Macrophages are a major source of pro-inflammatory factors in the arterial intima and play a central role in the development of atherosclerotic plaque. Macrophages express toll-like receptor 4 (TLR4), a plasma membrane receptor, which when activated triggers the nuclear factor κB (NFκB) and mitogen-activated protein kinase signaling pathways leading to the production of pro-inflammatory cytokines. TLR4 expression and signaling have been positively associated with atherosclerotic lesion formation. Very long-chain polyunsaturated fatty acids, specifically, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have anti-inflammatory effects on macrophages, while saturated fatty acids have pro-inflammatory effects. However, the effect of enriching macrophages with EPA, DHA, or a saturated fatty acid on TLR4 cell surface expression and TLR4-mediated production of pro-inflammatory cytokines is not well characterized. We hypothesized that the production of pro-inflammatory cytokines would be downregulated in EPA- or DHA-enriched macrophages stimulated with TLR4 ligand, which may be mediated by a reduction in cell surface expression of TLR4 and its associated molecules CD14 and MD2, while enrichment of macrophages with a saturated fatty acid would have the opposite effect.
The objective of this thesis was to use the murine macrophage cell line, RAW 264.7 to determine the effect of enriching the cell membrane with EPA, DHA, or a saturated fatty acid, myristic acid (MA), on TNFα and IL-6 production, cell surface expression of TLR4, and associated molecules CD14 and MD2 induced by ultra-pure LPS stimulation (a TLR4-specific agonist). The involvement of cAMP response element-binding protein (CREB), prostaglandin E2 (PGE2) and nuclear factor κB (NFκB) in mediating the differential effect of DHA on TNFα and IL-6 production were also studied.
EPA- and DHA-enrichment decreased the inflammatory response of RAW 264.7 cells to ultra-pure LPS stimulation relative to control cells: a reduction in TNFα, IL-6 and PGE2 production, as well as NFκB activity was observed. In contrast, MA-enrichment did not potentiate the effect of ultra-pure LPS relative to control cells. EPA and DHA had a greater inhibitory effect on IL-6 compared to TNFα in both secretion and mRNA expression. This suggests an interference of signaling downstream of TLR4. Focusing on DHA, we found no effect on cell surface expression of TLR4, TLR4-MD2 complex or CD14, or the level of LPS-cell binding. Since NFκB is a major positive regulator of both TNFα and IL-6 gene transcription, we hypothesized that the weaker inhibitory effect of DHA on TNFα compared to IL-6 production may be due to the decrease in PGE2 production, since PGE2 has been previously reported to inhibit TNFα (possibly through the activation of CREB), and enhance IL-6 production. Addition of exogenous PGE2 had a dose-dependent inhibitory effect on TNFα mRNA expression after 3 h of stimulation, but only at concentrations higher than that found to be secreted by our cells. However, inhibiting PGE2 production by a cyclooxygenase 2 inhibitor also resulted in a small reduction in TNFα mRNA levels after 3 h but not 6 h of stimulation, suggesting that PGE2 had a minor stimulatory effect (if any) on TNFα production under the conditions evaluated in our system. Neither increasing nor decreasing PGE2 concentration had any effect on IL-6 mRNA expression. Although these data confirm differential regulation of TNFα and IL-6 by PGE2, it does not seem to be likely that a reduced PGE 2 production potentially induced by DHA is a significant contributing factor to the observed weak inhibitory effect of DHA on TNFα production. Since DHA had no significant effect on CREB activity, the involvement of this transcription factor in the DHA-induced inhibition of TNFα and IL-6 was not pursued. The effect of chemically reducing NFκB activity resulted in a larger inhibitory effect on IL-6 compared to TNFα mRNA expression, which is similar to the effect of DHA. These data suggest that the differential effect of DHA on TNFα and IL-6 mRNA expression may be mediated primarily by a reduction in NFκB activity, and that regulatory mechanisms are partially different between the TNFα and IL-6 genes.
The results of this research add to the current understanding of the effect of very-long chain polyunsaturated fatty acids and saturated fatty acids on TLR4 activation and signaling, and address the cytokine-specific effects of EPA and DHA in TLR4-activated macrophages. These data will advance the efforts to develop more specifically defined anti-inflammatory effects of EPA and DHA, which will lead to better understanding of the influence of EPA and DHA on atherosclerotic lesion progression.
|Advisor:||Lichtenstein, Alice H.|
|Commitee:||Lamon-Fava, Stefania, Matthan, Nirupa R., Wu, Dayong|
|School:||Tufts University, Gerald J. and Dorothy R. Friedman School of Nutrition Science and Policy|
|Department:||Biochemical and Molecular Nutrition|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Keywords:||Docosahexaenoic acid, Eicosapentaenoic acid, Inflammation, Macrophages, Myristic acid, Raw 264.7 cells|
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