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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.

SUBMITTER: Machado M

PROVIDER: S-EPMC8005646 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Methionine presents a pivotal role in the regulation of many cellular events with crucial impact on the immune system, such as in processes involved in the control of inflammation and polyamines synthesis. Accordingly, the present study aimed to assess the modulatory effects of dietary methionine on the European seabass (Dicentrarchus labrax) immune status, inflammatory response and disease resistance to Photobacterium damselae subsp. piscicida (Phdp). For this purpose, fish were randomly distributed in three independent groups (three replicates per group) and each was fed the corresponding diet: a control diet (CTRL) formulated to meet the established amino acid requirements for the species; a diet supplemented with methionine at 0.5% of feed weight relative to the CTRL diet (8.2% of methionine concentration above CTRL); and one supplemented with methionine at 1% of feed weight to the CTRL diet (11.8% of methionine concentration above CTRL). To evaluate the immune status of fish fed with each of the diets before being submitted to bacterial infection fish were sampled from each group at 2 and 4 weeks after the beginning of feeding. Non-sampled fish were injected intraperitoneally with Phdp (5 × 103 cfu/fish) at 4 weeks after initiation of feeding and the inflammatory response (at 4, 24, and 48 h post-infection) and survival (lasting 21 days post-infection) evaluated. Fish hematological profile, peripheral cell dynamics, plasma humoral immune parameters, leucocyte migration to the inflammatory focus and head-kidney gene expression were evaluated. Results show that methionine dietary supplementation improves seabass cellular immune status without evidence of activation of pro-inflammatory mechanisms. Additionally, the observed enhanced immune status provided by methionine supplementation translated into an improved immune response to infection, as higher cellular differentiation/proliferation and recruitment to the inflammatory focus, improved plasma humoral immune parameters and modulation of key immune-related genes was observed. Lastly, after a bacterial challenge, higher survival was observed in fish fed supplemented diets, ultimately corroborating the positive effect of methionine administration for 4 weeks in the cellular immune status.

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

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