Department of Vascular and Inflammatory Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu- cho, Hirosaki, Aomori, 036-8562, Japan.
Department of Respiratory Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
Mol Biol Rep. 2024 Sep 11;51(1):974. doi: 10.1007/s11033-024-09904-9.
One of the causes of tubulointerstitial nephritis is viral infection, with innate immune responses affecting its pathogenesis. Toll-like receptor 3 (TLR3) recognizes viral infections and acts antivirally by activating signaling to produce inflammatory cytokines/chemokines, including C-C motif chemokine ligand 5 (CCL5) and interferon-β (IFN-β). Although cylindromatosis lysine 63 deubiquitinase (CYLD) is known to be associated with tubulointerstitial nephritis and renal function, its role in the antiviral innate immune response in tubular epithelial cells remains unknown. In this study, we investigated the association between CYLD and TLR3-mediated CCL5 production in cultured human renal proximal tubular epithelial cells (hRPTECs).
Polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand, was used to stimulate hRPTECs. mRNA expression was measured using reverse transcription-quantitative polymerase chain reaction. Protein expression was assayed using western blotting or an enzyme-linked immunosorbent assay. Knockdown of IFN-β, nuclear factor-kappa B (NF-κB) p65, and CYLD was performed by transfecting cells with specific small interfering RNAs. The intracellular localization of CYLD in hRPTECs was analyzed using immunofluorescence. Poly IC induced CCL5 expression in a time- and concentration-dependent manner, and knockdown of either IFN-β or p65 reduced poly IC-induced CCL5 expression. CYLD knockdown increased the poly IC-induced CCL5, phosphorylated IκB kinase α/β (IKK complex), and phosphorylated p65 expression. The CYLD protein was localized in the cytoplasm, and poly IC did not alter its expression.
CYLD may prevent excessive inflammation due to an antiviral innate immune response by suppressing IKK complex and NF-κB activation downstream of TLR3 in hRPTECs.
肾小管间质性肾炎的病因之一是病毒感染，固有免疫反应影响其发病机制。Toll 样受体 3（TLR3）识别病毒感染，并通过激活信号产生炎症细胞因子/趋化因子，包括 C-C 基序趋化因子配体 5（CCL5）和干扰素-β（IFN-β）来发挥抗病毒作用。虽然已知圆柱瘤赖氨酸 63 去泛素化酶（CYLD）与肾小管间质性肾炎和肾功能有关，但它在肾小管上皮细胞的抗病毒固有免疫反应中的作用尚不清楚。在这项研究中，我们研究了 CYLD 与 TLR3 介导的培养的人肾近端小管上皮细胞（hRPTEC）中 CCL5 产生之间的关联。
多聚肌苷酸-多聚胞苷酸（poly IC），一种合成的 TLR3 配体，用于刺激 hRPTEC。使用逆转录定量聚合酶链反应测量 mRNA 表达。使用 Western 印迹或酶联免疫吸附试验测定蛋白质表达。通过转染细胞特异性小干扰 RNA 来敲低 IFN-β、核因子-κB（NF-κB）p65 和 CYLD。使用免疫荧光分析 hRPTEC 中 CYLD 的细胞内定位。poly IC 以时间和浓度依赖的方式诱导 CCL5 表达，并且 IFN-β 或 p65 的敲低减少了 poly IC 诱导的 CCL5 表达。CYLD 敲低增加了 poly IC 诱导的 CCL5、磷酸化 IKK 复合物和磷酸化 p65 的表达。CYLD 蛋白定位于细胞质中，poly IC 不会改变其表达。
CYLD 可能通过抑制 TLR3 下游的 IKK 复合物和 NF-κB 激活，在 hRPTEC 中防止抗病毒固有免疫反应引起的过度炎症。
