Anti-inflammatory properties of prostaglandin E2: Deletionof microsomal prostaglandin E synthase-1 exacerbates non-immuneinflammatory arthritis in mice

Andrey Frolov a, Lihua Yang a, Hua Dong b, Bruce D. Hammock b, Leslie J. Crofford a,n

a Division of Rheumatology, Department of Internal Medicine, University of Kentucky, Lexington, KY 40536, USAb Department of Entomology and UCD Cancer Center, University of California, Davis, CA 95616, USA

a r t i c l e i n f o

Article history:Received 21 June 2013Received in revised form7 August 2013Accepted 14 August 2013

Keywords:ProstaglandinArthritisInflammationmPGES-1

a b s t r a c t

Prostanoids and PGE2 in particular have been long viewed as one of the major mediators of inflammationin arthritis. However, experimental data indicate that PGE2 can serve both pro- and anti-inflammatoryfunctions. We have previously shown (Kojima et al., J. Immunol. 180 (2008) 8361–8368) that microsomalprostaglandin E synthase-1 (mPGES-1) deletion, which regulates PGE2 production, resulted in thesuppression of collagen-induced arthritis (CIA) in mice. This suppression was attributable, at least in part,to the impaired generation of type II collagen autoantibodies. In order to examine the function of mPGES-1 and PGE2 in a non-autoimmune form of arthritis, we used the collagen antibody-induced arthritis(CAIA) model in mice deficient in mPGES-1, thereby bypassing the engagement of the adaptive immuneresponse in arthritis development. Here we report that mPGES-1 deletion significantly increased CAIAdisease severity. The latter was associated with a significant (�3.6) upregulation of neutrophil, but notmacrophage, recruitment to the inflamed joints. The lipidomic analysis of the arthritic mouse paws byquantitative liquid chromatography/tandem mass-spectrometry (LC/MS/MS) revealed a dramatic (�59-fold) reduction of PGE2 at the peak of arthritis. Altogether, this study highlights mPGES-1 and its productPGE2 as important negative regulators of neutrophil-mediated inflammation and suggests that specificmPGES-1 inhibitors may have differential effects on different types of inflammation. Furthermore,neutrophil-mediated diseases could be exacerbated by inhibition of mPGES-1.

& 2013 Published by Elsevier Ltd.

1. Introduction

Prostaglandins (PG) are important lipid mediators known toregulate a broad spectrum of physiological functions, includingimmunity and inflammation [1,2]. PG are produced from arachi-donic acid (ARA), which is released from membrane phospholipidsby phospholipases [3] or synthesized from linoleic acid (LA) [4].

The ARA metabolic cascade includes a rate-limiting step of ARAconversion into PGH2 which is controlled by the cyclooxygenase(COX) isoforms 1 and 2 [5]. PGH2 is then quickly processed toprostanoids, such as PGF2, PGD2, and PGE2, by their respective PGisomerases [6] (Fig. 1).

Prostanoids and PGE2 in particular, have been shown to play animportant role in as mediators of symptoms of inflammatory arthri-tidies including rheumatoid arthritis (RA) as demonstrated by thewidespread use of treatments that inhibit PG production [7–10].However, the current strategies to inhibit PGs by using non-steroidalanti-inflammatory drugs (NSAID) and selective COX-2 inhibitors areoften inadequate in providing symptomatic relief of pain, swelling, andstiffness [11,12]. Setting aside the incomplete blocking efficiency ofNSAID and COX-2 selective inhibitors in vivo, the limited ability ofthese agents to curb inflammation could be explained, at least in part,by two-faceted and opposing roles of PG, especially PGE2, in theregulation of the inflammatory process. Indeed, the lipidomic analysisof animals with Lyme arthritis revealed that among major PG, onlyPGE2 production in arthritic joints was undergoing a cyclic change andwas rising to a similar level during both the initiation and theresolution stage of the inflammatory disease [13]. This would be

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Prostaglandins, Leukotrienes and EssentialFatty Acids

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Abbreviations: ARA, arachidonic acid; RA, rheumatoid arthritis;MPO, myeloperoxidase; IL-6, interleukin 6; LC/MS/MS, liquid chromatography/tandem mass spectrometry; PGE2, prostaglandin E2; PGD2, prostaglandin D2;PGF2α, prostaglandin F2α; 5-HETE, 5-hydroxyeicosatetraenoic acid; 9-HETE,9-hydroxyeicosatetraenoic acid; 11-HETE, 11-hydroxyeicosatetraenoic acid;15-HETE, 15-hydroxyeicosatetraenoic acid; 9,10-DiHOME,(7)9,10-dihydroxy-12Z-octadecenoic acid; 14,15-DiHETrE,(7)14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid; 9,10-EpOME,(7)9(10)-epoxy-12Z-octadecenoic acid; 14,15-EpETrE,(7)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid

n Correspondence to: Division of Rheumatology & Immunology, Department ofInternal Medicine, Vanderbilt University, 1161 21st Ave S, T3113 MCN, Nashville, TN37232. Tel.: þ1 615 322 4746; fax: þ1 615 322 6248.

E-mail address: leslie.j.crofford@vanderbilt.edu (L.J. Crofford).

