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Extracellular vesicles secreted by Giardia duodenalis regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways.

ABSTRACT: Giardia duodenalis, also known as G. intestinalis or G. lamblia, is the major cause of giardiasis leading to diarrheal disease with 280 million people infections annually worldwide. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism participating in cells communications. The aim of this study is to explore the roles of G. duodenalis EVs (GEVs) in host-pathogen interactions using primary mouse peritoneal macrophages as a model. Multiple methods of electron microscopy, nanoparticle tracking analysis, proteomic assays, flow cytometry, immunofluorescence, qPCR, western blot, ELISA, inhibition assays, were used to characterize GEVs, and explore its effects on the host cell innate immunity as well as the underlying mechanism using primary mouse peritoneal macrophages. Results showed that GEVs displayed typical cup-shaped structure with 150 nm in diameter. GEVs could be captured by macrophages and triggered immune response by increasing the production of inflammatory cytokines Il1β, Il6, Il10, Il12, Il17, Ifng, Tnf, Il18, Ccl20 and Cxcl2. Furthermore, activation of TLR2 and NLRP3 inflammasome signaling pathways involved in this process. In addition, CA-074 methyl ester (an inhibitor of cathepsin B) or zVAD-fmk (an inhibitor of pan-caspase) pretreatment entirely diminished these effects triggered by GEVs exposure. Taken together, these findings demonstrated that GEVs could be internalized into mouse peritoneal macrophages and regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways.

SUBMITTER: Zhao P

PROVIDER: S-EPMC8046354 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Extracellular vesicles secreted by Giardia duodenalis regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways.

Zhao Panpan P Cao Lili L Wang Xiaocen X Dong Jingquan J Zhang Nan N Li Xin X Li Jianhua J Zhang Xichen X Gong Pengtao P

PLoS neglected tropical diseases 20210402 4

Giardia duodenalis, also known as G. intestinalis or G. lamblia, is the major cause of giardiasis leading to diarrheal disease with 280 million people infections annually worldwide. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism participating in cells communications. The aim of this study is to explore the roles of G. duodenalis EVs (GEVs) in host-pathogen interactions using primary mouse peritoneal macrophages as a model. Multiple methods of electron microscopy, nanoparticl ...[more]

PMID: 33798196

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Project description:BackgroundGiardia duodenalis is a parasitic organism that can cause giardiasis, an intestinal infection, particularly prevalent in young children, with clinical symptoms of diarrhea. We previously reported that extracellular G. duodenalis triggers intracellular nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs). However, the exact pathogen-associated molecular patterns in G. duodenalis EVs (GEVs) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated.MethodsRecombinant eukaryotic expression plasmids of pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEVs were constructed, transfected into primary mouse peritoneal macrophages and screened by measuring the expression levels of the inflammasome target molecule caspase-1 p20. The preliminary identification of G. duodenalis alpha-2 and alpha-7.3 giardins was further verified by measuring the protein expression levels of key molecules of the NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1, and caspase-1 p20), the secretion levels of IL-1β, the level of apoptosis speck-like protein (ASC) oligomerization and the immunofluorescence localization of NLRP3 and ASC. The roles of the NLRP3 inflammasome in G. duodenalis pathogenicity were then evaluated using mice in which NLRP3 activation was blocked (NLRP3-blocked mice), and body weight, parasite burden in the duodenum and histopathological changes in the duodenum were monitored. In addition, we explored whether alpha-2 and alpha-7.3 giardins triggered IL-1β secretion in vivo through the NLRP3 inflammasome and determined the roles of these molecules in G. duodenalis pathogenicity in mice.ResultsAlpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vitro. This led to caspase-1 p20 activation, upregulation of the protein expression levels of NLRP3, pro-IL-1β and pro-caspase-1, significant enhancement of IL-1β secretion, ASC speck formation in the cytoplasm and also induction of ASC oligomerization. Deletion of the NLRP3 inflammasome aggravated G. duodenalis pathogenicity in mice. Compared to wild-type mice gavaged with cysts, mice gavaged with cysts in NLRP3-blocked mice displayed increased trophozoite loads and severe duodenal villus damage, characterized by necrotic crypts with atrophy and branching. In vivo assays revealed that alpha-2 and alpha-7.3 giardins could induce IL-1β secretion through the NLRP3 inflammasome and that immunization with alpha-2 and alpha-7.3 giardins decreased G. duodenalis pathogenicity in mice.ConclusionsOverall, the results of the present study revealed that alpha-2 and alpha-7.3 giardins trigger host NLRP3 inflammasome activation and decrease G. duodenalis infection ability in mice, which are promising targets for the prevention of giardiasis.

Project description:Microtubules play critical roles in regulating the activation of NLRP3 inflammasome and microtubule-destabilizing agents such as colchicine have been shown to suppress the activation of this inflammasome. However, it remains largely unknown whether paclitaxel, a microtubule-stabilizing agent being used in cancer therapy, has any influences on NLRP3 inflammasome activation. Here we showed that paclitaxel pre-treatment greatly enhanced ATP- or nigericin-induced NLRP3 inflammasome activation as indicated by increased release of cleaved caspase-1 and mature IL-1β, enhanced formation of ASC speck, and increased gasdermin D cleavage and pyroptosis. Paclitaxel time- and dose-dependently induced α-tubulin acetylation in LPS-primed murine and human macrophages and further increased ATP- or nigericin-induced α-tubulin acetylation. Such increased α-tubulin acetylation was significantly suppressed either by resveratrol or NAD+ (coenzyme required for deacetylase activity of SIRT2), or by genetic knockdown of MEC-17 (gene encoding α-tubulin acetyltransferase 1). Concurrently, the paclitaxel-mediated enhancement of NLRP3 inflammasome activation was significantly suppressed by resveratrol, NAD+, or MEC-17 knockdown, indicating the involvement of paclitaxel-induced α-tubulin acetylation in the augmentation of NLRP3 inflammasome activation. Similar to paclitaxel, epothilone B that is another microtubule-stabilizing agent also induced α-tubulin acetylation and increased NLRP3 inflammasome activation in macrophages in response to ATP treatment. Consistent with the in vitro results, intraperitoneal administration of paclitaxel significantly increased serum IL-1β levels, reduced bacterial burden, dampened infiltration of inflammatory cells in the liver, and improved animal survival in a mouse model of bacterial infection. Collectively, our data indicate that paclitaxel potentiated NLRP3 inflammasome activation by inducing α-tubulin acetylation and thereby conferred enhanced antibacterial innate responses, suggesting its potential application against pathogenic infections beyond its use as a chemotherapeutic agent.

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Extracellular vesicles secreted by Giardia duodenalis regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways.

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Extracellular vesicles secreted by Giardia duodenalis regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways.

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