INFECTION AND IMMUNITY, June 2010, p. 2868–2876 Vol. 78, No. 60019-9567/10/$12.00 doi:10.1128/IAI.00018-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Mitogen-Activated Protein Kinase Phosphatase 2 Regulates theInflammatory Response in Sepsis�

Timothy T. Cornell,* Paul Rodenhouse, Qing Cai, Lei Sun, and Thomas P. ShanleyDivision of Pediatric Critical Care Medicine, Department of Pediatrics and Communicable Diseases, University of

Michigan School of Medicine, and C.S. Mott Children’s Hospital, Ann Arbor, Michigan 48109-0243

Received 8 January 2010/Returned for modification 16 February 2010/Accepted 24 March 2010

Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatoryresponse to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signaltransduction 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 importantnegative 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, alsoa dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known toregulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. Wehypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had asurvival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) ora polymicrobial infection via cecal ligation and puncture. The MKP-2�/� mice also exhibited decreasedserum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-�], interleukin-1�[IL-1�], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bonemarrow-derived macrophages (BMDMs) from MKP-2�/� mice showed increased phosphorylation of theextracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increasedinduction 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 targetsERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 oncytokine production.

Severe sepsis has a significant impact on public health, withan estimated incidence of nearly 800,000 cases per year, result-ing in over 200,000 deaths at an annual cost of over $17 billion(2). The pathophysiology of sepsis involves a dysregulation ofthe inflammatory response, leading to an imbalance betweenpro- and anti-inflammatory mediators (1, 4, 15, 21). In thesetting of sepsis, this imbalance is a result of complex interac-tions of signal transduction pathways, triggered by host expo-sure to microbe-associated molecular patterns (MAMPs), suchas lipopolysaccharide (LPS). These MAMPs bind Toll-like re-ceptors (TLR) on the cell surface and utilize a variety ofpathways to propagate signals to the nucleus, triggering geneexpression responses.

Among the most active signal transduction pathways in-volved in the immune response are the mitogen-activated pro-tein kinase (MAPK) pathways. Three major families of theMAPK pathway exist in mammalian species: c-Jun NH2-ter-minal kinases (JNK), the extracellular signal-regulated proteinkinase (ERK), and the p38 mitogen-activated protein kinase(p38). These MAPKs are activated by dual phosphorylation ontyrosine and threonine residues through a conserved cascadeof upstream kinases, termed MAPK kinases (MKK) andMAPK kinase kinases (MKKK). The activation of the terminal

kinases results in the nuclear translocation and promoter bind-ing of transcription factors resulting in the gene expression ofnumerous mediators involved in the inflammatory response(12).

The kinase-mediated phosphorylation involved in theMAPK pathways is balanced by the presence of a dephosphor-ylating system comprised of phosphatases to create a dichot-omous regulatory process (17). The observation that theMAPKs are phosphorylated on tyrosine and threonine resi-dues makes them unique targets for the dual-specific phos-phatases (DUSPs), which specifically dephosphorylate theseresidues (18). The best-studied dual-specific phosphatase,MAP kinase phosphatase-1 (MKP-1), has been shown to be anegative regulator of the innate immune response (10, 14, 23,29). MKP-2 is another DUSP, which is closely related toMKP-1 (13, 19). MKP-2 is a 42-kDa inducible phosphataseknown to be upregulated in response to growth factors, onco-genes, phorbol 12-myristate 13-acetate (PMA), oxidativestress, and UV light as well as LPS (5, 11, 13, 16, 19, 26–28);however, the role of MKP-2 in regulating the innate immuneresponse has not been elucidated. We hypothesized thatMKP-2 would play a complementary and redundant role toMKP-1 in negatively regulating the MAPK pathways followinginfectious 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 forMKP-2 in that in its absence, a significant downregulation ofinflammation and a survival advantage occur in response toLPS and polymicrobial peritonitis.

* Corresponding author. Mailing address: Division of Pediatric Crit-ical Care Medicine, C.S. Mott Children’s Hospital, F-6882, 1500 EastMedical Center Drive, Ann Arbor, MI 48109-0243. Phone: (734) 764-5302. Fax: (734) 647-5624. E-mail: ttcornel@med.umich.edu.

� Published ahead of print on 29 March 2010.

