kinase signaling network. Science 297:1018-1023. [PubMed]
4. Bone, R. C. 1996. Sir Isaac Newton, sepsis, SIRS, and CARS. Crit. Care Med. 24:1125-1128. [PubMed]
5. Brondello, J. M., A. Brunet, J. Pouyssegur, and F. R. McKenzie. 1997. The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade. J. Biol. Chem. 272:1368-1376. [PubMed]
6. Brondello, J. M., J. Pouyssegur, and F. R. McKenzie. 1999. Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 286:2514-2517. [PubMed]
7. Caunt, C. J., S. P. Armstrong, C. A. Rivers, M. R. Norman, and C. A. McArdle. 2008. Spatiotemporal regulation of ERK2 by dual specificity phosphatases. J. Biol. Chem. 283:26612-26623. [PMC free article] [PubMed]
8. Chen, P., D. Hutter, P. Liu, and Y. Liu. 2002. A mammalian expression system for rapid production and purification of active MAP kinase phosphatases. Protein Expr. Purif. 24:481-488. [PubMed]
9. Chen, P., J. Li, J. Barnes, G. C. Kokkonen, J. C. Lee, and Y. Liu. 2002. Restraint of proinflammatory cytokine biosynthesis by mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages. J. Immunol. 169:6408-6416. [PubMed]
10. Chi, H., S. P. Barry, R. J. Roth, J. J. Wu, E. A. Jones, A. M. Bennett, and R. A. Flavell. 2006. Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses. Proc. Natl. Acad. Sci. U. S. A. 103:2274-2279. [PubMed]
11. Chu, Y., P. A. Solski, R. Khosravi-Far, C. J. Der, and K. Kelly. 1996. The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J. Biol. Chem. 271:6497-6501. [PubMed]
12. Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55-72. [PubMed]
13. Guan, K. L., and E. Butch. 1995. Isolation and characterization of a novel dual specific phosphatase, HVH2, which selectively dephosphorylates the mitogen-activated protein kinase. J. Biol. Chem. 270:7197-7203. [PubMed]
14. Hammer, M., J. Mages, H. Dietrich, A. Servatius, N. Howells, A. C. Cato, and R. Lang. 2006. Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock. J. Exp. Med. 203:15-20. [PMC free article] [PubMed]
15. Hotchkiss, R. S., and I. E. Karl. 2003. The pathophysiology and treatment of sepsis. N. Engl. J. Med. 348:138-150. [PubMed]
16. Jeffrey, K. L., T. Brummer, M. S. Rolph, S. M. Liu, N. A. Callejas, R. J. Grumont, C. Gillieron, F. Mackay, S. Grey, M. Camps, C. Rommel, S. D. Gerondakis, and C. R. Mackay. 2006. Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1. Nat. Immunol. 7:274-283. [PubMed]
17. Keyse, S. M. 2000. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr. Opin. Cell Biol. 12:186-192. [PubMed]
18. Lang, R., M. Hammer, and J. Mages. 2006. DUSP meet immunology: dual specificity MAPK phosphatases in control of the inflammatory response. J. Immunol. 177:7497-7504. [PubMed]
19. Misra-Press, A., C. S. Rim, H. Yao, M. S. Roberson, and P. J. Stork. 1995. A novel mitogen-activated protein kinase phosphatase. Structure, expression, and regulation. J. Biol. Chem. 270:14587-14596. [PubMed]
20. Nimah, M., B. Zhao, A. G. Denenberg, O. Bueno, J. Molkentin, H. R. Wong, and T. P. Shanley. 2005. Contribution of MKP-1 regulation of p38 to endotoxin tolerance. Shock 23:80-87. [PubMed]
21. Oberholzer, A., C. Oberholzer, R. M. Minter, and L. L. Moldawer. 2001. Considering immunomodulatory therapies in the septic patient: should apoptosis be a potential therapeutic target? Immunol. Lett. 75:221-224. [PubMed]
22. Ramesh, S., X. J. Qi, G. M. Wildey, J. Robinson, J. Molkentin, J. Letterio, and P. H. Howe. 2008. TGF beta-mediated BIM expression and apoptosis are regulated through SMAD3-dependent expression of the MAPK phosphatase MKP2. EMBO Rep. 9:990-997. [PubMed]
23. Salojin, K. V., I. B. Owusu, K. A. Millerchip, M. Potter, K. A. Platt, and T. Oravecz. 2006. Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J. Immunol. 176:1899-1907. [PubMed]
24. Sohaskey, M. L., and J. E. Ferrell, Jr. 2002. Activation of p42 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase, induces phosphorylation and stabilization of MAPK phosphatase XCL100 in Xenopus oocytes. Mol. Biol. Cell 13:454-468. [PMC free article] [PubMed]
25. Stanley, E. R. 1997. Murine bone marrow-derived macrophages. Methods Mol. Biol. 75:301-304. [PubMed]
26. Wang, J., W. H. Shen, Y. J. Jin, P. W. Brandt-Rauf, and Y. Yin. 2007. A molecular link between E2F-1 and the MAPK cascade. J. Biol. Chem. 282:18521-18531. [PubMed]
27. Zhang, T., J. M. Mulvaney, and M. S. Roberson. 2001. Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone. Mol. Cell Endocrinol. 172:79-89. [PubMed]
28. Zhang, T., and M. S. Roberson. 2006. Role of MAP kinase phosphatases in GnRH-dependent activation of MAP kinases. J. Mol. Endocrinol. 36:41-50. [PubMed]
29. Zhao, Q., X. Wang, L. D. Nelin, Y. Yao, R. Matta, M. E. Manson, R. S. Baliga, X. Meng, C. V. Smith, J. A. Bauer, C. H. Chang, and Y. Liu. 2006. MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock. J. Exp. Med. 203:131-140. [PMC free article] [PubMed]

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