The A_2b adenosine receptor negatively regulates proinflammatory responses in Japanese flounder (Paralichthys olivaceus) head kidney macrophages

The A_2b adenosine receptor (A_2bR) is a critical modulator of inflammatory responses. Despite its high expression in Japanese flounder (Paralichthys olivaceus) macrophages, the expression characteristics and immunomodulatory functions of A_2bR in this species remain poorly understood. Here, we molecularly characterized flounder A_2bR and established its function as a potent negative regulator of teleost inflammation. The flounder A_2bR gene displays an exon-intron architecture analogous to that of its mammalian orthologs and is broadly expressed across tissues, with maximal abundance in the skin. A_2bR expression was induced by adenosine and significantly modulated by LPS stimulation in HKMs in vitro, as well as by Edwardsiella piscicida infection in vivo. Pharmacological activation of A_2bR markedly attenuated the expression of proinflammatory cytokines and chemokines and reduced nitric oxide (NO) production, while concomitantly enhancing the expression of anti-inflammatory il-10 and the negative-feedback regulator socs1 in HKMs stimulated with LPS or poly(I:C). These immunosuppressive effects were recapitulated in A_2bR-overexpressing flounder FG-9307 cells and murine RAW264.7 macrophages, underscoring an evolutionarily conserved function. Furthermore, A_2bR activation significantly attenuated PAMP-induced pyroptosis-related gene expression in HKMs. Conversely, both pharmacological inhibition and siRNA-mediated knockdown of A_2bR produced opposite effects. Collectively, we demonstrate for the first time in teleosts that A_2bR orchestrates anti-inflammatory responses by suppressing the production of inflammatory mediator and the expression of proinflammatory genes, while promoting IL-10- and SOCS1-mediated negative feedback. These results suggest that A_2bR is a promising target for modulating immune responses to infectious and inflammatory diseases in teleosts.