8,9-Epoxyeicosatrienoic Acid Inhibits Antibody Production of B Lymphocytes in Mice

Epoxyeicosatrienoic acids (EETs), synthesized from arachidonic acid by cytochrome P450 epoxygenases, are converted to dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. EETs exert anti-inflammatory effects. However, the effect of EETs on humoral immunity is poorly understood. The present study is to investigate the potential role of EETs on B cell function and mechanisms. We examined the role of EETs on antibody production of splenic B cells from C57BL/6 and apolipoprotein E-deficient (ApoE−/−) mice by means of ELISA. Of the 4 EET regioisomers, 8,9-EET decreased basal and activation-induced B cell antibody secretion. As well, 8,9-EET significantly inhibited B-cell proliferation and survival, plasma cell differentiation and class-switch recombination. Western blot analysis revealed that lipopolysaccharide-induced nuclear translocation of NF-κB could be attenuated by 8,9-EET. Furthermore, germinal center formation was impaired by 8,9-EET in mice in vivo. 8,9-EET may inhibit B-cell function in vitro and in vivo, which suggests a new therapeutic strategy for diseases with excess B cell activation.


Introduction
B cells play an essential role in immunity [1]. The differentiation of B cells into antibody-secreting plasma cells is essential for humoral immune responses. After antigen-stimulation in secondary lymphoid organs, naive B cells proliferate and undergo maturation and differentiation that includes class switch recombination, affinity maturation, and differentiation into plasma cells or memory B cells [1,2]. Effective antibody response depends on the integration of multiple signals. Although engagement of B cell receptor by specific antigens initiates the cascade, non-antigen-specific stimuli, such as lipopolysaccharide (LPS) and CD40L, have a profound effect on the quantity and quality of the response [3,4]. Activation of mouse B cells by LPS induces B cell differentiation as well as increased antibody production [3]. In addition, the maturation and differentiation of B cell depend on concerted action of panoply of transcription factors, most notably interferon regulatory factor 4 (IRF-4), Xbox binding protein 1 (XBP-1) and activation-induced cytidine deaminase (AICDA), which leads to the gene expressions necessary for plasma cell differentiation and class switch recombination of B cells [5][6][7].
Arachidonic acid is abundant in immune cells and is composed of 15,20% of fatty acid in phospholipids of the plasma membrane [18]. Many studies have showed that cyclooxygenase and lipoxygenase metabolites of arachidonic acid contribute to cell and humoral immunity [19][20][21][22], but the effect of the third major class of arachidonic acid metabolites is little known. CYP 450 epoxygenases and sEH are found in lymphoid tissues, such as spleen and lymph nodes [23], which suggest the biological roles of EETs in immunity. In addition, two studies have shown that CYP450 epoxygenase product 5,6-EET is responsible for hypotonicity-induced responses in B cells [24,25]. However, the effect of EETs on humoral immunity is little known.
In the present study, we investigated the potential role of EETs in the function of splenic B cells from C57BL/6 and ApoE2/2 mice. 8,9-EET inhibited B cell to proliferate, survival, plasmacytoid cell generation, class-switch recombination, and antibody secretion, which might be mediated by the inhibition of NF-kB activation. This knowledge might contribute to the treatment of diseases induced by overactivated B cells.

