Effects of docosahexaenoic supplementation and in vitro vitamin C on the oxidative and inflammatory neutrophil response to activation

Xavier Capó, Miquel Martorell, Antoni Sureda, Josep Antoni Tur, Antoni Pons, Xavier Capó, Miquel Martorell, Antoni Sureda, Josep Antoni Tur, Antoni Pons

Abstract

We studied the effects of diet supplementation with docosahexaenoic (DHA) and in vitro vitamin C (VitC) at physiological concentrations on oxidative and inflammatory neutrophil response to phorbol myristate acetate (PMA). Fifteen male footballers ingested a beverage enriched with DHA or a placebo for 8 weeks in a randomized double-blind study. Neutrophils were isolated from blood samples collected in basal conditions at the end of nutritional intervention. Neutrophils were cultured for 2 hours at 37°C in (a) control media, (b) media with PMA, and (c) media with PMA + VitC. PMA induces neutrophil degranulation with increased extracellular myeloperoxidase and catalase activities, nitric oxide production, expression of the inflammatory genes cyclooxygenase-2, nuclear factor κβ, interleukin 8 and tumor necrosis factor α, and interleukin 6 production. DHA diet supplementation boosts the exit of CAT from neutrophils but moderates the degranulation of myeloperoxidase granules induced by PMA. VitC facilitates azurophilic degranulation of neutrophils and increases gene expression of myeloperoxidase induced by PMA. VitC and DHA diet supplementation prevent PMA effects on inflammatory gene expression, although together they do not produce additional effects. DHA diet supplementation enhances antioxidant defences and anti-inflammatory neutrophil response to in vitro PMA activation. VitC facilitates neutrophil degranulation but prevents an inflammatory response to PMA.

Figures

Figure 1
Figure 1
Effects of in vitro PMA activation, vitamin C, and DHA diet supplementation on total CAT and MPO enzyme activities and on their distribution in the extracellular compartment of neutrophils. Statistical analysis: two-way ANOVA, p < 0.05. S: significant effect of DHA dietary supplementation; A: significant effect of PMA activation; S × A: significant interaction between DHA dietary supplementation and PMA activation effects. ∗ indicates differences between placebo and experimental groups; # indicates difference with respect to the control group; $ indicates differences between PMA and PMA + VitC group. When interaction S × A exists between supplementation and activation factors, different lowercase letters reveal significant differences. Results are the mean ± SEM.
Figure 2
Figure 2
Effects of PMA activation, in vitro vitamin C, and DHA diet supplementation on the percentage of extracellular NOx and the total NOx production. Statistical analysis: two-way ANOVA, p < 0.05. S: DHA diet supplementation effect; A: PMA activation effect; S × A: interaction between supplementation and activation effects. # indicates difference with respect to the control group; $ indicates differences between PMA and PMA + VitC group. When interaction S × A exists between supplementation and activation factors, different lowercase letters reveal significant differences. Results are the mean ± SEM.
Figure 3
Figure 3
Effects of PMA activation, in vitro vitamin C, and DHA diet supplementation on IL6 and TNFα production rate. Statistical analysis: two-way ANOVA, p < 0.05. S: DHA diet supplementation effect; A: PMA activation effect; S × A: interaction between supplementation and activation effects. When interaction S × A exists between supplementation and activation factors, different lowercase letters reveal significant differences. Results are the mean ± SEM.

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