https://orcid.org/0000-0001-7043-5593
Figures
Abstract
Although they represent the cornerstone of analgesic therapy, opioids, such as morphine, are limited in efficacy by drug tolerance, hyperalgesia and other side effects. Activation of microglia and the consequent production of proinflammatory cytokines play a key pathogenic role in morphine tolerance, but the exact mechanisms are not well understood. This study aimed to investigate the regulatory mechanism of epidermal growth factor receptor (EGFR) on microglial activation induced by morphine in mouse microglial BV-2 cells. In this research, BV-2 cells were stimulated with morphine or pretreated with AG1478 (an inhibitor of EGFR). Expression levels of cluster of differentiation molecule 11b (CD11b), EGFR, and phospho-EGFR were detected by immunofluorescence staining. Cell signaling was assayed by Western blot. The migration ability of BV-2 cells was tested by Transwell assay. The production of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in the cell supernatant was determined by ELISA. We observed that the expression of CD11b induced by morphine was increased in a dose- and time- dependent manner in BV-2 cells. Phosphorylation levels of EGFR and ERK1/2, migration of BV-2 cells, and production of IL-1β and TNFα were markedly enhanced by morphine treatment. The activation, migration, and production of proinflammatory cytokines in BV-2 cells were inhibited by blocking the EGFR signaling pathway with AG1478. The present study demonstrated that the EGFR/ERK signaling pathway may represent a novel pharmacological strategy to suppress morphine tolerance through attenuation of microglial activation.
Citation: Yang Y, Sun Y, Hu R, Yan J, Wang Z, Li W, et al. (2021) Morphine promotes microglial activation by upregulating the EGFR/ERK signaling pathway. PLoS ONE 16(9): e0256870. https://doi.org/10.1371/journal.pone.0256870
Editor: Michael Bader, Max Delbruck Centrum fur Molekulare Medizin Berlin Buch, GERMANY
Received: April 19, 2021; Accepted: August 17, 2021; Published: September 14, 2021
Copyright: © 2021 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was funded by the National Natural Science Foundation of China [grant numbers 81801094 by Yaqiong Yang, 82071177 by Hong Jiang].
Competing interests: The authors have declared that no competing interests exist.
Introduction
Opioids, such as morphine, continue to be powerful analgesic drugs for managing chronic pain with a high rate of abuse potential [1]. Their analgesic efficacy is limited by drug tolerance, hyperalgesia and other side effects, which hinder the prolonged clinical use of opioid drugs [2]. Therapeutic strategies that can bolster the analgesia effects while reducing tolerance and side effects are urgently needed to improve patient quality of life.
Although the exact mechanisms of opioid tolerance are complex and not well understood, compelling evidence has recently shown that the activation of microglia and consequent production of proinflammatory cytokines play a key pathogenic role [3, 4]. Activated microglia undergo a dramatic morphological changes from quiescent to a macrophage-like accompanied by increased expression of cell surface markers, such as cluster of differentiation molecule 11b (CD11b), cluster of differentiation (CD14), major histocompatibility complex (MHC) molecules, chemokine receptors, and several other markers, producing large numbers of proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, which could enhance the reactivity of dorsal horn neurons, induce central sensitization, and reduce the antinociceptive effect of morphine [5, 6]. Although the inhibition of microglial activation may represent a potential therapeutic strategy for improving the analgesic effect of morphine [7], the specific regulation mechanism is still unclear.
The epidermal growth factor receptor (EGFR) is a member of the ErbB family with four different receptor tyrosine kinases and has been successfully targeted for cancer therapy [8, 9]. EGFR can be activated by the Mu opioid receptors (MOR) [10, 11]. Inhibition of EGFR/MAPK signaling pathway attenuates microglial inflammatory response and secondary damage after spinal cord injury in rats [12]. EGFR may also be involved in pain and analgesia signaling in neuropathic pain rat models [13, 14]. However, none of these previous studies examined the role of EGFR in morphine induced microglial activation. Herein, we demonstrate that morphine induces activation of microglia in a dose- and time- dependent manner. Moreover, we found the impact of morphine on microglia was due to EGFR and its downstream ERK signaling pathway activation and that inhibition of EGFR suppresses the activation and migration of microglia and reduces the secretion of proinflammatory cytokines. These data suggest that EGFR may have potential as a novel target for the treatment of morphine tolerance.
Materials and methods
Cell culture
The mouse microglial BV-2 cell line was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (High Glucose, Gibco, China) with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Sangon Biotech, Shanghai, China) at 37°C in a humidified atmosphere of 95% air and 5% CO2. When cells reached approximately 80% confluence, they were used for experiments.
Cell treatments
Morphine hydrochloride (10 mg/mL) was obtained from Shenyang First Pharmaceutical Factory, Northeast Pharmaceutical Group Company (Shenyang, China) and diluted in phosphate-buffered saline (PBS, Gibco, China) to 1 mg/mL. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (USA) and dissolved in PBS at a final concentration of 1 μg/ml according to previous studies [12, 15–19]. AG1478, a specific tyrosine kinase inhibitor of EGFR, was purchased from MedChemExpress (USA) and dissolved in 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) at a final concentration of 2.5, 5, and 10 μM according to previous study [12]. Cells were pretreated with AG1478 for 30 min before treatment with other drugs.
