Research On The Mechanisms Of Giardia Duodenalis-Induced Programmed Cell Death In Macrophages And Intestinal Epethelial Cells

Giardia duodenalis is a zoonotic protozoan with a worldwide distribution.Giardia trophozoites can adhere to intestinal epithelial cells(IECs)and its infection lead to occurrence of diarrhea.Although Giardia was reported to induce immune response of macrophages and apoptosis of IECs,the detailed underlying molecular mechanism remains largely unclear.The present study aims to explore the mechanisms of immune response of macrophages and apoptosis of IECs through establishing models of interaction between Giardia and macrophages or IECs,which provides new insights into the fundamental understanding of giardiasis pathogenesis and macrophage-mediated host defense mechanisms against Giardia infection.During interaction between Giardia and macrophages(J774A.1 cell line and peritoneal macrophages(PMs)of C57BL/6 mice),lactate dehydrogenase(LDH)release in the culture supernatant was measured.The results showed Giardia could induce LDH release from macrophages,which reflected the changes in the cell membrane permeability.In CCK-8 assays and flow cytometry analysis,Giardia stimulation caused reduction in cell viability and increase of Annexin V positive cells.Transmission electron microscope(TEM)was used to examine the ultrastructure of Giardia-infected macrophages,the results showed that mitochondrial swelling,disruption of cristae structure and damage of cell membrane were present in cells after treatment.Reactive oxygen species(ROS)over-production and mitochondrial membrane potential loss in Giardia-treated cells were found by use of DCFH-DA and JC-1 staining.The release of LDH and membrane damage are the key markers of macrophage pyroptosis,thus whether Giardia could induced macrophage pyroptosis was further explored.Western blot analysis demonstrated that Giardia infection could activate NLRP3 infammasome,which promote caspase-1(CASP1)cleavage.Activated CASP1 could cleave gasdermin-D(GSDMD)and cause release of its N-terminal fragment(GSDMD-N),which would promote the pore formation in the cell membrane to induce pyroptosis.Moreover,activated CASP1 also could induce the cleavage of pro-IL-1? and pro-IL-18 in macrophages to its mature form,which was further secreted into the extracellular environment through the gasdermin pore.In the following experiments,we found Giardia-induced macrophage pyroptosis and pro-inflammatory cytokine release were all inhibited by NLRP3inhibitor(MCC950),suggesting that NLRP3 activation play a crucial role in regulating Giardia-induced macrophage pyroptosis.Based on these results,we further explored the mechanisms of NLRP3 activation.The K63 ubiquitination level of NLRP3 in macrophages was detected through co-immunoprecipitation experiment.The results showed that Giardia infection could promote K63 de-ubquitination of NLRP3 in macrophages.Subsequently expression levels of six candidate deubiquitinating enzymes were exaimined,the results showed that only A20 was significantly increased upon stimulation of Giardia.Si RNA transfection experiments was conducted to knockdown the expression of A20,the results showed that knockdown of A20 could significantly recover K63 ubiquitination of NLRP3 and inhibit NLRP3 activation in Giardia-treated cells,indicating that A20 up-regulation induced by Giardia could remove K63-linked ubiquitin chains from NLRP3,which directly cause NLRP3 activation and then recruit ASC and CASP1 to form inflammasome,thus leading to pyroptosis.As an extracellular parasite,Giardia might activate inflammatory response of macrophages through activating pattern recognition receptor(TLRs)on macrophage surface.The expressions of TLRs(TLR2,TLR4 and TLR5)on the surface of infected macrophages were detected by immunofluorescence analysis.The results showed TLR2 and TLR4 were significantly elevated in Giardia-treated cells.We then used TLR2 inhibitor TLR2-IN-C29(C29)and TLR4 inhibitor TAK-242(TAK)to pretreat PMs to inhibit activation of TLR2 or TLR4,the results showed that Giardia-induced NLRP3 activation,GSDMD-driven macrophage pyroptosis and release of inflammatory cytokines were all markedly suppressed by TAK,while this was not the case for C29.Next,the relationship between TLR4 and NLRP3 de-ubiquitination was further explored,we found that TAK could inhibit production of ROS induced by Giardia,and TAK and NAC could also inhibit A20 up-regulation,thus impeding K63 de-ubiquitination of NLRP3.These results together demonstrated that TLR4 activation play a key role in regulating Giardia-induced macrophage pyroptosis.To further explore the effects of Giardia secreted proteins on TLR4 activation,five recombinant Giardia proteins(pyridoxamine5'-phosphate oxidase(PNPO,GL50803?5810),peptidyl-prolyl cis-trans isomerase B(PPIB,GL50803?17163)and three tenascins(GL50803?114815,GL50803?10330 and GL50803?16833)were expressed using the constructed prokaryotic protein expression system.The purified protein was used to stimulate macrophages,aiming to detect the effects of stimulation on macrophage pyroptosis.The results of immunofluorescence experiments revealed that the recombinant protein PPIB stimulation could up-regulate TLR4 expression.Similar to