Prostaglandins, Leukotrienes and Essential Fatty Acids 89 (2013) 351–358

indicative of a dual role of PGE2 in regulation of inflammation: itcould mediate inflammation at the initiation stage while inhibitinginflammation during resolution or by triggering a resolution pro-gram. The requirement of PGE2 for the resolution of inflammationwas confirmed in the recent studies where mice at the chronic stageof the autoimmune arthritis, when treated with the specific COX-2inhibitor, displayed an increased disease severity which was cor-rected downwards upon repletion with a synthetic PGE2 analog [14].A growing body of evidence indicates that PGE2 apparently exerts itsanti-inflammatory functions at both a cellular and molecular levels.The cellular mode of PGE2 anti-inflammatory functions includes sup-pression of neutrophil functions [15], reprogramming the classicallyactivated macrophages (M1) into the alternatively activated macro-phages (M2) [16], and skewing monocyte-to-macrophage differen-tiation toward M2 [17]. At the molecular level, PGE2 has been shownto negatively regulate inflammation by inhibiting CCL5 expression inactivated macrophages [18], suppressing macrophage and synovialfibroblast (SF) TNF-α expression induced by T cell-derived IL-17 [19],blocking NF-κB activation and NF-κB/DNA binding in response to LPSstimulation [20], as well as by differentially regulating nucleartranslocation of p65 and p50 NF-κB subunits following SF stimulationwith IL-1β and TNF-α. In the latter case, PGE2 stimulated p50 nuclearaccumulation and inhibited that of p65 thereby promoting formationof p50p50 homodimer and reducing p50p65 heterodimer formationthus efficiently blocking expression of the inflammatory genes [21]. Itshould be noted that sustained p50p50 homodimer nuclear accu-mulation was shown to play a central role in the resolution ofinflammation [22–24]. Finally, PGE2 can exert its anti-inflammatoryfunction by switching ARA metabolism toward production of anti-inflammatory and pro-resolution lipids, including lipoxin A4 (LXA4)as well as E- and D-series resolvins and protectins [25].

PGE2 production during acute and chronic inflammation isdependent on the enzyme mPGES-1, an inducible enzyme whoseexpression is upregulated strongly under inflammatory conditions.mPGES-1 functions in the ARA metabolic cascade downstream fromCOX-2 by catalyzing the isomerization of the endoperoxide PGH2

into PGE2 [26,27] (Fig. 1). mPGES-1 works alongside two other PGH2isomerases, cytosolic PGES (cPGES) and mPGES-2, but the latter twoenzymes are constitutively expressed and are mostly responsible forbasal production of PGE2 [26]. Studies on the deletion of mPGES-1gene in mice revealed its essential role in female fertility, pain,atherosclerosis, tumorigenesis, and inflammation which made thisenzyme an attractive therapeutic target [28].

Our group [29] and others [30] by using CIA model havedemonstrated a significant reduction in the incidence and theseverity of this autoimmune arthritis in mPGES-1 null mice. TheCIA model is dependent on developing cellular and humoralimmune responses to type II collagen. We found that themPGES-1 null mice had a profound defect in their ability togenerate collagen-II autoantibodies [29]. Therefore, we reasonedthat an anti-inflammatory role of PGE2 might be masked by astrong effect of PGE2 in the regulation of humoral immunity. Wenow test this hypothesis by using microsomal PGE synthase-1(mPGES-1) KO mice. In order to bypass the autoantibody produc-tion step in arthritis development and correctly ascertain a role forPGE2 in the regulation of systemic and local inflammation, wechose to induce arthritis in mPGES-1 KO mice through directinjection of collagen-II antibody, i.e. utilizing the well establishedCAIA model [31,32]. In this model, the inflammatory response istriggered after the ligation of Fcγ receptors (FcγR) on polymorpho-nuclear cells and macrophages by immune complexes [31,33].

In the present study we confirmed our hypothesis and demon-strated that mPGES-1, and consequently, PGE2 deficiency, signifi-cantly increased the incidence and severity of CAIA in mice.Furthermore, analysis of specific inflammatory cell markers in thearthritic paws pointed toward a major involvement of neutrophils,and not macrophages, in the maintenance of local inflammation inthe mPGES-1 KO animals. Lipidomic profiling of the inflamed jointsof arthritic mice revealed changes in the ARA metabolic pathway thatwere not limited to COX-dependent PGE2 production and couldinclude all branches of the ARA metabolic pathway.

Altogether, our studies further emphasize the pleiotropic activ-ities of mPGES-1-PGE2 in inflammatory arthritis. The developmentof mPGES-1 inhibitors for treatment of inflammation and pain willneed to account for diversity in the mediators of different forms ofarthritis that are likely to influence efficacy of these agents.

2. Materials and methods

2.1. Animals

DBA1/lac J mPGES-1 þ/� mice were obtained from Pfizer andwere described elsewhere [29]. mPGES-1 �/� mice wereobtained by mating mPGES-1 þ/� animals. Wild type littermateswere used as control. Mice were housed in microisolator cages in apathogen-free barrier facility, and all experiments were performedunder the Institutional Animal Care and Use Committee guidelinesas set forth by the University of Kentucky, Lexington KY.

2.2. Arthritis model

CAIA was induced in 8–12 week old, randomly selected maleand female mice using mouse monoclonal anti-type II collagen5 clone antibody cocktail kit with lipopolysaccharide (LPS) fromEscherichia coli 0111:B4 (Chondrex) following the manufacturer′sinstructions. Mice were immunized on day 0 by intraperitoneal(IP) injection of 150 μg of the antibody cocktail and boosted with25 μg of LPS on day 3. The CAIA severity was assessed usingChondrex mouse arthritis scoring system and was shown to reachits peak on day 7 (http://www.chondrex.com/animal-models/athrogen-cia-arthritogenic-monoclonal-antibody-cocktail) [32].