Immunofluorescence staining
Different groups of BV-2 cells were cultured at 2×105 cells/cm2 on glass coverslips in 6-well culture plates. Coverslips were fixed with 4% paraformaldehyde for 15 min, blocked with 5% FBS for 30 min, and then incubated with CD11b antibody (1:200, ab8878, Abcam, UK), anti-EGFR (1:250, ab52894, Abcam, UK) or anti-phospho-EGFR (Tyr1068) (1:500, 3777, Cell Signaling Technology, USA) at -4°C overnight. After being washed, cells were incubated with the corresponding fluorescent-conjugated IgG secondary antibody (Proteintech Group, PTG, USA) at 37°C for 1 h and then counterstained with Hoechst (Beyotime Biotechnology, Shanghai, China) at room temperature in the dark for 15 min. Images were acquired under a fluorescence microscope (OLYMPUS IX71, Tokyo, Japan) and were analyzed using ImageJ software.
Western blot analysis
Different groups of BV-2 cells were seeded and incubated in 6-well plates in DMEM with 10% FBS. For CD11b expression experiments, cells were incubated with 0, 25, 50, 100, and 200 μM morphine for 24 h or 200 μm morphine for 0, 2, 4, 6 and 8 h. For EGFR inhibition experiments, cells were incubated with DMSO, AG1478 (10 μM), morphine (200 μM), or AG1478 (10 μM) + morphine (200 μM) for 24 h. After treatments, total proteins were extracted after lysis in RIPA lysis buffer (Beyotime, China). Lysates were centrifuged at 12,000 g for 10 min and supernatants were collected for protein concentration assay, performed using a BCA kit (Pierce, Rockford, IL). Proteins (20 μg) were separated on 8% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore Billerica, MA). The membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: anti-CD11b (1:1000, A1581, ABclonal, China), anti-EGFR (1:1000, ab52894, Abcam, UK), anti-phospho-EGFR (Tyr1068) (1:1000, 3777, Cell Signaling Technology, USA), anti-ERK1/2 (1:5000, ab184699, Abcam, UK), anti-phospho-ERK1/2 (1:2000, 4370, Cell Signaling Technology, USA), anti-TLR4 (1:1000, AF7017, Affinity Biosciences, China) and anti-GAPDH (1:2000, AF7021, Affinity Biosciences, China). Next, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch, USA) for 1 h at room temperature with subsequent exposure of membranes to enhanced chemiluminescence reagents (Millipore Billerica, MA).
Migration assay
Transwell® migration assays were performed in 24-well Transwell® polycarbonate membrane inserts with 8.0-mm pore size (diameter 6.5 mm, Costar, Corning Incorporated, USA). BV-2 cells (100 μl, 1x105 cells/well) were loaded into the upper chamber and allowed to adhere to the polycarbonate filters for 10 min at 37°C in a humidified atmosphere of 95% air and 5% CO2. According to the grouping, 200 μM morphine was added to the upper chamber in the morphine group and morphine + AG1478 group, and 10 μM AG1478 was added to the upper and lower chambers in both the AG1478 group and morphine + AG1478 group. Equal amount of DMSO was added in the control group. Cells were cultured at 37°C for 48 h. Then, migrated cells were fixed with 4% paraformaldehyde, prechilled on ice for 30 min, and stained with 0.1% crystal violet for 10 min. Six fields of view were randomly selected under a microscope (OLYMPUS IX71, Tokyo, Japan), and the number of migrated cells was counted at a 20 μm scale bar.
Enzyme-linked immunosorbent assay (ELISA)
BV-2 cells (1×104 cells/well) were cultured in 96-well plates overnight. Cells were treated with PBS, DMSO, LPS (1 μg/mL), morphine (200 μM), LPS (1 μg/mL) + morphine (200 μM), morphine (200 μM) + AG1478 (10 μM), and LPS (1 μg/mL) + morphine (200 μM) + AG1478 (10 μM) for 3, 6, 12, and 24 h, respectively. Levels of IL-1β and TNF-α in cell supernatants of different groups were measured using a Mouse Interleukin-1β ELISA Kit (LianShuo Biological, Shanghai, China) and a Mouse TNF-α ELISA Kit (LianShuo Biological, Shanghai, China), according to the manufacturer’s protocols. The absorbance of each well was measured at 450 nm using a microplate leader.
Statistical analysis
All experiments were completed at least three times and data are presented as the mean ± standard error of the mean (SEM). Data were analyzed using analysis of variance (ANOVA) and the least significant differences method for multiple comparisons. p < 0.05 was considered statistically significant.
