Involvement of LTC4 and NADPH oxidase 5 in extracellular trap formation in Ruditapes philippinarum

NADPH oxidase 5 (NOX5) is a calcium-dependent enzyme that generates reactive oxygen species (ROS) and plays critical roles in immune responses, the functions of which have been shown to be regulated by the pro-inflammatory mediator leukotriene C4 (LTC4). In the present study, a novel NOX5 homology was identified from Ruditapes philippinarum (designated as RpNOX5). Phylogenetic analysis and conserved motif characterization supported RpNOX5 as a new member of the invertebrate NOX5 family. RpNOX5 contains typical NOX5 structural domains, including EF-hand motifs, ferric reductase domain, FNR-like domain and NAD-binding domain. Spatial expression analysis revealed that RpNOX5 was ubiquitously expressed in all examined tissues with the highest expression level in gills. LPS stimulation upregulated RpNOX5 expression, as well as induced LTC4 production. Both LPS and LTC4 enhanced RpNOX5 expression, whereas LTC4 receptor antagonist pranlukast abolished this effect. Functional assays showed that LTC4 and LPS significantly induced superoxide anion (by 3.84-fold and 3.90-fold, respectively) and ROS production (by 2.73-fold and 3.24-fold, respectively), whereas pretreatment with pranlukast or knockdown of RpNOX5 markedly attenuated these effects. In hemocytes, LTC4 and LPS stimulation induced the release of extracellular trap (ET) structures, accompanied by elevated extracellular double-stranded DNA (dsDNA) levels. Abundant fibrous ET-like structures were observed in gills of LPS- and LTC4-treated groups with surrounding tissues remaining intact. These effects in hemocytes and gills were reduced after pranlukast or knockdown of RpNOX5. Collectively, LTC4 and RpNOX5 promote ET formation in R. philippinarum through ROS production, providing novel insights into the regulatory network between lipid mediators and innate immunity in clams.