Effect of omega-3 fatty acids on toll-like receptor 4-mediated macrophage inflammation and its regulation.
Honda, Kaori.
2014
-
Abstract: 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 &kappaB; (NFκB) and mitogen-activated protein kinase signaling pathways
leading to the production of ... read morepro-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 êB
(NFêB) 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 PGE2 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 TNFá 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.
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Biochemical and Molecular Nutrition.
Advisor: Alice Lichtenstein.
Committee: Nirupa Matthan, Stefania Lamon-Fava, and Dayong Wu.
Keyword: Nutrition.read less - ID:
- cv43p827f
- Component ID:
- tufts:20370
- To Cite:
- TARC Citation Guide EndNote