Dendritic cells (DC) play a key role in the initiation of the inflammatory response, directing the adaptive immune response and determining the nature of the T-helper cell response to inflammatory stimuli. Long-chain n-3 PUFA, EPA and DHA have the potential to modulate the immune response⁽Reference Wang, Hao, Li, Yan, Ye, Li, Li and Li¹⁾. However, little is known about the ability of EPA and DHA to modify the interaction between PPARγ and NF-κB.

The present study has investigated the effects of EPA and DHA on expression of PPARγ and on components of the NF-κB pathway in DC isolated from BALB/c mice. Bone marrow-derived immature DC were cultured in RPMI 1640 medium supplemented with fetal calf serum and granulocyte-macrophage colony-stimulating factor for 7 d. DC were treated with either dimethyl suphoxide (vehicle control), EPA (25 μm) or DHA (25 μm) during the length of the culture period. On day 7 DC were stimulated with lipopolysaccharide (LPS; 100 ng/ml) for either 2 or 5 h. At the end of the incubation period, nuclear and cytosolic protein was extracted using the TransAm Nuclear Extraction Kit (Active Motif, Rixensart, Belgium) and levels of NF-κB, PPARγ, inhibitory subunit of NF-κB (IκB) kinaseα and IκBα were measured by Western blot. PPARγ and NF-κB TransAm™ kits (Active Motif) were used to assess the ability of PPARγ and NF-κB to bind target DNA sequences. In order to understand the interaction between PPARγ and NF-κB within the cell, DC cultured under similar conditions were immunostained for PPARγ and NF-κB, and their nuclear and cytoplasmic expression were quantified using a confocal microscope (Improvision Software; Improvision, Coventry, UK).

Western blot and TransAm™ analysis showed that EPA and DHA supplementation was associated with significant down-regulation of components of the NF-κB pathway and decreased affinity to bind target DNA. Treatment with EPA and DHA also lead to an increase in PPARγ levels and an increase in PPARγ:DNA binding affinity.

Figure. EPA and DHA modulate PPARγ and NFκBp65.

Confocal microscopy confirmed that pre-LPS stimulation, nuclear and cytoplasmic NF-κB expression was down regulated in both DHA- and EPA-treated cells. In LPS-stimulated DC DHA markedly reduced nuclear NF-κB localisation.

The study shows important nutrient-sensitive interactions between NF-κB and PPARγ in DC, wherein DHA and EPA mediate their anti-inflammatory effects in PPARγ-dependent and -independent mechanisms. Given the important role of fatty acids and DC in innate immunity this observation requires further investigation.