The Role Of TLR9 In The Inflammatory Response Induced By Giardia Duodenalis In Mouse Macrophages

Giardia duodenalis,also known as Giardia lamblia or Giardia intestinalis(simply named Giardia),is an important zoonotic protozoan parasite that predominantly parasitizes the duodenum and causes giardiasis.The clinical manifestations of giardiasis are mainly watery diarrhea,steatorrhea,abdominal pain,vomiting,malnutrition absorption and weight loss.G.duodenalis is also a lethal factor in people with immunocompromised conditions like AIDS patients,organ transplant patients,and cancer patients.Currently,metronidazole is the most used drug for clinical treatment of giardiasis,but there exist problems like side effects and drug resistance,and there is no effective vaccine available for giardiasis prevention.The reason for this is the lack of clarity on the pathogenesis of G.duodenalis and searching for key molecular targets is particularly crucial.Toll-like receptors(TLRs),as one of the most important pattern recognition receptors(PRRs)in the mammalian innate immune system,play a critical role in resistance to parasitic infections and pathogenesis.TLRs can recognize various parasites,such as Leishmania,Plasmodium,Trichomonas vaginalis,and G.duodenalis,which were previously investigated.It has been demonstrated previously in laboratory that Giardia lamblia virus(GLV)-free Giardia can upregulate TLR2,TLR4 and TLR9 expression in mouse macrophages,while GLV-containing Giardia can trigger an increase in TLR3 expression in addition to enhancing TLR2,TLR4 and TLR9 expression.Among them,TLR2,TLR3 and TLR4 have been reported and relevant specific molecular mechanisms have been revealed.However,the role of TLR9 in G.duodenalis infection have not been reported so far.This study explored the effect and molecular recognition mechanism of TLR9 in the infection process of GLV-free and GLV-containing Giardia,in order to provide theoretical basis and novel targets for the prevention and treatment of giardiasis.The main research contents were as follows:1.The effect of host TLR9 in G.duodenalis infection1.1 GLV-free and GLV-containing Giardia stimulation triggered TLR9 activation in mouse peritoneal macrophages Mouse peritoneal macrophages were separated and G.duodenalis trophozoites were cultivated in vitro to establish the G.duodenalisstimulated cell model,the expression level of TLR9 m RNA was detected by reverse transcriptase-quantitative PCR(RT-q PCR).The results demonstrated that G.duodenalis stimulation caused a significant upregulation of TLR9 m RNA expression in mouse peritoneal macrophages,and there was no statistically significant difference in TLR9 m RNA expression levels between GLV-free and GLV-containing Giardia stimulation groups.1.2 GLV-free and GLV-containing Giardia mediated the production of proinflammatory cytokines via TLR9 in mouse peritoneal macrophages The expression of TLR9 in mouse peritoneal macrophages was interfered by si RNA transfection,then macrophages were stimulated with GLV-free and GLV-containing Giardia,and the protein expression of TLR9 and production of pro-inflammatory cytokines were measured by Western blot and ELISA,respectively.The results showed that TLR9 si RNA treatment significantly diminished the expression level of TLR9 induced by G.duodenalis in mouse peritoneal macrophages,while the secretion levels of proinflammatory cytokines IL-6,TNF-α and IL-12 p40 induced by G.duodenalis were also significantly decreased,and GLV-containing Giardia elicited significantly greater production of these proinflammatory cytokines than GLV-free Giardia.2.The role of TLR9 and its related inflammatory signaling pathways in G.duodenalis infection2.1 G.duodenalis triggered activation of p38/ERK MAPKs,AKT and NF-κB p65 signaling pathways G.duodenalis-stimulated cell model was established,the activation of macrophage signaling pathways and secretion of pro-inflammatory cytokines by G.duodenalis