Involvement of LTB4 and a CXCR4-like protein in extracellular trap formation in Ruditapes philippinarum.

Leukotriene B4 (LTB4) is a pro-inflammatory lipid mediator that cooperates with CXC chemokine receptor type 4 (CXCR4) in regulating immune responses in vertebrates, but their function in bivalve innate immunity remains largely unknown. This study investigated the involvement of LTB4 and a CXCR4-like protein in extracellular traps (ET) formation in Ruditapes philippinarum. Metabolomics revealed that LTB4 was a key differential metabolite in gill tissues following LPS stimulation, and ELISA further confirmed that LTB4 levels were significantly elevated upon LPS challenge. A CXCR4 homolog designated RpCXCR4-like was further characterized, which possesses typical seven transmembrane (7-TM) and the DRY motif. In unstimulated clams, RpCXCR4-like transcripts were constitutively expressed in all examined tissues, especially in gills. After LPS stimulation, expression of RpCXCR4-like mRNA in hemocytes and gills was up-regulated. Furthermore, LTB4 treatment also induced RpCXCR4-like expression, whereas pretreatment with the LTB4 receptor antagonist docosatrienoic acid (DTA) significantly reduced this upregulation. Functional assays demonstrated that LTB4 and LPS significantly induced reactive oxygen species (ROS) production and ET formation in gill cells, whereas pretreatment with DTA or knockdown of RpCXCR4-like significantly attenuated these effects. Collectively, LTB4 and RpCXCR4-like promote ET formation in gills through ROS production, providing novel insights into the regulatory network between lipid mediators and innate immunity in clams.