Fig. 1. Eicosanoid biosynthetic pathway. The plain symbols depict proteins andbiosynthetic pathways. The boxed symbols identify ARA and LA as well as theirmetabolites that were measured in the current study.

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2.3. Real-time RT-PCR

Total RNA was obtained from the synovial capsules that wereisolated on day 7 from the front paws of arthritic mice with anarthritis score of Z3. The RNA isolation was performed withTRIzol reagent (Ambion) following the manufacturer′s instruc-tions. Total RNA was quantitatively converted into the singlestranded cDNA by using High Capacity cDNA Archive Kit (AppliedBiosystems). Particular genes were detected using the respectiveTaqMan Gene Expression Assays (Applied Biosystems) on a 7300Real-Time RT-PCR system from the same manufacturer by relativequantitation employing glyceraldehyde 3-phosphate dehydrogen-ase (GAPDH) as the reference gene.

2.4. Lipid extraction

Lipids were extracted from pulverized front mouse paws asdescribed [13] with the following modifications. After pulveriza-tion, the respective frozen powder was immediately placed intothe Pyrex 10 ml screw cap analytical tube with the 3 ml of ice-cold50% ethanol and weighted. The sample wet weight was later usedfor the normalization of eicosanoid levels. 10 μl of antioxidantmixture (0.2 mg/ml butylated hydroxytoluene and 0.2 mg/mlEDTA in 2:1:1 methanol:ethanol:H2O solution) was added to eachtube and samples were kept at �80 1C for a few days prior to theirovernight shipment on dry ice to the lipid analytical facility.

2.5. Analysis of eicosanoids by LC/MS/MS

This was carried out as previously described in detail [34]. Briefly,an Agilent 1200 SL liquid chromatography series (Agilent Corporation,Palo Alto, CA) with an Agilent Eclipse Plus C18 2.1�150mm, 1.8 mmcolumn were used for the eicosanoid separation. The mobile phase Awas water with 0.1% acetic acid while the mobile phase B wascomposed by acetonitrile/methanol (80/15, v/v) and 0.1% acetic acid.Gradient elution was performed at a flow rate of 250 ml/min. Theinjection volume was 10 ml and the samples were kept at 4 1C in theauto sampler. Analytes were detected by negative MRM mode using a4000 QTrap tandem mass spectrometer (Applied Biosystems)equipped with an electrospray ionization source (Turbo V). Calibrationcurves were generated by 10 ml injections of seven standards contain-ing each analyte, internal standard I (d4-PGE2, d11-14,15-DiHETrE, d8-5-HETE, and d11-11(12)-EpETrE), and internal standard II (1-cyclo-hexyl-dodecanoic acid urea, CUDA) for quantification purpose.

3. Results

3.1. CAIA is exacerbated in mPGES-1KO mice

Using the described protocol, CAIA in mice is a highly reprodu-cible experimental arthritis model the inflammation reaches amaximum at D7 and persists for two weeks [32]. Because we wereinterested in examining the effect of mPGES-1 deficiency on inflam-mation when the latter reaches its maximum, we did not extendour analysis of experimental animals with CAIA beyond D7 (Fig. 2).As shown in this figure, twomajor indicators of arthritis, its incidenceand clinical score, were significantly higher in mPGES-1 KO com-pared to WT mice on D7 (Fig. 2A and B). It is worth noting thatarthritis was numerically though not statistically more severe inmPGES-1 KO mice throughout the course of the experiment (Fig. 2Aand B). The animal body weights, however, were not differentcomparing mPGES-1 null and WT mice (Fig. 2C). The data wouldbe consistent with the requirement of mPGES-1, and consequentlyPGE2, primarily for the suppressive effect on local, and not systemic,inflammation in the CAIA model of inflammatory arthritis.

3.2. Increased neutrophil infiltration into the inflamed jointsof mPGES-1 KO mice with CAIA

It was previously shown that CAIA was driven by the recruitmentof neutrophils and macrophages to the inflamed joints [31,33]. There-fore, in order to elucidate a mechanism which is responsible for theobserved increased CAIA severity in mPGES-1 mice (Fig. 2), weassessed the infiltration of neutrophils and macrophages into thearthritic joints of WT and mPGES-1 KO mice with CAIA at its peak bymeasuring the expression of the respective specific markers on D7 by

Fig. 2. Increased severity of inflammatory arthritis in mPGES-1 KO mice. WT andmPGES-1 KO mice were injected with mouse monoclonal anti-type II collagen5 clone antibody cocktail kit with lipopolysaccharide (LPS) from Escherichia coli0111:B4 on D0 and boosted with LPS on D3. The arthritis severity was assessed byarthritis score (A) and by the number of inflamed paws (B). The animal body weight(C) was used as a surrogate, whole-body inflammation marker. Data shown aremean7SE. (n) depicts Pr0.05 as compared to control (Student t-test, n¼9). (nn)depicts Pr0.05 as compared to control (Mann–Whitney rank sum test, n¼9).

