Methamphetamine induced neuroinflammation in mouse brain and microglial cell line BV2: Roles of the TLR4/TRIF/Peli1 signaling axis

doi:10.1016/j.toxlet.2020.07.028

Toxicology Letters

Volume 333, 15 October 2020, Pages 150-158

https://doi.org/10.1016/j.toxlet.2020.07.028 Get rights and content

Highlights

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Downregulation of Peli1 strikingly suppresses Meth-induced neuroinflammation.
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The TLR4-TRIP axis is important for Peli1 modulation in Meth treated microglial cells.
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Cytokines and signaling are dramatically decreased after TLR4-TRIP-Peli1 inhibition.
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The TLR4-TRIP-Peli1 axis is obviously activated in the Meth-treated mouse brain.

Abstract

Methamphetamine (Meth), a highly addictive drug, can induce irreversible neuronal damage and cause neuropsychiatric and cognitive disorders. Meth's effects on modulating microglial neuroimmune functions and eliciting neuroinflammation have attracted considerable attention in recent years. Recent evident of the effect of the non-dependent domain containing adaptor inducing interferon (TRIF)/Pellino1 (Peli1) signaling axis on pro-inflammatory cytokine production provides novel clues for inflammation. Therefore, our study investigated Meth-induced neurotoxicity from a neuropathological perspective by examining TLR4-TRIF-Peli1 axis signaling activation. Meth significantly activated microglia accompanied by marked increase of TLR4 and TRIF expression, NF-kB and MAPK pathways activation and the production of IL-1β, TNF-α and IL-6. Peli1 was involved in Meth-mediated neuroinflammation and knockdown of Peli1 strongly reversed NF-kB and MAPK pathways activation and pro-inflammatory cytokine excretion. Intriguingly, Peli1 upregulation induced by Meth was dependent on TRIF rather than the myloid differentiation factor 88 (MyD88) pathway, since the silencing of TRIF significantly suppressed Meth-induced Peli1 upregulation, while MyD88 knockdown had no obvious impact. Additionally, an in vivo study verified TLR4-TRIF-Peli1 axis activation and an enhanced level of downstream cytokine expression in the cortex after Meth treatment. Therefore, these findings provide new insight regarding the specific contributions of the TRIF-Peli1 pathway to Meth-mediated neuroinflammation.

Introduction

Methamphetamine (Meth) abuse has become a global public health problem, with more than 33 million people using the drug worldwide; this number continues to grow due to its availability, ease of use, low price, and high potential for addiction (Courtney and Ray, 2014). Mounting reports have revealed that Meth exposure can induce irreversible neuronal damage and cause neuropsychiatric and cognitive disorders in which neuroinflammation may be involved (Kaushal et al., 2013; Xu et al., 2017). However, the mechanism underlying Meth induced neuroinflammation has yet to be clarified.

As resident innate immune cells in the central nervous system (CNS) (Yang and Zhou, 2019), microglial cells are the most motile cells and serve as the first line of defense in the CNS. Microglial activation may occur as a consequence of exogenous or endogenous stimuli, accompanied by the excretion of cytokines, including IL-1β, TNF-α and IL-6. Neuroinflammation is through to underlie numerous types of CNS injury and neurodegenerative diseases, including depression, anxiety, Parkinson's disease and Alzheimer's disease (Xu et al., 2018; Yang and Zhou, 2019).

Meth exposure has been proposed to induce neural damage in a direct or indirect manner. Our previous study showed that Meth treatment contributes to Alzheimer's disease like changes, including p-tau and amyloid precursor protein (APP) enhancement, via the inhibition of insulin signaling, which has suppressive effects on inflammation (Xu et al., 2018; Chen et al., 2019). Increasing evidence has indicated that Meth induces disturbances in microglial homeostasis through microglial activation in the brain (Kaushal et al., 2013; Xu et al., 2017) in which the Toll-like receptors (TLRs) exert important roles.

TLRs are highly expressed in glial cells and mediate microglia activation. Toll-like receptor 4 (TLR4) is one of the most important receptors in the TLR family because it is the only one that can activate the MyD88 and the TRIF pathway (Trudler et al., 2010; Shirjang et al., 2017). TRIF is a TLR adapter and is unique to TLR3 and TLR4-mediated signaling pathways (Ullah et al., 2016). It mediates signaling from TLR4 through the activation of the TRIF-related adaptor molecule (TRAM) followed by regulating interferon regulatory factor 3 (IRF3) and NF-κB expression, which may potentiate the production of inflammatory factors in the late stage of inflammatory response process (Lundberg et al., 2013; Zhu et al., 2019). Recent evidence has suggested that blocking the TLR4 receptor abolishes the inflammatory signaling response in microglial cells, indicating the critical importance of TLR4 in microglia-mediated neuroinflammation (Kerfoot et al., 2004). Regarding the potential roles of TLR4 in Meth-mediated neuroinflammation, our recent work revealed a pronounced increase in TLR4 expression as well as inflammatory factor release in microglial cells after Meth treatment (Wan et al., 2017), however, the activation process linking TLR4 and inflammatory factors, remains poorly understood.

