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.

Giardia duodenalis and Its Secreted PPIB Trigger Inflammasome Activation and Pyroptosis in Macrophages through TLR4-Induced ROS Signaling and A20-Mediated NLRP3 Deubiquitination

“…A recent study found that Giardia EVs trigger IL-1β, IL-6, and TNF-α secretion from mouse PMs via promoting the nuclear translocation of the NF-κB p65 subunit [ 53 ]. Another recent study identified TLR2 as a mediator for pyroptotic signaling in mouse PMs induced by Giardia EVs [ 25 ]. In contrast, our study revealed a significant increase of both TLR2 and TLR4 expression in Giardia -treated macrophages.…”

“…The VSPs of Giardia were reported to activate TLR2 and TLR4 in HEK293 cells [ 24 ]. The latest research has shown that Giardia extracellular vesicles (EVs) activate NLRP3 inflammasome signaling via TLR2 [ 25 ]. Growing evidence shows that activation of the NLRP3 inflammasome involves its deubiquitination or phosphorylation [ 26 , 27 ].…”

Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways

“…Located on the cell membrane and in intracellular endosomes, TLRs can monitor and recognize a range of PAMPs derived from bacteria, parasites, viruses, and fungi. Importantly, studies have revealed the functions and mechanisms associated with the innate immune responses mediated by TLR2 and TLR4 during Giardia infection (Lee et al, 2014;Li et al, 2017;Serradell et al, 2019;Zhao et al, 2021). Additionally, it has been shown that the Plasmodium falciparum-derived metabolite, hemozoin, can activate TLR9 in host macrophages and DCs, leading to the massive secretion of proinflammatory factors and chemokines (Kawai and Akira, 2009;Kumar et al, 2009).…”

“…Melting curves were analyzed following the PCR run to validate the homogeneity of PCR products. All the primers were synthesized by Sangon (Shanghai, China) and contained the following sequences: TLR9, 5 -CTGCCCAAACTCCACACTCT-3 forward primer and 5 -ACAAGTCCACAAAGCGAAGG-3 reverse primer (Zhao et al, 2021); and β-actin, 5 -TGCTGTCCCTGTATGCCTCT-3 forward primer and 5 -GGTCTTTACGGATGTCAACG-3 reverse primer (Li et al, 2017).…”

Vibrio alginolyticus Triggers Inflammatory Response in Mouse Peritoneal Macrophages via Activation of NLRP3 Inflammasome

“…The culture supernatants were collected and concentrated as previously described ( Zhao et al., 2021 ). Cell pellets were washed in ice-cold PBS and lysed with RIPA containing the protease inhibitor of phenylmethanesulfonyl fluoride (PMSF, 1 mM).…”