ORIGINAL RESEARCH

Soluble Epoxide Hydrolase Inhibitor Attenuates Inflammation andAirway Hyperresponsiveness in MiceJun Yang1, Jennifer Bratt2, Lisa Franzi2, Jun-Yan Liu1, Guodong Zhang1, Amir A. Zeki2, Christoph F. A. Vogel3,4,Keisha Williams2, Hua Dong1, Yanping Lin1, Sung Hee Hwang1, Nicholas J. Kenyon2, and Bruce D. Hammock1

1Department of Entomology and Nematology and Comprehensive Cancer Center; 2Department of Internal Medicine, Division ofPulmonary, Critical Care, and Sleep Medicine; 3Department of Environmental Toxicology; and 4Center for Health and the Environment.University of California, Davis, California

Abstract

Control of airway inflammation is critical in asthma treatment.Soluble epoxide hydrolase (sEH) has recently been demonstrated asa novel therapeutic target for treating inflammation, including lunginflammation. We hypothesized that pharmacological inhibition ofsEH can modulate the inflammatory response in a murine ovalbumin(OVA) model of asthma. BALB/c mice were sensitized andexposed to OVA over 6 weeks. A sEH inhibitor (sEHI) wasadministered for 2 weeks. Respiratory system compliance,resistance, and forced exhaled nitric oxide were measured. Lunglavage cell counts were performed, and selected cytokines andchemokines in the lung lavage fluid were measured. A LC/MS/MSmethod was used to measure 87 regulatory lipids mediators inplasma, lung tissue homogenates, and lung lavage fluid. Thepharmacological inhibition of sEH increased concentrationsof the antiinflammatory epoxy eicosatrienoic acids andsimultaneously decreased the concentrations of theproinflammatory dihydroxyeicosatrienoic acids anddihydroxyoctadecenoic acids. All monitored inflammatorymarkers, including FeNO levels, and total cell and eosinophil

numbers in the lung lavage of OVA-exposedmice were reduced bysEHI. The type 2 T helper cell (Th2) cytokines (IL-4, IL-5) andchemokines (Eotaxin and RANTES) were dramatically reducedafter sEHI administration. Resistance and dynamic lungcompliance were also improved by sEHI. We demonstrated thatsEHI administration attenuates allergic airway inflammation andairway responsiveness in a murine model. sEHI may have potentialas a novel therapeutic strategy for allergic asthma.

Keywords: soluble epoxide hydrolase; asthma; inflammation; lipidmediators; type 2 T helper cell cytokines

Clinical Relevance

We demonstrated that inhibition of soluble epoxide hydrolaseattenuates allergic airway inflammation and airwayresponsiveness in a murine model. Therefore, sEH inhibitorsmay have potential as a novel therapeutic strategy for allergicasthma.

Three hundred million people worldwidesuffer from episodic or persistentasthma (1). The cornerstones oftreatment for persistent asthma are

inhaled corticosteroids and b-agonistbronchodilators; however, a significantminority of patients with asthma does notrespond well to these therapies (2). Thus,

there are ongoing efforts to develop noveltreatment strategies (3), such as specificantagonists of type 2 T helper cell (Th2)cytokines and mediators.

(Received in original form October 15, 2013; accepted in final form May 23, 2014 )

This work was supported by American Asthma Association grant AAF 09-0269, National Institute of Environmental Health Sciences grant R01 ES002710, andNational Institute of Environmental Health Sciences Superfund Research Program grant P42 ES004699. Analytical work was partially supported by theNational Institutes of Health and by National Institute of Diabetes and Digestive and Kidney Diseases grant U24 DK097154. National Institutes of Health grantHL105573 provided the NO work equipment. This work was also supported by a Fellowship from Cystic Fibrosis Research, Inc. (J.Y.), by #UL1TR000002 andCTSC NIH KL2 (K12) Award TR000134 (A.A.Z.), and by T32HL007013 (J.B.).

Author Contributions: Conception and design: B.D.H., J.Y., and N.J.K. Analysis and interpretation: J.Y., J.B., L.F., J.-Y.L., G.Z., A.A.Z., C.F.A.V., K.W., H.D.,Y.L., and S.H.H. Drafting the manuscript: J.Y., B.D.H., and N.J.K.

