Evidence of the Autophagic Process during the Fish Immune Response of Skeletal Muscle Cells against Piscirickettsia salmonis

SIMPLE SUMMARY: In mammals, autophagy plays a fundamental role in the defense against intracellular pathogens; however, in fish, this noncanonical function has not been described. In this context, it was proposed to study whether autophagy was modulated/activated in muscle cells challenged with a bacterial pathogen. Muscle cell cultures were performed and challenged with Piscirickettsia salmonis, the main threat to the salmon industry. Genes associated with immune response and autophagy were evaluated. In addition, the protein content of the LC3-II-specific marker of the autophagic process was evaluated via Western blot. Additionally, genes associated with vesicular traffic and endocytosis were evaluated, finding that P. salmonis promotes these processes. The results show a concomitant modulation of the genes associated with the immune response, vesicular trafficking, and autophagy, suggesting an early intracellular response by the muscle cell against this bacterium. Due to the necessity of seeking and discovering new alternatives and strategies to fight intracellular pathogens in the salmon industry, a better understanding of how autophagy participates in immune system responses may lead to the development of new technologies that allow for the effective control of intracellular pathogens, improving animal welfare and contributing to the sustainability of the global fish industry. ABSTRACT: Autophagy is a fundamental cellular process implicated in the health of the cell, acting as a cytoplasmatic quality control machinery by self-eating unfunctional organelles and protein aggregates. In mammals, autophagy can participate in the clearance of intracellular pathogens from the cell, and the activity of the toll-like receptors mediates its activation. However, in fish, the modulation of autophagy by these receptors in the muscle is unknown. This study describes and characterizes autophagic modulation during the immune response of fish muscle cells after a challenge with intracellular pathogen Piscirickettsia salmonis. For this, primary cultures of muscle cells were challenged with P. salmonis, and the expressions of immune markers il-1β, tnfα, il-8, hepcidin, tlr3, tlr9, mhc-I and mhc-II were analyzed through RT-qPCR. The expressions of several genes involved in autophagy (becn1, atg9, atg5, atg12, lc3, gabarap and atg4) were also evaluated with RT-qPCR to understand the autophagic modulation during an immune response. In addition, LC3-II protein content was measured via Western blot. The challenge of trout muscle cells with P. salmonis triggered a concomitant immune response to the activation of the autophagic process, suggesting a close relationship between these two processes.