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real-time RT-PCR. We used myeloperoxidase (MPO) as the neutrophilspecific marker [35,36] as well as IL-6 [37,38] and PTGS2 (COX-2)[39,40] as the dual neutrophil/macrophage inflammatory markers. Wedetailed further the phenotype of activated macrophages in thearthritic joints by probing the expression of the classically activated(M1, inflammatory) and alternatively activated (M2, anti-inflamma-tory) specific markers. We used CCL3 and NOS2 to detect M1macrophages as well as CD163 and CD206 for the M2 macrophageidentification [38,41]. The respective data are presented in Figs. 3–5.As it is seen from Fig. 3, CAIA in mPGES-1 KO mice at its peak on D7 ischaracterized by significantly increased MPO (Fig. 3A, �3.6-fold) and

PTGS2 (Fig. 3C, �2.5-fold) expression as compared to the WT mice.Expression of IL-6 demonstrated a trend toward upregulation (Fig. 3B,�2.5-fold), although this change did not reach statistical significance.Given that PTGS2 could be expressed by both neutrophils andmacrophages, the above data while being clearly indicative of theincreased infiltration of neutrophils into the arthritic joints of mPGES-1 KO mice with CAIA, leave in question the respective macrophageinfiltration. We addressed the latter by measuring expression of theM1 and M2 macrophage specific markers. (Figs.4 and 5). As in thesefigures, neither M1 (Fig. 4) nor M2 (Fig. 5) macrophage markersdemonstrated any change, thereby efficiently ruling out upregulatedmacrophage infiltration as a dominant force for the increased severityof the inflammatory arthritis in mPGES-1 KO mice.

3.3. Lipidomic profiling of CAIA in mPGES-1 KO mice

Although a role of ARA metabolites, such as lipoxins, resolvins,and protectins in resolution of inflammation is well established[25], very little is known regarding the function of eicosanoids asanti-inflammatory agents in arthritis [13]. In order to gain moreinsight into the role of ARA metabolites, and particularly PGE2, ininflammatory arthritis, we performed lipidomic analysis of frontpaws from the unelicited (non-immunized) WT and mPGES-1 KOmice as well as from the mice with CAIA when the latter reachedits peak on D7. The results are presented in Figs. 6 and 7 where theARA metabolites are grouped according to their respective biosyn-thetic pathways (Fig. 1),

Fig. 3. Analysis of inflammatory markers in arthritic paws of WT and mPGES-1 KOanimals by real-time RT-PCR. Total RNA was obtained from a synovial capsuleisolated from the front arthritic paw on D7. Relative mRNA expression of MPO (A),IL-6 (B), and PTGS2 (C) was measured using the respective TaqMan Gene Expres-sion Assays from Applied Biosystems. Each datum point represents a single animal.Two animal groups were compared by Student t-test with the Pr0.05 consideredto be significant (n¼9).

Fig. 4. Analysis of the classically activated (M1) macrophage markers in arthriticpaws of WT and mPGES-1 KO animals by real-time RT-PCR. Total RNA was obtainedfrom a synovial capsule isolated from the front arthritic paw (arthritis score Z3)on D7. Relative mRNA expression of CCL3 (A) and NOS2 (B) was measured using therespective TaqMan Gene Expression Assays from Applied Biosystems. Each datapoint represents a single animal. Two animal groups were compared by Studentt-test with the Pr0.05 considered to be significant (n¼9).

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3.3.1. COX-dependent pathwayAs seen in Fig. 6A, genetic deletion of mPGES-1 did not result in

the statistically significant differences in the basal production ofCOX-dependent ARA metabolites. At the same time, there was atrend toward �2.2-fold reduction in the basal PGE2 productionwith the concomitant trends toward �2.7-fold upregulation ofPGD2 and �2-fold reduction of 15-deoxy-PGJ2 (Fig. 6A). Moreimportantly, in the setting of inflammatory arthritis, mPGES-1 KOas compared to WT animals displayed a dramatic (�59-fold) andstatistically significant reduction in PGE2 production, whereas thesizable downregulation of PGD2 (�3.2-fold), 6-keto-PGF1α (�2.2-fold), and PGF2α (�19-fold) levels can be only viewed as a trend(Fig. 6A). We were unable to measure a change in 15-deoxy-PGJ2levels during arthritis induction in either the WT or mPGES-1 nullmice (Fig. 6A). These data are consistent with mPGES-1 being adominant PGH2 isomerase which is responsible for PGE2 produc-tion in both the naïve and arthritic mouse joints.

3.3.2. LOX-dependent pathwayThere was no significant difference in 5-HETE, 12-HETE, and

15-HETE levels between naïve WT and mPGES-1 KO mice, whereastheir production in arthritic mPGES-1 KO mice vs WT mice witharthritis showed a trend toward reduction by respectively �3.4,3.8, and 2.6-fold (Fig. 6B).

3.3.3. CYP450-dependent pathwayThis data set revealed that only ARA metabolites, 14(15)-EET

and 11(12)-EET, and not the LA derivatives, 12,13DiHOME, 9,10-DiHOME, 9(10)-EpOME, and 12(13)-EpOME could have beenaffected by mPGES-1 deletion in arthritic animals. As compared

Fig. 5. Analysis of the alternatively activated (M2) macrophage markers in arthriticpaws of WT and mPGES-1 KO animals by real-time RT-PCR. Total RNA was obtainedfrom a synovial capsule isolated from the front arthritic paw (arthritis score Z3)on D7. Relative mRNA expression of CD163 (A) and CD206 (B) was measured usingthe respective TaqMan Gene Expression Assays from Applied Biosystems. Eachdatum point represents a single animal. Two animal groups were compared byStudent t-test with the Pr0.05 considered to be significant (n¼9).

Fig. 6. Quantitative eicosanoid profiling of arthritic paws from WT and mPGES-1KO animals by LC/MS/MS. Lipids were extracted from the front paws of WT control,WT arthritic (arthritis score Z3 on D7), mPGES-1 KO control, mPGES-1 KO arthritic(arthritis score Z3 on D7). The lipid analytes were grouped based on theirrespective metabolic pathways: COX (A), LOX (B), and CYP450 (C) (Fig. 1). Datashown are mean7SE. (*) depicts Pr0.05 as compared to WT arthritic (n¼3–5).