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MATERIALS AND METHODS

Mice. MKP-2�/� mice were provided by Jeffery Molkentin (Cincinnati Chil-dren’s Hospital Research Foundation, Cincinnati, OH). The mutation was gen-erated in embryonic stem cells from the C57BL/6 mouse strain (Jackson Labo-ratories, 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 obtainingtail 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 micereceived a total of 1 ml (approximately 40 ml/kg) of i.p. fluid. Mice were exam-ined every 12 h for a total of 5 days.

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

Cecal ligation and puncture model. Anesthesia of age- and sex-matched WTand 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 wasidentified, and a 3-0 silk suture was used to ligate the cecum at its proximal aspectwithout occlusion of the intestinal lumen. A 21-gauge needle was used to maketwo complete (through-and-through) punctures of the cecum. Fecal material wasexpressed to ensure the patency of both punctures. The peritoneum and skinwere sutured closed. Mice were resuscitated with 1 ml of warmed, normal salineinjected subcutaneously and placed in a warming bed until ambulatory and fullyrecovered from anesthesia. Sham animals received the same treatment as theexperimental animals, but without ligation or puncture of the cecum. Mice wereexamined every 12 h for a total of 7 days.

While we were aware of the role of antibiotics and fluid administration inaffecting survival from cecal ligation and puncture (CLP), antibiotics were notprovided because the specific objective of these experiments was to determinethe impact of the presence of polymicrobial organisms in MKP-2�/� mice.

All animal care and procedures were conducted under the guidelines andpolicies of the University of Michigan’s Unit for Laboratory and Animal Medi-cine in compliance with the University Committee on the Use and Care ofAnimals.

Isolation of bone marrow-derived macrophages (BMDMs). Bone marrow cellswere harvested and cultured in RPMI supplemented with 10% fetal bovineserum (FBS), penicillin-streptomycin, glutamine, and 30% L929 cell supernatantas previously reported (25). Briefly, following harvest, cells were allowed to growfor 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 wellin six-well plates. Cells were allowed to adhere for 4 to 6 h. The media werechanged to RPMI supplemented with 0.5% FBS, penicillin-streptomycin, andglutamine, and the cells were incubated overnight. The following morning, cellswere stimulated with ultrapure LPS (100 ng/ml), lipoteichoic acid (LTA) (10�g/ml) or poly(I:C) (1 �g/ml) (InvivoGen, San Diego, CA) for the times indi-cated below.

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

Immunoblotting. Following LPS stimulation (100 ng/ml), BMDMs were har-vested 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 �l Halt protease inhibitor cocktail (Pierce, Rockford, IL)added for each 1 ml of buffer. Samples were separated using SDS-PAGE andtransferred to the nitrocellulose. Nitrocellulose blots were probed with antibod-ies 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-phos-phate dehydrogenase; Abcam, Cambridge, MA) as indicated in the results sec-tion. 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 ImmobilonWestern chemiluminescent HRP kit (Millipore, Billerica, MA). Blots were im-aged using a Gel-Doc XR system (Bio-Rad, Hercules, CA), and densitometrymeasurements 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 indicatedbelow. Quantitative reverse transcription-PCR (qRT-PCR) was performed on thesamples 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; catalognumber D-001810-10) from Dharmacon (Lafayette, CO) via electroporationusing the mouse macrophage Nucleofector kit (Lonza, Basel, Switzerland) forBMDMs from C57BL/6 mice. Briefly, BMDMs were isolated as described aboveand differentiated for 7 days. Cells were harvested, and 1 � 106 cells wereresuspended in 100 �l of Nucleofector solution. A concentration of 40 nMsiMKP-1 or control siRNA was added to the cell suspension, and the cellsunderwent electroporation per the manufacturer’s program, using the Nucleo-fector device (Lonza, Basel, Switzerland). Cell suspensions were then added to 2ml 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 (100ng/ml) for 2 h. Control cells were exposed to all conditions except siRNA.

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

Statistical analysis. Results are reported as means � standard errors of themean (SEM). Log rank test (Mantel-Cox) was performed to determine statisticalsignificance for survival. Statistical significance for parametric data was deter-mined using an unpaired t test for experiments comprising two groups and aone-way analysis of variance (ANOVA) for experiments comprising three ormore groups. Statistical tests were conducted using GraphPad Prism 5.01 forWindows (GraphPad Software, Inc., La Jolla, CA).

