Mechanism Of The Interaction Between Host Macrophages And Toxoplasma Gondii

Background:Toxoplasma gondii is an obligate intracellular parasite of public health significance that infects a wide range of warm-blooded animals and there are multiple strains that vary in their virulence in different hosts.It is estimated that a third of the worldwide population is under the threat of T.gondii and the serum positive rate in some area even can be as high as 80%.In addition,toxoplasmosis is also a clinically important opportunistic pathogen Chronic infection with latent bradyzoite cysts is asymptomatic in immunocompetent individuals;however,upon host immunosuppression such as cancer or AIDS patient,the parasite reconverts into its proliferative tachyzoite form,which causes severe tissue damage that can result in organ failure and death.Therefore,T.gondii is a widely distributed,serious zoonosis pathogen.Macrophages are a type of white blood cell or resident tissue cells,of the immune system,that engulfs and digests cellular debris,foreign substances,pathogens,tumor cells,and anything else that does not have the type of proteins specific to healthy body cells on its surface in a process called phagocytosis.Those large phagocytes were found in essentially all tissues in the body,where they patrol for potential pathogens by movement.Besides phagocytosis,they play a critical role in nonspecific defense(innate immunity)and also help initiate specific defense mechanisms(adaptive immunity)by recruiting other immune cells such as lymphocytes.For example,they are important as antigen presenters to T cells.After digesting pathogens,macrophage will present the antigen of the pathogen to the corresponding helper T cell(Th1).The presentation was done by integrating it into the cell membrane and displaying it attached to an MHC class Ⅱ molecule(MHCⅡ),indicating to other white blood cells that the macrophage is not a pathogen,despite having antigens on its surface.Beyond increasing inflammation and activating the immune system,macrophage also plays an important role in anti-inflammation and can decrease immune reactions through the release of anti-inflamroation cytokines.Macrophage that encourages inflammation was called M1 macrophages,whereas that decrease inflammation and encourage tissue repair was called M2 macrophage.Their metabolism relected the difference;Ml macrophage had the unique ability to metabolize arginine to the "killer"molecule nitric oxide,whereas M2 macrophage could metabolize arginine to the"repair" molecule ornithine.However,this dichotomy has been recently questioned as further complexity has been discovered.Invasion of host cells by T.gondii is associated with three waves of proteins secreted sequentially from the rhoptries,micronemes,and dense granules into the host cell.These proteins have an important role in allowing T.gondii to establish the parasitophorous vacuole(PV)and replicate in the host cell and several of these proteins appear to alter host cell function and are important for immune evasion.Similarly,the cytokine signaling pathways initiated by Janus Associated kinases(JAK)and propagated by the Signal Transducer and Activator of Transcription(STAT)have been targeted by T.gondii,and other pathogens.Thus,the ability of IFNy to activate STAT1 and promote a population of macrophages termed M1,is required for the control of T.gondii.In macrophages infected with T.gondii the parasite protein TgIST present in the secretory granules traffics to the host cell nucleus where it recruits a repressive complex that blocks STAT1-mediated transcription.Interestingly,the ROP16 protein present in type Ⅰ and Ⅲ strains of T.gondii is required for the ability of T.gondii to activate these transcription factors and promotes an M2-like population of macrophage.Defining the impact of these specific events on the outcome of infection in vivo has not been straightforward.Parasites that express Cre recombinase in their rhoptries(Toxoplasma-Cre parasites)combined with Ai6 mice which express a sensitive Cre-dependent reporter(ZsGreenl fluorescent protein)have revealed that T.gondii can inject rhoptry proteins into cells that are not infected.The majority of the studies that have focused on how T.gondii interacts with macrophages have concentrated on infected cells but there is now the recognition that T.gondii can directly influence "bystander" populations.These transgenic parasites have provided unexpected insights into how T.gondii interacts with different host cell populations and revealed that