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to the WT mice with arthritis, the respective 14(15)-EET and 11(12)-EET displayed a tendency toward reduction in arthriticmPGES-1 KO mice by �2.9 and �2.0-fold (Fig. 6C).

3.3.4. Non-enzymatic pathwayWhile the 8-HETE level displayed no difference when it was

measured in WT and mPGES-1 under the basal and arthriticconditions, 11-HETE production in the naïve mPGES-1 KO vs WTmice showed a trend toward reduction by �1.7-fold and thisdisparity had reached �5.6-fold difference when the average11-HETE levels were compared between arthritic WT andmPGES-1 KO animals (Fig. 7A).

3.3.5. sEH pathwayThe strong anti-inflammatory properties of EETs are well-

established and their physiological levels are mainly regulated bytheir conversion into inactive or less active DHETs which iscatalyzed by sEH [42]. We have already noticed that m-PGES-1KO mice with arthritis were characterized by a significantly loweraverage levels of 11.12-EET and 14,15-EET as compared to their WTcounterparts (Fig. 6C). Since this apparent reduction could beattributable to the upregulated sEH activity, we have measured thelevels of the respective EET metabolites, 11,12-DHET and 14,15-DHET (Fig. 7B). The data in Fig. 7B show a tendency towardreduction of 11,12-DHET and 14,15-DHET levels in the arthriticmPGES-1 KO mice vs WT mice with arthritis by respectively �2.0and 3.4-fold. The calculated ratios of 11,12-DHET/11,12-EET for the

arthritic WT and mPGES-1 KO were quite similar (�0.41 and�0.44) so were the respective 14,15-DHET/14,15-EET values(�0.55 and �0.55). This supports the hypothesis that sEH activityremains intact in the arthritic mPGES-1 KO mice.

4. Discussion and conclusions

Prostanoids and particularly PGE2 have often been viewed assimply proinflammatory mediators in arthritis [7–10]. This issomewhat surprising because an important role of PGE2 in theresolution of inflammation where it switches ARA metabolismtoward production of anti-inflammatory and pro-resolving lipox-ins, resolvins, and protectins is well established [25]. Moreover,exogenous PGE1 was shown to curb nephritis and adjuvantarthritis in experimental animals [43,44]. However, there is agrowing body of clinical data [11,12] and experimental studies[13,14] pointing toward a more complicated and sophisticated roleof PGE2 in regulation of inflammatory arthritis. A recent study [14]identified an important role for PGE2 in resolving inflammation inthe autoimmune (CIA) model of experimental arthritis throughregulation of the anti-inflammatory lipid, lipoxin A4, therebyserving as pro-resolving lipid molecule. There is a growing under-standing of differences between anti-inflammatory and pro-resolving modes of action. The former mainly targets neutrophilrecruitment to the site of inflammation whereas the latter pro-motes the removal of apoptotic cells and infectious microorgan-isms by macrophages [25]. Therefore, we designed our study withthe aim of examining the anti-inflammatory functions of PGE2using mPGES-1 KO mice and CAIA as a model of inflammatoryarthritis.

The current study provides several important insights into theregulation of inflammatory arthritis by mPGES-1 and its product,PGE2. We established that mPGES-1 deficiency increased both theincidence and the severity of CAIA in mice. These results are in theagreement with those of Zurier and Quagliata [44] who demon-strated the anti-inflammatory effect of PGE1 in the adjuvantmodel of inflammatory arthritis. However, our results disagreewith those of Kamei et al. [45] who reported on the suppression ofCAIA in the mPGES-1 KO mice. The above discrepancy could beexplained by the different genetic background of mPGES-1 KOanimals, pure DBA1/lac J background used in our study and amixed C57BL/6�129/SvJ background used by Kamei et al. [45]. Yetthe different approach for mPGES-1 gene deletion and CAIAinduction could also add to the observed discrepancy. The factthat both arthritis score and incidence were increased in the PGES-1 KO animals with arthritis, but the loss of the body weight inarthritic mPGES-1 KO animals was similar to that of arthritic WTmice, would be consistent with local and not systemic inflamma-tion being primarily affected by the deletion of mPGES-1 gene.

The above conclusion is reinforced by our observation that ascompared to the WT mice, the peak of CAIA in the mPGES-1 KOanimals coincided with significantly (�3.6-fold) upregulated neu-trophil, and not macrophage infiltration. Therefore, in consideringanti-inflammatory versus pro-resolving modes of action [25],mPGES-1 and consequently PGE2, appear to exert primarily anti-inflammatory effects in the CAIA model of inflammatory arthritis.That said, mPGES-1 deletion results in other changes in eicosanoidmetabolism in addition to a dramatic reduction in PGE2 produc-tion. PGD2 and 6-keto-PGF1α increased in mPGES-1-deficientarthritic mice compared with control, albeit lower than levels inarthritis WT mice. Nevertheless, the ratio of PGE2 and other PG aredramatically altered by mPGES-1 deficiency and it is certainlyplausible that altered relative levels of eicosanoids could accountfor the increased arthritis severity in this model.

Fig. 7. Quantitative eicosanoid profiling of arthritic paws from WT and mPGES-1KO animals by LC/MS/MS. Lipids were extracted from the front paws of WT control,WT arthritic (arthritis score Z3 on D7), mPGES-1 KO control, mPGES-1 KO arthritic(arthritis score Z3 on D7). The lipid analytes were grouped based on theirrespective metabolic pathways: Non-Enzymatic (A) and sEH (B) (Fig. 1). Datashown are mean7SE (n¼3–5).