RESULTS

MKP-2 null mice have improved survival following endo-toxin challenge and cecal ligation and puncture. To investigatethe MKP-2’s regulation of the innate immune response trig-gered by TLR4 activation, we examined the effects of i.p.endotoxin injection in MKP-2�/� mice and compared this re-sponse to age- and sex-matched WT mice. As prior studiesemploying FVB/n and C57BL/6 strains of mice had shown a50% lethal dose (LD50) for mortality at 5 days of 30 mg/kg LPSdelivered i.p., MKP-2�/� mice were challenged with 30 mg/kgLPS i.p. MKP-2�/� mice (mortality rate of 20%; n 29)showed a 55% improved survival rate (P 0.01) compared tothat of WT mice (mortality rate of 65%; n 29) at 5 days (Fig.1A). Furthermore, the majority of WT mice (59%) died be-tween 24 and 48 h, with a median survival time of 36 h, whilethe majority (69%) of the MKP-2 mice was still alive at 5days. No deaths were noted in control mice in either group

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that were injected with equal volumes of 0.9% saline (datanot graphed).

While endotoxin challenge is an important in vivo model thatassists in elucidating the complex host inflammatory responseto activation of TLR4, it does not assess the ability of theinnate immune system to contain and/or eradicate viable,pathogenic organisms. Given our observation of improved sur-vival to endotoxin challenge in the MKP-2 null mice, we sus-pected that a dampened host immune response in the absenceof MKP-2 may be detrimental to pathogen clearance. There-fore, to examine this possibility, we used a cecal ligation andpuncture (CLP) model to assess the impact of MKP-2’s regu-lation of the innate immune response to polymicrobial perito-nitis. 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. 1B). No deaths were observed insham animals from either group (data not shown). These dataindicate a survival benefit for mice lacking MKP-2 in both asterile and viable pathogen LPS-induced inflammatory pro-cess.

MKP-2�/� mice have decreased serum cytokine levels fol-lowing endotoxin challenge. Since the survival benefits weresimilar in both our endotoxin and CLP models, we chose tofurther investigate the regulatory role of MKP-2 on cytokineproduction by using the endotoxin model, thus eliminating anyconfounding inflammatory response to the surgical procedure.

Consistent with the improved survival observed in this mousemodel of i.p. endotoxin challenge, a significantly attenuatedinflammatory cytokine response was observed in the MKP-2�/� mice compared to that in WT mice (Fig. 2). Serum levelsof IL-1�, IL-6, and TNF-� were significantly decreased in theMKP-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 WTmice. TNF-� levels were also significantly reduced in the MKP-2�/� mice (P 0.01) compared to those in WT mice 24 h afterLPS injection. This result did not appear to result from anaugmented anti-inflammatory response, as IL-10 productionwas also attenuated at 24 h in the MKP-2�/� mice (P 0.05)compared to that in WT mice (Fig. 2). In addition to these timepoints, attenuated proinflammatory cytokine production wasalso noted at earlier times (2 and 4 h after LPS injection) forseveral cytokines, including significant reductions in MIP-1�and MCP-1 and trends toward reduced TNF-� and IL-1� (datanot shown). This regulatory effect by MKP-2 appeared to in-volve specific cytokines, as there was no change observed inIFN-� levels. These data indicate overall decreased expressionof key inflammatory cytokines in response to LPS in the MKP-2�/� mice associated with improved survival.

MKP-2 influences activation of ERK, JNK, and p38. AsMKP-2 had been shown to modulate MAPK activation andsince macrophages and monocytes are key sources of cytokineproduction during the LPS-induced inflammatory response, wehypothesized that macrophages isolated from MKP-2�/� micewould have attenuated cytokine production in response toLPS. To test this hypothesis, we utilized BMDMs from MKP-2�/� and WT mice to investigate the effect of the absence ofMKP-2 on MAPK signaling and cytokine production. Consis-tent with the serum cytokine levels noted in the in vivo studies,macrophages from MKP-2�/� mice produced significantly lessTNF-� and IL-10 over time (Fig. 3A and B). The greatestdifference in the production levels of TNF-� was noted at 2 hafter LPS stimulation, with a 4-fold decrease in productionfrom MKP-2�/� BMDMs (P 0.01) compared to that fromWT BMDMs (Fig. 3A). The production of the anti-inflamma-tory cytokine IL-10 was also attenuated in MKP-2�/� BMDMs,albeit at 8 h (P 0.01) after LPS exposure (Fig. 3B).

