Docosahexaenoic acid differentially affects TNFα and IL-6 expression in LPS-stimulated RAW 264.7 murine macrophages

Abstract

Docosahexaenoic acid (DHA) is generally reported to have anti-inflammatory properties, however, prior work has documented differential effects on individual pro-inflammatory cytokines: reduced IL-6, but not TNFα, mRNA expression in macrophages. To elucidate the mechanism, the roles of prostaglandin E2 (PGE2), cyclic AMP response element-binding protein (CREB), and NFκB were examined in RAW 264.7 macrophages. DHA did not influence CREB activity, but significantly reduced PGE2 production by 41% and NFκB activity by 32%. Exogenous PGE2 inhibited TNFα mRNA expression dose dependently. Unexpectedly, inhibiting PGE2 production with NS-398 also decreased TNFα mRNA expression, suggesting a concentration-dependent dual role of PGE2 in regulating TNFα expression. IL-6 expression was unaffected by endogenous or exogenous PGE2. Partial block of NFκB activation (SN50; 46%, or, BAY-11-7082; 41%) lowered IL-6 to a greater extent than TNFα mRNA expression. The differential effect of DHA on TNFα and IL-6 mRNA expression may be mediated via reduction in NFκB activity.

Keywords: CREB; IL-6; Macrophages; PGE(2); TLR4; TNFα.

Fig. 1

Effect of fatty acid on TNFα (A) and IL-6 (B) gene expression. RAW 264.7 cells were pretreated with DHA or MA (100 μM, 24 h) then stimulated with ultra-pure LPS (100 ng/mL) in the presence of treatment fatty acid for the times indicated. Bars without common letters within the same time group statistically differ at P<0.05 determined by one-way ANOVA, adjusted with Tukey’s post-hoc test for multiple comparisons. Values are mean±SD of three independent experiments.

Fig. 2

Effect of fatty acid on PGE₂ secretion and CREB activity in RAW 264.7 cells. (A) Cells were pretreated with MA or DHA (100 μM, 24 h) and then stimulated with ultra-pure LPS (100 ng/mL, 6 h). PGE₂ concentration in culture supernatant was determined by ELISA. Values are mean±SD of three independent experiments. Bars without common letters statistically differ at P<0.05 determined by one-way ANOVA adjusted with Tukey’s post-hoc test for multiple comparisons. (B) Cells were pretreated with MA or DHA (100 μM, 24 h) and then stimulated with ultra-pure LPS (100 ng/mL, 30 min). P-CREB concentration in whole cell lysates was determined by ELISA. Values are mean±SD of four independent experiments. Bars without common letters within each group statistically differ at P<0.05 determined by two-way repeated measures ANOVA adjusted with Sidak’s post-hoc test for multiple comparisons.

Fig. 3

Effect of exogenous PGE₂ on (A) TNFα and (B) IL-6 gene expression. RAW 264.7 cells were incubated with exogenous PGE₂ at the concentrations indicated for 45 min, and then stimulated with ultra-pure LPS (100 ng/mL, 3 h). Bars without common letters statistically differ at P<0.05 determined by one-way ANOVA, adjusted with Tukey’s post-hoc test for multiple comparisons. Values are mean±SD of four independent experiments.

Fig. 4

Effect of NS-398 on (A) PGE₂ secretion, (B) TNFα, and (C) IL-6 gene expression. RAW 264.7 cells were pretreated with NS-398 (10 μM, 18 h) and then stimulated with ultra-pure LPS (100 ng/mL). (A) PGE₂ in culture supernatant was determined by ELISA after 6 h of ultra-pure LPS stimulation. **P <0.01 vs. control determined by unpaired Student t test. (B) TNFα and (C) IL-6 gene expression were determined after 3 or 6 h of ultra-pure LPS stimulation. Values are mean±SD of three independent experiments. *P<0.05 determined by two-way repeated measures ANOVA adjusted by Sidak’s test for multiple comparisons. Values are mean±SD of three independent experiments.

Fig. 5

NFκB activity in RAW 264.7 cells. (A) Western blot of nuclear p65 protein expression before and after 2 h of ultra-pure LPS stimulation relative to histone 3 (H3) protein expression (nuclear protein loading control). One representative experiment is shown out of 3 independent experiments that had similar results. (B) Cells were pretreated with DHA (100 μM, 24 h), then stimulated with ultra-pure LPS (100 ng/mL, 30 min). NFκB-DNA binding in nuclear extracts was determined by ELISA. Values are mean±SD of five independent experiments. *P<0.05 determined by Student t test.

Fig. 6

Effect of SN50 in RAW 264.7 cells. Cells were pretreated with SN50 (100 μM, 15 min) and then stimulated with ultra-pure LPS (100 ng/mL). (A) After 30 min of stimulation, nuclear protein expression of p50 and p65 were determined by western blot. TBP (TATA-binding protein) was used as a nuclear protein loading control. Values are mean of two independent samples of one experiment. (B) TNFα and (C) IL-6 mRNA expression after 3 h of stimulation was determined by RT-PCR. Values are mean±SD of three independent experiments. Bars without common letters statistically differ at P<0.05 determined by one-way ANOVA, adjusted with Tukey’s post-hoc test for multiple comparisons.

Fig. 7

Effect of BAY on inflammatory response of RAW 264.7 cells. Cells were pretreated with BAY (10 μM, 18 h) then stimulated with ultra-pure LPS (100 ng/mL). (A) NFκB-DNA binding determined by ELISA. Values are mean of triplicate samples of one experiment. (B) PGE₂ in culture supernatant was determined after 6 h of ultra-pure LPS stimulation. (C) TNFα and (D) IL-6 gene expression were determined after 3 or 6 h of stimulation. Values are mean±SD of three independent experiments. *P≤0.05 determined by two-way repeated measures ANOVA adjusted with Sidak’s post-hoc test for multiple comparisons.