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Finally, the involvement of PGE2 in promoting the anti-inflam-matory action of mPGES-1 is supported by our lipidomics data thatdemonstrated a staggering (�59-fold) PGE2 reduction at the peakof inflammatory arthritis. It should be noted, that mPGES-1 isconstitutively expressed at high level in neutrophils and isstrongly upregulated by inflammatory stimuli such as LPS [46].More importantly, mPGES-1 expression level correlates stronglywith PGE2 production [46]. These data provide a strong support tothe notion that PGE2 might exert its anti-inflammatory effectthrough the intracrine/autocrine negative regulation of neutrophilrecruitment and function, including the suppression of theneutrophil-mediated phagocytosis [15]. The anti-inflammatorymode of PGE2 could be masked in the autoimmune model ofarthritis (CIA) [14,29] by its strong positive regulation of autoanti-body formation [29]. Indeed, PGE2 may play a critical role at theintersection of innate and acquired immunity.

These conclusions are supported by the work of Serhan andcolleagues who demonstrate that early events in an inflammatoryresponse set the resolution program in motion. In this model,specialized pro-resolving mediators, including lipoxins, resolvins,and protectins, are actively generated and act on neutrophils andmacrophages to promote resolution of inflammation [25]. PG,including PGE2, involved in the initiation phase of inflammationactivate the translation of mRNAs encoding enzymes that arenecessary for production of these immunoresolvents. Inhibitorsof COX-2, in fact, delay the resolution of inflammation [47,48]. Insummary, all of these data point toward mPGES-1 as a pivotalregulator in the CAIA model of arthritis by inhibition of neutrophilfunction. When developing and using selective mPGES-1 inhibi-tors, attention must be paid to the specific mediators of differentforms of arthritis in evaluating potential clinical targets for theseagents.

Acknowledgment

This work was supported by RO1AR049010 (to L.J.C.) from theNational Institutes of Health. Partial support for analytical chem-istry was provided by NIEHS RO1 ES ES002710 and NIEHS Super-fund Research Program grant P42 ES004699, or NIH U24DK097154(to B.D.H.). B.D.H. is a George and Judy Marcus senior fellow of theAmerican Asthma Foundation.

References

[1] S.G. Harris, J. Padilla, L. Koumas, D. Ray, R.P. Phipps, Prostaglandins asmodulators of immunity, Trends Immunol. 123 (2002). (44-150).

[2] E. Ricciotti, G.A. Fitzgerald, Prostaglandins and inflammation, Arterioscler.Thromb. Vasc. Biol. 31 (2011) 986–1000.

[3] J.E. Burke, E.A. Dennis, Phospholipase A2 structure/function, mechanism, andsignaling, J. Lipid Res. (50 Suppl.) (2009) S237–S242.

[4] K.L. Fritsche, Too much linoleic acid promotes inflammation-doesn′t it?Prostaglandins Leukot. Essent. Fatty Acids 79 (2008) 173–175.

[5] C.A. Rouzer, L.J. Marnett, Cyclooxygenases: structural and functional insights, J.Lipid Res. (50 Suppl.) (2009) S29–S34.

[6] E.M. Smyth, T. Grosser, M. Wang, Y. Yu, G.A. Fitzgerald, Prostanoids in healthand disease, J. Lipid Res. (50 Suppl.) (2009) S423–S428.

[7] L.K. Myers, A.H. Kang, A.E. Postlethwaite, E.F. Rosloniec, S.G. Morham,B.V. Shlopov, S. Goorha, L.R. Ballou, The genetic ablation of cyclooxygenase2 prevents the development of autoimmune arthritis, Arthritis Rheum. 43(2000) 2687–2693.

[8] D.R. Robinson, A.H. Tashjian Jr., L. Levine, Prostaglandin-induced bone resorp-tion by rheumatoid synovia, Trans. Assoc. Am. Physicians 88 (1975) 146–160.

[9] L.J. Crofford, Is there a place for non-selective NSAIDs in the treatment ofarthritis? Joint Bone Spine 69 (2002) 4–7.

[10] T. Shibata-Nozaki, H. Ito, H. Mitomi, J. Akaogi, T. Komagata, T. Kanaji,T. Maruyama, T. Mori, S. Nomoto, S. Ozaki, H. Yamada, Endogenous prosta-glandin E2 inhibits aberrant overgrowth of rheumatoid synovial tissue and thedevelopment of osteoclast activity through EP4 receptor, Arthritis Rheum. 63(2011) 2595–2605.

[11] I. Melnikova, Future of COX2 inhibitors, Nat. Rev. Drug Discovery 4 (2005)453–454.

[12] R.F. van Vollenhoven, Treatment of rheumatoid arthritis: state of the art 2009,Nat. Rev. Rheumatol. 5 (2009) 531–541.

[13] V.A. Blaho, M.W. Buczynski, C.R. Brown, E.A. Dennis, Lipidomic analysis ofdynamic eicosanoid responses during the induction and resolution of Lymearthritis, J. Biol. Chem. 284 (2009) 21599–21612.

[14] M.M. Chan, A.R. Moore, Resolution of inflammation in murine autoimmunearthritis is disrupted by cyclooxygenase-2 inhibition and restored by prosta-glandin E2-mediated lipoxin A4 production, J. Immunol. 184 (2010)6418–6426.

