Molecular Mechanism Of Giardia Duodenalis Extracellular Vesicles Activating NLRP3 Inflammasome In Murine Macrophage

Giardia duodenalis is a kind of zoonotic parasitic protozoa with serious harm,and giardiasis is an important zoonosis caused by G.duodenalis parasitized in the intestinal tract of human and mammals.G.duodenalis cysts enter into the intestinal tract of the host through the mouth and then encapsulate to form trophozoites,which is an important pathogenic cause of inflammatory diarrhea,resulting in diarrhea,malnutrition,infant growth retardation and so on.G.duodenalis is also a fatal factor in people with low immunity,such as AIDS patients,organ transplant patients and cancer patients.Prevention and control of giardiasis mainly rely on metronidazole and other chemical drugs with the risk of teratogenic and carcinogenic.However,there is no effective vaccines.In recent years,there is an urgent need to find new targets for the control of giardiasis as the problem of giardiasis resistance intensifies.The natural immune system is the body's first defense line against pathogen infection.Pattern recognition receptors(PRRs)are widely distributed in various immune cells and activate the natural immune system by recognizing multiple types of pathogen-related molecular patterns(PAMPs)and damage-related molecular patterns(DAMPs).Membrane-bound Toll-like receptors(TLRs)TLR2 is involved in the recognition of G.duodenalis trophozoites and the regulation of host inflammatory responses,but the key molecules involved in this process remain unknown.Intracellular NLRs receptor is mainly expressed in immune cells and is characterized by transduction of NF-?B activation or secretion of pro-inflammatory factors IL-1?and IL-18.As an extracellular pathogen of G.duodenalis,there are few studies on the intracellular NLRs regulated immune responses.Whether G.duodenalis could deliver bioactive substances into host cells to activate NLRs? Extracellular vesicles are rich in peptides,proteins,lipids and nucleic acids,and are important biomolecules that mediate intercellular communication.Parasite extracellular vesicles can mediate information transfer between parasites and between parasites and hosts.Whether G.duodenalis could secrete extracellular vesicles(GEVs)and whether GEVs participate in the information transmission between hosts and parasites and regulate host immune response ?In this study,the GEVs were firstly isolated and identified,studied from the perspective of inducing inflammatory response of murine macrophages,and analyzed the key inflammasome and its activation mechanism involved in this process.The protein components in GEVs were analyzed to determine the single factor causing NLRP3 inflammasome activation.Finally,through animal experiments,the influence of key molecules of GEVs triggered NLRP3 inflammaome activation on parasite infection was determined,and its role in parasite pathogenesis was further revealed.1)Establishment of macrophage model stimulated by GEVs.The G.duodenalis trophozoite secreted GEVs were enriched through ultracentrifugation,and typical cup-type morphology and particle size of GEVs were observed by transmission electron microscopy(TEM)and nano-particle tracking analysis(NTA).Laser confocal showed that GEVs could enter into the host cells rapidly in a time-dependent manner,and this process depended on the active phagocytosis of macrophages.Results of real-time fluorescence quantitative PCR(q PCR)and enzyme-linked immunosorbent assay(ELISA)showed that GEVs could activate the inflammatory response of mouse peritoneal macrophages in a dose-dependent manner,accompanied by increased transcription levels of multiple inflammatory cytokines and chemokines and secretion levels of multiple inflammatory cytokines.These results suggest that G.duodenalis can secrete extracellular vesicles and enter host cells to activate the inflammatory response of host cells.2)Identification of molecular types of murine macrophage cells PRRs activated by GEVs.GEVs stimulation could increase the transcription levels of NLRs and TLRs,especially for NLRP3 and TLR2.GEVs stimulation can induce point-like activation of NLRP3 receptor around the nucleus and abundant expression of TLR2 receptor on the cell membrane.Blocking NLRP3 receptor through potassium outflow/lysosome injury pathway and TLR2 receptor significantly down-regulated the transcription levels of various inflammatory cytokines and chemokines,secretion levels of various inflammatory cytokines and IL-1? p17 protein expression levels.These results suggest that GEVs regulate the host cell inflammatory response by activating the host TLR2 and NLRP3 signaling pathways.3)Study on the activation of the second signal of murine