Results
Morphine induces microglial activation and enhances CD11b expression
To determine the effect of morphine on induced microglial activation, we used the immortalized murine microglial BV-2 cell line, which was derived from primary mouse microglial cells and has a similar responsive pattern to that of primary microglia after stimulation with LPS [20]. BV-2 cells were treated with different doses of morphine (0, 25, 50, 100, 200, and 400 μM) for 2, 4, 6, and 8 h, followed by immunofluorescence and Western blot analysis using CD11b, which is a microglial activation marker. From the results, we observed dose- and time- dependent effects of morphine on the expression of CD11b (Figs 1 and 2). EC50 values for each time points of morphine action were: 2 h, 145 μM; 4 h, 88.92 μM; 6 h, 78.89 μM; 8 h, 78.33μM (Fig 1B). Morphine, when used at a concentration of 200 μM for 6 h, caused microglia cells to exhibit an enlarged morphology and displayed intense CD11b immunoreactivity (Figs 1 and 2B). Previous studies have shown that morphine (200 μM, 6 h) significantly induced the activation of BV-2 cells [7, 21], which was consistent with our results. However, when BV-2 cells were treated with 200 μM morphine for 8 h, protein expression of CD11b was decreased (Fig 2B). Therefore, we chose a morphine concentration of 200 μM and a time of 6 h for subsequent experimental conditions.
BV-2 cells were treated with 0, 25, 50, 100, 200, and 400 μM morphine for 2, 4, 6, and 8 h and tested by immunocytochemistry staining. (a) Time- and dose-response effect of morphine on the expression of CD11b. Scale bar: 20 μm. (b) The time- and dose-response curve of BV-2 cells activated by morphine. Data were fit to a sigmoidal dose-response curve and EC50 values calculated using GraphPad Prism software. The mean optical densities of six randomly selected fields of cells from each group were analyzed by ImageJ software for quantitative analysis. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 were considered to be statistically significant compared to the control groups.
BV-2 cells were treated with 0, 25, 50, 100, and 200 μM morphine for 24 h (a) or with 200 μm morphine for 0, 2, 4, 6 and 8 h (b). GAPDH was used as a loading control. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 compared to the control group.
EGFR inhibition blocks the activation of microglia induced by morphine
To examine the activation of microglia by treatment with morphine or LPS, we detected the expression of CD11b in BV-2 cells by immunofluorescence. LPS has been the most extensively used microglia activator to induce inflammatory response [22]. Morphine also increased LPS-induced cytokine production in cultivated microglia [23]. Previous study has shown that EGFR inhibitors depressed inflammation after LPS stimulation by regulating the activation of EGFR/MAPK cascade in microglia [16], which may be a new neuroprotective mechanism after EGFR blockade. For these reasons, we used LPS as a positive control to study the effects of EGFR inhibitor on LPS- and morphine- activated microglia. Ours results demonstrated that treatment with morphine and LPS both increased the expression of CD11b on microglial membranes, and induced BV-2 cells activation and macrophage-like morphologically changes (Fig 3). We also found that the combination of morphine and LPS could further enhance the activation of microglia (Fig 3). Pretreatment with the EGFR inhibitor AG1478 abolished microglial activation in both the morphine and LPS + morphine groups [Fig 3, morphine (0.35 ± 0.03) vs. morphine + AG1478 (0.17 ± 0.02), p<0.01; LPS + morphine (0.45 ± 0.01) vs. LPS + morphine + AG1478 (0.28 ± 0.03), p<0.01]. AG1478 alone had no significant effect on the expression of CD11b at different concentrations (S1 Fig).
(a) BV-2 cells were treated with PBS, DMSO, LPS (1 μg/mL), morphine (200 μM), LPS (1 μg/mL) + morphine (200 μM), morphine (200 μM) + AG1478 (10 μM), and LPS (1 μg/mL) + morphine (200 μM) + AG1478 (10 μM) for 6 h. Cells were stained for the activation marker CD11b. Scale bar: 20 μm. (b) The mean optical densities of six randomly selected fields of cells from each group were analyzed by ImageJ software for quantitative analysis. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 compared to the control groups. # p < 0.05 and ## p < 0.01 morphine vs. morphine + AG1478. & p < 0.05 and && p < 0.01 LPS + morphine vs. LPS + morphine + AG1478.
Morphine enhances expression of EGFR in microglia
In this series of experiments, we sought to determine whether morphine directly induces EGFR expression in microglia. Accompanied by microglial activation, treatment with morphine (200 μM) or LPS (1 μg/mL) for 6 h significantly increased EGFR and p-EGFR expression, which was further increased by combined treatment of morphine and LPS (Fig 4, compared to the control group, p<0.01). Morphine-induced EGFR expression was reduced in response to pretreatment with AG1478 [Fig 4C, morphine (0.20 ± 0.03) vs. morphine + AG1478 (0.13 ± 0.01), p<0.05; LPS + morphine (0.32 ± 0.05) vs. LPS + morphine + AG1478 (0.18 ± 0.01), p<0.01]. Expression of p-EGFR was also inhibited by AG1478 [Fig 4D, morphine (0.22 ± 0.01) vs. morphine + AG1478 (0.13 ± 0.01), p<0.01; LPS + morphine (0.39 ± 0.01) vs. LPS + morphine + AG1478 (0.24 ± 0.03), p<0.01].
The expression of EGFR (a) and p-EGFR (b) in BV-2 cells was detected by immunocytochemical analysis. Scale bar: 20 μm. (c), (d) The mean optical densities of six randomly selected fields of cells from each group were analyzed by ImageJ software for quantitative analysis. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 compared to the control groups. # p < 0.05 and ## p < 0.01 morphine vs. morphine + AG1478. & p < 0.05 and && p < 0.01 LPS + morphine vs. LPS + morphine + AG1478.
