Serum-derived exosomes induce proinflammatory cytokines production in Cynoglossus semilaevis via miR-133-3p

Abstract

Exosomes are small membrane-enclosed vesicles secreted by various types of cells. In mammals, a wide range of physiological and pathological functions have been confirmed and attributed to EVs carrying a variety of molecular cargoes, including miRNAs. However, studies on the biological functions and related molecular mechanisms of serum exosomes isolated from teleost fish are limited. Indeed, the molecular mechanisms underlying the effects of serum exosomes on immune responses and inflammatory processes are unknown. Chinese tongue sole (Cynoglossus semilaevis) is an economically important species used widely in industrial aquaculture. Vibrio harveyi, a common bacterial pathogen that infects C. semilaevis and some other fish, causes excessive inflammatory reactions, which are characterized by skin ulceration. Here, we isolated serum-derived exosomes from C. semilaevis and investigated their effects on inflammatory processes following V. harveyi infection. We found that compared with uninfected fish, exosome abundance in infected fish blood increased with bacterial infection time, while expression of TNF-α increased, and that of IL-10 decreased, significantly. Moreover, artificial infection studies demonstrated that injection of serum exosomes isolated from infected fish increased expression of TNF-α, IL-6, and IL-8, which is consistent with the increase in proinflammatory cytokines induced by V. harveyi infection. To further investigate the mechanisms by which exosomes increase proinflammatory cytokine production, we performed miRNA expression profiling and found that 26 differentially expressed miRNAs were associated with bacterial infection and immune responses; of these, miR-133-3p was considerably more abundant in serum exosomes from infected fish. Bioinformatics analysis suggested that miR-133-3p inhibits NF-κB signaling pathways by targeting PP2A and affecting cytokine release. We also found that miR-133-3p increased expression of TNF-α, IL-6, and IL-8 in fish blood and kidney, whereas an miR-133-3p inhibitor showed the opposite results. Thus, the data suggest that serum exosomes participate in innate immunity in teleost fish by promoting inflammatory responses to bacterial infection. Exosome-mediated transfer of miR-133-3p increases expression of proinflammatory cytokines in C. semilaevis, resulting in excessive inflammatory responses during V. harveyi infection. These data may lead to development of methods and strategies that control skin ulceration in Chinese tongue sole.

Keywords: Cynoglossus semilaevis; Inflammation cytokines; Serum exosome; Vibrio harveyi; miR-133-3p.