Oxycodone induces HMGB1-mediated neuroimmune crosstalk between oligodendrocytes and microglia

• Oxycodone induces HMGB1 release from mature oligodendrocytes. • Oligodendrocyte-derived HMGB1 drives inflammatory activation in microglia. • HMGB1 from oligodendrocytes mediates inflammation via TLR and RAGE. Oxycodone induces HMGB1 release from mature oligodendrocytes. Oligodendrocyte-derived HMGB1 drives inflammatory activation in microglia. HMGB1 from oligodendrocytes mediates inflammation via TLR and RAGE. Oxycodone selectively upregulates HMGB1 in mature oligodendrocytes, leading to its cytoplasmic translocation and extracellular release. Extracellular HMGB1 interacts with TLR2 and RAGE receptors on microglia, activating NF-κB signaling and inducing pro-inflammatory cytokine expression (IL-6, IL-1β, CXCL10). Pharmacological inhibition of HMGB1 or receptor blockade attenuates this microglial inflammatory response, highlighting a novel oligodendrocyte–microglia signaling axis in opioid-induced neuroinflammation. Emerging evidence implicates opioid exposure in glial dysfunction and neuroimmune signaling. However, the role of oligodendrocytes in opioid-induced neuroinflammation remains poorly understood. Here, we demonstrate that oxycodone exposure upregulated high mobility group box 1 (HMGB1), a danger-associated molecular pattern (DAMP), in mature oligodendrocytes (OLs) but not oligodendrocyte precursor cells (OPCs). Oxycodone treatment significantly increased cytoplasmic translocation and extracellular release of HMGB1, which was blocked by opioid receptor antagonist naloxone. These effects occurred without compromising cell viability or differentiation, although a reduction in Ki67 + cells was observed, indicating decreased proliferation. Interestingly, while myelin-related gene expression remained unchanged, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) protein level was elevated, suggesting a possible myelin remodeling. To assess the functional consequences of extracellular HMGB1, we treated mouse and human microglial cells with recombinant HMGB1, which robustly elevated IL-6, IL-1β, and CXCL10 expression. Given the central role of NF-κB in regulating cytokine expression, we assessed phosphorylated p65 levels in the nuclear compartment of microglia exposed to conditioned media from oxycodone-treated OLs and found them to be elevated compared to controls. This pro-inflammatory phenotype was attenuated by co-treatment with the HMGB1 inhibitor glycyrrhizin. Pharmacological inhibition of HMGB1 receptors revealed that RAGE and TLR2, but not TLR4, were critical for IL-1β and IL-6 induction, highlighting distinct receptor-mediated inflammatory pathways. Collectively, we show OLs as an unrecognized source of HMGB1 during oxycodone exposure and establish a novel OL-microglia signaling axis underlying neuroinflammation. These insights emphasize the importance of glial crosstalk in opioid-related pathologies and may inform therapeutic strategies targeting HMGB1 signaling.