Correspondence and requests for reprints should be addressed to Bruce D. Hammock, Ph.D., Department of Entomology, University of California at Davis,One Shields Avenue, Davis, CA 95616. E-mail: bdhammock@ucdavis.edu

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Am J Respir Cell Mol Biol Vol 52, Iss 1, pp 46–55, Jan 2015

Copyright © 2015 by the American Thoracic Society

Originally Published in Press as DOI: 10.1165/rcmb.2013-0440OC on June 12, 2014

Internet address: www.atsjournals.org

46 American Journal of Respiratory Cell and Molecular Biology Volume 52 Number 1 | January 2015

Epoxyeicosatrienoic acids (EETs,or EpETrEs according to LIPIDMAPSnomenclature) are a class of importantlipid mediators with criticalphysiological functions that includevasodilation, antiinflammation,antihypertension, organ protection, andanalgesic effects (4). Specifically in lunghealth and lung disease, EETs arereported to affect lung epithelial iontransport (5–7), relax precontractedbronchi (8), reduce inflammation (9,10), regulate endothelial permeability inthe lung (11), and regulate pulmonaryvascular pressures (12, 13). Thus,modulation of endogenous EETs isan attractive approach to potentiallycontrol the symptoms of asthma,which include chronic airwayinflammation and airwayhyperresponsiveness (AHR).

The soluble epoxide hydrolase(sEH) hydrolyzes these bioactive EETsto their corresponding diols, whichare less beneficial and may be toxic.Using potent inhibitors of sEH tostabilize endogenous EETs (14–17),sEH has been recently demonstrated inanimal models as a novel therapeutictarget (4) for treating cardiovasculardiseases (18–20), inflammation (21, 22),pain (23–25), and pulmonary diseasessuch as pulmonary hypertension (26,27) and tobacco smoke–inducedchronic obstructive pulmonarydisease (9, 10).

In the present study, wehypothesized that pharmacologicalinhibition of sEH can modulate theinflammatory response in a well-established murine ovalbumin (OVA)-exposure asthma model. We found thatadministration of an sEH inhibitor(sEHI; t-TUCB) reduced totalinflammatory cell infiltration into theairway and lung and inhibited OVA-induced influx of eosinophils. Theprofiling of regulatory lipid mediatorsshows that t-TUCB administration notonly increased the antiinflammatorylipid mediators (EETs) but alsoincreased other antiinflammatorymediators, such as 17-hydroxydocosahexaenoic acid, and decreasedproinflammatory lipid mediators,including dihydroxyoctadecenoic acids(DHOMEs) and LTB4 in plasma, lungtissue, and lavage. Stabilization of theantiinflammatory epoxide lipid

mediators through pharmacologicalinhibition of sEH decreased productionof Th2 cytokines at the protein andmRNA levels after OVA induction.Furthermore, compliance andresistance of the respiratory system wereimproved after sEHI administration.These findings support the hypothesisthat sEH is a potential target totreat asthma.

Materials and Methods

AnimalsPathogen-free male BALB/c mice, aged8 to 10 weeks, were purchased fromCharles River Laboratory (Wilmington,MA). All mice were maintained in aHEPA-filtered laminar flow cage rackwith a 12-hour light/dark cycle andallowed free access to food and water.Figure 1A shows the animal protocol. Allprocedures with mice were performed inaccordance with an IACUC-approvedprotocol.

Drug Solutions and Exposure of Miceto OVA AerosolThe sEHI trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB)was synthesized as previously described(17). t-TUCB was dissolved in 0.05% (v/v)Tween-80 water solution. This solution(1 or 3 mg/kg) was administeredsubcutaneously to the mice every dayfor 14 days. On the last day, the drugswere administered 30 minutes before

OVA aerosol exposure. The exposureprocedures of OVA have been described indetail previously (28).

Lung Compliance andResistance MeasurementsDynamic lung compliance and respiratorysystem resistance were simultaneouslymeasured with a whole bodyplethysmograph for restrained animals(Buxco Inc., Troy, NY) 1 to 3 hours aftertermination of the final OVA exposure.Further details are provided in theonline supplement.

Measurement of Exhaled NOA 5-minute sample of exhaled gaseswas collected from the cannulated micethrough the ventilator exhalation portimmediately after insertion of the mouseinto a plethysmograph as previouslydescribed (29).

Cytokine and Chemokine AssaysThe concentrations of selected cytokinesand chemokines (Eotaxin, IFN-g, IL-1b,IL-2, IL-4, IL-5, IL-6, IL-10, IL-13,RANTES, and TNF-a) from thebronchoalveolar lavage fluid (BALF)supernatant were measured withcommercially available multipleximmunoassays according to themanufacturer’s instructions (Millipore,St. Charles, MO).

Measurement of t-TUCBConcentrationBlood (10 ml) was diluted with 0.1%aqueous EDTA (50 ml) and mixed

Figure 1. The protocol for exposure and treatments. BALB/c mice were sensitized byintraperitoneal injection of ovalbumin (OVA) and alum adjuvant solution for 4 weeks thenexposed to OVA aerosol six times. Depending on the treatment groups, mice wereinjected subcutaneously with vehicle solution, 1 mg/kg t-TUCB, or 3 mg/kg t-TUCB everyother day from Day 26 to Day 38. Previous pharmacokinetic studies with t-TUCB in miceindicated that this injection schedule maintains a sufficient plasma concentration toinhibit target activity.

