Abstract
BackgroundGiardia 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.MethodsIn 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.ResultsThe 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.ConclusionsThe 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
Highlights
Giardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals
The mRNA levels of proinflammatory cytokines were measured by quantitative PCR (qPCR) (Fig. 1a– c), and the protein secretion levels were measured with enzyme-linked immunosorbent assays (ELISAs) (Fig. 1d–f )
The results showed that both the G. intestinalis EVs (GEVs) and G. duodenalis treatment groups exhibited significantly upregulated proinflammatory cytokine transcription and expression compared to the phosphate-buffered saline (PBS)-negative analysis of the transcription levels of the proinflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α) in infected cells collected at 12 h. d–f Measurement of the secretion levels of proinflammatory cytokines in the supernatants collected 18 h after inoculation, by enzyme-linked immunosorbent assay (ELISA)
Summary
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. The ingestion of water or food contaminated with G. intestinalis cysts by susceptible mammalian hosts leads to giardiasis, especially in young children, with clinical presentations that range from no symptoms at all to severe diarrhea [2, 3]. Giardiasis has drawn public attention, and the World Health Organization lists it in the Neglected Diseases Initiative [5]. A better understanding of vital PAMPs from G. intestinalis and exploration of its roles in the immune mechanisms of host resistance to infection with G. intestinalis will provide new insights into effective strategies against giardiasis
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