Table1_Megalobrama amblycephala IL-22 attenuates Aeromonas hydrophila induced inflammation, apoptosis and tissue injury by regulating the ROS/NLRP3 inflammasome axis.doc

Mammalian interleukin-22 (IL-22) attenuates organismal injury by inhibiting reactive oxygen species (ROS) and impeding the NLRP3 inflammasome activation. However, the role of fish IL-22 in this process remains unclear. We characterized MaIL-22, an IL-22 homolog in blunt snout bream (Megalobrama amblycephala). Despite its low sequence identity, it shares conserved structures and close evolutionary relationships with other teleost IL-22s. Furthermore, Aeromonas hydrophila (A. hydrophila) infection leads to tissue injury in M. amblycephala immune organs and concomitantly altered Mail-22 mRNA expression, suggesting that MaIL-22 was involved in the antimicrobial immune response. To explore MaIL-22’s biological functions, we produced recombinant MaIL-22 (rMaIL-22) protein and demonstrated it significantly enhanced the survival of M. amblycephala post-A. hydrophila infection. To unravel its protective mechanisms, we explored the ROS/NLRP3 inflammasome axis and its downstream signaling responses. The results showed that rMaIL-22 treatment significantly elevated antioxidant enzyme (T-SOD, CAT and GSH-PX) activities to inhibit MDA activity and scavenge ROS in visceral tissues. Meanwhile, rMaIL-22 impeded the activation of NLRP3 inflammasome by suppressing NLRP3 protein and mRNA expression. This indicated that rMaIL-22 contributed to inhibit A. hydrophila-induced activation of the ROS/NLRP3 inflammasome axis. Consistent with these findings, rMaIL-22 treatment attenuated the expression of proinflammatory cytokines (il-1β, tnf-α and il-6) and proapoptotic genes (caspase-3 and caspase-8) while promoting antiapoptotic genes (bcl-2b and mcl-1a) expression, ultimately mitigating tissue injury in visceral tissues. In conclusion, our research underscores MaIL-22’s key role in microbial immune regulation, offering insights for developing IL-22-targeted therapies and breeding programs.

哺乳动物白细胞介素-22（interleukin-22, IL-22）可通过抑制活性氧（reactive oxygen species, ROS）生成及阻滞NLRP3炎性小体（NLRP3 inflammasome）活化，减轻机体损伤。然而，鱼类IL-22在该过程中的作用尚不清楚。本研究对团头鲂（Megalobrama amblycephala）中的IL-22同源基因MaIL-22进行了功能鉴定。尽管其序列同源性较低，但与其他硬骨鱼IL-22共享保守的结构特征及较近的进化亲缘关系。进一步研究发现，嗜水气单胞菌（Aeromonas hydrophila，简称A. hydrophila）感染可导致团头鲂免疫器官发生组织损伤，并伴随MaIL-22信使RNA（mRNA）表达水平的改变，提示MaIL-22参与了抗菌免疫应答。为探究MaIL-22的生物学功能，本研究制备了重组MaIL-22（rMaIL-22）蛋白，并证实其可显著提升嗜水气单胞菌感染后团头鲂的存活率。为阐明其保护作用机制，本研究围绕ROS/NLRP3炎性小体轴及其下游信号通路展开探索。结果显示，rMaIL-22处理可显著提高内脏组织中抗氧化酶（总超氧化物歧化酶T-SOD、过氧化氢酶CAT及谷胱甘肽过氧化物酶GSH-PX）的活性，抑制丙二醛（MDA）生成并清除ROS。同时，rMaIL-22可通过下调NLRP3的蛋白及mRNA表达，阻滞NLRP3炎性小体的活化，表明rMaIL-22可抑制嗜水气单胞菌诱导的ROS/NLRP3炎性小体轴激活。与上述结果一致，rMaIL-22处理可降低促炎细胞因子（白细胞介素-1β IL-1β、肿瘤坏死因子-α TNF-α及白细胞介素-6 IL-6）及促凋亡基因（半胱天冬氨酸蛋白酶-3 caspase-3、半胱天冬氨酸蛋白酶-8 caspase-8）的表达，同时上调抗凋亡基因（bcl-2b、mcl-1a）的表达，最终减轻内脏组织的损伤。综上，本研究证实MaIL-22在微生物免疫调控中发挥关键作用，为靶向IL-22的治疗策略开发及育种工作提供了理论依据。