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Yang, Bratt, Franzi, et al.: sEHI Attenuates Lung Inflammation in Asthma 47

vigorously. Samples were then extractedusing 200 ml of ethyl acetate twice anddried by Speedvac (Thermo Scientific,Waltham, MA). The residue wasreconstituted to 50 ml of internalstandard solution and measured byLC/MS/MS.

Regulatory Lipid Mediator ProfilingProfiles of regulatory mediators weremeasured using the LC/MS/MS methodas described previously (30). Aliquotsof plasma (250 ml), BAL supernatant(2 ml), or lung tissue (z 100 mg) wereused for the measurements, respectively.Further details are provided in the onlinesupplement.

Quantitative Real-Time ReverseTranscription PCRTotal RNA was isolated from lungtissue using Trizol and a Quick-RNA Miniprep isolation kit (Zymo Research, Irvine,CA). cDNA synthesis and the RT-PCRprocesses are described in the onlinesupplement.

Statistical AnalysisData are presented as means 6 SEM.Data were analyzed using unpairedvalues compared by two-tailed Student’s ttest or one-way or two-way ANOVA withTukey’s post test where appropriate,using the Prism 5.0 software package(GraphPad, Inc., San Diego, CA),with statistical significance defined asP < 0.05.

Results

sEHI Was Successfully Delivered andWell EngagedAfter 14 days of subcutaneous injectionof t-TUCB, concentrations of t-TUCB inblood (Figure 2A) reached 55.6 6 13.2nM (1 mg/kg) and 213 6 38 nM (3mg/kg), which are 42.8 and 164 timeshigher, respectively, than the IC50 oft-TUCB in mouse on the murinerecombinant sEH (1.3 nM) using trans-diphenylpropene oxide as substrate (17).As a result of enzyme inhibition, theconcentrations of 14,15-EpETrE, anendogenous substrate of sEH,

increased by 3- to 3.6-fold from 0.78 nMto 2.56 or 2.86 nM with theadministration of 1 or 3 mg/kg t-TUCBcompared with the vehicle control(Figure 2B). Increased concentrations of14,15-EpETrE after exposure to t-TUCBconfirmed the efficacy of t-TUCB asan sEHI in this study.

sEHI Administration IncreasedAntiinflammatory MediatorConcentrations and DecreasedProinflammatory MediatorConcentrationsWe analyzed the regulatory lipidmediators from BALF, plasma, and lungtissue homogenate using LC/MS/MS.Figure 3 shows the results presented asheatmaps, including a simplifiedarachidonic acid cascade listing themajor lipid mediators (Figure 3A) andthe significantly changed regulatory lipidmediators after t-TUCB administration(Figures 3B–3D). In general, for the P450and sEH pathways, sEHI administrationincreased epoxides in plasma andBALF and decreased diols in BALF andlung homogenates. In plasma, sEHIadministration increased some COX andLOX metabolites (11-HETE, 9-HETE,5-HETE, and 5-HEPE). The low doseof sEHI significantly reduced theproinflammatory mediators 6-keto-PGF1a (the metabolite and surrogate ofprostacyclin-PGI2) and LTB4. In BALF,sEHI administration increased LOXmetabolites, including 11-HETE, 9-HETE, 5-HETE, 15-HEPE, 17HDoHE,and 8-HETE. In lung homogenates, sEHIadministration increased the COXmetabolites 6-keto-PGF1a, TXB2,PGF2a, PGE3, PGJ2, and 11-HETE andincreased the LOX metabolites,including 15-HETE, 15(s) HETrE,15-HEPE, 17-hydroxy docosahexaenoicacid, and 8-HETE.

sEHI Administration Reduced Th2Cytokines and ChemokinesSeveral inflammatory cytokines wereinduced after OVA exposure (Figure 4 andFigure E1). After sEHI administration, IL-4and IL-5 in lung lavage fluid decreased toalmost the baseline level of the controlanimals (Figures 4A and 4B). By contrast,there were no clear trends for Th1 andinnate immune cytokines assayed (FigureE1). Because of the methodological issues

Figure 2. The concentration of t-TUCB in blood (A) and 14,15-EpETrE concentration inplasma (B) suggest that the inhibitor was delivered successfully in vivo and was well engaged.Blood was drawn 2 to 6 hours after administration of the last dose. *Significant differencefrom vehicle group. #Significant difference between the 1 mg/kg and 3 mg/kg t-TUCB groups.All three groups were exposed to OVA (n = 5 in all groups, except n = 4 in the Air1Vehiclegroup).

