TIPE2 Negatively Regulates NLRP3 Inflammasome Activation Induced By Toxoplasma Gondii Exosomes In Macrophages

Research backgrounds and purposesToxoplasma gondii is an obligate intracellular parasite that results in zoonotic toxoplasmosis,which distributes worldwide and infects almost all warm-blooded vertebrates,including humans,posing a huge threat to human health,animal husbandry and food safety.At present,there are no effective measures to control toxoplasmosis,since the infection and immune mechanism of T.gondii has not been fully elucidated.NLRP3 inflammasome is a multiprotein complex in the cytoplasm,and the activated NLRP3 inflammasome is closely related to many diseases,especially playing an important role in the process of inflammatory responses.Previous studies have shown that NLRP3 inflammasome pathway existed in both neutrophils and macrophages,and the activated NLRP3 inflammasome participated in the occurrence and development of T.gondii infection.However,whether exosomes derived from T.gondii that affect NLRP3 inflammasome has not been clearly reported.Numerous studies have proved that exosomes carried bioactive substances such as proteins,lipids,mRNA and miRNA,performing various biological functions through mediating intercellular communications and immune responses.Interestingly,T.gondii exosomes could promote the recruitment and activation of inflammatory cells such as neutrophils,monocytes and macrophages in the inflammatory foci,and then secreting cytokines and chemokines to cause inflammatory responses.In fact,however,the mechanism of inflammatory responses triggered by T.gondii exosomes remains unclear.TIPE2 is a natural anti-inflammatory protein that expresses in lymph nodes,spleen,thymus,small intestinal mucosa and other organs as well as immune cells such as macrophages and lymphocytes.TIPE2 protein could induce arginine metabolic conversion and reduce the synthesis of NO synthase to suppress NO production through inhibiting JNK,P38 MAPK and NF-κB signals,causing inflammation inhibition.In addition,TIPE2 promoted the differentiation of M2-type macrophages by activating the PI3K-AKT signaling pathway,and involved in the process of inflammation inhibition,parasite control and tissue repair.Nevertheless,it is unclear whether TIPE2 regulates NLRP3 inflammasome activation induced by T.gondii exosomes.Therefore,we focused on the influence of T.gondii exosomes on NLRP3 inflammasome in macrophages and explored the mediating effect of T.gondii exosomes in NLRP3 inflammasome pathway.Additionally,we illuminated the regulation mechanism of TIPE2 on NLRP3 inflammasome activation induced by T.gondii exosomes,aiming to provide theoretical basis for controlling T.gondii infection and screening drug targets.Research methods and resultsPart Ⅰ:T.gondii exosomes activated NLRP3 inflammasomeThe expression of NLRP3 inflammasome related molecules mRNA and protein was detected by qPCR and WB in RAW264.7 and BMDM cells that stimulated by T.gondii exosomes.The activity of caspase-1,the production of ROS and the release of LDH in macrophages stimulated by T.gondii exosomes were detected by ELISA and flow cytometry.The secretion of cytokines IL-1β and IL-18 in macrophages stimulated by T.gondii exosomes was detected by ELISA.These results proved that the expression of IL-6,TNF-α,NF-κB p-p65,NLRP3,pro-IL-1β,cleaved-caspase-1,ASC,secreted-IL-1β p17,secreted-IL-18,cleaved-GSDMD,and the activity of caspase-1,the production of ROS,the release of LDH,the secretion of cytokines IL-1β and IL-18 were significantly increased in macrophages stimulated by T.gondii exosomes for 6h.Part Ⅱ:TIPE2 negatively regulated NLRP3 inflammasome activation induced by T.gon.dii exosomes1.WB and qPCR were used to detect the expression of TIPE2 mRNA and protein in macrophages that infected with T.gondii and stimulated by T.gondii exosomes,respectively.The results showed that T.gondii and T.gondii exosomes both decreased the expression of TIPE2 in macrophages.2.RAW264.7 cells transfected with TIPE2 overexpressed lentivirus,BMDM cells transfected with TIPE2 overexpressed plasmids,BMDM cells transfected with TIPE2 siRNA and TIPE2-/-BMDM cells were used as experimental subjects.WB and qPCR were used to detect the expression of NLRP3 inflammasome related molecules mRNA and protein in macrophages stimulated by T.gondii exosomes.The secretion of cytokines IL-1β and IL-18 in macrophages stimulated by T.gondii exosomes was detected by ELISA.These data demonstrated that TIPE2 negatively regulated the expression of NLRP3,pro-IL-1β,cleaved-caspase-1,secreted-IL-1β p17,secreted-IL-18,cleaved-GSDMD,the secretion of cytokines IL-1β and IL-18 in macrophages stimulated by T.gondii exosomes for 6h.3.Co-IP was used to verify the interaction with TIPE2 and NLRP3 inflammasome.The results showed that TIPE2 interacted with the complex protein of NLRP3 inflammasome.Research conclusions and significance1.In this research,we investigated the effect of T.gondii exosomes on NLRP3 inflammasome pathway and found that T.gondii exosomes could activate NLRP3 inflammasome in macrophages,thereby promoting the communication between T.gondii and host cells,and providing a new molecular mechanism for the study of NLRP3 inflammasome activation by T.gondii.2.In this research,the negative immunoregulatory molecule TIPE2 was found to be abnormally expressed that stimulated by T.gondii exosomes.Remarkably,TIPE2 could play negative regulatory role in NLRP3 inflammasome activation induced by T.gondii exosomes,maintaining the balance to pro-inflammatory and anti-inflammatory.Collectively,this study illustrated the relationship between T.gondii exosomes and NLRP3 inflammasome for the first time,preliminarily verifying the regulatory mechanism of TIPE2 and NLRP3 inflammasome,which provided the theoretical foundation and experimental evidences for elucidating the mechanism of T gondii infection and immunoregulation.