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.

Tryptophan Modulatory Role in European Seabass (Dicentrarchus labrax) Immune Response to Acute Inflammation under Stressful Conditions

“…Engelsma et al [ 29 ] also described a similar effect in common carp ( Cyprinus carpio ). Despite being shy, the outcome of the inflammatory response observed in the present study is in agreement with the premise that tryptophan may prime immune suppression by promoting immune-tolerance signals during inflammation [ 2 , 3 , 29 , 30 ]. Upon inflammatory induction, tryptophan catabolism in macrophages through the kynurenine pathway contributes to a reduction of its availability for microorganisms consumption, deviates superoxide radicals for the modulation of cell oxidative status, and regulates T-cell function, thus creating conditions that favor immune suppression and tolerance [ 8 , 10 ].…”

“…Upon inflammatory induction, tryptophan catabolism in macrophages through the kynurenine pathway contributes to a reduction of its availability for microorganisms consumption, deviates superoxide radicals for the modulation of cell oxidative status, and regulates T-cell function, thus creating conditions that favor immune suppression and tolerance [ 8 , 10 ]. In addition, tryptophan supplementation may contribute to immune tolerance by promoting macrophage differentiation towards the M2/healing phenotype in an inflammatory scenario [ 3 ]. Nonetheless, no modulation of either kynurenine-related genes, such as the afmid and ido2 , was observed in response to tryptophan supplementation in an inflammatory scenario.…”

“…IDO2 is the first and a rate-limiting enzyme of tryptophan catabolism in macrophages, followed by the enzyme arylformamidase (AFMID), which is responsible for kynurenine formation. Through this route, tryptophan consumption sets up a protection system, mostly characterized by its immune suppressive effect and role during the immune response [ 3 , 8 , 10 ]. Nonetheless, the down-regulation of tnfα , cd8β , and tgfβ at earlier sampling points was perceived.…”

“…piscicida ( Phdp ). In fact, Machado et al’s [ 3 ] in vitro observations pointed to a more direct role of tryptophan in the limitation of self-damage in inflammatory scenarios by the modulation of pathways associated to the assemblage of anti-inflammatory machinery and induction of macrophages polarization towards an anti-inflammatory phenotype. Moreover, a scenario of dietary tryptophan deficiency may equally represent an handicap for the European seabass, since it may lead to a rise in plasma cortisol levels in response to infection, which may explain the observed compromised immune cell response and disease resistance to Phdp [ 2 ].…”

Comparative study on the effects of crystalline L-methionine and methionine hydroxy analogue calcium supplementations in the diet of juvenile Pacific white shrimp (Litopenaeus vannamei)

“…The utilization of different methionine sources was compared using the relative expression levels of associated key genes. Free methionine is absorbed and used for protein synthesis or is converted to S-adenosylmethionine (SAM), a methyl group donor for the methylation, transsulfuration, and aminopropylation ( Anstee and Day, 2012 ; Machado et al, 2021 ). In the methionine cycle, the synthesis of SAM requires a key molecule methionine adenosyltransferase (MAT) ( Froese et al, 2019 ).…”