8,9-EET Decreased Antibody Production by B Cells in C57BL/6 and ApoE2/2 Mice
To investigate the potential effect of EETs on peripheral B cells, purified B cells were cultured with or without EETs, and IgM and IgG production was detected in the presence of 5 mg/ml LPS and/ or 50 ng/ml IL-4. As compared with controls, 8,9-EET (1 mM) but not 5,6-, 11,12-or 14,15-EET significantly decreased the production of IgM (4.260.9 vs. 8.060.7 mg/ml) and IgG (35.661.8 vs. 57.461.0 ng/ml) ( Figure 1A and 1B). Compared with different classical stimuli including anti-IgM F(ab) 2 and sCD40L to activate B cells, the decreased IgM and IgG production by 8,9-EET was greater with LPS and LPS plus IL-4 stimulation (data not shown). In addition, antibody production was downregulated by 8,9-EET in a concentration-and timedependent manner ( Figure 1C-1F).
We examined the effect of 8,9-EET on B cell antibody production in ApoE2/2 mice, the spontaneous model of atherosclerosis, with a genetic background of C57BL/6 mice. First, we compared the antibody levels of naïve and LPSactivated B cells from ApoE2/2 mice and age-and sexmatched C57BL/6 mice. Secretion of IgG but not IgM by LPSactivated B cells was significantly higher, by more than 2-fold, in ApoE2/2 mice than C57BL/6 control mice ( Figure 1G and 1H). We then treated B cells of ApoE2/2 mice with 1 mM 8,9-EET and/or 5 mg/ml LPS with or without 50 ng/ml IL-4 for 3 days. As compared with the control, 8,9-EET markedly decreased the production of IgM (4.460.2 vs. 7.560.5 mg/ml) and IgG (34.063.1 vs. 55.265.0 ng/ml) in LPS-activated B cells from ApoE2/2 mice ( Figure 1I and 1J), so 8,9-EET inhibited antibody production from normal B-cells and hyper-responsive one from ApoE2/2 mice.

8,9-EET Inhibited B-cell Proliferation and Survival
To investigate the mechanism of decreased antibody production with 8,9-EET, we monitored cell division by carboxyfluorescein succinimidyl ester (CFSE) dilution assay. CFSE-labeled B cells from C57BL/6 mice were cultured under various conditions for 3 days and then analyzed by flow cytometry. The proportion of dividing cells was lower with 8,9-EET (1 mM) plus LPS (5 mg/mL) than with LPS alone (33.260.9% vs. 49.3% 61.7%) (Figure 2A and 2B). We next examined the effect of 8,9-EET on B-cell survival stimulated with LPS. B cells cultured for 2 days after stimulation were stained with annexin V (AnnV) and propidium iodide (PI) to identify live cells (AnnV and PI double-negative cells). The percentage of live cells was substantially lower with 8,9-EET plus LPS than with LPS alone (10.2% 63.0% vs. 29.9% 62.6%) ( Figure 2C and 2D). Similarly, 8,9-EET inhibited the LPS-induced proliferation and survival of B cells from ApoE2/2 mice (data not shown). Therefore, decreased B-cell proliferation and survival might contribute to the inhibition of antibody production by 8,9-EET.

8,9-EET Antagonized Class-switch Recombination
Upon encountering cognate antigens, B cells are activated to undergo 2 genetic alterations of their immunoglobulin genes, namely, somatic hypermutation and class-switch recombination, which allows for affinity maturation and the generation of different antibody classes (e.g., IgG, IgA and IgE) [26,27]. To determine whether 8,9-EET affects class-switch recombination, we first examined the expression of surface IgG 1 (sIgG 1 ) in B cells stimulated with LPS plus IL-4 for 3 days. The addition of 8,9-EET to these cultures decreased sIgG 1 + cell production as compared with the control (3.9% 60.3% vs. 7.8% 60.3%) ( Figure 4A and 4B). Molecular events involved in class-switch recombination include the expressions of germline transcripts (GLTs) and AICDA, followed by deletional switch recombination and expression of Im-CH mature transcripts [7]. Therefore, we measured the mRNA levels of GLTs, AICDA and Im-CH mature transcripts by real-time PCR. 8,9-EET significantly inhibited the expression of AICDA, Ic 1 -Cc 1 , Im-Cc 1 , Ic 2b -Cc 2b and Im-Cc 2b in B cells stimulated with LPS plus IL-4 ( Figure 4C). Thus, 8,9-EET inhibits the class-switch recombination of B cells.

