Dual role of homocysteine in enhancing metabolic activity and suppressing inflammatory cytokines in murine immune cells.

Homocysteine (HCy) is a sulfur-containing metabolic intermediate with largely unexplored potential as a damage-associated molecular pattern. This study investigated the immunomodulatory profile of HCy alone and in combination with the TLR4 agonist monophosphoryl lipid A (MPL). To model acute metabolic stress, we evaluated murine bone marrow-derived dendritic cells (DCs), macrophages, and splenocytes treated with HCy (6.25-600 μg/ml) in vitro, and assessed inflammatory cell recruitment in vivo in mice injected intraperitoneally with HCy (100 or 500 μg/mouse). HCy treatment significantly increased mitochondrial metabolic activity (CCK-8) without generalized necrosis. However, high concentrations (400-600 μg/ml) paradoxically suppressed MPL-induced splenocyte proliferation. HCy displayed distinct cell-specificity, acting as a direct agonist for macrophage maturation (upregulating CD40, CD86, and MHC-II) while failing to activate purified DCs. Notably, flow cytometry revealed that while metabolic signals increased, high-dose HCy induced late apoptosis in macrophages starting at 25 μg/ml, suggesting a state of metabolic stress rather than enhanced viability. Crucially, HCy exerted a regulatory effect on TLR4 signaling: co-treatment with HCy (200-600 μg/ml) dose-dependently suppressed MPL-induced pro-inflammatory cytokine secretion (TNF-α, IL-6) while maintaining phenotypic maturation. In vivo, HCy functioned as a chemotactic agent, recruiting DCs and monocytes to the peritoneal cavity and inducing an activated, M2-like (CD206+) macrophage phenotype. These findings demonstrate that HCy functions as a dual-action immune modulator that promotes phenotypic maturation and antigen-presenting cell recruitment while dampening excessive cytokine release through a stress-mediated regulatory mechanism.