Singh Ankita, Singh Vishal, Tiwari Rajiv L, Chandra Tulika, Kumar Ashutosh, Dikshit Madhu, Barthwal Manoj K
Pharmacology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Sitapur Road, Jankipuram Extension, Lucknow 226031, Uttar Pradesh, India.
Department of Transfusion Medicine and Blood Bank, King George's Medical University, Lucknow, Uttar Pradesh, India.
Cell Mol Immunol. 2016 Nov;13(6):745-763. doi: 10.1038/cmi.2015.62. Epub 2015 Aug 31.
In monocytic cells, Toll-like receptor 4 (TLR4)- and TLR2-induced reactive oxygen species (ROS) cause oxidative stress and inflammatory response; however, the mechanism is not well understood. The present study investigated the role of interleukin-1 receptor-associated kinase (IRAK), extracellular signal-regulated kinase (ERK), p67phox and Nox-2 in TLR4- and TLR2-induced ROS generation during interleukin-1 beta (IL-1β) transcription, processing, and secretion. An IRAK1/4 inhibitor, U0126, PD98059, an NADPH oxidase inhibitor (diphenyleneiodonium (DPI)), and a free radical scavenger (N-acetyl cysteine (NAC))-attenuated TLR4 (lipopolysaccharide (LPS))- and TLR2 (Pam3csk4)-induced ROS generation and IL-1β production in THP-1 and primary human monocytes. An IRAK1/4 inhibitor and siRNA-attenuated LPS- and Pam3csk4-induced ERK-IRAK1 association and ERK phosphorylation and activity. LPS and Pam3csk4 also induced IRAK1/4-, ERK- and ROS-dependent activation of activator protein-1 (AP-1), IL-1β transcription, and IL-1β processing because significant inhibition in AP-1 activity, IL-1β transcription, Pro- and mature IL-β expression, and caspase-1 activity was observed with PD98059, U0126, DPI, NAC, an IRAK1/4 inhibitor, tanshinone IIa, and IRAK1 siRNA treatment. IRAK-dependent ERK-p67phox interaction, p67phox translocation, and p67phox-Nox-2 interaction were observed. Nox-2 siRNA significantly reduced secreted IL-1β, IL-1β transcript, pro- and mature IL-1β expression, and caspase-1 activity indicating a role for Nox-2 in LPS- and Pam3csk4-induced IL-1β production, transcription, and processing. In the present study, we demonstrate that the TLR4- and TLR2-induced IRAK-ERK pathway cross-talks with p67phox-Nox-2 for ROS generation, thus regulating IL-1β transcription and processing in monocytic cells.
在单核细胞中，Toll样受体4（TLR4）和TLR2诱导的活性氧（ROS）会引发氧化应激和炎症反应；然而，其机制尚未完全明确。本研究调查了白细胞介素-1受体相关激酶（IRAK）、细胞外信号调节激酶（ERK）、p67phox和Nox-2在白细胞介素-1β（IL-1β）转录、加工和分泌过程中TLR4和TLR2诱导的ROS生成中的作用。一种IRAK1/4抑制剂、U0126、PD98059、一种NADPH氧化酶抑制剂（二苯基碘鎓（DPI））和一种自由基清除剂（N-乙酰半胱氨酸（NAC））可减弱TLR4（脂多糖（LPS））和TLR2（Pam3csk4）在THP-1细胞和原代人单核细胞中诱导的ROS生成和IL-1β产生。一种IRAK1/4抑制剂和小干扰RNA（siRNA）可减弱LPS和Pam3csk4诱导的ERK与IRAK1的结合以及ERK的磷酸化和活性。LPS和Pam3csk4还诱导了IRAK1/4、ERK和ROS依赖性的激活蛋白-1（AP-1）激活、IL-1β转录和IL-1β加工，因为在用PD98059、U0126、DPI、NAC、一种IRAK1/4抑制剂、丹参酮IIa和IRAK1 siRNA处理后，观察到AP-1活性、IL-1β转录、前体和成熟IL-β表达以及半胱天冬酶-1活性受到显著抑制。观察到IRAK依赖性的ERK与p67phox相互作用、p67phox易位以及p67phox与Nox-2相互作用。Nox-2 siRNA显著降低了分泌的IL-1β、IL-1β转录本、前体和成熟IL-1β表达以及半胱天冬酶-1活性，表明Nox-2在LPS和Pam3csk4诱导的IL-1β产生、转录和加工中发挥作用。在本研究中，我们证明TLR4和TLR2诱导的IRAK-ERK途径与p67phox-Nox-2相互作用以产生ROS，从而调节单核细胞中IL-1β的转录和加工。
