IMMUNOSTIMULATORY POTENTIAL OF NAD⁺ BOOSTERS: IN VITRO EFFECTS OF NMN AND NR ON HEAD-KIDNEY LEUKOCYTES OF GILTHEAD SEABREAM (SPARUS AURATA)

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) is a cofactor involved in metabolic processes and cellular functions. In fish, NAD⁺ helps manage oxidative stress, adapt to environmental changes, and mitigate the toxicity of pollutants. NAD⁺ can be synthesised via de novo and salvage pathways. Salvage pathways employ B3 derivatives, nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR) to produce NAD⁺. Consequently, strategies to elevate intracellular NAD⁺ levels through precursor supplementation have gained significant interest for their potential health benefits. Building on previous findings, where optimal concentrations were selected to enhance viability, mitochondrial bioenergetics, and migration in SAF-1 cells, this study evaluated the in vitro effects of NAD⁺ boosters at these selected concentrations on primary head-kidney leukocytes of gilthead seabream (Sparus aurata).

Methods

Head-kidney leukocytes were collected from gilthead seabream maintained and euthanised following ethical guidelines. The in vitro effects of the NAD⁺ boosters NMN and NR on these cells were tested for viability (using propidium iodide), phagocytosis, respiratory burst, peroxidase activity, and extracellular trap (ET) formation.

Results

Cell viability remained high across all conditions, showing no significant changes for NMN and only a minor decrease for NR at 0.75 mM. Both compounds significantly enhanced phagocytic ability without altering phagocytic capacity. Furthermore, NMN and NR boosted the innate immune response by increasing respiratory burst (NMN at 0.25 mM; NR at 0.75 mM) and significantly enhancing ET formation at all tested concentrations. Additionally, NR at 0.75 mM significantly elevated peroxidase activity, whereas NMN induced no changes in this parameter.

Conclusions

NMN effectively enhanced innate cellular immune parameters (phagocytosis, respiratory burst, and ETs) while preserving cell viability. Conversely, NR acted as a broader immunostimulant at higher doses, elevating peroxidase activity, albeit with a minor impact on cell viability. Overall, both NAD⁺ boosters emerged as promising functional additives to boost the innate immune response in gilthead seabream.