Modulation of Mu-Opioid Receptor Expression and Functional Impairment of Natural Killer Cells in Neuropathic Pain: Implications for Biomarker Discovery and Personalized Therapies

Background/Objectives: Chronic pain is a significant clinical challenge, in part due to the absence of reliable objective biomarkers for its evaluation and treatment. Growing evidence indicates that immune cells, including natural killer (NK) cells, are involved in the regulation of pain processes. NK cells are innate cytotoxic lymphocytes whose functional status may mirror underlying pathological pain states. In this study, we investigated μ-opioid receptor (MOR) expression and functional alterations of NK cells in a murine model of neuropathic pain induced by chronic constriction injury (CCI). Methods: Mice were divided into three groups: Sham (sciatic nerve exposure without ligation), CCI 14-day, and CCI 21-day groups. At the respective time points, animals were sacrificed and spleens were collected for analysis. Splenocytes were isolated by mechanical dissociation followed by centrifugation and erythrocyte lysis. Lymphocytes were analyzed by flow cytometry to evaluate MOR expression in NK cells and their degranulation activity (CD107a assay). Cells were incubated with fluorochrome-conjugated antibodies against NK cell markers (NK1.1, CD3, Ly49A, Ly49C/I) in combination with anti-MOR and anti-Interferon γ antibody (IFN-γ). Immunofluorescence and confocal microscopy analyses were performed to assess MOR localization and granzyme localization, supporting CD107a-mediated degranulation. Results: Flow cytometry analysis revealed a significant reduction in surface MOR expression on total NK cells from CCI mice compared with sham controls at 14 and 21 days post-injury, a finding corroborated by immunofluorescence evidence of MOR cellular internalization. Functionally, CCI induced a marked decrease in CD107a expression and impaired IFN-γ production both under basal conditions and following PMA/ionomycin stimulation, indicating a hyporesponsive state of NK cells. Consistently, confocal microscopy revealed extracellular release of Granzyme A following CCI, suggesting dysregulated degranulation. Conclusions: Neuropathic pain is associated with a remodeling of NK cell phenotype and effector functions, characterized by impaired cytotoxic activity and cytokine production, along with modulation of inhibitory receptor expression. Notably, MOR-reduced surface expression in NK cells emerges as a potential biomarker of neuropathic pain. Further studies are needed to elucidate the molecular mechanisms regulating MOR expression and its relationship with NK cell hyporesponsiveness and degranulation in chronic pain conditions.