Interleukin-17A/F1 Deficiency Reduces Antimicrobial Gene Expression and Contributes to Microbiome Alterations in Intestines of Japanese medaka (Oryzias latipes)
Interleukin-17A/F1 Deficiency Reduces Antimicrobial Gene Expression and Contributes to Microbiome Alterations in Intestines of Japanese medaka (Oryzias latipes)
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DOI：
10.3389/fimmu.2020.00425
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发表时间：
2020-03-17
影响因子：
7.3
通讯作者：
Hikima, Jun-ichi
中科院分区：
文献类型：
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作者：
Okamura, Yo;Morimoto, Natsuki;Hikima, Jun-ichi
在哺乳动物中，白细胞介素（IL）-17A和F是标志性的炎性细胞因子，在保护免受感染和肠粘膜免疫中发挥关键作用。在胃肠道（GI）中，通过潘氏细胞诱导抗菌肽（AMP）产生是IL-17 A和F在维持GI微生物组和健康的稳态中的基本作用。虽然哺乳动物IL-17A和F同源物（称为IL-17A/F1 - 3）已在几种鱼类中鉴定，但对其在肠道中的功能知之甚少。此外，鱼的肠道缺乏潘氏细胞，其GI结构与哺乳动物的GI结构非常不同。因此，通过IL-17 A/F基因的GI微生物组调节机制尚未完全阐明。在这项研究中，日本青鳉（Oryzias latipes）被用作硬骨鱼模型，并且使用CRISPR/Cas9基因组编辑技术建立IL-17A/F1-敲除（IL-17A/F1-KO）青鳉。此外，产生了两种IL-17 A/F1缺陷青鳉菌株，包括一种含有7-bp缺失（-7）的菌株和另一种含有11-bp添加（+11）的菌株。在建立F2纯合KO青鳉后，进行转录组分析（RNA-seq）以阐明肠中的IL-17A/F1依赖性基因诱导。RNA-seq和实时荧光定量PCR（qPCR）的结果表明，在IL-17 A/F1-KO青鳉中，免疫相关基因，包括白细胞介素-1 β（IL-1 β）、补体1q亚基C（C1qc）、转铁蛋白a（Tfa）和G型溶菌酶（LyzG）下调。有趣的是，蛋白质和脂质消化酶基因，包括磷脂酶A2，IB组（pla2g1b）和弹性蛋白酶-1-样（CELA 1），也在IL-17 A/F1-KO青鳉的肠道中下调。此外，为了揭示这些下调基因对IL-17 A/F1-KO中肠道微生物组的影响，进行了基于16S rRNA的宏基因组测序分析以分析微生物组构成。在非暴露状态下，IL-17 A/F1-KO青鳉的肠道微生物组在门水平上与野生型不同，其中疣微菌属和浮游菌属的水平显著更高。此外，在人类和鱼类病原体的操作分类单位（OTU）水平上，肠杆菌科类志贺邻单胞菌是IL-17 A/F1-KO青鳉中的优势种。这些发现表明IL-17A/F1参与维持健康的肠道微生物组。
In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1 beta (IL-1 beta), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.