AUTHOR=Chen Kangkang , Wang Xinyan , Wei Xiangyi , Chen Jiaqian , Wei Youheng , Jiang Haobo , Lu Zhiqiang , Feng Congjing 

TITLE=Nitric Oxide-Induced Calcineurin A Mediates Antimicrobial Peptide Production Through the IMD Pathway

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.905419

DOI=10.3389/fimmu.2022.905419

ISSN=1664-3224

ABSTRACT=<p>Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. <italic>Drosophila</italic> calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, we used a lepidopteran pest <italic>Ostrinia furnacalis</italic> and <italic>Drosophila</italic> S2 cells to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of <italic>nitric oxide synthase 1</italic>/<italic>2</italic> (<italic>NOS1/2</italic>), <italic>CanA</italic> and <italic>AMP</italic> genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected <italic>O. furnacalis</italic>. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed <italic>Escherichia coli</italic> induced <italic>NOS</italic> transcription and boosted NO production, whereas knockdown of <italic>NOS</italic> blocked the NO level increase caused by <italic>E. coli</italic>. As in <italic>O. furnacalis</italic> larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of <italic>CecropinA1</italic>, <italic>Defensin</italic>, <italic>Diptericin</italic>, and <italic>Drosomycin</italic> by killed <italic>E. coli</italic>. Knockdown of <italic>NOS</italic> also reduced the expression of <italic>CanA1</italic> and <italic>AMPs</italic> induced by <italic>E. coli</italic>, indicative of a role of NO in the <italic>AMP</italic> expression. Furthermore, <italic>CanA1</italic> RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced <italic>AMP</italic> expression, and knockdown of <italic>IMD</italic> suppressed NO-induced <italic>AMP</italic> expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 <italic>via</italic> the IMD pathway.</p>