were measured by Western blot and ELISA,respectively.The results showed that G.duodenalis can cause the phosphorylation of p38,ERK,AKT,p65 and IκBα,and elicit a gradual increase in the levels of pro-inflammatory cytokines IL-6,TNF-α and IL-12 p40 in macrophages.2.2 GLV-free and GLV-containing Giardia mediated the activation of p38/ERK MAPKs,AKT signaling pathway via TLR9 The TLR9 si RNA-treated mouse macrophages were stimulated separately with GLV-free and GLV-containing Giardia,the activation of signaling pathways and secretion of pro-inflammatory cytokines after TLR9 si RNA treatment were detected by Western blot and ELISA,respectively,and the localization of p65 in macrophages was observed by immunofluorescence assay.It was found that TLR9 si RNA treatment significantly diminished the phosphorylation levels of p38/ERK MAPKs and AKT induced by GLV-free and GLV-containing Giardia,while no significant changes were found in the phosphorylation levels of p65 and IκBα.Translocation of p65 into the nucleus was observed in both WT and TLR9 si RNA-treated mouse peritoneal macrophages induced by GLV-free and GLVcontaining Giardia.2.3 GLV-free and GLV-containing Giardia regulated host cell inflammatory response via TLR9-mediated p38/ERK MAPKs and AKT pathways Mouse peritoneal macrophages were pre-treated separately with p38,ERK and AKT inhibitors(SB203580,PD98059 and MK-2206 2HCl)followed by stimulation of macrophages with GLV-free and GLV-containing Giardia,respectively,the phosphorylation of p38,ERK and AKT were detected by Western blot and secretion of pro-inflammatory cytokines were measured by ELISA.The results indicated that p38,ERK and AKT inhibitors significantly reduced the phosphorylation levels of p38,ERK and AKT induced by GLV-free and GLV-containing Giardia,respectively.Both p38 and ERK inhibitors significantly lowered the secretion levels of pro-inflammatory cytokines IL-6,TNF-α and IL-12 p40,whereas AKT inhibitors significantly enhanced the secretion levels of these cytokines.3.The role of TLR9 in mouse macrophage pyroptosis induced by G.duodenalis3.1 G.duodenalis activated mouse macrophage pyroptosis mediated by GSDMD Mouse peritoneal macrophages were pretreated by LPS,and the expression or activation levels of key proteins related to pyroptosis,IL-1β secretion and LDH release were measured by Western blot,ELISA and LDH assay,respectively.The results revealed that stimulation of mouse peritoneal macrophages with G.duodenalis caused an increase in NLRP3 and IL-1β expression,IL-1β secretion level and LDH release and N-terminal release of GSDMD protein.3.2 GLV-free and GLV-containing Giardia activated mouse macrophage pyroptosis in a TLR9-independent way Mouse macrophages treated with TLR9 si RNA were stimulated with GLV-free and GLV-containing Giardia,and the expression or activation levels of key proteins related to pyroptosis,IL-1β secretion and LDH release were detected by Western blot,ELISA and LDH assay,respectively.The results showed that the expression levels of NLRP3 and IL-1β,IL-1β secretion level,Nterminal release of GSDMD and LDH release induced by GLV-free and GLVcontaining Giardia in TLR9 si RNA-treated groups were similar to those in negative controls.In conclusion,both GLV-free and GLV-containing Giardia could activate TLR9 in mouse peritoneal macrophages,and mouse macrophages produced pro-inflammatory cytokines to kill G.duodenalis via the TLR9-p38/ERK signaling pathways,while G.duodenalis attenuated host immune responses via the TLR9-AKT signaling pathway.Additionally,both GLV-free and GLV-containing Giardia activated NF-κB p65 signaling pathway in a TLR9-independent manner,promoted NLRP3 expression and activation,with enhanced inflammatory response via GSDMD-mediated pyroptosis to control G.duodenalis infection.This study suggested that the TLR9-mediated innate immune pathways could be potential molecular targets for the prevention and treatment of giardiasis.