Ivermectin modulates macrophage activity and enhances bacterial clearance in Pseudomonas aeruginosa acute pneumonia

The TLR4 receptor, together with the MD-2 co-receptor, is essential for macrophage recognition of LPS from Gram-negative bacteria such as Pseudomonas aeruginosa. Although ivermectin improves survival following LPS challenge in mice, its immunological mechanisms remain poorly understood. In silico molecular docking was performed to evaluate the binding of ivermectin to TLR4/MD-2. In vitro studies were conducted using RAW 264.7 macrophages and bone marrow-derived macrophages (BMDMs) treated with ivermectin and/or TLR4/MD-2 inhibitors, followed by LPS stimulation or infection with P. aeruginosa PA14. In vivo studies were carried out in C57BL/6 wild-type (WT) and TLR4 knockout (KO) mice treated with ivermectin or phosphate-buffered saline and subsequently intratracheally infected with PA14. Docking analysis demonstrated high-affinity binding of ivermectin to the MD-2 component of the TLR4/MD-2 complex. Ivermectin did not affect macrophage viability but impaired bacterial clearance, reduced NO and TNF-α secretion, and enhanced NF-κB activation in LPS-stimulated RAW 264.7 macrophages. These effects were reversed by TLR4/MD-2 complex inhibition with LPS/RS. In vivo, ivermectin treatment reduced the pulmonary bacterial load in TLR4 KO mice. Additionally, ivermectin decreased inflammatory infiltrates IL-6 and TNF-α levels while increasing IL-17 and IFN-γ production in infected lungs, with more pronounced effects in TLR4 KO mice. Ivermectin binds to MD-2 and suppresses macrophage microbicidal activity in vitro. In vivo, however-particularly in TLR4-deficient mice-ivermectin improved bacterial clearance, lung histopathology, and cytokine modulation. These findings highlight a complex, context-dependent immunomodulatory role of ivermectin.