EED226 treatment targets key immune genes to suppress LPS response in Chinese tongue sole splenocytes.

Epigenetic mechanisms governing immune responses to bacterial lipopolysaccharide (LPS) represent a critical yet underexplored frontier in immunology, particularly in teleost fish that lack canonical TLR4-mediated LPS recognition pathways. Here, we demonstrate that EED226, a selective inhibitor of embryonic ectoderm development (EED) protein, effectively modulates LPS-induced immune responses in Chinese tongue sole (Cynoglossus semilaevis) splenocytes. Phylogenetic analysis revealed that EED exhibits high sequence similarity across vertebrates, with the WD40 domain showing exceptional conservation, suggesting EED as a cross-species therapeutic target. Using transcriptomic approaches, we identified 213 immune genes that are upregulated by LPS stimulation and downregulated by co-treatment with LPS and EED226. Among these, we validated key immune mediators including clec4c, ltbr, mcp-1b, tnfrsf13b, tnfsf6, and tnfsf14 through quantitative RT-PCR analysis. Notably, single-cell RNA sequencing (scRNA-seq) revealed cell-type-specific co-expression patterns between eed and target immune genes: tnfrsf13b in B cells, mcp-1b in T cells, and tnfsf14 in both M2 macrophages and T cells. Functional pathway analysis demonstrated that EED226 primarily suppresses cytokine-cytokine receptor interaction and leukocyte transendothelial migration pathways, indicating broad immunomodulatory effects. This study represents a novel investigation into the immunoregulatory function of eed and its inhibitor EED226 in fish. These findings establish strong evidence for epigenetic regulation of LPS responses in teleost and provide mechanistic insights into EED-mediated immune response. Our results have significant implications for developing targeted epigenetic therapies to control bacterial infections and inflammatory diseases in aquaculture, while advancing our understanding of evolutionary conservation in innate immune regulation.