Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Meishan Campus, Ningbo University, Ningbo, 315832, China.
Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Meishan Campus, Ningbo University, Ningbo, 315832, China; Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Meishan Campus, Ningbo University, Ningbo, 315832, China.
Fish Shellfish Immunol. 2019 Apr;87:886-896. doi: 10.1016/j.fsi.2019.02.019. Epub 2019 Feb 21.
Chemokine (CC motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages (MO/MФ) in mammals. However, the functional repertoire of fish CCL2 remains unclear. Here, we identified a cDNA sequence encoding a novel CCL2-like protein (PaCCL2L) in ayu, Plecoglossus altivelis. Sequence analysis revealed that PaCCL2L grouped with CCL2 homologs, and is most closely related to Mexican tetra (Astyanax mexicanus) and zebrafish (Danio rerio) homologs. PaCCL2 transcripts were expressed in all tested tissues from healthy ayu, with the highest level in the spleen. Upon Vibrio anguillarum infection, PaCCL2L transcripts increased significantly in tested tissues, including the liver, spleen, and head kidney. We then produced the recombinant PaCCL2L mature peptide (rPaCCL2L) by prokaryotic expression and generated the corresponding antibodies (anti-PaCCL2L). A significant increase in PaCCL2L protein and mRNA expression was observed in ayu MO/MФ following V. anguillarum challenge. Intraperitoneal injection of rPaCCL2L resulted in significantly improved survival and reduced tissue bacterial load in V. anguillarum-infected ayu. rPaCCL2L had a positive effect on the chemotaxis of MO/MΦ and neutrophils both in vitro and in vivo. Meanwhile, rPaCCL2L exhibited a positive effect on the chemotaxis of LPS-stimulated MO/MΦ (M1 type) in vitro, whereas it exhibited no chemotaxis effect on cAMP-stimulated MO/MΦ (M2 type). In addition, rPaCCL2L treatment exhibited an enhanced effect on MO/MΦ phagocytosis, bacterial killing, respiratory burst, and mRNA expression of proinflammatory cytokines, whereas anti-PaCCL2L treatment had an inhibitory effect. Our study demonstrates that PaCCL2L might play a role in the immune response of ayu against V. anguillarum infection through chemotactic recruitment and activation of MO/MΦ.
趋化因子（CC 基序）配体 2（CCL2），也称为单核细胞趋化蛋白 1（MCP-1），是调节哺乳动物单核细胞/巨噬细胞（MO/MФ）迁移和浸润的关键趋化因子之一。然而，鱼类 CCL2 的功能谱尚不清楚。在这里，我们在牙鲆（Plecoglossus altivelis）中鉴定了一种编码新型 CCL2 样蛋白（PaCCL2L）的 cDNA 序列。序列分析表明，PaCCL2L 与 CCL2 同源物聚集在一起，与墨西哥四齿（Astyanax mexicanus）和斑马鱼（Danio rerio）同源物最为密切相关。PaCCL2 转录本在健康牙鲆的所有测试组织中均有表达，在脾脏中表达水平最高。在鳗弧菌感染后，PaCCL2L 转录本在包括肝脏、脾脏和头肾在内的测试组织中显著增加。然后，我们通过原核表达产生了重组 PaCCL2L 成熟肽（rPaCCL2L），并生成了相应的抗体（抗-PaCCL2L）。在鳗弧菌感染后，牙鲆 MO/MФ 中观察到 PaCCL2L 蛋白和 mRNA 表达显著增加。腹腔注射 rPaCCL2L 可显著提高鳗弧菌感染牙鲆的存活率并降低组织细菌负荷。rPaCCL2L 对体外和体内 MO/MΦ 和中性粒细胞的趋化作用具有积极影响。同时，rPaCCL2L 对 LPS 刺激的 MO/MΦ（M1 型）的趋化作用具有积极影响，而对 cAMP 刺激的 MO/MΦ（M2 型）则没有趋化作用。此外，rPaCCL2L 处理可增强 MO/MΦ 的吞噬作用、杀菌作用、呼吸爆发以及促炎细胞因子的 mRNA 表达，而抗-PaCCL2L 处理则具有抑制作用。我们的研究表明，PaCCL2L 可能通过趋化募集和激活 MO/MΦ 在牙鲆抵抗鳗弧菌感染的免疫反应中发挥作用。
