Abstract
Evodiamine is a major ingredient of the plant Evodia rutaecarpa, which has long been used for treating infection-related diseases including diarrhea, beriberi and oral ulcer, but the underlying mechanism is unclear. Here we aimed to explore whether evodiamine influenced NLRP3 (NLR family, pyrin containing domain 3) inflammasome activation in macrophages, which is a critical mechanism for defending the host against pathogenic infections. We uncovered that evodiamine dose-dependently enhanced NLRP3 inflammasome activation in lipopolysaccharide-primed macrophages, as indicated by increased interleukin (IL)-1β production and caspase-1 cleavage, accompanied by increased ASC speck formation and pyroptosis. Mechanistically, evodiamine induced acetylation of α-tubulin around the microtubule organization center (indicated by γ-tubulin) in lipopolysaccharide-primed macrophages. Such evodiamine-mediated increases in NLRP3 activation and pyroptosis were attenuated by activators of α-tubulin deacetylase, resveratrol and NAD+, or dynein-specific inhibitor ciliobrevin A. Small interfering RNA knockdown of αTAT1 (the gene encoding α-tubulin N-acetyltransferase) expression, which reduced α-tubulin acetylation, also diminished evodiamine-mediated augmentation of NLRP3 activation and pyroptosis. Evodiamine also enhanced NLRP3-mediated production of IL-1β and neutrophil recruitment in vivo. Moreover, evodiamine administration evidently improved survival of mice with lethal bacterial infection, accompanied by increased production of IL-1β and interferon-γ, decreased bacterial load, and dampened liver inflammation. Resveratrol treatment reversed evodiamine-induced increases of IL-1β and interferon-γ, and decreased bacterial clearance in mice. Collectively, our results indicated that evodiamine augmented the NLRP3 inflammasome activation through inducing α-tubulin acetylation, thereby conferring intensified innate immunity against bacterial infection.
Highlights
Macrophages are critical innate immune cells in tissues to sense pathogenic infections or tissue damages through multiple cytosolic pattern recognition receptors (PRRs) leading to the formation of inflammasomes, which are multimeric protein complexes that are induced in the cytosol as a platform for the activation of caspase-1 (Broz and Dixit, 2016)
Western blotting showed that LPS priming induced the expression of NLRP3 and pro-IL-1β proteins, whereas pro-caspase-1 and ASC were constitutively expressed in macrophages irrespective of LPS priming, which is consistent with previous studies (Kayagaki et al, 2013)
Immunofluorescence microscopy revealed that evodiamine significantly increased the formation of ASC specks in the cytosol (Figures 1F,G), which is another marker of NLRP3 inflammasome activation
Summary
Macrophages are critical innate immune cells in tissues to sense pathogenic infections or tissue damages through multiple cytosolic pattern recognition receptors (PRRs) leading to the formation of inflammasomes, which are multimeric protein complexes that are induced in the cytosol as a platform for the activation of caspase-1 (Broz and Dixit, 2016). The second signal is provided by a broad spectrum of stimuli including extracellular ATP, pore-forming toxins (e.g., nigericin), particulates (e.g., uric acid crystals, silica, and alum), and pathogens (bacteria, fungi, protozoan, and virus) (Martinon et al, 2006; Iwasaki and Medzhitov, 2015; Man and Kanneganti, 2015). Upon such second stimulation, NLRP3 recruits the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which in turn interacts with pro-caspase-1, leading to the formation of NLRP3 inflammasome. NLRP3 inflammasome activation is regarded as a critical host defense mechanism against pathogenic infections
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