Eicosanoid Imbalance in the NODMouse Is Related to a Dysregulationin Soluble Epoxide Hydrolaseand 15-PGDH Expression

MICHELLE RODRIGUEZ AND MICHAEL CLARE-SALZLER

Department of Pathology, Immunology, and Laboratory Medicine, College ofMedicine, University of Florida, Gainesville Florida 32610, USA

ABSTRACT: Eicosanoids promote or resolve inflammation dependingon the class produced. Macrophage from nonobese diabetic (NOD)mouse produce increased proinflammatory lipid mediators and low lev-els of antiinflammatory lipoxin A4 (LXA4). The enhanced proinflam-matory eicosanoids is secondary to increased cyclooxygenase-2 (Cox-2)expression and low levels of prostaglandin/leukotriene catabolic enzyme,15-hydroxyprostaglandin dehydrogenase (15-PGDH). Deficient LXA4production is not due to deficient lipoxygenase (LO) activity, but is relatedto increased soluble epoxide hydrolase (sEH), involved in metabolism ofanti-inflammatory epoxyeicosatrienoic acids (EET). These aberrationsin eicosanoid biology suggest that inflammation in the NOD mouse islikely to be prolonged and robust and may contribute to type 1 diabetes(T1D) pathogenesis.

KEYWORDS: epoxide hydrolase; 15-PGDH; lipoxin A4; inflammation;type 1 diabetes; eicosanoids

INTRODUCTION

The regulation of inflammatory and anti-inflammatory eicosanoids iscomplex and involves several enzyme systems and lipid intermediates.Cyclooxygenase-2 (Cox-2) is induced during inflammation and leads tohigh-level production of inflammatory prostaglandins and thromboxane.1

Leukotriene biosynthesis is initiated by the enzyme 5-lipoxygenase (LO),which converts arachidonic acid (AA) to highly proinflammatory medi-ator, leukotriene B4 (LTB4), while 15-LO is responsible for productionof 15-hydroxyeicosatetraenoic acid (15-HETE).2 Lipoxin A4 (LXA4), is

Address for correspondence: Michelle Rodriguez, Department of Pathology, Immunology, and Lab-oratory Medicine, College of Medicine, University of Florida, 1600 SW Archer Road No. D11-41,Gainesville, FL 32610. Voice: 352-392-4887; fax: 352-392-5393.

e-mail: rodrigm@pathology.ufl.edu

Ann. N.Y. Acad. Sci. 1079: 130–134 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1375.019

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generated from AA via sequential actions of 5-, and 15-LO and is a potentanti-inflammatory eicosanoid that acts to promote resolution of inflamma-tion.2 Other enzyme systems are involved in eicosanoid breakdown, such as15-hydroxyprostaglandin dehydrogenase (15-PGDH) enzyme which catabo-lizes prostaglandin E-2 (PGE-2), LTB4, and also converts LXA4 to inactiveoxo- and dihydro products.3 A critical lipid mediator pathway still relativelyunexplored is the cytochrome P450 epoxygenases. These enzymes are respon-sible for the production of epoxyeicosatrienoic acids (EET), which are derivedfrom AA and possess anti-inflammatory activity.4 Previous work demonstratedthat 14,15 EET inhibits PGE-2 production via suppression of Cox-2 activity.5

Interestingly, the soluble epoxide hydrolase (sEH) transforms EETs into theircorresponding diols (DHET),5 which have limited anti-inflammatory activity.Moreover, sEH inhibitors promote the formation of both EETs and LXA4, thussupporting inflammatory resolution.6

Previous studies in type 1 diabetes (T1D) demonstrated increased Cox-2 ex-pression in subjects with or at risk for T1D and in macrophages from nonobesediabetic (NOD) mouse, leading to enhanced inflammatory responses.7 Addi-tionally, products of the LO metabolism, such as LTB4, are also increased inthe NOD mouse.8 We have also recently found a relative deficiency in theproduction of anti-inflammatory LXA4 in this same model. The expressionof 15-PGDH and sEH, however, has not been examined. In this article wedemonstrate that macrophages from NOD mouse express heightened levels ofsEH in combination with decreased expression of 15-PGDH which together,may limit anti-inflammatory LXA4 production and enhance inflammatorylipid mediators, respectively, and thus promote inflammation and perhaps T1Dpathogenesis.

METHODS

Cell Culture and Eicosanoid Analysis

Bone marrow cells from NOD/LtJ and C57BL/6 were isolated from femurand tibia by standard procedures. Cells were cultured in enriched RPMI 1640at a concentration of 1.75 × 106/mL in the presence of M-CSF for 4 days.Supernatants were assayed for PGE-2, and LTB4 using commercially availablekits from Amersham Biosciences (Pittsburgh, PA). ELISA kits for LXA4 wereobtained from Cayman Chemical (Ann Arbor, MI).