As production of these cytokines is at least in part depen-dent on MAPK activation, we hypothesized that the pathwayswere modulated in the absence of MKP-2. We tested thishypothesis of MKP-2’s regulation of the MAPK signaling byfirst determining the kinetics of MKP-2 induction in BMDMsfollowing LPS stimulation. qRT-PCR for MKP-2 in WTBMDMs demonstrated a significant increase in MKP-2 tran-scription over the first 2 h of stimulation, with a maximumincrease in mRNA occurring at 1 h (Fig. 4A). Western blots ofLPS-stimulated WT BMDM lysates probed for MKP-2 showedmaximal protein production occurring between 1 and 2 h (Fig.4B). As expected, neither mRNA nor protein of MKP-2 wasdetected in the MKP-2�/� BMDM following LPS stimulation(data not shown). Of note, MKP-2 induction also occurred inresponse to other TLR agonists—LTA (TLR2 agonist; 10 �g/ml) and poly(I:C) (TLR3 agonist; 1 �g/ml)—although induc-tion was approximately 100-fold less than induction with LPSand peak induction occurred at 2 h following stimulation (Fig.4C and D).

We next performed Western blot analyses of both MKP-

FIG. 1. Improved survival in MKP-2�/� mice following LPS andCLP models of sepsis. (A) Age- and sex-matched WT (n 29) andMKP-2�/� (n 29) mice received i.p. injections of LPS (30 mg/kg).The percentage of alive mice was documented every 12 h for 5 daysfollowing injection. (B) Age- and sex-matched WT (n 20) andMKP-2�/� (n 20) mice underwent CLP (two punctures with a21-gauge needle). Survival was documented every 12 h for 7 daysfollowing injection. **, P 0.01.

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2�/� and WT BMDMs to determine the regulatory effect ofMKP-2 on the terminal kinases in the MAPK pathway. Con-sistent 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 WTBMDMs (Fig. 5). Under similar experimental conditions,phosphorylation of JNK and p38 was decreased in MKP-2�/�

BMDMs (Fig. 5) compared to that in WT BMDMs. These dataindicated that the absence of MKP-2 critically altered thephosphorylation state of ERK, JNK, and p38 in response toLPS, and we next sought to identify the link between thisregulation and the effect on cytokine production.

MKP-1 induction is increased in the absence of MKP-2. Asstated above, MKP-1 has been shown to be a negative regula-tor of the inflammatory response (10, 14, 23, 29), and interest-ingly, ERK activation results in decreased MKP-1 protein deg-radation (6, 9). Based on this knowledge, we suspected that

expression of MKP-2 via regulation of ERK activation couldaffect MKP-1 induction. Given our observations, we hypothe-sized that in the absence of MKP-2 and subsequent augmentedERK activation, MKP-1 expression would be significantly in-creased, resulting in reduced cytokine production. Consistentwith this hypothesis, we observed an increase in MKP-1 inMKP-2�/� BMDMs compared with that in WT BMDMs fol-lowing LPS stimulation (Fig. 6A). These data suggest a role forMKP-1 in the regulatory mechanism of MKP-2.

If increased expression of MKP-1 was responsible for theobserved reduction in MAPK activation and, thus, cytokineproduction in the MKP-2�/� cells, knockdown of MKP-1 inthis model should reverse this effect. Thus, we utilized RNAinterference directed at MKP-1 to determine if the increase inMKP-1 suppressed cytokine production in the MKP-2�/�

BMDMs. Consistent with our hypothesis, we detected a signifi-cant increase (�2-fold; P 0.01) in TNF-� production from

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�/�; at 24 h, n 11 for WT and n 11 forMKP-2�/�). Data are shown as mean � SEM. *, P 0.05; **, P 0.01.

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LPS-stimulated MKP-2�/� BMDMs transfected with siMKP-1compared to that from MKP-2�/� BMDMs transfected with non-targeting control siRNA (Fig. 6B). A similar 2-fold increase inTNF-� (P 0.05) production was also detected in LPS-stimu-lated WT BMDMs transfected with siMKP-1 compared to that inWT BMDMs transfected with nontargeting control siRNA (Fig.6C). However, absolute TNF-� production remained increased inWT BMDMs compared to that in MKP-2�/� BMDMs. Takentogether, these data suggest a strong mechanistic link betweenMKP-2 and regulation of MKP-1 expression in the modulation ofthe MAPK activation and cytokine production following LPSstimulation.

