The SPHK/S1P/S1PR axis promotes LPS challenge- and Edwardsiella piscicida infection-induced proinflammatory immune responses and bacterial killing activity in the head kidney macrophages of the Japanese flounder Paralichthys olivaceus

Sphingosine kinases (SPHK1 and SPHK2) are a family of isozymes that phosphorylate sphingosine to generate the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P), which subsequently binds to and activates S1P receptors (S1PRs). Although the SPHK/S1P/S1PR axis has been demonstrated to play crucial roles in modulating immune and inflammatory responses in mammals, its role in teleost immunity remains largely unexplored. In this study, we characterize and investigate the regulatory roles of two sphk isoforms, sphk1-like (sphk1-L) and sphk2, in the inflammatory response in head kidney macrophages (HKMs) from the Japanese flounder Paralichthys olivaceus. SPHK1-L and SPHK2 proteins display conserved domain organizations with their mammalian homologs, highlighting evolutionary conservation across vertebrate lineages. Gene expression analysis reveals that sphk1-L and sphk2 are highly induced in HKMs by LPS stimulation and Edwardsiella piscicida infection, suggesting their involvement in fish immune response. Pharmacological inhibition of SPHKs enzymatic activity or siRNA knockdown of either sphk1-L or sphk2 expression markedly attenuated LPS- and E. piscicida-induced expression of proinflammatory cytokines, chemokines, and inflammasome-related genes in HKMs. Conversely, treatment with the sphingosine analog FTY720 potentiated the expression of inflammatory immune response genes in LPS-treated and E. piscicida-infected HKMs, but this effect was significantly attenuated when SPHK enzymatic activity was pharmacologically inhibited, indicating that SPHK catalytic activity is essential for the enhanced expression of inflammatory response genes in fish macrophages. Additionally, we show that exogenous S1P treatment significantly enhances proinflammatory gene expression in LPS-stimulated and E. piscicida-infected HKMs, and multiple S1PRs are expressed in HKMs, suggesting that S1PR activation by extracellular S1P may involve in this process. Moreover, we demonstrate that SPHK inhibition facilitates the proliferation of intracellular bacteria E. piscicida, while S1P or FTY720 treatment markedly reduced the bacterial colonization in flounder HKMs. Collectively, our studies reveal that the SPHK/S1P axis promotes LPS challenge- and bacterial infection-induced proinflammatory immune responses and antibacterial activity in teleost fish macrophages, likely through the activation of multiple S1PRs.