8,9-EET Inhibited NF-kB Activation in B Cells
To determine the signaling pathway by which 8,9-EET affects B-cell activation and antibody production, we determined whether the activity of NF-kB transcription factor, a major pathway for Bcell activation [28], was involved in 8,9-EET-inhibited B-cell activation. In B-cells pretreated with 8,9-EET and LPS, we determined the LPS-induced nuclear translocation of NF-kB by western blot analysis with anti-p65 NF-kB antibody. Nuclear p65 NF-kB level was increased within 5 min after LPS stimulation and was inhibited by pretreatment with 1 mM 8,9-EET for 10 min ( Figure 5A and 5B). A similar result was observed by immunofluorescence analysis (data not shown). Therefore, 8,9-EET might inhibit LPS-induced B-cell activation at least in part by inhibiting the NF-kB signaling pathway.

Germinal-center Formation was Impaired by 8,9-EET in vivo
After antigen stimulation, naïve B cells enter into follicles and form germinal centers where B cells rapidly proliferate; germinal centers are the main sites for generation of high-affinity antibodysecreting plasma cells and memory B cells [1]. To determine whether 8,9-EET regulates B-cell function in vivo, sEH2/2 mice, with reduced EET degradation and treated with or without 8,9-EET were intraperitoneally injected with a T-cell-dependent antigen, 4-hydroxy-3-nitrophenyl acetyl-OVA (NP-OVA). The spleen size of mice with 8,9-EET infusion (15 ng/h) was smaller than that without 8,9-EET at day 6 pos-timmunization ( Figure 6A). The colocalization of CD19 and PNA from immunofluorescence analysis of spleens revealed fewer germinal centers and smaller structures with than without 8,9-EET ( Figure 6B). Therefore, 8,9-EET inhibited germinal center formation in vivo.

Discussion
EETs are widely considered as endothelium-derived hyperpolarizing factors that modulate a number of biological events in an autocrine or paracrine manner [29,30] and may play an important role in the regulation of inflammation [15,[31][32][33]. CYP2C9 and sEH are detected in lymphoid tissues [23]. As well, 5,6-EET is principally responsible for hypotonicity-induced responses in B cells [24,25], so EETs may play a biological role in humoral immunity. We have found that 8,9-EET inhibits B-cell activation induced by LPS with or without IL-4, as demonstrated by decreased IgM and IgG secretion, decelerated cell proliferation, deteriorated cell survival, inhibited plasma-cell formation and restrained class switch recombination. Therefore, B-cell humoral immunity is prevented by 8,9-EET in both normal and ApoE2/2 mice. This study indicates a new mechanism for the effect of EETs on immune response, which might be a new therapeutic target for excess B-cell activation.
The effect of 8,9-EET on B cells seems to be specific, because none of the other regioisomers inhibited the B-cell activation. Our findings are consistent with previous reports of the selective effect of different EETs including pre-glomerular endothelium-dependent vasoconstriction (5,6-EET), adenosine-induced vasodilation (11, in smooth muscle cells [34,35], and vasoconstriction (8, [36]. Therefore, the position of the epoxide group across the double bonds might be a required molecular conformation for the observed B-cell effect of 8,9-EET. We further explored the biochemical basis for the inhibition of 8,9-EET on B-cell activation. Many transcription factors such as the cyclic AMP-response element-binding protein [37], NF-kB [15], peroxisome proliferator-activated receptor a (PPARa) [38] and forkhead box O3a [39] are modulated by EETs. NF-kB plays  a crucial role in B cell proliferation and antibody production. We found that 8,9-EET inhibited LPS-induced translocation of p65 and enhanced the phosphorylation of IkB (data not shown), which might mediate the effects of 8,9-EET on B-cell function. However, the PPARs pathway might not be involved in the regulation of 8,9-EET in B cells ( Figure S1). Cellular EETs are present in free and phospholipid-bound forms. They are principally metabolized by sEH to corresponding DHETs. The inhibitory effects of 8,9-EET on B cells were demonstrated in vivo by direct infusion of 8,9-EET in sEH2/2 mice with a C57BL/6 genetic background. After 6 days of 8,9-EET infusion (15 ng/h), sEH2/2 mice immunized with NP-  Besides secreting antibodies, activated B cells can also provide co-stimulatory signals for T-cell activation [40,41]. We found that 8,9-EET decreased the mRNA expression of CD80 and CD86 in B cells ( Figure S2), which might lead to weakened and shortened cellular immunity and contribute to the multiple effects of 8,9-EET in vivo. However, the mechanism remains to be further investigated.
In conclusion, we have demonstrated that 8,9-EET, a naturally existing epoxide of arachidonic acid, inhibits the function of B cells. Development of more stable analogs of 8,9-EET may provide a new class of specific therapeutic tool for diseases mediated by excess B-cell activation.