Peli1, an E3 ubiquitin ligase, has been reported to be essential for the modulation of TLR signaling. Available evidence has shown that the knockdown of Peli1 markedly suppresses the activation of NF‑κB and the expression of pro-inflammatory genes (Chang et al., 2009; Jiang et al., 2003). In a mouse encephalomyelitis (EAE) model, Peli1 was proven to participate in TLR-induced pro-inflammatory and chemokine gene expression via the MyD88-induced activation of the ERK, JNK and p38 MAPK pathways. Of note, the Peli1 protein is highly expressed in the brain and predominantly in microglia, demonstrating the crucial role of Peli1 in neuroinflammation (Parikh et al., 2015). Peli1 was demonstrated to be upregulated after Meth treatment in our recent study; the potential mechanisms involving Peli1-mediated signaling induced by Meth, however, remain poorly understood.

Combining the critical roles of Peli1 in neuroinflammation and the effects of Meth on Peli1, the current study expanded and strengthened our understanding of the potential mechanisms underlying Meth induced neurotoxicity along the TLR4-MyD88/TRIF-Peli1 axis. Here, we provided the direct evidence that Peli1 was involved in Meth induced neuroinflammation, and the TLR4-TRIF axis rather than TLR4-MyD88 axis was participated in Peli1 modulation. Meanwhile, an in vivo study was also performed, with the significant activation of TLR4-TRIF-Peli1 axis in Meth exposure mouse model. Therefore, targeting the TLR4-TRIF-Peli1 axis may provide the novel strategies for Meth induced neuroinflammation.

Section snippets

Animals

Eight-week-old male C57BL/6 J mice were purchased from the Experimental Animal Center of Nanjing Medical University (China). All animals were housed in a pathogen-free environment [12 h light: dark cycle, room temperature (RT) of 21 ± 1 °C relative humidity 40∼70%] with sufficient food and water. All experiments were approved by the Animal Experiment Ethics Committee of Nanjing Medical University (permission number: IACUC-1801008). The mice were weight matched and randomly divided into the

Meth induces microglial cell activation and inflammatory cytokine expression

To assess Meth-induced neuroinflammation, BV2 cells were incubated with Meth (0,100, 300 and 900 μM) for 24 h, and CD11b was detected. Meth above 100 μM substantially enhanced the expression of CD11b, with a peak response at 900 μM (Fig. 1A). Considering the cell injury (Fig. S1A) and the concentrations of Meth levels (from ≤2 μM to 600 μM) found in the blood, urine and tissue of Meth users (Reynolds et al., 2007), 300 μM Meth was chosen for the subsequent experiments, this concentration of

Discussion

Previous studies have revealed that Meth exposure contributes to TLR4 upregulation and cytokine release which might be regulated by NF-κB and MAPK signaling (Murphy et al., 2015; Du et al., 2017). However, the potential link between TLR4 and inflammatory signaling mediated by Meth remains poorly understood. In this study, we validated the effects of Peli1 in Meth-induced neuroinflammation, and expanded our previous finding that the TRIF-Peli1 signaling axis is involved in the Meth-mediated

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81673213, 81701050), the Natural Science Foundation of Jiangsu Province (BK20191349), the Key Disease of Jiangsu Province Science and Technology Department (BL2014088), the Jiangsu Planned Projects for Postdoctoral Research Funds (2018K075B) and the Six talent peaks project in Jiangsu Province (WSW-329).

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¹: These authors contributed equally to this study.

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Methamphetamine induced neuroinflammation in mouse brain and microglial cell line BV2: Roles of the TLR4/TRIF/Peli1 signaling axis

Highlights

Abstract

Introduction

Section snippets

Animals

Meth induces microglial cell activation and inflammatory cytokine expression

Discussion

Declaration of Competing Interest

Acknowledgements

Toxicol In Vitro

International Immunopharmacology

Drug Alcohol Depend

J Biol Chem

J Biol Chem

Eur Neuropsychopharmacol

Int Rev Neurobiol

J Biol Chem

Brain Res

Biochim Biophys Acta

Cell Immunol

Toxicology

Peli1 facilitates TRIF-dependent Toll-like receptor signaling and proinflammatory cytokine production

Nat Immunol

The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity

Nat Immunol

Expression and function of the chemokine receptors CXCR1 and CXCR2 in sepsis

J Immunol

Peli1 controls the survival of dopaminergic neurons through modulating microglia-mediated neuroinflammation

Sci Rep

Toll-like receptor 4 mediates methamphetamine-induced neuroinflammation through caspase-11 signaling pathway in astrocytes

Front Mol Neurosci