Giardia duodenalis extracellular vesicles regulate the proinflammatory immune response in mouse macrophages in vitro via the MAPK, AKT and NF-κB pathways

Abstract

Background Giardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host’s innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored. Methods In this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs. Results The results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1β, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects. Conclusions The results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis–host interactions. Graphical abstract

Giardial lipid rafts share virulence factors with secreted vesicles and participate in parasitic infection in mice

“…It has been reported that giardial multicellular vesicles (MVs) are absorbed by dendritic cells, resulting in increased allostimulation ( Evans-Osses et al., 2017 ). In other reports, EVs released by Giardia have been shown to affect the TLR2 and NLRP3 inflammasome pathways and proinflammatory immune responses in mouse macrophages via the MAPK, AKT, and NF-κB pathways ( Zhao et al., 2021a ; Zhao et al., 2021b ).…”

“…In recent years, efforts have been made to understand the roles of giardial extracellular vesicles (GEVs) in modulating the host immune system ( Zhao et al., 2021a ; Zhao et al., 2021b ). There are three types of extracellular vesicles (EVs), each emanating from different cellular sites including the plasma membranes.…”

“…(2021a) reported that EVs released by trophozoites (giardial EVs or gEVs) are captured by macrophages, induce immune responses, and stimulate the production of proinflammatory cytokines along with Toll-like receptor 2 (TLR2) and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-signaling pathways. The authors have also demonstrated that the signaling cascade of p38 MAPK, p44/42 MAPK (Erk1/2), AKT, and NF-κB are affected in macrophages by gEVs ( Zhao et al., 2021b ). These studies have demonstrated the pivotal roles of gEVs that damage host cells during host -Giardia infections.…”

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

“…It was reported that Giardia infection can enhance the production of proinflammatory cytokines, such as TNF-α, IFN-γ, IL-6, and IL-12 p40 in PMs, through the TLR2-mediated activation of MAPK signaling [ 13 ]. 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 ].…”

The state of the art of extracellular vesicle research in protozoan infection

“…In addition, in the study, the roles played by the MAPK and AKT signaling pathways in EV-induced proinflammatory cytokine production were examined. After inoculating EVs into mouse macrophages, protein phosphorylation in the MAPK and AKT signaling pathways was detected at different times (Zhao et al, 2021b). The results showed that the level of p38 phosphorylation peaked 2 h after EV inoculation and began to decrease 4 h after inoculation.…”