Morphine induces inflammatory responses via both TLR4 and cGAS-STING signaling pathways

IF 3.7 3区 医学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Cytokine Pub Date : 2024-08-31 DOI:10.1016/j.cyto.2024.156737
Fei Xie , Yoshinori Kitagawa , Hiroki Ogata , Shingo Yasuhara , Zerong You , J.A. Jeevendra Martyn
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Abstract

Background

Opioid activation of the microglia or macrophage Toll-like receptor 4 (TLR4) and associated inflammatory cytokine release are implicated in opioid-induced hyperalgesia and tolerance. The cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway, activated by double-stranded DNA including mitochondrial DNA (mtDNA), has emerged as another key mediator of inflammatory responses. This study tested the hypothesis that morphine induces immune inflammatory responses in microglia and macrophages involving TLR4 and cGAS-STING pathway.

Methods

BV2 microglia and Raw 264.7 (Raw) macrophage cells were exposed to morphine with and without a STING inhibitor (C176) for 6 h or TLR 4 inhibitor (TAK242) for 24 h. Western blotting and RT-qPCR analyses assessed TLR4, cGAS, STING, nuclear factor-kappa B (NF-κB), and pro-inflammatory cytokine expression. Morphine-induced mitochondria dysfunction was quantified by reactive oxygen species (ROS) release using MitoSOX, mtDNA release by immunofluorescence, and RT-qPCR. Polarization of BV2 and Raw cells was assessed by inducible nitric oxide (iNOS) and CD86 expression. The role of mtDNA on morphine-related inflammation was investigated by mtDNA depletion of the cells with ethidium bromide (EtBr) or cell transfection of mtDNA extracted from morphine-treated cells.

Results

Morphine significantly increased the expression of TLR4, cGAS, STING, p65 NF-κB, and cytokines (IL-6 and TNF-α) in BV2 and Raw cells. Morphine-induced mitochondrial dysfunction by increased ROS and mtDNA release; the increased iNOS and CD86 evidenced inflammatory M1-like phenotype polarization. TLR4 and STING inhibitors reduced morphine-induced cytokine release in both cell types. The transfection of mtDNA activated inflammatory signaling proteins, cytokine release, and polarization. Conversely, mtDNA depletion led to the reversal of these effects.

Conclusion

Morphine activates the cGAS-STING pathway in macrophage cell types. Inhibition of the STING pathway can be an additional method to overcome immune cell inflammation-related morphine tolerance and opioid-induced hyperalgesia.

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吗啡通过 TLR4 和 cGAS-STING 信号通路诱导炎症反应
背景阿片激活小胶质细胞或巨噬细胞的 Toll 样受体 4 (TLR4) 以及相关炎性细胞因子的释放与阿片诱导的痛觉减退和耐受性有关。环GMP-AMP合成酶/干扰素基因刺激器(cGAS-STING)信号通路由包括线粒体DNA(mtDNA)在内的双链DNA激活,已成为炎症反应的另一个关键介质。本研究检验了吗啡诱导小胶质细胞和巨噬细胞免疫炎症反应涉及 TLR4 和 cGAS-STING 通路的假设。Western 印迹和 RT-qPCR 分析评估了 TLR4、cGAS、STING、核因子-kappa B (NF-κB) 和促炎细胞因子的表达。吗啡诱导的线粒体功能障碍通过使用 MitoSOX 的活性氧(ROS)释放、免疫荧光的 mtDNA 释放和 RT-qPCR 进行量化。通过诱导性一氧化氮(iNOS)和 CD86 表达评估了 BV2 和 Raw 细胞的极化。结果吗啡显著增加了 BV2 和 Raw 细胞中 TLR4、cGAS、STING、p65 NF-κB 和细胞因子(IL-6 和 TNF-α)的表达。吗啡通过增加 ROS 和 mtDNA 释放诱导线粒体功能障碍;iNOS 和 CD86 的增加证明了炎症 M1 样表型的极化。TLR4 和 STING 抑制剂减少了吗啡诱导的两种细胞类型的细胞因子释放。转染 mtDNA 激活了炎症信号蛋白、细胞因子释放和极化。结论吗啡激活巨噬细胞类型中的 cGAS-STING 通路。抑制 STING 通路可能是克服与免疫细胞炎症相关的吗啡耐受性和阿片诱导的痛觉减退的另一种方法。
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来源期刊
Cytokine
Cytokine 医学-免疫学
CiteScore
7.60
自引率
2.60%
发文量
262
审稿时长
48 days
期刊介绍: The journal Cytokine has an open access mirror journal Cytokine: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. * Devoted exclusively to the study of the molecular biology, genetics, biochemistry, immunology, genome-wide association studies, pathobiology, diagnostic and clinical applications of all known interleukins, hematopoietic factors, growth factors, cytotoxins, interferons, new cytokines, and chemokines, Cytokine provides comprehensive coverage of cytokines and their mechanisms of actions, 12 times a year by publishing original high quality refereed scientific papers from prominent investigators in both the academic and industrial sectors. We will publish 3 major types of manuscripts: 1) Original manuscripts describing research results. 2) Basic and clinical reviews describing cytokine actions and regulation. 3) Short commentaries/perspectives on recently published aspects of cytokines, pathogenesis and clinical results.
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