Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling-An In Vitro Study

Abstract

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes' immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1β and nf-κb expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-κb and p65. This study showed the ability of methionine surplus to improve leucocytes' response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.

Keywords: AIP56; Photobacterium damselae subsp. piscicida; amino acids; fish; inflammation.

Figure 1

Experimental design. L-15 (Leibowitz L-15 medium); M1x (Leibowitz L-15 medium supplemented with 1 mM of L-methionine); M2x (Leibowitz L-15 medium supplemented with 1.5 mM of L-methionine); T1x (Leibowitz L-15 medium supplemented 0.2 mM of L-tryptophan); T2x (Leibowitz L-15 medium supplemented 0.3 mM of L-tryptophan); MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay); Ø (unstimulated cells); LPSPhdp (Lipopolysaccharides extracted from Phdp); PhdpUV (UV-inactivated Phdp); ATP (Adenosine triphosphate); TAC (total antioxidant capacity); NO (nitric oxide).

Figure 2

Cell viability after 24 h incubation with the different AA treatments. Values are presented as means ± SD (n = 12). P-values from one-way ANOVA (p-value = 0.001). Tukey post hoc test was used to identify differences in the experimental treatments. Different letters indicate differences among AA treatments.

Figure 3

Extracellular ATP concentration in the supernatant of HKL subjected to the experimental treatments. Values are presented as means ± SD (n = 6). P-values from multifactorial ANOVA (p-value < 0.001). Tukey post hoc test was used to identify differences between the experimental treatments. Different low case letters indicate differences among AA treatments at the same time and stimuli, while capital letters denote statistically significant differences between stimuli, within the same AA treatment, at the same time. An asterisk indicates differences between times with the same stimulus.

Figure 4

Extracellular TAC (total antioxidants concentration) in the supernatant of HKL subjected to the experimental treatments. Values are presented as means ± SD (n = 6). P-values from multifactorial ANOVA (p-value < 0.001). Tukey post hoc test was used to identify differences in the experimental treatments. Capital letters denote statistically significant differences between stimuli, within the same AA treatment, at the same time.

Figure 5

Extracellular polyamines concentration in the supernatant of HKL subjected to the experimental treatments. Values are presented as means ± SD (n = 6). P-values from multifactorial ANOVA (p-value = 0.048). Tukey post hoc test was used to identify differences in the experimental treatments. Different low case letters indicate differences among AA treatments at the same stimuli, while capital letters denote statistically significant differences between stimuli within the same AA treatment.

Figure 6

Extracellular nitric oxide concentration in the supernatant of HKL subjected to the experimental treatments. Values are presented as means ± SD (n = 6). P-values from multifactorial ANOVA (p-value = 0.009). Tukey post hoc test was used to identify differences in the experimental treatments. Different low case letters indicate differences among AA treatments while capital letters denote differences between stimuli.

Figure 7

Quantitative expression of (A) DNA methyltransferase 1 (p-value = 0.003), (B) tumor necrosis factor α (p-value = 0.001), (C) ornithine decarboxylase (p-value = 0.028), (D) spermine synthase (p-value <0.001), (E) interleukin 10 (p-value= 0.037), (F) arginase 2 (p-value < 0.001) and (G) indoleamine dioxygenase 2 (p-value < 0.001) in HKL subjected to the experimental treatments. Values are presented as means ± SD (n = 6). Multifactorial ANOVA was followed by Tukey post hoc test was used to identify differences in the experimental treatments. Different low case letters indicate differences among AA treatments while capital letters denote statistically significant differences between stimuli. An asterisk indicates differences between times.

Figure 8

Example of a western blot of p65 cleavage in HKL lysates subjected to the experimental treatments and incubated for 2 h with Ø (-) or 2 µg ml^-1 of AIP56 (+).

Figure 9

Caspase 3 activity of HKL subjected to the experimental treatments. Values are presented as means ± SD (n = 6). P-values from multifactorial ANOVA (p- value < 0.001). Tukey post hoc test was used to identify differences in the experimental treatments. Different low case letters indicate differences among AA treatments while capital letters denote statistically significant differences between stimuli for the same AA treatment.

Figure 10

Quantitative expression of (A) caspase 3 (p-value = 0.011), (B) nuclear factor kappa B (p-value = 0.012), (C) interleukin 8 (p-value 0.015), (D) mechanistic target of rapamycin (p-value < 0.001), (E) interleukin 1 β (p-value = 0.032), (F) transcription factor p65 (p-value < 0.011), (G) arylformamidase (p-value < 0.025), (H) DNA (cytosine-5-)-methyltransferase 3 beta (p-value < 0.026) and (I) caspase 8 (p-value < 0.011), in HKL subjected to the experimental treatments. Values are presented as means ± SD (n = 6). Multifactorial ANOVA was followed by Tukey post hoc test was used to identify differences in the experimental treatments. Different low case letters indicate differences among AA treatments while capital letters denote statistically significant differences between stimuli. Symbols indicate differences between times.