[15] R.J. Smith, Modulation of phagocytosis by and lysosomal enzyme secretionfrom guinea-pig neutrophils: effect of nonsteroid anti-inflammatory agentsand prostaglindins, J. Pharmacol. Exp. Ther. 200 (1977) 647–657.

[16] K. Nemeth, A. Leelahavanichkul, P.S. Yuen, B. Mayer, A. Parmelee, K. Doi, P.G. Robey, K. Leelahavanichkul, B.H. Koller, J.M. Brown, X. Hu, I. Jelinek, R.A. Star, E. Mezey, Bone marrow stromal cells attenuate sepsis via prostaglan-din E(2)-dependent reprogramming of host macrophages to increase theirinterleukin-10 production, Nat. Med. 15 (2009) 42–49.

[17] M. Heusinkveld, Steenwijk PJ de Vos van, R. Goedemans, T.H. Ramwadhdoebe,A. Gorter, M.J. Welters, H.T. van, S.H. van der Burg, M2 macrophages induced byprostaglandin E2 and IL-6 from cervical carcinoma are switched to activated M1macrophages by CD4þ Th1 cells, J. Immunol. 187 (2011) 1157–1165.

[18] X. Qian, J. Zhang, J. Liu, Tumor-secreted PGE2 inhibits CCL5 production inactivated macrophages through cAMP/PKA signaling pathway, J. Biol. Chem.286 (2011) 2111–2120.

[19] W.H. Faour, N. Alaaeddine, A. Mancini, Q.W. He, D. Jovanovic, J.A. Di Battista,Early growth response factor-1 mediates prostaglandin E2-dependent tran-scriptional suppression of cytokine-induced tumor necrosis factor-alpha geneexpression in human macrophages and rheumatoid arthritis-affected synovialfibroblasts, J. Biol. Chem. 280 (9) (2005) 536–9546.

[20] F. D′Acquisto, L. Sautebin, T. Iuvone, R.M. Di, R. Carnuccio, Prostaglandinsprevent inducible nitric oxide synthase protein expression by inhibitingnuclear factor-kappaB activation in J774 macrophages, FEBS Lett. 440 (7)(1998) 6–80.

[21] P.F. Gomez, M.H. Pillinger, M. Attur, N. Marjanovic, M. Dave, J. Park, C.O. Bingham III, H. Al-Mussawir, S.B. Abramson, Resolution of inflammation:prostaglandin E2 dissociates nuclear trafficking of individual NF-kappaBsubunits (p65, p50) in stimulated rheumatoid synovial fibroblasts, J. Immunol.175 (2005) 6924–6930.

[22] H.W. Ziegler-Heitbrock, A. Wedel, W. Schraut, M. Strobel, P. Wendelgass,T. Sternsdorf, P.A. Bauerle, J.G. Haas, G. Riethmuller, Tolerance to lipopolysac-charide involves mobilization of nuclear factor kappa B with predominance ofp50 homodimers, J. Biol. Chem. 269 (1994) 17001–17004.

[23] C. Porta, M. Rimoldi, G. Raes, L. Brys, P. Ghezzi, L.D. Di, F. Dieli, S. Ghisletti,G. Natoli, B.P. De, A. Mantovani, A. Sica, Tolerance and M2 (alternative)macrophage polarization are related processes orchestrated by p50 nuclearfactor kappaB, Proc. Nat. Acad. Sci. U.S.A. 106 (2009) 14978–14983.

[24] J.R. Conner, I.I. Smirnova, A.P. Moseman, A. Poltorak, IRAK1BP1 inhibits inflam-mation by promoting nuclear translocation of NF-kappaB p50, Proc. Nat. Acad.Sci. U.S.A. 107 (2010) 11477–11482.

[25] C.N. Serhan, N. Chiang, T.E. Van Dyke, Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators, Nat. Rev. Immunol. 8 (2008)349–361.

[26] A.V. Sampey, S. Monrad, L.J. Crofford, Microsomal prostaglandin E synthase-1: theinducible synthase for prostaglandin E2, Arthritis Res. Ther. 7 (2005) 114–117.

[27] P.J. Jakobsson, S. Thoren, R. Morgenstern, B. Samuelsson, Identification ofhuman prostaglandin E synthase: a microsomal, glutathione-dependent,inducible enzyme, constituting a potential novel drug target, Proc. Nat. Acad.Sci. U.S.A. 96 (1999) 7220–7225.

[28] B. Samuelsson, R. Morgenstern, P.J. Jakobsson, Membrane prostaglandin Esynthase-1: a novel therapeutic target, Pharmacol. Rev. 59 (2007) 207–224.

[29] F. Kojima, M. Kapoor, L. Yang, E.L. Fleishaker, M.R. Ward, S.U. Monrad, P.C. Kottangada, C.Q. Pace, J.A. Clark, J.G. Woodward, L.J. Crofford, Defectivegeneration of a humoral immune response is associated with a reducedincidence and severity of collagen-induced arthritis in microsomal prosta-glandin E synthase-1 null mice, J. Immunol. 180 (2008) 8361–8368.

[30] C.E. Trebino, J.L. Stock, C.P. Gibbons, B.M. Naiman, T.S. Wachtmann, J.P. Umland,K. Pandher, J.M. Lapointe, S. Saha, M.L. Roach, D. Carter, N.A. Thomas, B.A. Durtschi, J.D. McNeish, J.E. Hambor, P.J. Jakobsson, T.J. Carty, J.R. Perez, L.P. Audoly, Impaired inflammatory and pain responses in mice lacking aninducible prostaglandin E synthase, Proc. Nat. Acad. Sci. U.S.A. 100 (2003)9044–9049.