Morphine induces microglial activation through the EGFR/ERK pathway
To further explore whether morphine upregulation of CD11b expression occurs through activation of the EGFR signaling pathway, BV-2 cells were pretreated with AG1478 for 30 min prior to treatment with morphine. Western blotting results revealed that protein expression of CD11b was significantly increased when cells were treated with morphine and markedly inhibited by AG1478 (Fig 5A). Morphine increased phosphorylation levels of EGFR but had no significant effect on total levels of EGFR. Treatment with AG1478 reversed the increase in EGFR levels induced by morphine (Fig 5B). We also found that expression of p-ERK1/2 was clearly enhanced by morphine treatment and decreased by AG1478. However, total levels of ERK1/2 did not obviously change in response to treatment (Fig 5C). These results suggest that morphine may activate microglia by regulating the EGFR/ERK pathway. Studies indicated that morphine could bind to Toll-like receptor 4 (TLR4) and induced the initiation of innate immune signaling cascade and the production of proinflammatory factors [23], which activated neuroinflammation in a manner parallel to endotoxin. Therefore, we also examined the effect of AG1478 on morphine induced TLR4 expression. The result found that AG1478 downregulated the expression of TLR4 induced by morphine (Fig 5A), which indicated that AG1478 might inhibit the activation of microglia by blocking innate immune signaling pathway.
BV-2 cells were incubated with DMSO, AG1478 (2.5, 5, and 10 μM), morphine (200 μM), and AG1478 (2.5, 5, and 10 μM) + morphine (200 μM) for 24 h. After treatment, cell lysates were examined by Western blot for expression levels of CD11b (a), EGFR and p-EGFR (b) and ERK1/2 and p-ERK1/2 (c). GAPDH was used as a loading control. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 were considered to be statistically significant.
EGFR inhibition reduces microglial migration induced by morphine
To confirm the effect of EGFR inhibition on the migration of microglia induced by morphine, the migration ability of cells was evaluated by Transwell assay. The results showed that after treatment with morphine, the number of migrated microglia was obviously increased [morphine (222 ± 18) vs. DMSO (100 ± 6), p <0.01], and AG1478 significantly inhibited microglial invasion [morphine (222 ± 18) vs. morphine+AG1478 (133 ± 8), p < 0.01] (Fig 6).
(a) BV-2 cells were treated with morphine or AG1478 for 48 h, and the number of migrated cells was evaluated by the Transwell assay. Scale bar: 20 μm. (b) Migration of BV-2 cells in six randomly selected fields from each group was quantified. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 compared to DMSO groups. ## p < 0.01 morphine vs. morphine + AG1478.
AG1478 inhibits morphine- and LPS- induced IL-1β and TNF-α production in BV-2 microglial cells
To further explore whether morphine- and LPS- mediated microglial activation is accompanied by an inflammatory response in BV-2 cells, we investigated the secretion of the key proinflammatory cytokines, IL-1β and TNF-α by ELISA analysis in cellular supernatants. Our results showed that LPS time-dependently increased the production of the inflammatory mediators IL-1β and TNF-α by BV-2 cells. Morphine increased the production of IL-1β at 6 h and 12 h compared to the PBS group, but there was no significant difference at 3 h or 24 h (Fig 7). Levels of TNF-α induced by morphine increased gradually from 6 h after administration. We also found that treatment with AG1478 markedly attenuated IL-1β and TNF-α production induced by morphine and LPS + morphine (Fig 7).
The effect of EGFR inhibition on morphine- and LPS- induced IL-1β (a) and TNF-α (b) production in BV-2 microglial cells. The supernatants of BV-2 cells were collected after centrifugation and measured by ELISA. The absorbance of each well was measured at 450 nm using a microplate leader. Data are presented as the means ± SEMs of three independent experiments. * p < 0.05 and ** p < 0.01 compared to PBS groups. # p < 0.05 and ## p < 0.01 morphine vs. morphine + AG1478. & p < 0.05 and && p < 0.01 LPS + morphine vs. LPS + morphine + AG1478.
Discussion
Although more and more pieces of literature show that spinal microglia play an important role in the pathogenesis of morphine tolerance [24, 25], but the exact regulatory mechanism is not clear. The study presented here assessed the signaling mechanisms by which morphine modulates microglial activation. A previous study suggested that morphine at concentrations >1 μM acts as a microglial chemoattractant in a rodent model of neuropathic pain [26]. Our results indicated that morphine potently enhanced expression of the microglial marker CD11b in a dose- and time-dependent manner, consistent with a previous study. It has been shown that there is remarkable resemblance between neuropathic pain and morphine tolerance. The activation of neuroimmunity was more obvious in rats with nerve injury and morphine than with injury alone, indicating that opioids and neuropathy may have cooperative effects on glial activation and cytokines [27]. In this study, we used morphine and LPS alone or in combination to treat BV-2 microglial cell. We found that morphine improved microglial reactivity, induced migration, and increased the expression of CD11b. LPS also upregulated the expression of CD11b, which was further increased by combined treatment with morphine. These results suggest that the activation process of microglia induced by morphine may have the same mechanism as that of inflammatory activation induced by LPS.
