Functional antagonism and underlying regulatory pathways of haptoglobin splicing isoforms in the immune response of half-smooth tongue sole (Cynoglossus semilaevis).

Haptoglobin (hp) is a conserved acute-phase protein with multiple functions, including immunomodulation, yet the roles of its splicing isoforms remain unclear. In this study, we investigated the functional dichotomy and underlying regulatory network of hp isoforms, hp-I and hp-E, in Cynoglossus semilaevis. In vivo challenge with Vibrio anguillarum revealed that hp-I overexpression was associated with a trend toward improved host survival, while hp-E overexpression tended to increase mortality. At the tissue level (liver, spleen, and intestine), hp-I broadly suppressed infection-induced expression of inflammatory cytokines, whereas hp-E amplified this inflammatory response. This antagonism was reproduced in vitro: in primary macrophages, hp-I inhibited LPS-induced phagocytosis, respiratory burst, and cytokine expression, while hp-E potentiated these functions. Similarly, in hepatocytes, hp-I attenuated and hp-E enhanced LPS-triggered cytokine production. Conventional enrichment analysis identified the MAPK signaling pathway as a core hub bidirectionally regulated, activated by hp-E and suppressed by hp-I. Complementary GSEA indicated hp-E promoted pro-inflammatory states (e.g., TLR signaling), whereas hp-I induced anti-inflammatory profiles. WGCNA identified a hp-E-correlated gene module enriched in immune-related pathways, including multiple receptor-mediated signaling pathways, and pinpointed 16 hub genes (e.g., irf8, spi1, hck). These multi-faceted analyses significantly expand the known regulatory scope of hp by defining a comprehensive receptors-MAPK-cytokine axis that is bidirectionally tuned by its splicing isoforms, moving beyond its previously recognized receptor interactions. This study unveils a novel layer of immunoregulation via hp alternative splicing and provides a framework for understanding its isoform-specific functions across vertebrates, with potential implications for genetic improvement and disease control in aquaculture.