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involved in IL-13 detection, we couldnot make firm conclusions regarding theinvolvement of IL-13 in this model system.The chemokine eotaxin was induced afterOVA exposure, and its levels were bluntedby inhibition of sEH (Figure 4C). Thesedata suggest that inhibition of sEH couldreduce the Th2-specific cytokines andchemokines, which are important ineosinophil trafficking, recruitment, andmaturation in airways. Lung expressionof IL-4 and IL-5 also showed that RNAlevels of these Th2 cytokines were down-regulated by sEHI administration (Figures4D and 4E).

sEHI Administration ReducedInflammatory Cell Infiltration in LungTissues and Lavage FluidSensitization and exposure of mice toOVA induced significant inflammatorycell infiltration into the airway(Figure 5A). The total cell count in BALFreached approximately 2.6 3 106 cells/ml. t-TUCB administration at 1 mg/kgdose decreased the number ofinflammatory cells infiltrated into theBALF. Furthermore, 3 mg/kg t-TUCBsignificantly reduced the totalcell number in BALF to approximately46.2%. ANOVA showed that there wassignificant reduction of total live cellnumbers after sEHI administration(P = 0.04).

Figure 5B shows the differential cellcounts determined by the Hema-3stain set. After OVA exposure, theeosinophil is the dominant inflammatorycell type in BALF, comprising up to75% of the total inflammatory cellinfiltrate. After sEHI administration,the percentage of eosinophils wasreduced to 65%. t-TUCB at a dose of 3mg/kg significantly reduced eosinophilinfiltration into lung lavage from 1.63 3106 to 7.05 3 105. ANOVA shows thatthere is significant reduction of totallive cell number after sEHIadministration (P = 0.049), indicatingthat sEHI not only reduced the totalinflammatory cell infiltration into theairway but also altered the ratio ofinflammatory cells present in the BALF(eosinophil/macrophage ratio from 4.39to 2.30). This result also correspondsto the reduction of Th2 cytokines in thelavage and lung tissue. Exhaled nitricoxide (NO) is a biomarker of airwayeosinophil inflammation and was

Figure 3. Heatmaps generated from regulatory lipid mediators show that t-TUCB administrationincreased antiinflammatory lipid mediators and decreased proinflammatory lipid mediators in vivo. (A)A simplified depiction of the arachidonic acid cascade. (B–D) Heatmaps based on regulatory lipidmediators in plasma (B), bronchoalveolar lavage fluid (BALF) (C), and lung homogenates (D). The colorcorresponds to the fold change as shown in the legend. Bright red means an increase of more than2 times significantly (P , 0.05); dark red means an increase of less than 2 times significantly; blackmeans no significant change; dark green means a decrease of less than 2 times significantly; andbright green means a decrease of more than 2 times significantly. For plasma and BALF, n = 5 inall groups, except n = 4 in the Air1Vehicle group; for lung homogenates results, n = 4 in all groups,except n = 3 in the Air1Vehicle group.

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Figure 4. t-TUCB administration dramatically decreased Th2 cytokines IL-4 and IL-5 (A and B) and chemokine (Eotaxin) (C) production in BALF anddown-regulated the gene expression of these Th2 cytokines (D and E) in lung homogenates. *Significant difference from the OVA1vehicle group (n = 5in all groups, except n = 4 in nonimmunized1Vehicle group).

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Figure 5. (A) t-TUCB administration reduced the infiltrated inflammatory cells (cells/ml) in BALF. (B) Differential cell counts by Hema-3 stain showsthat soluble epoxide hydrolase inhibitor (sEHI) reduced the number of eosinophil (cells/ml) in BALF. (C) t-TUCB administration reduced the inflammatorymarker FeNO. (D–G) Hematoxylin and eosin stain results of lung tissues shows that sEHI reduces the infiltration of inflammatory cells into lungtissues. Shown are results from the air control group (D), the vehicle control group after OVA exposure (E), the 1 mg/kg t-TUCB–treated group (F),and the 3 mg/kg t-TUCB–treated group (G). *Significant difference from the OVA1vehicle group.

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increased from 5.43 to 15.1 ppb afterOVA exposure (Figure 5C). Treatmentwith 1 and 3 mg/kg of t-TUCB decreasedthe induction of this inflammatorybiomarker to 3.68 and 6.57 ppb,respectively. ANOVA with Bonferronipost test shows that there is significantreduction of FeNO after sEHI administration(P = 0.0006). In lung tissues, there wasmarked inflammatory cell influx inthe peribronchiolar space after OVAexposure, as shown in the hematoxylin andeosin stain result (Figures 5D and 5E),which is consistent with the lung lavagedata (Figures 5A and 5B). After inhibitionof sEH, inflammatory cells in the lung

tissue were reduced in a dose-dependentmanner (Figures 5F and 5G). Thesefigures show that sEHI administrationreduced the lung inflammatory cellinfiltration, which is consistent with theinflammatory cell results in the BALFcell counts.