EGFR, a well-known receptor, has been attracting much attention due to its potency in mediating microglial activation [28]. Binding to EGFR ligands, such as epidermal growth factor (EGF) and tumor necrosis factor α (TNF-α), causes microglial activation by transactivation of mitogen-activated protein kinase (MAPK) and other downstream signaling pathways [29, 30]. Inhibition of the EGFR/MAPK signaling pathway has been proven to suppress the microglial inflammatory response and associated secondary injury in rats after spinal cord injury [12], and rapid phosphorylation of EGFR is attributed to LPS-triggered intracellular calcium mobilization and migration of microglia [16]. However, the EGFR signaling pathway is essential for microglial activation and cytokine production, making it a potential therapeutic target for the treatment of inflammatory diseases, but the regulatory role of EGFR in morphine-induced microglial activation remains unclear. LPS, a natural TLR4 ligand, has been the most widely used microglia activator to induce inflammatory response [22]. Study has shown that LPS-induced microglia activation is accompanied by EGFR transactivation [12], but there is no evidence that LPS is an effective ligand for EGFR [16]. In this study, we found that morphine and LPS both remarkably upregulated levels of EGFR and p-EGFR in BV-2 cells and were further increased by combined treatment of morphine and LPS. Morphine- and LPS- induced EGFR and p-EGFR overexpression were reduced by pretreatment with AG1478, an inhibitor of EGFR (Fig 4). Furthermore, we found that protein phosphorylation levels of EGFR and ERK1/2 were both increased by morphine and decreased by AG1478 (Fig 5). The increased expression of CD11b induced by morphine was also inhibited by pretreatment with AG1478 (Figs 3 and 5A). These results indicate that EGFR pathway plays an important role in the process of microglia activation induced by morphine and LPS. Extensive studies have shown that opioids cause activation of signaling by the innate immune receptor TLR4, leading to proinflammatory glial activation [23, 31–33]. Blockade of TLR4 inhibited the microglia activation and attenuated morphine tolerance [34]. In this study, AG1478 also inhibited the expression of TLR4 induced by morphine, suggesting that EGFR inhibitors depress microglia activation through regulating innate immune signaling pathway.
Morphine-induced activation of microglia may lead to elevated local concentrations of proinflammatory cytokines and chemokines [2, 35, 36], hindering the efficiency of morphine analgesia [37]. Proinflammatory cytokines secreted by continuously activated microglia include IL-1β and TNF-α [38]. IL-1β is one of the most crucial members of IL-1 family and an angiogenic agent involved in the mechanism of neuropathic pain. In addition, the recent research has shown that IL-1β is the first cytokine involved in the development of morphine tolerance [39]. Blockade of IL-1 or IL-1β signaling can obviously reduce the development of morphine tolerance [40, 41]. TNF-α is an effective proinflammatory cytokine that can be expressed by glial cells [42]. Inhibition of TNF-α signaling suppresses the development of morphine tolerance in animal experiments [43–45]. Interestingly, a previous study found that morphine activates Toll-like receptor-4 (TLR-4) to elevate the production of proinflammatory cytokines in BV-2 cells in a way similar to LPS [46]. In the present study, we demonstrated that morphine treatment notably increased IL-1β and TNF-α produced by BV-2 cells, which is parallel to the previous reports [7, 47]. Morphine and LPS promoted activated microglia to produce increased proinflammatory factors, which was abolished by AG1478 pretreatment. Thus, our data suggest that EGFR inhibitors might suppress morphine-induced microglial activation primarily by inhibiting the production of proinflammatory cytokines.
In summary, we report that the EGFR signaling pathway is essential for microglial activation, metastasis and cytokine production, making it a potential therapeutic target for the treatment of morphine-induced drug tolerance. Due to the pleiotropic cellular function of the receptor family, the mechanism of EGFR mediated or promoted the occurrence and maintenance of neuropathic pain in a variety of mechanisms [48]. In our previous experiments, we found that cetuximab, an anti-EGFR monoclonal antibody, combined with celecoxib, a highly selective nonsteroidal anti-inflammatory agent, may help relieve cancer pain through reduced tumor-derived mediators in vitro [49]. In addition, in our recent study, we established cancer induced bone pain rat model with morphine tolerance and found that EGFR and its downstream ERK pathway were significantly activated in spinal cord after morphine administration [50]. EGFR is one of the most important targets of antitumor therapy, and EGFR antagonists may eventually play an indispensable clinical role not only in the treatment of malignant tumors but also in the treatment of pain, especially cancer pain, which is resistant to opioid treatment.
Supporting information
S1 Fig. The expression of CD11b in BV2 microglia treated with AG1478.