Giardia treatment,recombinant PPIB stimulation also could induce TLR4-dependent pyroptosis.The present study not only elucidated the molecular mechanism of macrophage pyroptosis induced by Giardia,but also revealed that Giardia-secreted PPIB might serve as the potential PAMP in activating pattern recognition receptor TLR4 on macrophage surface.During interaction between Giardia and IECs(Caco-2 cell line),cell viability was significantly reduced upon Giardia stimulation.Giardia-induced IEC apoptosis was initially determined through AO/EB staining.Upon trophozoite stimulation,typical cell apoptotic characteristics were also observed,including nucleolus margination and nuclear membrane shrinkage through TEM detection,which serve as typical cell apoptotic characteristics.The differential expression genes(DEGs)between infected and non-infected cells were determined through transcriptomic analysis,some of which(e.g.,TNFR1,RIP1,and CASP8)were mapped to the extrinsic apoptosis pathway.Based on this result,it was preliminarily speculated that Giardia could induce apoptosis in IECs through extrinsic pathways.In the study of apoptosis mechanism,CASP8/3 signaling pathways screened by transcriptomic analysis were verified.Western blot analysis showed Giardia could induce cleavage of CASP8/3,and the effector caspase(cleaved CASP3)increased time-dependently in immunofluorescence analysis.Moreover,pretreatment of Caco-2 cell with CASP8/3 inhibitor Q-VD-OPH could significantly impede Giardia-induced CASP8/3 activation and IEC apoptosis.In the following experiments,we further detected expression of the membrane death receptor TNFR1 according to RNA-sequencing data analysis.Total TNFR1 protein expression was elevated after simulation of Giardia in western blotting analysis.Subsequently,TNFR1 on the cell surface was fluorescently labeled with a specific antibody.Flow cytometry analysis showed that expression of TNFR1 on the cell membrane was increased gradually after trophozoite treatment.TNFR1 m RNA expression showed the same trend with the protein expression.It was reported that TNFR1-mediated extrinsic apoptosis involved K63 deubiquitination status of RIP1.The present study demonstrated K63 ubiquitination of RIP1 in Caco-2 cells was reduced time-dependently after Giardia treatment.Several candidate deubiquitinases and ubiquitin ligases were screened,among which A20 and CYLD were significantly up-regulated in Giardia-infected cells.A20 or CYLD silencing increased the levels of K63 ubiquitination of RIP1 in trophozoite-treated cells.Moreover,we also found that TNFR1 knockdown not only inhibited A20 and CYLD up-regulation and RIP1 deubiquitination induced by Giardia,but also suppressed CASP8/3-mediated apoptosis.Finally,the activation mechanism of TNFR1 induced by Giardia was further explored.The results showed that Giardia could induce production and secretion of TNF-? through western blotting analysis of cell lysates and the culture supernatant.After trophozoite treatment,more TNF-? were determined on Caco-2 cell surface in immunofluorescence.We then detected the expression and distribution of TLR2,TLR4 and TLR5 on cell surface through western blot and immunofluorescence analysis,the results showed Giardia stimulation could significantly up-regulated expression of TLR2 and TLR4.When the Caco-2 cells were pretreated with C29 or TAK,we found that only TAK could inhibit Giardia-induced TNF-?secretion and TNFR1 activation.These results demonstrated that Giardia stimulation preferentially activate TLR4-dependent inflammatory response that mainly promoted TNF-? secretion,which then triggered TNFR1-mediated apoptosis.The recombinant Giardia protein PPIB has been determined to exert a crucial function on triggering TLR4-mediated macrophage pyrptosis,we hazard a guess that the recombinant PPIB may function as a trigger for TLR4-dependent inflammatory response.As expected,the results showed that stimulation of IECs with recombinant PPIB could significantly activate TLR4,promote TNF-? secretion and induce apoptosis.Moreover,pretreatment with TAK could suppress the above effects,suggesting Giardia-secreted PPIB might play a role in activating TLR4-mediated apoptosis.This study reveals the molecular mechanism of Giardia-induced IEC apoptosis through four perspectives including virulence proteins,recognition of receptors,inflammatory response and indirect induction of apoptosis.Overall,the present study first proposed that Giardia and its secreted protein PPIB could activate TLR4-dependent macrophage pyroptosis and IEC apoptosis.In one hand,PPIB could trigger macrophage surface receptor TLR4-regulated NLRP3 deubiquitination and then activate NLRP3 inflammasome,leading to CASP1/GSDMD-mediated macrophage pyroptosis;in the other hand,PPIB also could trigger IEC surface receptor TLR4-mediated inflammatory response that mainly promoted TNF-? secretion.Extracellular TNF-? activated surface receptor TNFR1 and then regulated RIP1 deubiquitination,which caused activation of CASP8/3 and occurrence of apoptosis.The present study not only provided a potent basis for exploring giardiasis pathogenesis and host defense mechanism during Giardia infection,but also provided some new insights in the prevention and treatment of giardiasis.