Our results reveal severe eosinophil-dominant inflammation in the airwayand alveolar of the mice after OVAexposure. sEHI administration reducedthis inflammation, as shown intotal live cell number, inflammatorycell differentiation, hematoxylinand eosin–stained lung tissue, andFeNO.

sEHI Reduced Methacholine-InducedChanges in Resistance and DynamicCompliance of the Respiratory SystemExposure to OVA reduced the baselinecompliance from 0.036 ml/cm H2O(filter air group) to 0.023 ml/cm H2O(OVA1vehicle). Administration of 3mg/kg sEHI rescued the baselinecompliance to 0.03 ml/cm H2O(Figure 6B). Although 1 mg/kg sEHI didnot rescue the baseline compliance,both sEHI treatment groups helped toprevent the change in compliance withmethacholine challenge (P , 0.0001for overall effect by treatment groupand P = 0.0005 for the doses; assessed bytwo-way ANOVA).

OVA exposure increased baselineresistance and the slope of resistancechange in response to methacholinechallenge(Figure 6A). sEHIadministration rescued the baselineresistance in a dose-dependent manner(P = 0.0035 for overall effect bytreatment group and P = 0.0018 for thedoses of methacholine, as assessed bytwo-way ANOVA). At 1 mg/kg, sEHIreduced the slope of resistance changealong with methacholine, whereas sEHIat a dose of 3 mg/kg shows a morecomplex pattern: a shallow initial slopewith low methacholine dose, thena rapid increase in slope with the higherdose of methacholine.

Discussion

We demonstrated that administration ofan sEHI markedly attenuates theallergic airway inflammation and AHRcaused by exposing mice to OVA.Specifically, the sEHI reduced the totalinflammatory cell number by 50%.Compared with two clinically availablecompounds (montelukast anddexamethasone) tested in the similaranimal model, sEHI had a greater effecton reduction of the inflammatorycells infiltration than montelukast anddexamethasone did (40 and 28%) (31).In addition, sEHI reduced IL-5 levelsto 12.5% of those mice treated withOVA and vehicle. Taken together, thesefindings indicate that sEHI is a promisingpotential candidate drug to treat allergicasthma.

We and others have previouslyshown the antiinflammatory effects of

Figure 6. Airway hyperreacitivity (A) and Dynamic lung compliance (B) results show that t-TUCBadministration rescued OVA-induced asthmatic airway hyperresponsiveness (n = 4 for OVA1Vehiclegroup and the OVA13 mg/kg t-TUCB group; n = 3 for the nonummunized1Vehicle group and theOVA11 mg/kg t-TUCB group).

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sEHIs in different disease models (9, 21,22, 32). The mechanism is believedto be through stabilizing of theantiinflammatory EETs, which regulateNF-kB translocation (33), or throughreducing production of proinflammatorydiols, including DHOMEs (34) anddihydroxy eicosatrienoic acids (DHETs)(35). Here, our data show that theconcentration of sEHI in the plasmasignificantly altered the circulating EETand DHET levels present in plasma,BALF, and lung homogenates (Figures3C and 3D). Node (33) reported thatEETs (EpETrEs) can reduce endothelialcell VCAM-1 expression in response toTNF-a, IL-1a, and LPS. At the sametime, sEHI administration significantlyreduced proinflammatory DHETs inBALF and lung homogenate (Figures 3Cand 3D). The DHETs were reported asessential for monocyte chemotaxis toMCP-1 (35). In particular, sEHI reducedthe DHOMEs, metabolites of leukotoxin,and iso-leukotoxin. It was found thatthese DHOMEs are more toxic thanEpOMEs and are associated withmultiple organ failure and adultrespiratory distress syndrome (34).Taken together, these findings indicatethat the effect t-TUCB on theconcentrations of EETs and DHETs maycontribute to the antiinflammatoryeffects of sEHI in this murineasthmatic model.

In this study, we also observed thatadditional lipid mediators were affectedupon t-TUCB administration. Theproinflammatory lipid mediator LTB4

was decreased in the plasma after low-dose sEHI administration. It wasreported that LTB4 participates in theallergic sensitization process in animalmodels (36). Therefore, the effect ofinhibition of sEH might also benefit froma reduction of proinflammatory LTB4.Another lipid mediator, 17HDoHE,which is a precursor to resolvins andpossesses biological activity that inhibitsTNF-a–induced IL-1b expression (37),was increased in lavage and lunghomogenates after sEHI administration.Resolvins have been reported to promotethe resolution of the allergic airwaysresponse (38).

Our findings add to those reportedelsewhere by describing the effects of

sEH inhibition on allergic airwayinflammation. Previous studies (10, 21)have demonstrated that sEHI can reducethe inflammatory cytokines IL-1b, IL-6,and INF-g. In the present study, wefound that sEHI reduced Th2 cytokinesand chemokines, which are known toplay major roles in the asthmaticimmune response (39). Specifically, thepronounced effect of sEHI on IL-5and eotaxin-1, a key cytokine anda chemokine responsible for therelease of eosinophil from the bonemarrow and homing of eosinophilto the lung, is rather intriguing.Indeed, mepolizumab, a monoclonalantibody against IL-5 used for thetreatment of severe asthma, is garneringsignificant attention in the clinicalrealm (40). The inhibition of sEHmay be an alternative strategy fordecreasing IL-5 levels in concertwith other key mediators of lunginflammation.