(a) BV-2 cells were treated with DMSO, AG1478 (2.5, 5, and 10 μM), morphine (200 μM), and AG1478 (2.5, 5, and 10 μM) + morphine (200 μM) for 6 h and tested by immunocytochemistry staining (red fuorescence; × 200 magnifcation). (b) The mean optical densities of six randomly selected fields of cells from each group were analyzed by ImageJ software for quantitative analysis. Data are expressed as the means ± SEMs. All data were analyzed using ANOVA. * p < 0.05 and ** p < 0.01 compared to the DMSO group. # p < 0.05 and ## p < 0.01 compared to morphine group.
https://doi.org/10.1371/journal.pone.0256870.s001
(TIF)
References
- 1. Volkow ND, McLellan AT. Opioid Abuse in Chronic Pain—Misconceptions and Mitigation Strategies. N Engl J Med. 2016;374(13):1253–63. Epub 2016/03/31. pmid:27028915.
- 2. Horvath RJ, DeLeo JA. Morphine enhances microglial migration through modulation of P2X4 receptor signaling. J Neurosci. 2009;29(4):998–1005. Epub 2009/01/30. pmid:19176808; PubMed Central PMCID: PMC2727471.
- 3. Ferrini F, Trang T, Mattioli TA, Laffray S, Del’Guidice T, Lorenzo LE, et al. Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl(-) homeostasis. Nat Neurosci. 2013;16(2):183–92. Epub 2013/01/08. pmid:23292683; PubMed Central PMCID: PMC4974077.
- 4. Vacca V, Marinelli S, Luvisetto S, Pavone F. Botulinum toxin A increases analgesic effects of morphine, counters development of morphine tolerance and modulates glia activation and mu opioid receptor expression in neuropathic mice. Brain Behav Immun. 2013;32:40–50. Epub 2013/02/14. pmid:23402794.
- 5. Berta T, Liu YC, Xu ZZ, Ji RR. Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1beta and ERK signaling in the spinal cord of mice. Neuroscience. 2013;247:376–85. Epub 2013/05/28. pmid:23707980; PubMed Central PMCID: PMC3722295.
- 6. Taves S, Berta T, Chen G, Ji RR. Microglia and spinal cord synaptic plasticity in persistent pain. Neural Plast. 2013;2013:753656. Epub 2013/09/12. pmid:24024042; PubMed Central PMCID: PMC3759269.
- 7. Pan Y, Sun X, Jiang L, Hu L, Kong H, Han Y, et al. Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation. J Neuroinflammation. 2016;13(1):294. Epub 2016/11/20. pmid:27855689; PubMed Central PMCID: PMC5114746.
- 8. Garrett TP, McKern NM, Lou M, Elleman TC, Adams TE, Lovrecz GO, et al. Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor alpha. Cell. 2002;110(6):763–73. Epub 2002/09/26. pmid:12297049.
- 9. Yang Y, Jiang H, Gao H, Kong J, Zhang P, Hu S, et al. The monoclonal antibody CH12 enhances the sorafenib-mediated growth inhibition of hepatocellular carcinoma xenografts expressing epidermal growth factor receptor variant III. Neoplasia. 2012;14(6):509–18. Epub 2012/07/13. pmid:22787432; PubMed Central PMCID: PMC3394193.
- 10. Belcheva MM, Szucs M, Wang D, Sadee W, Coscia CJ. mu-Opioid receptor-mediated ERK activation involves calmodulin-dependent epidermal growth factor receptor transactivation. J Biol Chem. 2001;276(36):33847–53. Epub 2001/07/18. pmid:11457825.
- 11. Belcheva MM, Tan Y, Heaton VM, Clark AL, Coscia CJ. Mu opioid transactivation and down-regulation of the epidermal growth factor receptor in astrocytes: implications for mitogen-activated protein kinase signaling. Mol Pharmacol. 2003;64(6):1391–401. Epub 2003/12/03. pmid:14645669.
- 12. Qu WS, Tian DS, Guo ZB, Fang J, Zhang Q, Yu ZY, et al. Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury. J Neuroinflammation. 2012;9:178. Epub 2012/07/25. pmid:22824323; PubMed Central PMCID: PMC3418570.
- 13. Martin LJ, Smith SB, Khoutorsky A, Magnussen CA, Samoshkin A, Sorge RE, et al. Epiregulin and EGFR interactions are involved in pain processing. J Clin Invest. 2017;127(9):3353–66. Epub 2017/08/08. pmid:28783046; PubMed Central PMCID: PMC5669538.
- 14. Puig S, Donica CL, Gutstein HB. EGFR Signaling Causes Morphine Tolerance and Mechanical Sensitization in Rats. eNeuro. 2020;7(2). Epub 2020/03/01. pmid:32111605; PubMed Central PMCID: PMC7218007.
- 15. Labuzek K, Liber S, Gabryel B, Buldak L, Okopien B. Ambivalent effects of compound C (dorsomorphin) on inflammatory response in LPS-stimulated rat primary microglial cultures. Naunyn Schmiedebergs Arch Pharmacol. 2010;381(1):41–57. Epub 2009/11/27. pmid:19940979.
- 16. Qu WS, Liu JL, Li CY, Li X, Xie MJ, Wang W, et al. Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia. Neurochem Int. 2015;90:85–92. Epub 2015/07/26. pmid:26209152.
