Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells
Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates μ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cel...
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MDPI AG
2024-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/4/2137 |
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| author | Frida L. Martínez-Cuevas Silvia L. Cruz Claudia González-Espinosa |
| author_facet | Frida L. Martínez-Cuevas Silvia L. Cruz Claudia González-Espinosa |
| author_sort | Frida L. Martínez-Cuevas |
| collection | DOAJ |
| description | Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates μ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express μ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca<sup>2+</sup>]<i>i</i>) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca<sup>2+</sup>]<i>i</i>. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of μ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between μ-opioid and TLR4 receptors. |
| first_indexed | 2024-03-07T22:29:30Z |
| format | Article |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-07T22:29:30Z |
| publishDate | 2024-02-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3bf2cc4dfd73497e9da320629e6528d72024-02-23T15:19:54ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-02-01254213710.3390/ijms25042137Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast CellsFrida L. Martínez-Cuevas0Silvia L. Cruz1Claudia González-Espinosa2Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav, IPN), Unidad Sede Sur, Calzada de los Tenorios No. 235, Col. Rinconada de las Hadas, México City CP 14330, MexicoDepartamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav, IPN), Unidad Sede Sur, Calzada de los Tenorios No. 235, Col. Rinconada de las Hadas, México City CP 14330, MexicoDepartamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav, IPN), Unidad Sede Sur, Calzada de los Tenorios No. 235, Col. Rinconada de las Hadas, México City CP 14330, MexicoMethadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates μ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express μ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca<sup>2+</sup>]<i>i</i>) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca<sup>2+</sup>]<i>i</i>. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of μ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between μ-opioid and TLR4 receptors.https://www.mdpi.com/1422-0067/25/4/2137methadoneµ-opioid receptorsTLR4 receptorsmast cellsETosismast cell extracellular traps |
| spellingShingle | Frida L. Martínez-Cuevas Silvia L. Cruz Claudia González-Espinosa Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells International Journal of Molecular Sciences methadone µ-opioid receptors TLR4 receptors mast cells ETosis mast cell extracellular traps |
| title | Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells |
| title_full | Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells |
| title_fullStr | Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells |
| title_full_unstemmed | Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells |
| title_short | Methadone Requires the Co-Activation of μ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells |
| title_sort | methadone requires the co activation of μ opioid and toll like 4 receptors to produce extracellular dna traps in bone marrow derived mast cells |
| topic | methadone µ-opioid receptors TLR4 receptors mast cells ETosis mast cell extracellular traps |
| url | https://www.mdpi.com/1422-0067/25/4/2137 |
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