Castro Isabel, Carvajal Patricia, Aguilera Sergio, Barrera María-José, Matus Soledad, González Sergio, Molina Claudio, González María-Julieta
Departamento de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Independencia 1027, 8380453, Independencia, Santiago, Chile.
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, 8380453, Independencia, Santiago, Chile.
J Autoimmun. 2025 May;153:103412. doi: 10.1016/j.jaut.2025.103412. Epub 2025 Apr 1.
Labial salivary glands (LSG) from Sjögren's disease (SjD) patients are characterized by increased levels of pro-inflammatory cytokines, such as type I interferons (IFN-I). These LSG also show activation of the integrated stress response (ISR) with overexpression of protein kinase R (PKR), a known IFN-stimulated gene. In vitro, IFN-I stimulation reproduces the downregulation of hsa-miR-145-5p, which is associated with TLR4 overexpression observed in LSG of SjD patients. MicroRNA levels depend on its biogenesis, which is a multi-step process involving several protein complexes. It is not known whether altered miRNA biogenesis is associated with the activation of the ISR induced by IFN-I in LSG from SjD. The aim of this study was to characterize the expression and localization of components of the miRNA biogenesis machinery in LSG of SjD patients, to assess the effect of pro-inflammatory cytokines on these components, and to test whether inhibition of the IFN-β-induced ISR restores the levels of hsa-miR-145-5p. In LSG from 12 SjD patients and 11 non-SjD sicca controls, we determined mRNA fold changes, relative protein levels, and the localization of the ISR and miRNA biogenesis machinery components by RT-qPCR, Western blot, and immunofluorescence, respectively. Pro-inflammatory cytokines, the ISR inhibitor ISRIB, and the PKR inhibitor C16 were used for in vitro assays. In LSG from SjD patients, PKR and its activator PACT colocalized in the cytoplasm, whereas the PKR inhibitor TRBP was observed in the nuclei. IFN-β activates PKR, increases p-eIF2α and ATF4 levels, and increases PACT and AGO2 detection in stress granules. C16 inhibits PKR phosphorylation but increases ATF4 by activating GCN2. ISRIB restores levels of hsa-miR-145-5p and its target TLR4 mRNA upon IFN-β stimulation. These findings suggest an association between inflammation, cellular stress, and miRNA biogenesis, where modulation of the ISR emerges as a potential strategy to restore cellular homeostasis in LSG from SjD patients.
干燥综合征（SjD）患者的唇腺（LSG）具有促炎细胞因子水平升高的特征，如I型干扰素（IFN-I）。这些唇腺还表现出整合应激反应（ISR）的激活，蛋白激酶R（PKR）过表达，PKR是一种已知的IFN刺激基因。在体外，IFN-I刺激可重现hsa-miR-145-5p的下调，这与在干燥综合征患者唇腺中观察到的TLR4过表达有关。微小RNA水平取决于其生物合成，这是一个涉及多种蛋白质复合物的多步骤过程。尚不清楚miRNA生物合成的改变是否与干燥综合征患者唇腺中IFN-I诱导的ISR激活有关。本研究的目的是表征干燥综合征患者唇腺中miRNA生物合成机制成分的表达和定位，评估促炎细胞因子对这些成分的影响，并测试抑制IFN-β诱导的ISR是否能恢复hsa-miR-145-5p的水平。在12例干燥综合征患者和11例非干燥综合征口干对照者的唇腺中，我们分别通过RT-qPCR、蛋白质印迹和免疫荧光法测定了mRNA倍数变化、相对蛋白水平以及ISR和miRNA生物合成机制成分的定位。促炎细胞因子、ISR抑制剂ISRIB和PKR抑制剂C16用于体外实验。在干燥综合征患者的唇腺中，PKR及其激活剂PACT在细胞质中共定位，而PKR抑制剂TRBP在细胞核中被观察到。IFN-β激活PKR，增加p-eIF2α和ATF4水平，并增加应激颗粒中PACT和AGO2的检测。C16抑制PKR磷酸化，但通过激活GCN2增加ATF4。ISRIB在IFN-β刺激后恢复hsa-miR-145-5p及其靶标TLR4 mRNA的水平。这些发现表明炎症、细胞应激和miRNA生物合成之间存在关联，其中调节ISR成为恢复干燥综合征患者唇腺细胞稳态的潜在策略。
