Supplementary Material for: Chloroquine enhances MAVS-mediated cytokine induction and alters mitochondrial morphology

Introduction: Chloroquine (CQ), a well-known antimalarial agent, has been proposed as a potential antiviral compound due to its ability to interfere with multiple cellular pathways critical for viral replication. Although CQ exhibits broad-spectrum antiviral activity, its effect on host innate immune responses remains incompletely understood. The timing of CQ administration, whether before or after infection, may lead to different immunological outcomes. Therefore, the immunomodulatory effects of CQ should be carefully evaluated before antiviral therapy. Methods: To investigate the immunomodulatory role of CQ (50 M), we used immunofluorescence staining, western blotting, and reporter assays to evaluate innate immune activation in A549 cells. We established a doxycycline-inducible system to activate MAVS-mediated signaling without viral infection. Plaque assays and antiviral tests were performed to measure viral replication, while cytokine array and RT-qPCR were used to quantify cytokine production. Mitochondrial morphology was assessed using immunofluorescence microscopy. Results: CQ enhanced innate immune responses triggered by dengue virus infection and Poly(I:C) stimulation. This enhancement was associated with the activation of the mitochondrial antiviral signaling protein (MAVS) and its upstream receptors, including retinoic acid-inducible gene I and melanoma differentiation-associated protein 5. CQ strengthened MAVS-dependent antiviral signaling and increased IL-6 induction more than 13-fold. Alterations in mitochondrial morphology may contribute to this immunostimulatory effect. Conclusion: CQ promotes MAVS-mediated antiviral and inflammatory cytokine responses, potentially through its effect on mitochondrial dynamics. These findings indicate that while CQ may enhance antiviral defense, its immune-stimulating properties should be carefully evaluated prior to its use as an antiviral agent in treating RNA virus infections.