There are reports indicating thatsome of these regulatory lipid mediators,such as EETs, have direct function on thebronchi (6, 41). We observed that sEHIadministration increased EETs in theBALF (Figure 5C), which may directlyrescue airway hyperreactivity. However,inflammation may also play a role inregulating lung compliance andresistance. The antiinflammatory effectsof sEHI might have contributed to theimprovement of lung function aftersEHI administration in this acute modelof asthma. In addition, lipid mediatorssuch as HETEs are reported to haveeffects on the airways (42). Alterationsin the levels of various lipidmediators may explain why treatmentwith 3 mg/kg of sEHI did not showimproved rescue (in comparison totreatment with 1 mg/kg) of theresistance induced by 2.0 mg/ml ofmethacholine (Figure 6B). Directpulmonary administration of a sEHIwould provide additional evidence onhow sEHI regulates lung compliance andresistance. We have not developed aneffective system for administering sEHIdirectly to the lung.

In this study, neither COX-2 nor5-LOX in lung homogenates wassignificantly suppressed by sEHIadministration in OVA-exposed mice

(Figure E2). These findings suggest, atleast in lung homogenates, that the majoreffects of sEHI are unlikely due to theNF-kB pathway.

sEHI administration increased theantiinflammatory mediators systemicallyand in the airways, as indicated by thelipid mediator levels from the lavage,while simultaneously decreasingproinflammatory mediators in the lungtissues and airways. These changes inlipid mediators influenced the reductionand down-regulation of Th2 cytokinesand chemokine expression in the airways.The reduction of these Th2 cytokinesand chemokines further decreasedthe recruitment of inflammatory cellsinfiltration into the lungs and airways.The reduction in overall lunginflammation and the increase of EETsin airway contributes to the alleviationof AHR.

The data are consistent with the sEH(EPHX2) being primarily responsiblefor the conversion of fatty acid epoxidesto the corresponding diols in the lungand with t-TUCB inhibiting thiscatalytic activity. The activity of mEH(EPHX1) on this substrate is low, as arepulmonary levels of the mEH. We havefound no evidence for catalytic activityof EH3 (EPHX3) or EH4 in the lung.Although there is no evidence ofinhibition of EPHX1, -3, or -4 or anunknown enzyme by t-TUCB, we cannotexclude the possibility of off-targeteffects.

Among the limitations of thisstudy is that we used only prophylacticand not therapeutic treatment. Long-term pulmonary inflammation,including asthma, leads to chronicchanges in the lung. However, ourshort-term model did not assess theeffects of sEHI on fibrotic biomarkersbecause this would require a longer-termexposure to inflammation, particularlyone that is associated with chronicdiseases involving the use of multipledrugs with different mechanismsof action. Future studies need toaddress the possible beneficial anddetrimental effects of long-termsEHI use. n

Author disclosures are available with the textof this article at www.atsjournals.org.

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ORIGINAL RESEARCH

Yang, Bratt, Franzi, et al.: sEHI Attenuates Lung Inflammation in Asthma 55

Supplementary Files

Soluble Epoxide Hydrolase Inhibitor Attenuates Inflammation and Airway

Hyperresponsiveness in Mice

Jun Yang1, Jennifer Bratt2, Lisa Franzi2, Junyan Liu1, Guodong Zhang1, Amir A. Zeki2,

Christoph F. A. Vogel3,4, Keisha Williams2, Hua Dong1, Yanping Lin1, Sung Hee

Hwang1, Nicholas J. Kenyon2, Bruce D. Hammock1 *

1 Department of Entomology and Comprehensive Cancer Center, 2 Department of Internal

Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, 3 Department of

Environmental Toxicology, 4 Center for Health and the Environment. University of

California, Davis, CA 95616.

Address correspondence to: Bruce D. Hammock

Department of Entomology

University of California at Davis

One Shields Avenue, Davis, CA 95616.

Tel: (530) 752-7519

Fax: (530) 751-1537

E-mail: bdhammock@ucdavis.edu

SUPPLEMENTARY MATERIALS AND METHODS

Lung compliance and resistance measurements

The dynamic lung compliance (Cdyn) and resistance of the respiratory system (Rst) was

measured with a plethysmograph for restrained animals (Buxco Inc., Troy, NY) 1–3

hours after termination of the final (6th) OVA exposure. Mice were deeply anesthetized

and sedated with medetomidine, 0.5 mg/kg (Domitor, Orion Pharma, Finland), and

tiletamine/zolpidem, 50 mg/kg (Telazol, Fort Dodge Laboratories, Fort Dodge, IA) and

surgically cannulated and ventilated at 7–8 cc/kg using a mouse ventilator (MiniVent,

Harvard Apparatus, Cambridge, MA) for the duration of the procedure. Cdyn (mL/cm of

H2O) and Rst (cm of H2O*sec/mL) measurements were made at baseline and

immediately following serial 3 minute nebulizations of saline and methacholine (0.5, 1.0

and 2.0 mg/ml), with 2-minute recovery periods allowed after each exposure.