macrophage cells inflammasome by GEVs.GEVs stimulation mouse peritoneal macrophages led to an increase in the transcription levels of NLRP3 inflammasome key proteins.The activation of caspase-1 p20 and secretion of IL-1? p17 increased from 6 h to 12 h after infection and then decreased.The activation of caspase-1 p20 and secretion of IL-1? p17 increased in a dose-dependent manner in the range of 12.5 ?g to 25 ?g.IL-1? gene transcription,protein secretion and expression levels were down-regulated by blocking potassium outflow pathway,lysosomal injury pathway,caspase-1activation pathway,and pan-caspase-1 activation pathway,which were more significant.Blockade of IL-1? significantly down-regulated the transcription and secretion levels of other inflammatory cytokines of IL-6 and TNF-?.These results suggest that G.duodenalis can activate the host NLRP3 inflammasome through lysosomal damage pathway,regulate IL-1? p17 secretion and mediate the host inflammatory response through extracellular vesicles.4)Study on the activation of the first signal of murine macrophage cells inflammasome by GEVs.GEVs prestimulation of mouse macrophages cells and THP-1 cells could promote G.duodenalis-mediated transcription and secretion of pro-IL-1? and other inflammatory factors IL-6/TNF-?.The phosphorylation of p38 peaked at 2 h and the phosphorylation of ERK and AKT peaked at 4 h in GEVs stimulated host cells within 0 h to 4 h.Blocking the p38 and ERK pathways could significantly down-regulate the transcription and secretion of pro-IL-1? and other inflammatory factors IL-6/TNF-?.Blocking AKT pathway could significantly up-regulate the transcription and secretion of these inflammatory factors.GEVs induced NF-?B p65 to enter into the nucleus in host cells,and the phosphorylation of p-p65 and p-IKK?? reached the peak at 1 h and the degradation of T-I?B? reached the lowest level within 0 h-4 h.Inhibition of NF-?B pathway by blocking I?B?phosphorylation significantly down-regulated the transcription and secretion of pro-IL-1?,the first signal of NLRP3 inflammasome,and other inflammatory factors IL-6/TNF-?.These results suggest that GEVs can promote G.duodenalis-mediated NLRP3 inflammasome first signaling activation,which depends on the positive regulation of p38,ERK,NF-?B and negative regulation of AKT,and that the p38,ERK,AKT,NF-?B signaling pathways are involved in GEVs-mediated host inflammatory response.5)Key molecules screening and functional study of murine inflammasome activated by GEVs.The proteomic analysis showed that proteins in GEVs ranged from 18.4 k Da to 116 k Da and were concentrated at 66 k Da.After enzymatic hydrolysis,a total of 153 proteins and 399 peptides were identified,and 131 known proteins were matched with G.duodenalis database,including key proteins involved in EV generation and pathogenesis of G.duodenalis.These proteins were involved in129 signaling pathways,including: ribosome,glycolysis,NOD-like receptor,MAPK,PI3K-AKT,TOLL pathways.It has been preliminarily screened that multiple giardins can induce the activation of caspase-1 p20,alpha-2 giardin and alpha-7.3 giardin can up-regulate key protein expression of NLRP3 inflammasome,activation of caspase-1,and secretion of IL-1?,oligomerization of ASC,speck-like ASC protein and point-like aggregation of NLRP3 protein;Alpha-2 giardin and alpha-7.3 giardin can induce host IL-1? secretion through NLRP3 inflammasome.Blocking NLRP3 inflammasome significantly reduced the mice body weight at the early stage of infection,significantly increased the number of G.duodenalis trophozoites,damaged intestinal villus structure to necrosis,and crypt atrophy.G.duodenalis infection in mice pretreated with alpha-2 and alpha-7.3 in GEVs significantly alleviated weight loss,G.duodenalis trophozoites number,intestinal villi damage and crypt atrophy to different degree.The protective effect of alpha-7.3 giardin on mice was more significant than that of alpha-2 giardin.These results suggest that a variety of proteins in the GEVs can be involved in EV generation and giardiasis,and activate cell death,MAPK,PI3K/AKT,NOD-like receptor and other signaling pathways.Alpha-2giardin and alpha-7.3 giardin in GEVs can activate hosts' NLRP3 inflammasome in vitro and in vivo.NLRP3 inflammasome recognizes alpha-2 giardin and alpha-7.3giardin against G.duodenalis infection.In conclusion,this study elucidates the molecular recognition mechanism by which G.duodenalis stimulates the activation of NLRP3 inflammasome in host cells and regulates the host inflammatory response by secreting GEVs.NLRP3 inflammasome recognizes alpha-2 giardin and alpha-7.3 giardin against G.duodenalis infection,providing potential new targets for treatment and prevention of giardiasis.