Targeting phospholipase A2 with arachidonyl trifluoromethyl ketone modulates macrophage activation during Japanese...

Phospholipase A₂ (PLA₂) is a crucial enzyme in lipid metabolism and inflammatory signaling, playing a significant role in macrophage activation and neuroinflammation. However, its role in Japanese encephalitis virus (JEV)-mediated immune responses remains unclear. In this study, we show that PLA₂ and its associated receptors, lysophosphatidic acid receptors (LPAR1 and LPAR3), are significantly upregulated at 24 h post-JEV infection, suggesting their involvement in JEV-induced inflammatory responses. To investigate the effect of PLA₂ inhibition, we used arachidonyl trifluoromethyl ketone (AACOCF₃). Molecular docking using AutoDock Vina 1.1.2, supported by Protein Data Bank and AlphaFold structures, indicated that AACOCF₃ interacts with key inflammatory targets including gp91^phox (- 7.9 kcal/mol) and LPAR3 (- 7.8 kcal/mol), suggesting its potential to modulate inflammatory and oxidative pathways. In vitro studies in JEV-infected macrophages demonstrated that AACOCF₃ reduced activation of calcium-dependent cytosolic (c) PLA₂ expression and phosphorylation, LPAR1, 3, MAPK signaling (JNK, ERK1/2, p38), and NF-κB protein levels, along with a reduction in JE viral RNA levels, likely reflecting indirect effects mediated through host pathway modulation. AACOCF₃ also decreased proinflammatory cytokines (IL-1β, TNF-α) and autophagy-related markers (pAkt, LC3-II/I, SQSTM1/p6₂), and reduced expression of the ROS-generating enzyme gp91^phox (NOX2), indicating partial attenuation of inflammatory and oxidative stress-associated pathways. Oxidative stress was assessed via gp91^phox expression, and NF-κB was evaluated at protein level. These findings suggest that PLA₂ contributes to JEV-induced inflammatory signaling, and that its inhibition modulates host responses during infection in vitro, rather than exerting a definitive therapeutic effect.