Ethics Statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Health Science Center of Peking University. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Health Science Center of Peking University (Permit Number: 12125). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

B-cell Isolation
B cells from mouse spleens were purified by positive selection according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, single-cell suspensions of spleen tissue were centrifuged. After lysis of erythrocytes, singlecell suspensions were incubated with anti-CD19-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and subjected to a magnetic field to separate B cells. Purified B cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 mM b-ME, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were kept at 37uC in a 5% CO 2 incubator for different times.

Proliferation
Carboxyfluorescein succinimidyl ester (CFSE) staining was used to analyze cell division according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Briefly, B cells (10 7 /ml) were labeled with 2.5 mM CFSE for 10 min at 37uC in the dark, stopped by 25% FBS, washed 2 times with RPMI 1640 medium containing 10% FBS, then resuspended in RPMI 1640 medium containing 10% FBS. Cells were then plated at 2610 6 cells/well in 48-well round-bottom plates. CFSE-labeled cells were cultured under various conditions. Three days later, cells underwent detection by FACS Calibur flow cytometry (BD Biosciences, San Jose, CA).

Survival Assay
To measure apoptosis, cells stimulated for 2 days under the aforementioned conditions were incubated for 15 min with annexin V (AnnV) and for 5 min with propidium iodide (PI) by use of the Vybrant Apoptosis Assay Kit (Molecular Probes, Eugene, OR) in the dark, and the fraction of subdiploid cells was measured by FACS Calibur flow cytometry.

Western Blot Analysis
Whole-cell extracts were collected with cell lysis buffer (Beyotime, Jiangsu, China) plus 1 mM PMSF. Cytoplasmic and nuclear protein extracts from B cells were prepared by use of the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce, Rockford, IL). Total protein was quantified by bicinchoninic acid protein assay (Pierce, Rockford, IL). Proteins were separated by 10% SDS-PAGE, then electrophoretically transferred onto nitrocellulose membranes, which were incubated with the indicated primary antibody, washed, then incubated with an appropriate IRDyeTM-conjugated second antibody. Specific immunofluorescence bands were detected by use of the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

Quantitative Real-time RT-PCR Analysis
Total RNA from primary mouse spleen B cells was isolated by use of Trizol reagent (Applygen Technologies, Beijing) and reverse transcribed with the reverse transcription system (Promega, Madison, WI). Then the reaction mixture underwent PCR. The nucleotide sequences of primers are shown in Table S1. The amount of PCR products formed in each cycle was evaluated by SYBR Green I fluorescence. Amplification reactions involved the Mx3000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA). Data were analyzed with use of Stratagene Mx3000 software.

Statistical Analysis
All data were expressed as mean 6 SEM. Data analysis involved unpaired Student t test and one-way and two-way ANOVA with GraphPad Prism software (GraphPad, La Jolla, CA) followed by Student-Newman-Keuls tests. P,0.05 was considered statistically significant.  Table S1 The nucleotide sequences of primers. Total RNA from primary mouse spleen B cells was isolated and reverse transcribed with the reverse transcription system. Then the reaction mixture underwent PCR. The nucleotide sequences of primers are shown in this Table S1. (TIF)