Nortriptyline exerts context-dependent cytokine modulation and reduces pHrodo-associated phagocytic response in activated J774.2 macrophages.

BACKGROUND: Major depressive disorder is frequently associated with immune-inflammatory activation that influences treatment outcomes. Although tricyclic antidepressants like nortriptyline are extensively utilized for depression and neuropathic pain management, their direct immunomodulatory effects on core innate immune executors require further characterization. Therefore, this study evaluated the condition-dependent immunomodulatory activities of nortriptyline by assessing its integrated effects on pro-inflammatory cytokine secretion and functional phagocytic capacity in J774.2 murine macrophages. METHODS: Cells were treated with non-cytotoxic concentrations of nortriptyline (1, 3, and 5 µg/mL) under basal or Lipopolysaccharide (LPS, 1 µg/mL)-stimulated conditions. Extracellular secretion profiles of TNF-α, IL-12p40, IL-6, and GM-CSF were quantified via ELISA, while the functional phagocytic response was evaluated using fluorogenic pHrodo™ Green E. coli BioParticles™ via flow cytometry. RESULTS: Nortriptyline sustained cellular viability above 85% across all evaluated concentrations (1, 3, and 5 µg/mL). Under LPS challenge, nortriptyline drove a highly distinct, cytokine-specific regulatory trajectory, showing a numerical concentration-dependent downward trend in TNF-α (down to ~ 6,342 pg/mL, P > 0.05) and a significant concentration-dependent suppression of IL-12p40 (down to ~ 5,767 pg/mL, P < 0.01). Regarding IL-6 levels, low-dose (1 µg/mL) nortriptyline co-treatment resulted in a higher IL-6 secretion compared to LPS alone (~ 5,700 pg/mL), whereas the maximum dose (5 µg/mL) significantly diminished its secretion to ~ 3,100 pg/mL relative to the LPS control. Functionally, 5 µg/mL nortriptyline significantly reduced the pHrodo-associated phagocytic response, decreasing the proportion of active effector cells from 77% down to 45%. CONCLUSION: These findings demonstrate that nortriptyline exerts context-dependent cytokine modulation and reduces pHrodo-associated phagocytic response in J774.2 murine macrophages, highlighting the direct capacity of this tricyclic compound to alter specific innate inflammatory outputs in vitro.