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Dimethyl itaconate alleviates the pyroptosis of macrophages through oxidative stress

ABSTRACT: Macrophages are involved in the pathophysiology of many diseases as critical cells of the innate immune system. Pyroptosis is a form of macrophage death that induces cytokinesis of phagocytic substances in the macrophages, thereby defending against infection. Dimethyl itaconate (DI) is an analog of itaconic acid with anti-inflammatory effects. However, the effect of dimethyl itaconate on macrophage pyroptosis has not been elucidated clearly. Thus, the present study aimed to analyze the effect of DI treatment on a macrophage pyroptosis model (Lipopolysaccharide, LPS + Adenosine Triphosphate, ATP). The results showed that 0.25 mM DI ameliorated macrophage pyroptosis and downregulated interleukin (IL)-1β expression. Then, real-time quantitative polymerase chain reaction (RT-qPCR) was used to confirm the result of RNA-sequencing of the upregulated oxidative stress-related genes (Gclc and Gss) and downregulated inflammation-related genes (IL-12β and IL-1β). In addition, Gene Ontology (GO) enrichment analysis showed that differential genes were associated with transcript levels and DNA replication. Kyoto encyclopedia of genes and genomes (KEGG) enrichment showed that signaling pathways, such as tumor necrosis factor (TNF), Jak, Toll-like receptor and IL-17, were altered after DI treatment. N-acetyl-L-cysteine (NAC) reversed the DI effect on the LPS + ATP-induced macrophage pyroptosis and upregulated the IL-1β expression. Oxidative stress-related protein Nrf2 is involved in the DI regulation of macrophage pyroptosis. Taken together, these findings suggested that DI alleviates the pyroptosis of macrophages through oxidative stress.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12865-021-00463-3.

SUBMITTER: Huang S

PROVIDER: S-EPMC8573905 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background Exosomes derived from cardiac microvascular endothelial cells (CMECs) under hypoxia can mediate cardiac repair functions and alleviate pyroptosis and oxidative stress during ischemia-reperfusion (I/R) injury. This study is aimed at investigating the effect and mechanism of miR-27b-3p underlying hypoxic CMECs-derived exosomes against I/R injury. Methods CMECs were isolated from the left ventricle of Sprague-Dawley rats, followed by culturing under hypoxic conditions or pretreatment with the miR-27b-3p inhibitor. CMECs-derived exosomes were added into H9C2 cells before hypoxia/reoxygenation (H/R) or injected into the rat heart before I/R injury. An in vivo I/R injury model was established by ligating and releasing the left anterior descending coronary artery. Expression of pyroptosis-related factors was detected using Western blot, and heart infarcted size was determined by the 2,3,5-triphenyl-2H-tetrazpinolium chloride staining method. Dual-Luciferase Reporter assays were performed to analyze the interactions of nmiR-27b-3p-forkhead box O1 (Foxo1) and Gasdermin D- (GSDMD-) Foxo1. Chromatin-immunoprecipitation (ChIP) assays were performed to validate the interactions between forkhead box O1 (Foxo1) and Gasdermin D (GSDMD) and Foxo1-mediated histone acetylation of GSDMD. Results CMECs were successfully identified from left ventricle of Sprague-Dawley rats. The expressions of Foxo1 and pyroptosis-related proteins (GSDMD, NLPR3, cleaved caspase 1, IL-1β, and IL-18) were upregulated in the rat heart after I/R injury. Treatment of CMEC-derived exosomes, especially that under hypoxic conditions, significantly reduced pyroptosis in the rat heart. miR-27b-3p was significantly upregulated in CMEC-derived exosomes under hypoxic conditions, and miR-27b-3p inhibition in exosomes alleviated its cytoprotection and inhibited oxidative stress in H9C2 cells. Treatment with Foxo1 overexpression plasmids aggravated in vitro H/R and in vivo I/R injury by upregulating pyroptosis-related proteins. Further experiments validated that miR-27b-3p negatively targeted Foxo1, which bound to the promoter region of GSDMD. Conclusions These results demonstrated a great therapeutic efficacy of miR-27b-3p overexpression in hypoxic CMEC-derived exosomes in preventing the development of myocardial damage post I/R injury through inhibiting Foxo1/GSDMD signaling-induced oxidative stress and pyroptosis.

Project description:BackgroundInflammatory stimuli induce immunoresponsive gene 1 (IRG1) expression that in turn catalyzes the production of itaconate from the tricarboxylic acid cycle. Itaconate has recently emerged as a regulator of immune cell functions, especially in macrophages. Studies show that itaconate is required for the activation of anti-inflammatory transcription factor Nrf2 by LPS in mouse and human macrophages, and LPS-activated IRG1-/- macrophages that lack endogenous itaconate production exhibit augmented inflammatory responses. Moreover, dimethyl itaconate (DMI), an itaconate derivative, inhibits IL-17-induced I?B? activation in keratinocytes and modulates IL-17-I?B? pathway-mediated skin inflammation in an animal model of psoriasis. Currently, the effect of itaconate on regulating macrophage functions and peripheral inflammatory immune responses is well established. However, its effect on microglia (MG) and CNS inflammatory immune responses remains unexplored. Thus, we investigated whether itaconate possesses an immunomodulatory effect on regulating MG activation and CNS inflammation in animal models of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE).MethodsChronic C57BL/6 EAE was induced followed by DMI treatment. The effect of DMI on disease severity, blood-brain barrier (BBB) disruption, MG activation, peripheral Th1/Th17 differentiation, and the CNS infiltration of Th1/Th17 cells in EAE was determined. Primary MG was cultured to study the effect of DMI on MG activation. Relapsing-remitting SJL/J EAE was induced to assess the therapeutic effect of DMI.ResultsOur results show DMI ameliorated disease severity in the chronic C57BL/6 EAE model. Further analysis of the cellular and molecular mechanisms revealed that DMI mitigated BBB disruption, inhibited MMP3/MMP9 production, suppressed microglia activation, inhibited peripheral Th1/Th17 differentiation, and repressed the CNS infiltration of Th1 and Th17 cells. Strikingly, DMI also exhibited a therapeutic effect on alleviating severity of relapse in the relapsing-remitting SJL/J EAE model.ConclusionsWe demonstrate that DMI suppresses neuroinflammation and ameliorates disease severity in EAE through multiple cellular and molecular mechanisms, suggesting that DMI can be developed as a novel therapeutic agent for the treatment of MS/EAE through its immunomodulatory and anti-inflammatory properties.

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Dimethyl itaconate alleviates the pyroptosis of macrophages through oxidative stress

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