DISCUSSION

We aimed to study the role of MKP-2 in regulating theinflammatory response in sepsis by utilizing the MKP-2 nullmouse. Initially, we anticipated that MKP-2 would play a sim-ilar and perhaps redundant role to MKP-1 in negatively regu-lating cytokine production in response to a canonical inflam-matory trigger (e.g., LPS). Contrary to our initial hypothesis,we observed that mice lacking the DUSP MKP-2 exhibited adecreased inflammatory response following LPS stimulation.Furthermore, MKP-2�/� mice were conferred a survival ben-efit not only in this “sterile” in vivo model but also in thepolymicrobial CLP model of sepsis (Fig. 1B). This survivalbenefit correlated with significantly reduced production of pro-and anti-inflammatory cytokines (Fig. 2). These findings areintriguing, as it suggests that the functional role of MKP-2 inregulating the host’s immune response can involve modulatingthe cytokine response, but preliminarily, without affecting the

FIG. 3. MKP-2 regulates cytokine production in BMDMs. ELISAsfor immunoreactive TNF-� (A) and IL-10 (B) performed on MKP-2�/� and WT BMDM media following stimulation with LPS (100ng/ml) for the times indicated. Data are shown as mean � SEM (**,P 0.01). These data are representative of three independent bonemarrow harvests.

FIG. 4. MKP-2 is induced by TLR ligands. qRT-PCR (A) and Western blot analysis (B) results showing mRNA and protein induction ofMKP-2 in WT BMDMs following stimulation with LPS (100 ng/ml) for the times indicated. (C and D) qRT-PCR data indicating an increase inMKP-2 mRNA following stimulation of WT BMDMs with LTA (10 �g/ml) (C) and poly(I:C) (1 �g/ml) (D). qRT-PCR data are shown as mean �SEM. These data are representative of three independent BMDM preparations.

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ability to contain and/or eradicate a viable pathogen. Ongoingstudies are examining the effect of MKP-2 in regulating com-ponents of this immune response that are beyond the scope ofthese current studies, including antigen presentation, phago-cytosis, and oxidative burst. Immune responsive cells in theform of isolated BMDMs also exhibited decreased production

of archetypal pro- and anti-inflammatory cytokines TNF-� andIL-10 in in vitro studies. Mechanistically, this regulation ofcytokine production by MKP-2 was associated with alteredphosphorylation of ERK, JNK, and p38 as well as an increasedproduction of the negative regulatory DUSP, MKP-1. LPSstimulation of MKP-2�/� BMDMs increased phosphorylation

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) forthe times indicated. (A) Western blots of cell lysates probed with antibodies to the phosphorylated forms of JNK, p38, and ERK indicatingincreased phosphorylation of ERK and decreased phosphorylation of JNK and p38. (B) Graphs showing the percentages of phosphorylated MAPKgenerated by normalizing densitometry of phosphorylated MAPK to the densitometry of the signal for the corresponding nonphosphorylatedMAPK obtained after stripping and reprobing Western blots. These data are reported as the percentages of phosphorylated MAPK, and they arerepresentative of three experiments employing independent BMDM preparations.

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of ERK and decreased phosphorylation of both JNK and p38compared to that of BMDMs derived from WT mice (Fig. 5).Additionally, MKP-1 was increased in MKP-2�/� BMDMsstarting at 30 min following LPS stimulation (Fig. 6). Thisincrease in MKP-1, which is a negative regulator of both pro-and anti-inflammatory cytokine production via JNK and p38regulation (10, 14, 20, 23, 29) likely explains the decreasedcytokine production in the MKP-2�/� cells compared to WTcells. As further evidence of this mechanistic link betweenMKP-2 and MKP-1 expression, we also found that silencingMKP-1 in the MKP-2 null BMDMs reversed this phenotype, aswe observed increased production of TNF-� from MKP-1-silenced MKP-2�/� BMDMs (Fig. 6B). These data suggest thatincreased expression of MKP-1 driven by augmented ERKactivation in the absence of MKP-2 was involved in the atten-uated cytokine production.

Given these observations, we believe that our data demon-strate a critical mechanism of cross talk between MKP-2 andMKP-1 that is mediated through ERK activation. This is highlyplausible given prior findings that ERK activation is importantfor maximal induction of MKP-1 (24) and stabilization of theMKP-1 protein (6, 9). Furthermore, this link likely explains our

