Amino acids as modulators of the European seabass, Dicentrarchus labrax, innate immune response: an in vitro approach

Abstract

Teleost innate immune system is a most developed and powerful system in which fish highly rely throughout their lives. Conditions in aquaculture farms are particularly prone to disease, thus, health and welfare ensuring strategies are an urgent call to which nutrition is gradually becoming a most regarded achievement tool. This study intended to evaluate different amino acids' effect on immune-related mechanisms as well as their potential as enhancers of European seabass, Dicentrarchus labrax, leucocyte functioning. To achieve these goals, primary cultures of head-kidney leucocytes were established and kept in amino acid (glutamine, arginine, tryptophan or methionine) supplemented culture media in two doses. The effects of amino acids treatments were then evaluated after stimulation with either Vibrio anguillarum or Vibrio anguillarum lipopolysaccharides by measuring nitric oxide production, extracellular respiratory burst, ATP and arginase activities, and expression of immune-related genes. Glutamine, arginine and tryptophan showed to be particularly relevant regarding cell energy dynamics; arginine and tryptophan supplementation also resulted in down-regulation of important immune-related genes. Immune responses in cells treated with methionine were generally enhanced but further studies, particularly those of enzymes activity, are essential to complement gene expression results and to better understand this nutrient's immune role in fish.

Figure 1

Nitric oxide content measured as total nitrites in the supernatant of HKL subjected to AA treatments under CTRL (), vaLPS () or Vang () stimulation conditions, regardless time of incubation. The AA treatments depicted in the X-axis are as follows: L-15, control; G1, 4.1 mM _L-glutamine; G2, 5.1 mM _L-glutamine; A1, 5.7 mM _L-arginine; A2, 7.2 mM _L-arginine; T1, 0.2 mM _L-tryptophan; T2, 0.25 mM _L-tryptophan; M1, 1 mM _L-methionine; M2, 1.25 mM _L-methionine. Values represent means ± SD (n = 6 biological replicates). Different symbols stand for statistically significant differences between stimuli within the same AA treatment, regardless time of incubation. Different letters denote statistically significant differences between AA treatments, within the same stimulus (Multifactorial ANOVA; Tukey post-hoc test; P ≤ 0.05).

Figure 2

Extracellular superoxide anion (O₂ ⁻) production in HKL subjected to the experimental treatments. The AA treatments depicted in the X-axis are as follows: L-15, control; G1, 4.1 mM _L-glutamine; G2, 5.1 mM _L-glutamine; A1, 5.7 mM _L-arginine; A2, 7.2 mM _L-arginine; T1, 0.2 mM _L-tryptophan; T2, 0.25 mM _L-tryptophan; M1, 1 mM _L-methionine; M2, 1.25 mM _L-methionine. Values represent means ± SD (n = 6 biological replicates). Different low case letters denote statistically significant differences between AA treatments, regardless stimulation or incubation time (Multifactorial ANOVA; Tukey post-hoc test; P ≤ 0.05).

Figure 3

Extracellular ATP concentration in the supernatant of HKL subjected to the experimental treatments for 4 (a) or 24 h (b) under CTRL () or vaLPS () conditions. The AA treatments depicted in the X-axis are as follows: L-15, control; G1, 4.1 mM _L-glutamine; G2, 5.1 mM _L-glutamine; A1, 5.7 mM _L-arginine; A2, 7.2 mM _L-arginine; T1, 0.2 mM _L-tryptophan; T2, 0.25 mM _L-tryptophan; M1, 1 mM _L-methionine; M2, 1.25 mM _L-methionine. Values represent means ± SD (n = 6 biological replicates). * and ^# stand for statistically significant differences between stimuli within the same AA treatment and time of incubation. Different low case letters denote statistically significant differences between AA treatments within the same stimuli and incubation time. x and y stand for statistically significant differences between incubation time, within the same AA treatment and stimuli (Multifactorial ANOVA; Tukey post-hoc test; P ≤ 0.05).