JunD functions as a transcription factor of IL-10 to regulate bacterial infectious inflammation in grass carp (Ctenopharyngodon idella)

Abstract

IL-10 is a key anti-inflammatory mediator ensuring the protection of a host from excessive inflammation in response to pathogen infections, whose transcription or expression levels are tightly linked to the onset and progression of infectious diseases. An AP-1 family member called CiJunD was shown to be a transcription factor of IL-10 in grass carp (Ctenopharyngodon idella) in the current study. CiJunD protein harbored the conserved Jun and bZIP domains. Mutant experiments demonstrated that CiJunD bound to three specific sites on IL-10 promoter, i.e., 5'-ATTATTCATA-3', 5'-AGATGAGACATCT-3', and 5'-ATTATTCATC-3', mainly relying on the bZIP domain, and initiated IL-10 transcription. Expression data from the grass carp spleen infected by Aeromonas hydrophila and lipopolysaccharide (LPS) challenged spleen leukocytes indicated that the expressions of CiJunD and IL-10 were positively correlated, while the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, IFN-γ, and TNF-α, showed an overall downward trend when CiJunD and IL-10 peaked. The ability of CiJunD to down-regulate the production of pro-inflammatory cytokines and up-regulate the expression of IL-10, both with and without LPS stimulation, was confirmed by overexpression experiments. Meanwhile, the subcellular fractionation assay revealed that the nuclear translocation of CiJunD was significantly enhanced after the LPS challenge. Moreover, in vivo administration of grass carp with Oxamflatin, a potent agonist of JunD activity, could promote IL-10 but suppress the expression of pro-inflammatory cytokines. Intriguingly, tissue inflammation lesions and the survival rates of grass carp infected with A. hydrophila were also significantly improved by Oxamflatin administration. This work sheds light on the regulation mechanism by JunD of IL-10 expression and bacterial infectious inflammation for the first time, and it may present a viable method for preventing infectious diseases in fish by regulating IL-10 expression and inflammatory response.

Keywords: Excessive inflammation; Grass carp; IL-10 expression; JunD; Pro-inflammatory cytokines.