Table_2_Novel Insight Into Nutritional Regulation in Enhancement of Immune Status and Mediation of Inflammation Dynamics Integrated Study In Vivo and In Vitro of Teleost Grass Carp (Ctenopharyngodon idella): Administration of Threonine.docx

This study aims to investigate the effects of threonine (Thr) on immunoregulation in vivo and in vitro of teleost grass carp (Ctenopharyngodon idella). Juveniles (9.53 ± 0.02 g) were reared for 8 weeks with respective Thr diet (3.99, 7.70, 10.72, 14.10, 17.96, and 21.66 g/kg) and then challenged with Aeromonas hydrophila for in vivo study. Macrophages isolated from head kidney were treated in vitro for 48 h with L-Thr (0, 0.5, 1.0, 2.0, 4.0, and 8.0 mM) after 6 h of lipopolysaccharide induction. The results showed that, compared with Thr deficiency (3.99 g/kg), the optimal dietary Thr (14.10g/kg) affected the immunocyte activation in the head kidney (HK) and spleen (SP) by downregulating the mRNA expressions of MHC-II and upregulating CD4 (not CD8), and it mediated the innate immune by enhancing the activities of lysozyme (LZ), acid phosphatase content of complement 3 (C3) and C4, increasing the mRNA abundances of hepcidin, liver expressed antimicrobial peptide-2A (LEAP-2A), LEAP-2B, β-defensin1, downregulating tumor necrosis factor α (TNF-α), IL-6, IL-1β, IL-12p35, IL-12p40, IL-17AF1, and IL-17D partly by attenuating RORγ1 transcriptional factor and nuclear factor kappa B p65 (NF-κBp65) signaling cascades [IKKβ/IκBα/NF-κBp65] and upregulating transforming growth factor β1 (TGF-β1), IL-4/13A, -4/13B, IL-10, and IL-22 partly by GATA-3. Besides these, the optimal dietary Thr regulated the adaptive immune by upregulating the mRNAs of immunoglobulin M (IgM) and IgZ (not IgD). Moreover, 2 mM Thr downregulated in vitro the mRNA abundances of colony stimulating factor-1, inducible nitric oxide synthase, mannose receptor 1, matrix metalloproteinase2 (MMP-2), and MMP-9 significantly (P < 0.05), indicating that Thr could attenuate the M1-type macrophages’ activation. Moreover, L-Thr downregulated the mRNA transcripts of TNF-α, IL-6, and IL-1β associated with impairing the SOCS1/STAT1 signaling and upregulated IL-10 and TGF-β1 partly by accentuating the SOCS3/STAT3 pathway. The above-mentioned observations suggested that Thr improved the immune status in the immune organs of fish by enhancing the immune defense and mediating the inflammation process. Finally, based on the immune indices of LZ activity in HK and C3 content in SP, the optimal Thr for immune enhancement in juvenile grass carp (9.53–53.43 g) was determined to be 15.70 g/kg diet (4.85 g/100 g protein) and 14.49 g/kg diet (4.47 g/100 g protein), respectively.

本研究旨在探究苏氨酸（threonine, Thr）对硬骨鱼类（teleost）草鱼（Ctenopharyngodon idella）体内外免疫调控的影响。选取初始体质量为(9.53±0.02)g的草鱼幼鱼，饲喂分别添加3.99、7.70、10.72、14.10、17.96及21.66 g/kg苏氨酸的配合饲料8周，随后以嗜水气单胞菌（Aeromonas hydrophila）攻毒开展体内免疫相关实验。分离头肾巨噬细胞（macrophages），经脂多糖（lipopolysaccharide）诱导6小时后，分别用浓度为0、0.5、1.0、2.0、4.0及8.0 mM的L-苏氨酸处理48小时，进行体外免疫实验。

与苏氨酸缺乏组（3.99 g/kg）相比，适宜饲粮水平的苏氨酸（14.10 g/kg）可通过下调主要组织相容性复合体II（MHC-II）的mRNA表达、上调CD4（而非CD8）的转录水平，影响头肾（HK）与脾脏（SP）中的免疫细胞活化；同时可通过增强溶菌酶（LZ）活性、补体3（C3）与补体4（C4）的含量，上调铁调素（hepcidin）、肝脏表达抗菌肽-2A（LEAP-2A）、LEAP-2B及β-防御素1（β-defensin1）的mRNA丰度，下调肿瘤坏死因子α（TNF-α）、IL-6、IL-1β、IL-12p35、IL-12p40、IL-17AF1及IL-17D的表达，该调控过程部分依赖于减弱视黄酸相关孤儿受体γ1（RORγ1）转录因子与核因子κB p65（NF-κBp65）信号通路[IKKβ/IκBα/NF-κBp65]的活化；此外，该适宜苏氨酸水平还可通过GATA-3介导上调转化生长因子β1（TGF-β1）、IL-4/13A、IL-4/13B、IL-10及IL-22的表达，从而调控先天性免疫应答。

除此之外，适宜饲粮苏氨酸还可通过上调免疫球蛋白M（IgM）与IgZ（而非IgD）的mRNA水平，调控适应性免疫应答。

体外实验结果进一步显示，2 mM L-苏氨酸可显著下调集落刺激因子1（colony stimulating factor-1）、诱导型一氧化氮合酶（inducible nitric oxide synthase）、甘露糖受体1（mannose receptor 1）、基质金属蛋白酶2（MMP-2）及MMP-9的mRNA表达量（P < 0.05），表明苏氨酸可减弱M1型巨噬细胞的活化。同时，L-苏氨酸可通过损害SOCS1/STAT1信号通路下调TNF-α、IL-6及IL-1β的mRNA转录，并通过增强SOCS3/STAT3通路部分上调IL-10与TGF-β1的表达。

上述结果表明，苏氨酸可通过增强免疫防御、调控炎症进程，改善鱼类免疫器官的免疫状态。最后，基于头肾溶菌酶活性与脾脏补体3含量这两项免疫指标，本研究确定体质量为9.53~53.43 g的草鱼幼鱼，其饲粮中用于增强免疫的最适苏氨酸添加量分别为15.70 g/kg饲料（即4.85 g/100 g蛋白质）与14.49 g/kg饲料（即4.47 g/100 g蛋白质）。