- 17. Horvath RJ, Nutile-McMenemy N, Alkaitis MS, Deleo JA. Differential migration, LPS-induced cytokine, chemokine, and NO expression in immortalized BV-2 and HAPI cell lines and primary microglial cultures. J Neurochem. 2008;107(2):557–69. Epub 2008/08/23. pmid:18717813; PubMed Central PMCID: PMC2581646.
- 18. Li C, Zhao B, Lin C, Gong Z, An X. TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF-kappaB signaling. Cell Biol Int. 2019;43(4):360–72. Epub 2018/04/18. pmid:29663649; PubMed Central PMCID: PMC7379930.
- 19. Cai Y, Kong H, Pan YB, Jiang L, Pan XX, Hu L, et al. Procyanidins alleviates morphine tolerance by inhibiting activation of NLRP3 inflammasome in microglia. J Neuroinflammation. 2016;13(1):53. Epub 2016/03/05. pmid:26931361; PubMed Central PMCID: PMC4774188.
- 20. Henn A, Lund S, Hedtjarn M, Schrattenholz A, Porzgen P, Leist M. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX. 2009;26(2):83–94. Epub 2009/07/01. pmid:19565166.
- 21. Wang L, Yin C, Xu X, Liu T, Wang B, Abdul M, et al. Pellino1 Contributes to Morphine Tolerance by Microglia Activation via MAPK Signaling in the Spinal Cord of Mice. Cell Mol Neurobiol. 2020;40(7):1117–31. Epub 2020/01/29. pmid:31989355.
- 22. Qin L, Li G, Qian X, Liu Y, Wu X, Liu B, et al. Interactive role of the toll-like receptor 4 and reactive oxygen species in LPS-induced microglia activation. Glia. 2005;52(1):78–84. Epub 2005/05/28. pmid:15920727.
- 23. Hutchinson MR, Lewis SS, Coats BD, Rezvani N, Zhang Y, Wieseler JL, et al. Possible involvement of toll-like receptor 4/myeloid differentiation factor-2 activity of opioid inactive isomers causes spinal proinflammation and related behavioral consequences. Neuroscience. 2010;167(3):880–93. Epub 2010/02/25. pmid:20178837; PubMed Central PMCID: PMC2854318.
- 24. Cui Y, Chen Y, Zhi JL, Guo RX, Feng JQ, Chen PX. Activation of p38 mitogen-activated protein kinase in spinal microglia mediates morphine antinociceptive tolerance. Brain Res. 2006;1069(1):235–43. Epub 2006/01/13. pmid:16403466.
- 25. Horvath RJ, Romero-Sandoval EA, De Leo JA. Inhibition of microglial P2X4 receptors attenuates morphine tolerance, Iba1, GFAP and mu opioid receptor protein expression while enhancing perivascular microglial ED2. Pain. 2010;150(3):401–13. Epub 2010/06/25. pmid:20573450; PubMed Central PMCID: PMC2921455.
- 26. Tawfik VL, LaCroix-Fralish ML, Nutile-McMenemy N, DeLeo JA. Transcriptional and translational regulation of glial activation by morphine in a rodent model of neuropathic pain. J Pharmacol Exp Ther. 2005;313(3):1239–47. Epub 2005/03/04. pmid:15743926.
- 27. Jokinen V, Sidorova Y, Viisanen H, Suleymanova I, Tiilikainen H, Li Z, et al. Differential Spinal and Supraspinal Activation of Glia in a Rat Model of Morphine Tolerance. Neuroscience. 2018;375:10–24. Epub 2018/02/09. pmid:29421434.
- 28. Yan P, Wu X, Liu X, Cai Y, Shao C, Zhu G. A Causal Relationship in Spinal Cord Injury Rat Model Between Microglia Activation and EGFR/MAPK Detected by Overexpression of MicroRNA-325-3p. J Mol Neurosci. 2019;68(2):181–90. Epub 2019/03/27. pmid:30911940.
- 29. Fischer OM, Hart S, Ullrich A. Dissecting the epidermal growth factor receptor signal transactivation pathway. Methods Mol Biol. 2006;327:85–97. Epub 2006/06/20. pmid:16780214.
- 30. Panigone S, Hsieh M, Fu M, Persani L, Conti M. Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway. Mol Endocrinol. 2008;22(4):924–36. Epub 2008/01/12. pmid:18187604; PubMed Central PMCID: PMC2276469.
- 31. Lacagnina MJ, Watkins LR, Grace PM. Toll-like receptors and their role in persistent pain. Pharmacol Ther. 2018;184:145–58. Epub 2017/10/11. pmid:28987324; PubMed Central PMCID: PMC5858962.
- 32. Hutchinson MR, Shavit Y, Grace PM, Rice KC, Maier SF, Watkins LR. Exploring the neuroimmunopharmacology of opioids: an integrative review of mechanisms of central immune signaling and their implications for opioid analgesia. Pharmacol Rev. 2011;63(3):772–810. Epub 2011/07/15. pmid:21752874; PubMed Central PMCID: PMC3141878.
- 33. Jacobsen JH, Watkins LR, Hutchinson MR. Discovery of a novel site of opioid action at the innate immune pattern-recognition receptor TLR4 and its role in addiction. Int Rev Neurobiol. 2014;118:129–63. Epub 2014/09/02. pmid:25175864.
