Functional characterization of colony-stimulating factors in cytokine modulation and phagocytic cell activation in Japanese flounder.

Colony-stimulating factors (CSFs), including macrophage CSF (MCSF) and granulocyte CSF (GCSF), play central roles in the regulation and activation of phagocytic cells in vertebrates. However, their functions and regulatory mechanisms in teleosts remain partially understood. In this study, the functional characteristics of MCSF and GCSF were analyzed in Japanese flounder (Paralichthys olivaceus) to examine their tissue distribution, evolutionary relationships, and immunoregulatory roles in peripheral blood leukocytes (PBLs). MCSF and GCSF were highly expressed in the spleen and gill, supporting their potential involvement in immune surveillance and hematopoiesis. Phylogenetic analysis revealed that flounder CSFs clustered into species-specific clades within teleosts, indicating lineage-specific duplications and functional divergence. In vivo plasmid DNA-driven activation resulted in significant upregulation of CSF genes and downstream immune-related markers (AP-1, PU.1, and CXCR1), particularly by 7 days postinfection. In vitro, HINAE cells transfected with CSF-expression plasmids effectively expressed both genes, indicating an efficient platform for cytokine production. When these CSF-producing HINAE cells were indirectly cocultured with PBL, they induced time-dependent activation of phagocyte-related transcription factors: AP-1 showed an early transient peak, PU.1 peaked at 24 h, and CXCR1 expression was progressively upregulated. GCSF promoted early granulocyte responses, while MCSF enhanced delayed macrophage differentiation. These findings reveal distinct time-discrete effects of CSFs on phagocyte activation, highlighting their reciprocal roles in modulating innate immune dynamics in teleosts. This study offers insights into the molecular regulation of cytokine responses, enabling targeted disease resistance strategies in aquaculture and paving the way for more effective, species-specific infectious disease management methods.