Abstract
Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01–100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)–induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter–like/myeloid differentiation primary response 88– and TRIF-related adaptor molecule/TIR domain–containing adapter-inducing interferon-β–dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain–containing adapter-inducing interferon-β knockout, and MyD88 adapter–like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)–induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.
Footnotes
- Received March 13, 2015.
- Accepted August 26, 2015.
P.V.B and K.H.M. are co-first authors of this work.
This research was supported by the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro and by the National Council for Scientific and Technological Development. P.V.B. and K.H.M. were supported by fellowships from the Coordination for the Improvement of Higher Education Personnel Foundation and the National Council for Scientific and Technological Development as students of the Oswaldo Cruz Institute (Fiocruz) Graduate Program in Cellular and Molecular Biology.
↵This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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