Lung Tissue Processing

Immediately after lung physiology measurements, mice were killed with an overdose of

Beuthanasia-D. After blood collection, lungs were lavaged twice with 1 ml of PBS (pH

7.4), and then centrifuged at 2,500 rpm on a tabletop centrifuge for 10 minutes. The total

lavage live cell number and differential cell counts were determined.

Mice had their lungs fixed or immediately frozen and stored at -80 °C. Lungs were fixed

for histological evaluation with 1% paraformaldehyde. After fixation and paraffin

embedding, the lungs were stained with hematoxylin and eosin to qualitatively assess

peribronchiolar inflammation. Half of right lungs were immersed in RNA Later solution

(Life Technology, Carlsbad, CA) for the followed RT-PCR measurements.

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Airway Inflammatory Cell Populations

Total cell counts in the lung lavage fluid were determined using a hemocytometer and

trypan blue exclusion; 100 μL of the remaining cell suspension was processed onto slides

using a cytocentrifuge at 1650 rpm for 7 minutes for determination of the cell differential

counts. Slides were air-dried, stained with a Hema3 stain set as described in the

manufacturer's instructions (Fisher Scientific, Kalamazoo, MI).

LC/MS/MS analysis for t-TUCB

The liquid chromatography system used for analysis was an Agilent 1200 SL liquid

chromatography series (Agilent Corporation, Palo Alto, CA). The autosampler was kept

at 4 °C. Liquid chromatography was performed on an ACQUITY UPLC BEH C18, 1.7

µm, 2.1× 50 mm column (Waters, Milford, MA). Mobile phase A was water with 0.1%

glacial acetic acid. Mobile phase B consisted of acetonitrile with 0.1% glacial acetic acid.

Gradient elution was performed at a flow rate of 400 µL/min. The gradient began with

50% B and reached 95% B at 3 min. After holding at 95% for 0.5 min, the composition

of mobile phase went back to 50% at 3.5 min for another 0.5 min. The column was

connected to a 4000 QTrap tandem mass spectrometer (Applied Biosystems Instrument

Corporation, Foster City, CA) equipped with an electrospray source (Turbo V). The

instrument was operated in negative MRM mode. Individual analyte standards were

infused into the mass spectrometer and MRM transitions and source parameters

optimized for t-TUCB. The MRM transition for t-TUCB is 437.2/137.1; the transition for

CUDA is 339.2/214.3.

Extraction Protocol for Plasma Oxylipin Profile

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The Oasis cartridges (Waters,) were washed with ethyl acetate and methanol, and

equilibrated to the initial condition with the solution containing H2O and methanol.

Diluted supernatant was loaded on the cartridge and washed with SPE solution (H2O

with 5% methanol with 0.1% acetic acid) twice. The cartridges were dried by vacuum

and analytes were eluted with 0.5 mL of methanol and 1.5 mL of ethyl acetate to tubes

with 6 µL of trapping solution (30% glycerol in methanol). The elutes were dried by

vacuum (SpeedVac) and reconstitute with internal solution (200 nM CUDA methanol

solution).

Extraction Protocol for BALF Oxylipin Profile

Ten microLiters of antioxidant solution (0.2 mg/mL of BHT and EDTA) were added to 2

mL BAL fluid supernatant followed by the addition of 10 uL of surrogate solutions, in

which 9 deuterated internal standards were included. BAL fluids were then loaded on the

Oasis cartridges as mentioned above. The followed SPE protocol is same with the plasma

extraction protocol described above.

Extraction Protocol for Lung Homogenates Oxylipin Profile

Ten microLiters of anti-oxidant solution were added to 100mg of lung tissue followed by

the addition of 10 µL of surrogate solution. After addition of 400 µL of ice-cold

methanol with 0.1 % of acetic acid and 0.1% of BHT, the lung tissues were stored at -

80°C freezer for 30 min. Then, the lung tissue samples were homogenized using Mixer

Mill MM301 (Retsch, Haan, Germany) at 30 Hz for 10 min. The homogenates were

stored at -80°C freezer overnight. After centrifuged at 10,000 rpm for 10 min, the

supernatant were collected and the remaining pellets were washed with 100 µL of ice-

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cold methanol with 0.1 % of acetic acid and 0.1% of BHT and centrifuged again. The

supernatants of each sample were combined and diluted with 2 mL of H2O and load onto

SPE cartridges. Then the SPE protocol is the same as the one for plasma extraction

protocol.

