Table_3_Interleukin-1 Ligands and Receptors in Lumpfish (Cyclopterus lumpus L.): Molecular Characterization, Phylogeny, Gene Expression, and Transcriptome Analyses.docx

The interleukin (IL)-1 family play a fundamental role as immune system modulators. Our previous transcriptome-analyses of leukocytes from lumpfish (Cyclopterus lumpus L.) showed that IL-1β was among the most highly upregulated genes following bacterial exposure. In the present study, we characterized IL-1 signaling pathways, identified and characterized four ligands of the IL-1 family in lumpfish; IL-1β type I and type II, IL-18, and the novel IL-1 family members (nIL-1F), both at mRNA and gene levels. The two IL-1β in lumpfish is termed IL-1β1 (type II) and IL-1β2 (type I). Furthermore, a comprehensive phylogenetic analysis of 277 IL-1 ligands showed that nIL-1F, in common with IL-1β, likely represents an ancestral gene, as representatives for nIL-1F were found in cartilaginous and lobe-finned fish, in addition to teleosts. This shows that nIL-1F is not exclusively present in teleosts as previously suggested. Our analyses of exon-intron structures, intron phases, phylogeny and synteny clearly show the separation of IL-1β into groups; type I and type II, which likely is a result of the third whole genome duplication (3R WGD). The phylogenetic analysis shows that most teleosts have both type I and type II. Furthermore, we have determined transcription levels of the IL-1 ligands in leukocytes and 16 different tissues, and their responses upon in vitro stimulation with seven different ligands. In addition, we have identified the IL-1 receptors IL-1R1, IL-1R2, IL-1R4 (ST2/IL-33 receptor/IL-1RL), IL-1R5 (IL-18R1), and partial sequences of DIGIRR and IL-1R3 (IL-RAcP). Identification of immune molecules and description of innate responses in lumpfish is interesting for comparative and evolutionary studies and our study constitutes a solid basis for further functional analyses of IL-1 ligands and receptors in lumpfish. Furthermore, since lumpfish are now farmed in large numbers to be used as cleaner fish for removal of sea lice on farmed salmon, in-depth knowledge of key immune molecules, signaling pathways and innate immune responses is needed, as the basis for design of efficient immune prophylactic measures such as vaccination.

白细胞介素（interleukin, IL）家族作为免疫系统的核心调控因子，发挥着基础性作用。我们此前对圆鳍鱼（Cyclopterus lumpus L.）白细胞开展的转录组分析显示，白细胞介素-1β（IL-1β）是细菌暴露后上调幅度最高的基因之一。本研究对圆鳍鱼的IL-1信号通路进行了系统表征，并从信使RNA（mRNA）和基因两个层面，鉴定并表征了圆鳍鱼体内的4种IL-1家族配体：I型IL-1β、II型IL-1β、IL-18，以及新型IL-1家族成员（novel IL-1 family members, nIL-1F）。圆鳍鱼体内的两种IL-1β分别被命名为IL-1β1（II型）和IL-1β2（I型）。此外，针对277种IL-1配体的全面系统发育分析显示，nIL-1F与IL-1β类似，很可能是一类祖先基因——因为除硬骨鱼外，在软骨鱼和肉鳍鱼中也发现了nIL-1F的同源序列。这表明nIL-1F并非如此前研究所提出的那样仅存在于硬骨鱼中。我们对外显子-内含子结构、内含子相位、系统发育及同线性的分析结果清晰表明，IL-1β可分为I型和II型两个类群，这一分化很可能源于第三次全基因组复制（third whole genome duplication, 3R WGD）。系统发育分析显示，多数硬骨鱼同时拥有I型和II型IL-1β。此外，我们还测定了圆鳍鱼白细胞与16种不同组织中IL-1配体的转录水平，以及它们在7种不同配体体外刺激下的应答反应。另外，我们还鉴定出了IL-1受体IL-1R1、IL-1R2、IL-1R4（ST2/IL-33受体/IL-1RL）、IL-1R5（IL-18R1），以及DIGIRR和IL-1R3（IL-RAcP）的部分序列。对圆鳍鱼免疫分子的鉴定以及固有免疫应答的描述，可为比较免疫学与进化生物学研究提供重要参考，本研究也为后续开展圆鳍鱼IL-1配体与受体的功能分析奠定了坚实基础。此外，当前圆鳍鱼已被大规模养殖，作为清洁鱼用于清除养殖三文鱼体表的海虱，因此亟需深入解析其关键免疫分子、信号通路与固有免疫应答，以此为基础开发疫苗接种等高效免疫预防策略。