Contribution Of P62-Mediated Nrf2 Pathway To The Antioxidant Reaction In Toxoplasma Gondii Infection

Toxoplasma gondii(T.gondii)is an obligate intracellular parasite of the Apicomplexa.T.gondii can infect a wide range of warm-blooded hosts in the worldwide including humans and cause the disease toxoplasmosis.This disease is considered to wide-sprided in animals in China,which can cause high fever,neurological symptoms,dyspnea in the infected animals.Preganent abortion,abnormal fetus,and stillbirth also occur in acute infection.Toxoplasmosis is also an important zoonotic disease and highly prevalent in the world.It is estimated that over one-third of the human population was infected with T.gondii in the world and sometimes cause serious problem for public health.Host cells produce excessive amounts of reactive oxygen species(ROS)after sensing T.gondii-derived proteins or upon stimulation by cytokines,which are important processes for innate immune responses and cellular resistance to parasites.However,excessive ROS is toxic to cell survival.But,the mechanism regulates the ROS level of host cells infected with T.gondiii is unclear.Nuclear factor erythroid 2-related factor 2(Nrf2)is the master transcription factor for antioxidant genes.Nrf2-regulated expression of cytoprotective gene play an important role in the defense system against antioxidant stress to maintain the balance of intracellular redox.P62-Keap1-Nrf2 signal pathway can promote transcription and expression of many cell-protective antioxidant genes.In this study,RAW264.7 cells were used to analyse the signaling pathways involved in the antioxidant process of the cells infected with T.gondii.The ROS level induced by T.gondii infection was detected by different MOI and time dose.We further investiged the role of Nrf2 and P62 in the regulation of antioxidant pathways.The main results are as follows:(1)T.gondii infection can induce upregulation of ROS level in RAW264.7 cells in a dose-dependent manner.(2)T.gondii infection activates Nrf2 antioxidant signaling pathway in RAW264.7 cells.The transcription of Nrf2-targetted genes were detected by using real-time PCR,the results showed that T.gondii infection could up-regulate the transcription of downstream genes,HO-1(P<0.05),GSTA-1(P<0.01)and NQO-1(P<0.05).(3)Compared with the expression of Nrf2-targetted genes in wide type cells,GSTA-1(P<0.001)and NQO-1(P<0.001)were significantly down-regulated,and ROS level was up-regulated in Nrf2 knockout cell lines infected with T.gondii.Knockout of Nrf2 significantly inhibit the growth and replication of T.gondii.(4)T.gondii infection can result in significant down-regulation of P62 expression in NIH/3T3(P<0.001)and HEK293T(P<0.001)and up-regulation of P62 expression in RAW264.7(P<0.001),THP-1(P<0.05).We also found similar results when primary mouse peritoneal macrophages were used.Deletion of Nrf2 did not affect P62 expression in the cells infected with T.gondii.(5)Successful construction of Nrf2 knockout cell line.T.gondii infection activates P62-regulated Nrf2 antioxidant signaling pathway in RAW264.7 cells.Compared with the expression of Nrf2-targetted genes in the wide-type cells,HO-1(P<0.05),GSTA-1(P<0.001)and NQO-1(P<0.001)were significantly down-regulated,in p62 knockout cell lines infected with T.gondii.(6)T.gondii infection can increase autophagy level in RAW264.7.The results of Western Blot analysis showed that the expression of autophagic marker protein,LC3-II,increased significantly in cells infected with T.gondii.(7)Successful construction of p62 knockout cell line.Raw cells infected with T.gondii can activate Nrf2 antioxidant pathway by p62-mediated selective autophagy to degrade Keap1 protein.The results of western blot analysis showed that the degradation of Keap1 protein was inhibited in P62 knockout cells infected with T.gondii.In conclusion,we found that the Nrf2 signaling pathway is important for maintaining redox homeostasis in activated macrophages,as well as for survival of T.gondii in these cells.Importantly,expression of p62 by RAW cells contribute to the antioxidant reaction linked to Nrf2-Keap1 signaling during T.gondii infection.