An insect serotonin receptor mediates cellular immune responses and its inhibition by phenylethylamide derivatives from bacterial secondary metabolites

Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune responses and modulates nerve signal in insects. Se-5HTR, a specific receptor of serotonin, has been identified in the beet armyworm, Spodoptera exigua. It is classified into subtype 7 among known 5HTRs. Se-5HTR was expressed in all developmental stages of S. exigua. It was expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited significant immunosuppression by preventing cellular immune responses such as phagocytosis and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in significant immunosuppression. Such immunosuppression was also induced by bacterial secondary metabolites derived from Xenorhabdus and Photorhabdus. To determine specific bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites with respect to cellular immune responses associated with Se-5HTR and selected 10 potent inhibitors. These 10 selected compounds competitively inhibited cellular immune responses against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA derivatives were screened and resulting potent chemicals were used to design a compound to be highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It showed high immunosuppressive activities along with specific and competitive inhibition activity for Se-5HTR. This study reports the first 5HT receptor from S. exigua and provides its specific inhibitor designed from bacterial metabolites and their derivatives. Author SummarySerotonin (5-hydroxytryptamine: 5-HT) plays a crucial role in mediating nerve and immune signals in insects. Interruption of 5-HT signal leads to malfunctioning of various insect physiological processes. Se-5HTR, a 5-HT receptor of beet armyworm, Spodoptera exigua, was identified and classified as subtype 7 (5-HT7) of 5-HT receptors. A specific inhibitor (SB-269970) for 5-HT7 highly inhibited immune responses such as phagocytosis and nodulation mediated by Se-5HTR. Two entomopathogenic bacteria, Xenorhabdus and Photorhabdus, could secrete potent inhibitors against immune responses mediated by 5-HTR. Bacterial secondary metabolites were screened against Se-5HTR-mediating immune responses. Most of resulting compounds shared phenylethylamide (PEA) chemical skeleton. Subsequent screening using PEA derivatives supported the importance of this chemical skeleton. Based on their relative inhibitory activities, a compound was designed and synthesized. This novel compound possessed high inhibitory activities against Se-5HTR-mediating immune responses and exhibited competitive inhibition with 5-HT.