[31] D.L. Asquith, A.M. Miller, I.B. McInnes, F.Y. Liew, Animal models of rheumatoidarthritis, Eur. J. Immunol. 39 (2009) 2040–2044.

[32] K. Kannan, R.A. Ortmann, D. Kimpel, Animal models of rheumatoid arthritisand their relevance to human disease, Pathophysiology 12 (2005) 167–181.

[33] N. Ozaki, S. Suzuki, M. Ishida, Y. Harada, K. Tanaka, Y. Sato, T. Kono, M. Kubo,D. Kitamura, J. Encinas, H. Hara, H. Yoshida, Syk-dependent signaling path-ways in neutrophils and macrophages are indispensable in the pathogenesisof anti-collagen antibody-induced arthritis, Int. Immunol. 24 (2012) 539–550.

[34] R.L. Charles, J.R. Burgoyne, M. Mayr, S.M. Weldon, N. Hubner, H. Dong,C. Morisseau, B.D. Hammock, A. Landar, P. Eaton, Redox regulation of solubleepoxide hydrolase by 15-deoxy-delta-prostaglandin J2 controls coronaryhypoxic vasodilation, Circ. Res. 108 (2011) 324–334.

[35] P.P. Bradley, D.A. Priebat, R.D. Christensen, G. Rothstein, Measurement ofcutaneous inflammation: estimation of neutrophil content with an enzymemarker, J. Invest. Dermatol. 78 (1982) 206–209.

A. Frolov et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 89 (2013) 351–358 357

[36] L.M. van, M.J. Gijbels, A. Duijvestijn, M. Smook, M.J. van de Gaar, P. Heeringa,M.P. de Winther, J.W. Tervaert, Accumulation of myeloperoxidase-positiveneutrophils in atherosclerotic lesions in LDLR�/� mice, Arterioscler.Thromb.Vasc. Biol. 28 (2008) 84–89.

[37] C. Melani, G.F. Mattia, A. Silvani, A. Care, L. Rivoltini, G. Parmiani, M.P. Colombo, Interleukin-6 expression in human neutrophil and eosinophilperipheral blood granulocytes, Blood 81 (1993) 2744–2749.

[38] F.O. Martinez, S. Gordon, M. Locati, A. Mantovani, Transcriptional profiling ofthe human monocyte-to-macrophage differentiation and polarization: newmolecules and patterns of gene expression, J. Immunol. 177 (2006)7303–7311.

[39] C.G. Maloney, W.A. Kutchera, K.H. Albertine, T.M. McIntyre, S.M. Prescott, G.A. Zimmerman, Inflammatory agonists induce cyclooxygenase type 2 expres-sion by human neutrophils, J. Immunol. 160 (1998) 1402–1410.

[40] A. Tomlinson, I. Appleton, A.R. Moore, D.W. Gilroy, D. Willis, J.A. Mitchell, D.A. Willoughby, Cyclo-oxygenase and nitric oxide synthase isoforms in ratcarrageenin-induced pleurisy, Br. J. Pharmacol. 113 (1994) 693–698.

[41] C.A. Gleissner, I. Shaked, K.M. Little, K. Ley, CXC chemokine ligand 4 induces aunique transcriptome in monocyte-derived macrophages, J. Immunol. 184(2010) 4810–4818.

[42] J.D. Imig, B.D. Hammock, Soluble epoxide hydrolase as a therapeutic target forcardiovascular diseases, Nat. Rev. Drug Discovery 8 (2009) 794–805.

[43] R.B. Zurier, D.M. Sayadoff, A.B. Torrey, N.F. Rothfield, Prostaglandin E treat-ment of NZB/NZW mice, Arthritis Rheum. 20 (1977) 723–728.

[44] R.B. Zurier, F. Quagliata, Effect of prostaglandin E 1 on adjuvant arthritis,Nature 234 (1971) 304–305.

[45] D. Kamei, K. Yamakawa, Y. Takegoshi, M. Mikami-Nakanishi, Y. Nakatani,S. Oh-Ishi, H. Yasui, Y. Azuma, N. Hirasawa, K. Ohuchi, H. Kawaguchi,Y. Ishikawa, T. Ishii, S. Uematsu, S. Akira, M. Murakami, I. Kudo, Reduced painhypersensitivity and inflammation in mice lacking microsomal prostaglandine synthase-1, J. Biol. Chem. 279 (2004) 33684–33695.

[46] M. Mosca, N. Polentarutti, G. Mangano, C. Apicella, A. Doni, F. Mancini, B.M. De,I. Coletta, L. Polenzani, G. Santoni, M. Sironi, A. Vecchi, A. Mantovani,Regulation of the microsomal prostaglandin E synthase-1 in polarized mono-nuclear phagocytes and its constitutive expression in neutrophils, J. Leukoc.Biol. 82 (2007) 320–326.

[47] MM-Y. Chan, A.R. Moore, Resolution of inflammation in murine autoimmunearthritis is disrupted by cyclooxygenase-2 inhibition and restored by prosta-glandin E2-mediated lipoxin A4 production, J. Immunol. 184 (2010)6418–6426.

[48] D.W. Gilroy, P.R. Colville-Nash, D. Willis, J. Chivers, M.J. Paul-Clark, D.A. Willoughby, Inducible cyclooxygenase may have anti-inflammatory proper-ties, Nat. Med. 5 (1999) 698–701.

A. Frolov et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 89 (2013) 351–358358