Quantitative Real-time Reverse Transcription–PCR (RT-PCR)

Total RNA was isolated from tissue using Trizol and a Quick-RNA Mini prep isolation

kit (Zymo Research, Irvine, CA), and cDNA synthesis and RT-PCR process are fully

described in the supplementary files. was done as previously described (1). Quantitative

detection of β-actin and differentially expressed genes was performed with LightCycler

LC480 (Roche, Indianapolis, IN) using the Fast SYBR Green Master Mix (Life

Technologies, Grand Island, NY) according to the manufacturer’s instructions. DNA-free

total RNA (1.0 μg) was reverse-transcribed using 4 U Omniscript reverse transcriptase

(RT; Qiagen) and 1 μg oligo(dT)15 in a final volume of 40 μl. The primers for each gene

were designed on the basis of the respective cDNA or mRNA sequences using OLIGO

primer analysis software provided by Steve Rozen and the Whitehead Institute/MIT

Center for Genome Research (2). So, that the targets were 100–200 bp in length. The

following primer sequences were used: mouse β-actin (forward primer, 5′-

AGCCATGTACGTAGCCATCC-3′; reverse primer, 5′-

CTCTCAGCTGTGGTGGTGAA-3′), mouse IL-4 (forward primer, 5’-

TCAACCCCCAGCTAGTTGTC-3’; reverse primer, 5’-

TGTTCTTCGTTGCTGTGAGG-5’), mouse IL-5 (forward primer, 5’-

GAAGTGTGGCGAGGAGAGAC-3’; reverse primer, 5’-

GCACAGTTTTGTGGGGTTTT-3’) and mouse IL-13 (forward primer:5’-

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CAGCTCCCTGGTTCTCTCAC-3’); reverse primer: 5’-

CCACACTCCATACCATGCTG-3’). PCR amplification was carried out in a total

volume of 20 μl containing 2 μl cDNA, 10 μl 2 × Fast SYBR Green Master Mix, and 0.2

μM of each primer. The PCR cycling conditions were 95°C for 30 sec followed by 40

cycles of 94°C for 3 sec, and 60°C for 30 sec. Detection of the fluorescent product was

performed at the end of the 72°C extension period. Negative controls were concomitantly

run to confirm that the samples were not cross-contaminated. A sample with DNase- and

RNase-free water instead of RNA was concomitantly examined for each of the reaction

units described above. To confirm the amplification specificity, the PCR products were

subjected to melting curve analysis.

Western blot method

The lung tissue were solicited in a homogenization buffer containing 0.1% SDS, protease

inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 574 μM phenylmethanesulfonyl

fluoride (PMSF) in PBS. Homogenates were centrifuged at 10,000 rpm for 20 minutes

and the resulting supernatant was stored at -80 °C until further use.

Antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) unless

otherwise stated. Total protein concentration of homogenate samples was determined

using the Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Samples

containing 20 μg of protein were incubated at 65 °C for 15 minutes, electroporated under

reducing conditions, and transferred to a polyvinylidene difluoride (PVDF) membrane.

Membranes were blocked in 5% dry milk in PBS for 1 hour at 25 °C, then incubated in

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0.4 μg/ml of goat, anti-mouse Arg1, 0.4 μg/ml of goat, anti-mouse Arg2, or 0.4 μg/ml of

rabbit anti-human α-actinin overnight at 4 °C, then incubated for 1 hour at 25 °C in 40

ng/ml of horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (Pierce

Biotechnology, Rockford, IL) or 40 ng/ml of horseradish peroxidase (HRP) conjugated

goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL). Bands were visualized using

the Immobilon western chemiluminescent HRP substrate kit (Millipore, Billerica, MA)

and images captured using Image Reader LAS-3000 version 2.1 (FUJIFILM, Cypress,

CA).

References

1. Vogel, C. F., Sciullo, E., Park, S., Liedtke, C., Trautwein, C., Matsumura, F.,

2004. Dioxin increases C/EBPbeta transcription by activating cAMP/protein

kinase A. J Biol Chem 279, 8886-94.

2. Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for

biologist programmers. Methods Mol Biol 132, 365-86.

SUPPLEMENTARY FIGURE LEGENDS

Figure E1. The inflammatory cytokine panel as well as the IL-4, IL-5, eotaxin shown in

Figure 4 responded differently to the OVA exposure and administration of sEHI.

Figure E2. COX2 (A) and 5-LOX (B) induced by OVA challenge but were not reduced

by administration of sEHI.

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Supplementary Table E1. The concentrations of lipid mediators in Plasma, BALF and lung homogenates. The units in plasma and BALF are in nmol/L. The unit in lung homogenates is in pmol/g tissue.

Supplementary Table E2. The epoxides to diols ratios supported the engagement of sEHI in plasma, BALF and lung homegenates.

This table is in Excel format. It can be accessed from this issue’s table of contents online at www.atsjournal.org.

Figure E1.

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Figure E2.

(A)

(B)

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