The haemocyte highly-expressed E-type prostanoid receptor regulates TNF expression during immune response of oyster Crassostrea gigas

Prostaglandin E2 imparts diverse physiological effects on multiple cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4), among which the EP4 is one of subtypes known to mediate the immune response in mammalian monocytes and macrophages. However, the precise characteristics and functions of EP4 in mollusks remain unclear. In the present study, an EP4 homologue (designated as CgEP4) was identified from oyster Crassostrea gigas. CgEP4 contained a seven-helix transmembrane domain, shared significant homology with its vertebrate homologs, and clustered with EP4s from other Mollusca by phylogenetic analysis. When CgEP4 was transfected and expressed in HEK293 cell line, the concentration of intracellular Ca²⁺ was significantly increased after treatment with its agonist PGE2, while that of cAMP was not obviously changed. The mRNA transcripts of CgEP4 were dominantly expressed in haemocytes, which was 28.37-fold (p < 0.05) higher than that in hepatopancreas. By immunofluorescence analysis, CgEP4 was found to be mainly expressed in the agranulocyte subpopulation, and CgEP4⁺ agranulocyte accounted for 18% of total haemocytes. After Vibrio splendidus stimulation, the mRNA expression of CgEP4 in haemocytes was not obviously changed at the first 12 h, then significantly up-regulated at 24 h, which was 2.7-fold (p < 0.01) of that in control group. After PGE2 treatment in vivo, the phosphorylation of JNK (p-JNK) in haemocytes, but not the phosphorylated ERK (p-ERK), was significantly decreased, which was 0.64-fold (p < 0.05) of that in control group. Simultaneously, the mRNA expression levels of CgTNF-1 and CgTNF-2 in haemocytes dramatically down-regulated, which were 0.06-fold (p < 0.05) and 0.73-fold (p <0.05) of that in control group at 24 h. When the EP4 antagonist Grapiprant was added with PGE2 treatment in vivo, the p-JNK in haemocytes significantly increased, concomitant with the up-regulation of expressions of CgTNF-1 and CgTNF-2, which were 2.86-fold (p < 0.05) and 1.31-fold (p <0.05) of that in control group at 24 h. Collectively, these results provides the experimental evidence of a haemocyte highly-expressed EP4 receptor CgEP4 regulating TNFs' expression through MAPK pathway in the innate immune response in C. gigas, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes.