Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, 266003, PR China.
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, 266003, PR China; Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, PR China.
Mol Immunol. 2021 Jul;135:170-182. doi: 10.1016/j.molimm.2021.04.011. Epub 2021 Apr 23.
The polymeric immunoglobulin receptor (pIgR) transports secretory immunoglobulins across mucosal epithelial cells into external secretions, playing critical roles in mucosal surface defenses, but the regulation mechanism of pIgR expression is not clarified in teleost fish. In this study, the dynamic changes of flounder (Paralichthys olivaceus) pIgR (fpIgR) and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) mRNA expression in mucosal tissues were first analyzed post inactivated Vibrio anguillarum immunization, and increased production of TNF-α was found to correlate with increased expression of fpIgR. To determine that cytokine TNF-α influenced fpIgR expression, following confirming that natural fpIgR expressed on flounder gill (FG) cells, FG cells were incubated with various concentrations of recombinant TNF-α for different time, the results showed that the expressions of fpIgR were significantly upregulated at gene and protein levels in a dose-dependent and time-dependent manner, and similar change trend was observed for free secretory component (SC) secreted by fpIgR into the culture supernatant. After FG cells were treated with TNF-α, specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, nuclear factor kappa-B (NF-κB) inhibitor Bay11-7082, and the mixtures of TNF-α and wortmannin / Bay11-7082 respectively, the fpIgR protein and mRNA levels, together with SC secretion, obviously decreased in wortmannin- and Bay11-7082-treated cells compared with the untreated control, and cotreatment with wortmannin / Bay11-7082 plus TNF-α resulted in lower expression compared with that upon treatment with TNF-α alone, indicating that the inhibition of PI3K and NF-κB both blocked the ability of TNF-α to increase cellular fpIgR and SC levels. Furthermore, the gene expressions of PI3K and NF-κB were upregulated and present a tendency to increase first and then decrease after TNF-α treatment of FG cells; However, the expression of PI3K mRNA was inhibited significantly by wortmannin but not by Bay11-7082, and the expression of NF-κB mRNA was suppressed obviously by Bay11-7082 but not by wortmannin, suggesting that inhibition of PI3K or NF-κB had no influence on each other. All these results collectively revealed that TNF-α could transcriptionally upregulate fpIgR expression and SC production, and this TNF-α-induced pIgR expression was regulated by complex mechanisms that involved PI3K and NF-κB signaling pathways, which provided evidences for pro-inflammatory cytokine TNF-α acting as a regulator in pIgR expression and better understanding of regulation mechanism of pIgR expression in teleost fish.
聚合物免疫球蛋白受体（pIgR）将分泌型免疫球蛋白转运穿过黏膜上皮细胞进入外分泌液，在黏膜表面防御中发挥关键作用，但在鱼类中，pIgR 表达的调节机制尚不清楚。在这项研究中，我们首先分析了灭活鳗弧菌免疫后牙鲆（Paralichthys olivaceus）pIgR（fpIgR）和促炎细胞因子肿瘤坏死因子-α（TNF-α）mRNA 在黏膜组织中的动态变化，发现 TNF-α 的产生增加与 fpIgR 表达增加相关。为了确定细胞因子 TNF-α 是否影响 fpIgR 的表达，在证实天然 fpIgR 表达在牙鲆鳃（FG）细胞上后，FG 细胞用不同浓度的重组 TNF-α 孵育不同时间，结果显示 fpIgR 的表达在基因和蛋白水平上均呈剂量依赖性和时间依赖性显著上调，并且 fpIgR 分泌到培养上清液中的游离分泌成分（SC）也表现出相似的变化趋势。用 TNF-α 处理 FG 细胞后，用特定的磷酸肌醇 3-激酶（PI3K）抑制剂渥曼青霉素、核因子 kappa-B（NF-κB）抑制剂 Bay11-7082 以及 TNF-α 与渥曼青霉素/Bay11-7082 的混合物分别处理，与未处理的对照相比，渥曼青霉素和 Bay11-7082 处理的细胞中 fpIgR 蛋白和 mRNA 水平以及 SC 分泌明显降低，并且与单独用 TNF-α 处理相比，用 TNF-α 联合渥曼青霉素/Bay11-7082 处理导致表达水平更低，表明 PI3K 和 NF-κB 的抑制均阻断了 TNF-α 增加细胞内 fpIgR 和 SC 水平的能力。此外，在 FG 细胞用 TNF-α 处理后，PI3K 和 NF-κB 的基因表达上调，呈现先增加后减少的趋势；然而，渥曼青霉素显著抑制 PI3K mRNA 的表达，但不抑制 Bay11-7082 的表达，Bay11-7082 明显抑制 NF-κB mRNA 的表达，但不抑制渥曼青霉素的表达，表明抑制 PI3K 或 NF-κB 对彼此没有影响。所有这些结果共同表明，TNF-α 可以转录上调 fpIgR 的表达和 SC 的产生，这种 TNF-α 诱导的 pIgR 表达受涉及 PI3K 和 NF-κB 信号通路的复杂机制调节，为促炎细胞因子 TNF-α 作为 pIgR 表达的调节剂提供了证据，并更好地理解了鱼类中 pIgR 表达的调节机制。
