Table_3_Interleukin-22 Deficiency Contributes to Dextran Sulfate Sodium-Induced Inflammation in Japanese Medaka, Oryzias latipes.xlsx

Mucosal tissue forms the first line of defense against pathogenic microorganisms. Cellular damage in the mucosal epithelium may induce the interleukin (IL)-22-related activation of many immune cells, which are essential for maintaining the mucosal epithelial barrier. A previous study on mucosal immunity elucidated that mammalian IL-22 contributes to mucus and antimicrobial peptides (AMPs) production and anti-apoptotic function. IL-22 has been identified in several teleost species and is also induced in response to bacterial infections. However, the roles of IL-22 in teleost immunity and mucus homeostasis are poorly understood. In this study, Japanese medaka (Oryzias latipes) was used as a model fish. The medaka il22, il22 receptor A1 (il22ra1), and il22 binding protein (il22bp) were cloned and characterized. The expression of medaka il22, il22ra1, and il22bp in various tissues was measured using qPCR. These genes were expressed at high levels in the mucosal tissues of the intestines, gills, and skin. The localization of il22 and il22bp mRNA in the gills and intestines was confirmed by in situ hybridizations. Herein, we established IL-22-knockout (KO) medaka using the CRISPR/Cas9 system. In the IL-22-KO medaka, a 4-bp deletion caused a frameshift in il22. To investigate the genes subject to IL-22-dependent regulation, we compared the transcripts of larval medaka between wild-type (WT) and IL-22-KO medaka using RNA-seq and qPCR analyses. The comparison was performed not only in the naïve state but also in the dextran sulfate sodium (DSS)-exposed state. At the transcriptional level, 368 genes, including immune genes, such as those encoding AMPs and cytokines, were significantly downregulated in IL-22-KO medaka compared that in WT medaka in naïve states. Gene ontology analysis revealed that upon DSS stimulation, genes associated with cell death, acute inflammatory response, cell proliferation, and others were upregulated in WT medaka. Furthermore, in DSS-stimulated IL-22-KO medaka, wound healing was delayed, the number of apoptotic cells increased, and the number of goblet cells in the intestinal epithelium decreased. These results suggested that in medaka, IL-22 is important for maintaining intestinal homeostasis, and the disruption of the IL-22 pathway is associated with the exacerbation of inflammatory pathology, as observed for mammalian IL-22.

黏膜组织是抵御病原微生物的第一道防线。黏膜上皮细胞损伤可诱导白细胞介素（interleukin, IL）-22相关的多种免疫细胞活化，而该活化过程对维持黏膜上皮屏障功能至关重要。既往针对黏膜免疫的研究表明，哺乳动物IL-22可促进黏液与抗菌肽（antimicrobial peptides, AMPs）的产生，并发挥抗凋亡功能。目前已在多种硬骨鱼物种中鉴定出IL-22，且其表达可因细菌感染而被诱导。然而，IL-22在硬骨鱼免疫与黏液稳态中的作用仍有待阐明。

本研究以日本青鳉（Oryzias latipes）作为模式实验鱼类。我们克隆并鉴定了青鳉il22、il22受体A1（il22 receptor A1, il22ra1）以及il22结合蛋白（il22 binding protein, il22bp）的基因序列。采用实时荧光定量PCR（quantitative real-time PCR, qPCR）检测了上述基因在青鳉各组织中的表达情况，结果显示其在肠道、鳃及皮肤等黏膜组织中均呈高表达。通过原位杂交（in situ hybridization）技术，我们验证了il22与il22bp mRNA在鳃与肠道中的定位分布。

本研究借助CRISPR/Cas9系统构建了IL-22敲除（IL-22-knockout, KO）青鳉模型：在该敲除青鳉中，il22基因因4个碱基的缺失而发生移码突变。为探究受IL-22依赖性调控的靶基因，我们分别采用RNA测序（RNA-seq）与qPCR技术，比较了野生型（wild-type, WT）与IL-22敲除青鳉幼鱼的转录组表达谱，该比较实验分别在未刺激的稳态与硫酸葡聚糖钠（dextran sulfate sodium, DSS）刺激状态下开展。

在未刺激稳态下，与野生型青鳉相比，IL-22敲除青鳉体内共有368个基因的转录水平显著下调，其中包括编码抗菌肽与细胞因子等的免疫相关基因。基因本体（Gene Ontology, GO）富集分析结果显示，经DSS刺激后，野生型青鳉体内与细胞死亡、急性炎症反应、细胞增殖等相关的基因均呈现上调表达。

此外，经DSS刺激的IL-22敲除青鳉出现了伤口愈合延迟、凋亡细胞数量增多以及肠道上皮杯状细胞数量减少的表型。

上述结果表明，在青鳉中IL-22对维持肠道稳态具有重要作用，且IL-22信号通路的缺陷会加剧炎症病理损伤，这与哺乳动物中IL-22的功能特征一致。