findings that all three terminal MAPKs were altered in theabsence of MKP-2. This was initially not expected, as severalstudies have shown that MKP-2 has much a higher specificityfor ERK than for either JNK or p38 (8, 11, 13, 19). Thus, it isnot surprising that in the absence of MKP-2, we observedincreased activation of ERK; however, given that we also ob-served increased expression of MKP-1, a known regulator ofJNK and p38, it is likely that MKP-1 is responsible for thesignificant reduction in phosphorylation of JNK and p38 ob-served in the LPS-stimulated MKP-2 null BMDMs. We there-fore propose a mechanism of MKP-2 regulation of cytokineproduction in which, following LPS binding to TLR4, the threearms of the MAPK pathway are simultaneously activated (Fig.7). JNK and p38 are directly involved in the induction andproduction of proinflammatory cytokines, while ERK activa-tion leads to induction and stabilization of MKP-1, whichserves as a negative regulator of proinflammatory cytokineproduction by inhibiting the action of JNK and p38 (10, 14, 20,23, 29). Subsequently, MKP-2 is induced to deactivate ERKand thus destabilize MKP-1, resetting the cellular mechanismfor cytokine production.

Since MKPs remove phosphates on activated MAPKs, this

FIG. 6. Increased levels of MKP-1 in MKP-2�/� BMDMs attenuate cytokine production. (A) Western blots of WT and MKP-2�/� BMDMwhole-cell lysates probed with antibodies to MKP-1 indicate increased levels of MKP-1 in MKP-2�/� BMDMs compared to that in WT BMDMs.(B) ELISA for immunoreactive TNF-� in cell media after transfection of MKP-2�/� BMDMs with control siRNA or siMKP-1 and subsequent LPSstimulation (100 ng/ml) for 2 h shows that cytokine production is restored in MKP-2�/� BMDMs following MKP-1 knockdown (as indicated bydecreased protein levels detected by Western blotting). (C) ELISA for immunoreactive TNF-� in cell media after transfection of BMDMs withcontrol siRNA or siMKP-1 and subsequent LPS stimulation (100 ng/ml) for 2 h shows similar fold increases in WT and MKP-2�/� BMDMsfollowing MKP-1 knockdown. Data are representative of three independent BMDM isolations and are shown as mean � SEM (*, P 0.01).

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putative role of a phosphatase as a positive regulator of theinflammatory response is surprising; however, such a role hasbeen demonstrated for a related MKP, PAC-1 (16). Similar toour results, Jeffrey et al. showed decreased cytokine produc-tion by PAC-1�/� macrophages in response to LPS. MKP-2 ispresent in a variety of tissues (19) whereas PAC-1 is limited toimmune cells (16). It is plausible that the redundancy of thepositive regulation on the inflammatory response for these twoMKPs is necessary in immune cells, but the ubiquitous natureof MKP-2 may provide a more global positive regulation. Thecomplex nature of regulation of the MAPK pathway by theDUSPs is not completely understood but may impact the reg-ulation of scaffolding and cellular trafficking (7) as well as theflexibility of the cellular response to amount and type of stimuli(3). Although our data support a positive regulatory role forMKP-2 involving ERK-mediated cross talk with MKP-1, fur-ther studies are under way to define the precise interactionsinvolved between MKP-2, ERK, and MKP-1. We are addition-ally pursuing the effects of other TLR agonists on MKP-2induction as well as the effects of MKP-2 overexpression inorder to gain more insight into the regulatory role of MKP-2.Finally, we acknowledge that the altered cytokine productionmay be the result of preconditioned or altered phenotypicmacrophages. Further investigation is needed to understandthe impact MKP-2 has on macrophage development as well asinvolvement of other regulators besides MKP-1 that are re-sponsible for the altered cytokine response.

An additional limit to the current studies is the complexnature of cytokine production in vivo in response to LPS.Although we used BMDMs to investigate the regulatory role ofMKP-2 in cytokine production, the exact role of macrophagesin the improved survival noted in our MKP-2�/� mice is un-known. Further studies are under way investigating both po-tential alterations in the hematopoietic differentiation and im-pact 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 theregulatory role of MKP-2 in both hematopoietic differentiationand inflammatory/immune response.

In summary, our studies demonstrated a survival benefitassociated with decreases in both pro- and anti-inflammatorycytokines in the absence of MKP-2 and an increase in thephosphorylation of ERK, while there is a decrease in phosphor-ylation of JNK and p38 concomitant with an increase in MKP-1induction. These data suggest a cross talk mechanism (Fig. 7)by which MKP-1 is involved in the global regulation of cytokineproduction by MKP-2.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grantsK12GM076344 (T.T.C.) and RO1GM66839 (T.P.S.). We thank thePediatric Critical Care Scientist Development Program (PCCSDP) forsupporting the work of T.T.C.

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

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