RNA Quantification by PCR

Macrophages received lipopolysaccharide (LPS) (1�g/mL) challenge for12 h. The RNeasy� Mini Kit (QIAgen, Valencia, CA) was used for the ex-

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FIGURE 1. (A) and (B) Quantitation of soluble sEH, and 15-PGDH by reverse tran-sciption polymerase chain reaction (RT-PCR) from bone marrow-derived macrophages fromC57BL/6 (gray bars) and NOD/LtJ (black bars). Data is a mean ± SEM of at least threeindependent experiments. The data were analyzed by two-tailed Student’s t-test. P < 0.05was considered statistically significant. ∗P < 0.05.

traction of total RNA from cell pellets. A total of 0.4 �g total RNA concen-tration was measured with the Jenway 6305 spectrophotometer (Model 6305UV/Visible range spectrophotometer [190–1000 nm]; Barloworld ScientificLtd. T/AS, Jenway, England). cDNA was obtained using the TaqMan� ReverseTranscription Kit (Applied Biosystems, Foster City, CA). For the TaqMan� as-say the TaqMan� Universal PCR Master Mix Kit (Applied Biosystems) wasused. The probe was labelled with the 5′ reporter dye 6-carboxyfluorescein(FAM). �-Actin was used as internal control. The reactions were run on the

RODRIGUEZ & CLARE-SALZLER: EICOSANOID IMBALANCE 133

ABI PRISMTM 7700 Sequence Detection System (Applied Biosystems). Theamplification program was: 50◦C for 2min, 95◦C for 10 min, followed by 45cycles of 95◦C for 15 s, 60◦C for 1 min.

RESULTS AND CONCLUSIONS

Our previous studies and those of others demonstrated increased PGE-2and LTB4 production with a relative deficiency in LXA4. We found markedlyreduced levels of 15-PGDH (FIG. 1 A) in macrophages from NOD mice incomparison to C57BL/6. This is significant because 15-PGDH is the key en-zyme responsible for metabolic inactivation of PGE-2, LTB4, and LXA4. De-spite low levels of 15-PGDH, the production of LXA4 is still limited in theNOD mouse suggesting that LXA4 deficiencies are not secondary to increased15-PGDH. However, quantitation of sEH showed increased mRNA expressionin macrophages from NOD mouse, which would result in enhanced metabolismof anti-inflammatory EET, for example, 14,15 EET, to less active products(FIG. 1 B), thus failing to suppress COX-2 activity, and allowing for increasedPGE-2 production as reported. Interestingly, limited LXA4 production wasovercome by in vitro administration of a specific sEH inhibitor. Although notdetermined, this inhibitor would also increase anti-inflammatory EETs. Takentogether, these results suggest the combinatorial effect of sustained expressionof sEH, with decreased expression of 15-PGDH contributes to limited LXA4synthesis and elevated levels of PGE2, and LTB4, respectively. These defectswould therefore sustain the enhanced or prolonged inflammatory responsesand potentially contribute to islet inflammation. Further studies are needed todetermine whether treatment with selective sEH inhibitors and or replacementof LXA4 with stable analogues alters T1D.

REFERENCES

1. FITZPATRICK, F.A. 2004. Cyclooxygenase enzymes: regulation and function. Curr.Pharm. Des. 10: 577–588.

2. NASSAR, G.M. & K.F. BADR. 1995. Role of leukotrienes and lipoxygenases inglomerular injury. Miner. Electrolyte Metab. 21: 262–270.

3. CLISH, C.B., B.D. LEVY, et al. 2000. Oxidoreductases in lipoxin A4 metabolic in-activation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation. J. Biol. Chem. 275: 25372–25380.

4. NODE, K., Y. HUO, et al. 1999. Anti-inflammatory properties of cytochrome P450epoxygenase-derived eicosanoids. Science 285: 1276–1279.

5. SPECTOR, A.A., X. FANG, et al. 2004. Epoxyeicosatrienoic acids (EETs): metabolismand biochemical function. Prog. Lipid Res. 43: 55–90.

6. SCHMELZER, K.R., L. KUBALA, et al. 2005. Soluble epoxide hydrolase isa therapeutic target for acute inflammation. Proc. Natl. Acad. Sci. USA.

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102: 9772–9777. Available at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?orig db=PubMed&db=PubMed&cmd=Search&term=Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%5BJour%5D+AND+102%5Bvolume%5D+AND+9772%5Bpage%5D+AND+2005%5Bpdat%5D+AND+Schmelzer+K%5Bauthor%5D.

7. LITHERLAND, S., T. XIE, et al. 2000. Aberrant prostaglandin synthase 2 expressiondefines an antigen-presenting cell defect for insulin-dependent diabetes mellitus.J. Clin. Invest. 104: 515-523.

8. LETY, M.A., J. COULAUD, et al. 1992. Enhanced metabolism of arachidonic acidby macrophages from nonobese diabetic (NOD) mice. Clin. Immunol. Im-munopathol. 64: 188–196.