Introduction
The immune system in teleost fish, similar to other vertebrates, consists of both innate and adaptive immunity. Immunological memory has long been believed to be a unique characteristic of the adaptive immune system that generates specific and long-lasting responses to antigens. However, a novel understanding has emerged recently demonstrating that the innate immune system also possesses memory-like capabilities, a phenomenon referred to as "trained immunity". This is a significant finding because the innate immune system was previously considered a rapid but non-specific defence lacking immunological memory. Studies in fish have suggested that innate immune cells, such as macrophages and neutrophils, can exhibit an enhanced response upon re-exposure to stimuli, highlighting a new layer of complexity in host defence. Studying "trained immunity" in fish is particularly novel and relevant due to their evolutionary position as early vertebrates with a well-developed innate immune system. This capacity for innate immune cells to "remember" prior encounters, potentially through metabolic modulation and epigenetic reprogramming, offers a promising direction for understanding broad-spectrum protection and developing sustainable disease management strategies in aquaculture.
Materials and Methods
In this research, leukocytes were isolated from the head kidney of 100 g Atlantic salmon (Salmo salar) in freshwater stage and cultured under standardized conditions for 2 days. Cells were first stimulated for 24 hours with β-glucan (10 μg/mL) to induce a training response, followed by a five-day resting period in the same cultured condition. Subsequently, cells received secondary stimulation with phosphate-buffered saline (PBS, control), β-glucan (100 µg/mL, homologous stimulus), and lipopolysaccharide (LPS, 100 µg/mL, heterologous stimulus). Cell’s immune activity was assessed by phagocytosis, nitric oxide (NO), and reactive oxygen species (ROS) production. To evaluate metabolic regulation, intracellular levels of lactate and fumarate were quantified. In addition, quantitative PCR was performed to assess gene expression related to immunity, glycolysis metabolism, and epigenetic regulation.
Results
Preliminary results showed that trained cells exhibited a noticeable enhancement in phagocytic activity upon secondary stimulation with both β-glucan and LPS compared to untrained cells. ROS production increased in β-glucan-stimulated cells, while NO levels remained relatively consistent among groups. Besides, fumarate concentrations were higher in β-glucan treatments and appeared to decline in trained groups, meanwhile, intracellular lactate levels remained relatively stable across both trained and untrained groups. Gene expression analysis of cells after the resting period showed elevated immune and metabolic gene expression in the trained group, with significant differences mainly in metabolic genes. While gene expression profiles were largely similar between trained and untrained leukocytes, several genes demonstrated distinct expression levels in the trained group, indicating potential targets of trained immune regulation. In addition, LPS stimulation elicited higher immune and metabolic gene expressions compared to PBS and β-glucan within the same group. In contrast, most epigenetic-related genes tend to exhibit minimal variation in expression levels across treatments and groups.
Conclusion
These findings provide preliminary evidence that β-glucan can induce trained immunity in Atlantic salmon leukocytes. The observed functional and metabolic shifts suggest that trained innate responses in fish may contribute to broad-spectrum immune protection. This study highlights the potential of trained immunity as a novel approach for improving disease resilience and supporting sustainable health strategies in aquaculture.
Acknowledgements
The work was supported by the SIS Frontiers project funded by the Research Council of Norway through the Strategic Institute Initiative.