Protective effect of gedunin on TLR-mediated inflammation by modulation of inflammasome activation and cytokine production: Evidence of a multitarget compound.

Activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) triggers an innate immune response, via cytokine production and inflammasome activation. Herein, we have investigated the modulatory effect of the natural limonoid gedunin on TLR activation in vitro and in vivo. Intraperitoneal (i.p.) pre- and post-treatments of C57BL/6 mouse with gedunin impaired the influx of mononuclear cells, eosinophils and neutrophils, as well as the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and nitric oxide (NO), triggered by lipopolysaccharide (LPS) in mouse pleura. Accordingly, in vitro post-treatment of immortalized murine macrophages with gedunin also impaired LPS-induced production of such mediators. Gedunin diminished LPS-induced expression of the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) on pleural leukocytes in vivo and in immortalized macrophages in vitro. In line with this, gedunin inhibited LPS-induced caspase-1 activation and the production of IL-1ß in vivo and in vitro. In addition, gedunin treatment triggered the generation of the anti-inflammatory factors IL-10 and heme oxigenase-1 (HO-1) at resting conditions or upon stimulation. We also demonstrate that gedunin effect is not restricted to TLR4-mediated response, since this compound diminished TNF-α, IL-6, NO, NLRP3 and IL-1ß, as well as enhanced IL-10 and HO-1, by macrophages stimulated with the TLR2 and TLR3 agonists, palmitoyl-3-Cys-Ser-(Lys)4 (PAM3) and polyriboinosinic:polyribocytidylic acid (POLY I:C), in vitro. In silico modeling studies revealed that gedunin efficiently docked into caspase-1, TLR2, TLR3 and to the myeloid differentiation protein-2 (MD-2) component of TLR4. Overall, our data demonstrate that gedunin modulates TLR4, TLR3 and TLR2-mediated responses and reveal new molecular targets for this compound.

Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.

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-ß-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-ß 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.

Gedunin, a natural tetranortriterpenoid, modulates T lymphocyte responses and ameliorates allergic inflammation.

T lymphocytes are critical cells involved in allergy. Here, we report that the natural tetranortriterpenoid gedunin impaired allergic responses primarily by modulating T lymphocyte functions. The intraperitoneal (i.p.) administration of gedunin inhibited pleural leukocyte accumulation triggered by intra-pleural (i.pl.) challenge with ovalbumin (OVA) in previously sensitized C57BL/6 mice; this inhibition was primarily due to the impairment of eosinophil and T lymphocyte influx. Likewise, i.pl. pre-treatment with gedunin inhibited eosinophil and T lymphocyte migration into mouse lungs 24 h after OVA intra-nasal (i.n.) instillation. Pre-treatment with gedunin diminished the levels of CCL2, CCL3, CCL5, CCL11, Interleukin-5 and leukotriene B(4) at the allergic site. In vitro pre-treatment with gedunin failed to inhibit T lymphocyte adhesion and chemotaxis towards pleural washes recovered from OVA-challenged mice, suggesting that gedunin inhibits T lymphocyte migration in vivo via the inhibition of chemotactic mediators in situ. In vivo pre-treatment with gedunin reduced the numbers of CD69(+) and CD25(+) T lymphocytes in the pleura and CD25(+) cells in the thoracic lymph nodes 24 h after OVA i.pl. challenge. In accordance, in vitro treatment of T lymphocytes with gedunin inhibited α-CD3 mAb-induced expression of CD69 and CD25, proliferation, Interleukin-2 production and nuclear translocation of NFκB and NFAT. Notably, post-treatment of mice with gedunin reverted OVA-induced lung allergic inflammation by decreasing the T lymphocyte and eosinophil counts and the levels of eosinophilotactic mediators in bronchoalveolar lavage fluid. Our results demonstrate a remarkable anti-allergic effect of gedunin due to its capability to modulate T cell activation and trafficking into the airways.

Efeito da gedunina sobre a ativação de macrófagos induzida por LPS / Gedunin effect on the activation of macrophages induced by LPS

Os macrófagos são células do sistema imune com perfil predominantementeinflamatório, também capazes de desempenhar um papel regulatório. O lipopolissacarídeo(LPS) presente na membrana externa de bactérias Gram-negativas é um potente ativador demacrófagos, reconhecido pelo complexo de receptores membranares TLR4/MD-2/CD14, cujaativação leva a uma cascata de sinalização intracelular, dependente das proteínas adaptadorasMAL/MyD88 (via aguda) e TRAM/TRIF (via tardia). A ativação do TLR4 induz atranslocação do NFκB, a geração de diversos mediadores inflamatórios e a expressão deenzimas responsßveis pela geração de mediadores lipídicos. A gedunina, um limonóide deorigem natural presente em espécies vegetais da família Meliaceae, é um conhecidomodulador da proteína de choque térmico (Hsp)90, uma chaperona que apresenta umimportante papel na resposta imune por estabilizar diferentes proteínas clientes, tais comofatores de transcrição e tirosinas quinases, dentre as quais diversas estão associadas àsinalização do TLR4. O objetivo geral deste estudo foi avaliar o efeito da gedunina sobre aresposta inflamatória induzida por LPS in vitro e in vivo, investigando o seu papel nasinalização do TLR4 e o envolvimento da Hsp90 nesta via. O pré-tratamento in vitro demacrófagos wild type (WT) imortalizados a partir de medula óssea de camundongos C57BL/6com gedunina, em concentrações não tóxicas, inibiu o influxo de Ca2+ intracelular e aprodução de TNF-alfa e NO de forma concentração dependente, 6 h e 24 h após estímulo comLPS. Nossos dados também demonstraram que a gedunina modula tanto a via aguda quanto avia tardia da sinalização de TLR4, inibindo a produção de TNF-alfa e IL-6 em macrófagos TRIFknockout (KO) e MAL KO, 6 h e 24 h após o estímulo com LPS...

Macrophages are inflammatory cells that also play a key regulatory role in immuneresponses. Lipopolysaccharide (LPS) present on the outer membrane of Gram-negativebacteria is a potent activator of macrophages, which is recognized by the TLR4/MD-2/CD14receptor complex, triggering an intracellular signaling cascade dependent on MAL/MyD88(early phase) and TRAM/TRIF (late phase) adaptor proteins. TLR4 activation induces NFκBtranslocation into the nucleus, the production of several inflammatory mediators and theexpression of enzymes responsible for the generation of lipid mediators. Gedunin, a limonoidfrom plant species of Meliaceae family, is a modulator of the chaperone heat shock protein(Hsp)90, which in turn presents an important role in immune responses by stabilizing andactivating several eukaryotic proteins, such as transcriptional factors and tyrosine kinasesassociated with the TLR4 signaling pathway. The aim of this study was to evaluate the effectof gedunin on the inflammatory response induced by LPS in vitro and in vivo, investigating itsimpact on TLR4 cell signaling and the involvement of Hsp90 in this phenomenon. In vitropretreatment of wild type (WT) macrophages with gedunin, at non-cytotoxic concentrations,inhibited intracellular Ca2+ influx and the production of TNF-alpha and NO in a concentrationdependentmanner, 6 h and 24 h after stimulation with LPS. Our data also demonstrate thatgedunin modulates both acute and late TLR4 signaling pathways, since it inhibited TNF-alpha andIL-6 production by TRIF knockout (KO) and MAL KO macrophages, at 6 h and 24 h afterLPS stimulation...