- 34. Eidson LN, Murphy AZ. Blockade of Toll-like receptor 4 attenuates morphine tolerance and facilitates the pain relieving properties of morphine. J Neurosci. 2013;33(40):15952–63. Epub 2013/10/04. pmid:24089500; PubMed Central PMCID: PMC3787504.
- 35. Zhang L, Berta T, Xu ZZ, Liu T, Park JY, Ji RR. TNF-alpha contributes to spinal cord synaptic plasticity and inflammatory pain: distinct role of TNF receptor subtypes 1 and 2. Pain. 2011;152(2):419–27. Epub 2010/12/17. pmid:21159431; PubMed Central PMCID: PMC3022092.
- 36. Dominguini D, Steckert AV, Michels M, Spies MB, Ritter C, Barichello T, et al. The effects of anaesthetics and sedatives on brain inflammation. Neurosci Biobehav Rev. 2021;127:504–13. Epub 2021/05/17. pmid:33992694.
- 37. Matsushita Y, Omotuyi IO, Mukae T, Ueda H. Microglia activation precedes the anti-opioid BDNF and NMDA receptor mechanisms underlying morphine analgesic tolerance. Curr Pharm Des. 2013;19(42):7355–61. Epub 2013/03/02. pmid:23448475.
- 38. Jia J, Li C, Zhang T, Sun J, Peng S, Xie Q, et al. CeO2@PAA-LXW7 Attenuates LPS-Induced Inflammation in BV2 Microglia. Cell Mol Neurobiol. 2019;39(8):1125–37. Epub 2019/07/01. pmid:31256326.
- 39. Tu H, Chu H, Guan S, Hao F, Xu N, Zhao Z, et al. The role of the M1/M2 microglia in the process from cancer pain to morphine tolerance. Tissue Cell. 2020;68:101438. Epub 2020/11/22. pmid:33220596.
- 40. Johnston IN, Milligan ED, Wieseler-Frank J, Frank MG, Zapata V, Campisi J, et al. A role for proinflammatory cytokines and fractalkine in analgesia, tolerance, and subsequent pain facilitation induced by chronic intrathecal morphine. J Neurosci. 2004;24(33):7353–65. Epub 2004/08/20. pmid:15317861; PubMed Central PMCID: PMC6729781.
- 41. Shavit Y, Wolf G, Goshen I, Livshits D, Yirmiya R. Interleukin-1 antagonizes morphine analgesia and underlies morphine tolerance. Pain. 2005;115(1–2):50–9. Epub 2005/04/20. pmid:15836969.
- 42. de Oliveira CM, Sakata RK, Issy AM, Gerola LR, Salomao R. Cytokines and pain. Rev Bras Anestesiol. 2011;61(2):255–9, 60–5, 137–42. Epub 2011/04/09. pmid:21474032.
- 43. Shen CH, Tsai RY, Shih MS, Lin SL, Tai YH, Chien CC, et al. Etanercept restores the antinociceptive effect of morphine and suppresses spinal neuroinflammation in morphine-tolerant rats. Anesth Analg. 2011;112(2):454–9. Epub 2010/11/18. pmid:21081778.
- 44. Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, et al. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat Neurosci. 2013;16(9):1211–8. Epub 2013/07/23. pmid:23872599; PubMed Central PMCID: PMC3977045.
- 45. Zeng X, Lin MY, Wang D, Zhang Y, Hong Y. Involvement of adrenomedullin in spinal glial activation following chronic administration of morphine in rats. Eur J Pain. 2014;18(9):1323–32. Epub 2014/03/26. pmid:24664661.
- 46. Wang X, Loram LC, Ramos K, de Jesus AJ, Thomas J, Cheng K, et al. Morphine activates neuroinflammation in a manner parallel to endotoxin. Proc Natl Acad Sci U S A. 2012;109(16):6325–30. Epub 2012/04/05. pmid:22474354; PubMed Central PMCID: PMC3341002.
- 47. Han Y, Jiang C, Tang J, Wang C, Wu P, Zhang G, et al. Resveratrol reduces morphine tolerance by inhibiting microglial activation via AMPK signalling. Eur J Pain. 2014;18(10):1458–70. Epub 2014/04/24. pmid:24756886.
- 48. Borges JP, Mekhail K, Fairn GD, Antonescu CN, Steinberg BE. Modulation of Pathological Pain by Epidermal Growth Factor Receptor. Front Pharmacol. 2021;12:642820. Epub 2021/06/01. pmid:34054523; PubMed Central PMCID: PMC8149758.
- 49. Yang Y, Yan J, Huang Y, Xu H, Zhang Y, Hu R, et al. The cancer pain related factors affected by celecoxib together with cetuximab in head and neck squamous cell carcinoma. Biomed Pharmacother. 2015;70:181–9. Epub 2015/03/18. pmid:25776499.
- 50. Yang Y, Chen Z, Hu R, Sun Y, Xiang L, Yan J, et al. Activation of the spinal EGFR signaling pathway in a rat model of cancer-induced bone pain with morphine tolerance. Neuropharmacology. 2021:108703. Epub 2021/07/15. pmid:34260958.