2-Carba cyclic phosphatidic acid inhibits lipopolysaccharide-induced prostaglandin E2 production in a human macrophage cell line

Abstract

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that contains a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Using mouse models for multiple sclerosis (cuprizone-induced demyelination and experimental autoimmune encephalomyelitis) and traumatic brain injury, we revealed that cPA and its metabolically stabilized cPA derivative, 2-carba-cPA (2ccPA), have potential to protect against neuroinflammation. In this study, we investigated whether 2ccPA has anti-inflammatory effect on peripheral immune function or not using inflammation-induced macrophages-like cell line, THP-1 monocytes differentiated by phorbol 12-myristate 13-acetate (PMA). Lipopolysaccharide (LPS)-stimulated THP-1 cells were found to have higher expression of the mRNAs of several inflammation-related cytokines and of the enzyme cyclooxygenase-2 (Cox-2); however, when THP-1 cells were stimulated by LPS in the presence of 2ccPA, the increase in the expression of pro-inflammatory cytokine and Cox-2 mRNA was attenuated. 2ccPA treatment also decreased the amount of prostaglandin E2 (PGE2) produced by LPS-stimulated THP-1 cells and decreased expression of the mRNA of prostaglandin E receptor 2 (EP2, PTGER2), a PGE2 receptor that mediates inflammation. These results indicate that 2ccPA has anti-inflammatory properties.

Keywords: 2ccPA; Anti-inflammatory; Prostaglandin E2; THP-1 monocytes.

Fig. 1

CD11 and LPAR expression in PMA-differentiated THP-1 cells. (A) The expression level of CD11b mRNA was determined using real-time RT-PCR. Data are presented as the mean ± SEM. Statistical analysis was performed using the Student's t test. (*p < 0.0001, vs. DMSO vehicle control). (B) The protein levels of CD11b and GAPDH were determined using Western blot analysis. Real-time RT-PCR analyses of LPA1, LPA2, LPA3, LPA4, LPA5, LPA6, GPR87, and P2Y10 mRNA expression levels in (C) THP-1 cells and (D) differentiated THP-1 cells. The mRNA expression level of each gene was normalized relative to that of GAPDH.

Fig. 2

Variation in cytokine and Cox-2 mRNA expression after LPS stimulation in differentiated THP-1 cells. To measure mRNA expression, differentiated THP-1 cells were cultured with LPS (1 μg/mL) for the time indicated. (A) IL-1RA, TGF-β, IL-10, IL-1α, IL-1β, IL-6, IL-8, and (B) TNF-α and Cox-2 mRNA levels were determined using quantitative real-time PCR. Data represent the mean ± SEM of triplicate independent experiments. One-way ANOVA followed by Bonferroni's post-hoc comparisons tests were performed. (*p < 0.001, **p < 0.0001 vs. 0 time).

Fig. 3

Effect of 2ccPA on LPS-induced cytokines and Cox-2 mRNA expression and PGE2 secretion in differentiated THP-1 cells. Differentiated THP-1 cells were incubated with LPS (1 μg/mL) along with or without 2ccPA (1, 10 μM). Cells were collected after 4 h of incubation and mRNA expressions were analyzed by real-time PCR. The data were calculated based on the Cq values, and the expression of each gene was normalized to the levels of GAPDH. The data were calculated as the relative induction compared to that of controls (without LPS treatment). Data are presented as the mean ± SEM, n = 4. Statistical analysis was performed using a Bonferroni's test. (*p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Vehicle).

Fig. 4

Effect of 2ccPA on LPS-induced PGE2 secretion and PGE2 receptor mRNA expression in differentiated THP-1 cells. (A) Effect of 2ccPA on PGE2 secretion in differentiated THP-1 treated with LPS. Differentiated THP-1 cells were incubated with LPS (1 μg/mL) along with 2ccPA (0.1, 1, 3, or 10 μM) for 6 h. The concentrations of PGE2 in culture media were determined using ELISA. One-way ANOVA followed by Bonferroni's post-hoc comparisons tests were performed. (****p < 0.0001 vs. Vehicle). (B&C) Differentiated THP-1 cells were incubated with LPS (1 μg/mL) in the presence of 2ccPA (10 μM) for 4 h. The cells were collected and the Ep2 (B) and Ep4 (C) mRNA expressions were analyzed by real-time PCR. (*p < 0.05; **p < 0.01 vs. Vehicle without LPA, ^#P < 0.05; vs. Vehicle with LPA).