injection alone was sufficient to activate STAT6.Furthermore,while direct interaction with T.gondii has been considered a key event required for the production of IL-12,these Cre parasites revealed that the major cellular source of IL-12 were dendritic cells that had neither phagocytosed T.gondii or been infected by this organism.Thus,the development of these Cre-expressing parasites provide an opportunity to dissect the impact of injection alone on host cell functions and how this compares to cells that are productively infected with T.gondii.Aims:1.Dissecting the impact of classical activation on Type Ⅲ(CEP)toxoplasma gondii.2.Constructing the in vitro and in vivo host-pathogen interaction model,studying the biology and immulogy meaning on "Kiss and Run" model.3.Defining the character of CEP secreted ROP16 induced macrophage activation and the outcome of infection with ROP16 absence.4.Revealing the impact of ROP16 on T cell activation and response.Methods:1.Infecting Barf3-/-,IL-12-/-,LysmCreSTAT1 mice and anti-IFNγ antibody treated WT mice.Comparing with WT mice in the live death curve,parasite burden and the marker of typical macrophage by flow cytometry.2.Constructing the Cre-ZsGreen reporter model in vitro.Using the Type Ⅱ(PRU-Cre-tdTomato)and the Type Ⅲ(CEP-Cre-tdTomato)together with bone marrow-derived macrophage(BMDM)from Ai6 mouse and Ai6xSTAT6-/-mouse.3.FACS sorting the bystand cells,infected cells and injected cells in each cell type.Analyzing the microarray data from each group,identify injection-specific and infection-specific genes and find the cluster of those genes.Validation those typical M2 macrophage activation genes and phosphrylation of STAT6 in each population.4.LP.infecting Ai6 reporter mouse with CEP-Cre-tdTomato,build in vivo model for studying host-pathogen interaction.FACS sorts byastand,infected and injected large peritoneal macrophages.Using low cell number RNAseq method setting up the libraries,R program was used for data analyzing.5.Comparing the exiting altermative activating(M2)macrophage data set,define Toxoplasma specific induced M2 macrophage.And validate with FACS.6.Using DAVID GO term and GSEA analysis,find biology and immunology characteristic in each population.Compare the M1 and M2 response with bystand,infected and injected cells.7.Infecting WT and STAT6 mice with CEP strain,compare parasite burden,serum IL-12 and IFNγ.8.Infecting BMDM from WT mouse with CEP and CEP△ ROP16,test phospholation of STAT6,alternative activation macrophage markers and iNOS production.Count parasite burden with in vitro killing assay.9.Infecting WT,anti-IFNγ Treated、IL-12-/-mice with CEP and CEP △ROP16 through I.P.,at day 4 post infection compare parasite burden in the peritoneal cells.Compare large peritoneal macrophage in the WT mice with CEP and CEP△ROP16 at d.p.i 1.At day 10,compare parasite DNA in the different tissues,serum IL-12 and IFNγ.10.Compare T cell activation,effector T cell,cytokines production and CTL efficience in spleencyte at 10 d.p.i with CEP and CEP△ ROP16.Results:1.Classical activation(M1)macrophage plays a vitro role in controlling Type ⅢToxoplasma gondii in acute infection.2.Type Ⅲ Toxoplasma gondii can directly activate alternative activation(M2)macrophage by phospholate STAT6 both in infected and injected BMDM.3.Type Ⅲ Toxoplasma gondii can alter infected and injected macrophage polarization to M2 macrophage in mouse.4.The injected nacrophages in mouse are in an intermediate status between infected and bystand(uninfected)cells.The STAT1 pathway in the injected macrophages are not completely blocked as which in the infected macrophages.5.No significant difference between global STAT6 KO and WT mouse on parasite burden and also cytokines production.6.The ROPl6 protein from Type Ⅲ Toxoplasma directly phospholates STAT6 and activate M2 macrophage genes in BMDM,which attenuates the IFNγ induced iNOS production to protect the parasite in the macrophage.7.CEP infected mouse can activate more alternativae activation macropahges and has less IL-12 and IFNγ in the serum compated to ROP 16 mutant parasite,which helps the parasite survive in the mouse.Once blocked IL-12 or IFNγ in the mouse,the parasite burden shows no difference with CEP strain and ROP 16 mutant.8.Although the ROP 16 mutant infection resulted in less parasite burden in the mouse,it induces more T cell activation,effecting,cytokines production and also specific cytotoxic T cells killing.Conclusion:The ability of 7bxoplas/ma gondii to inject rhoptry proteins into host cells is a maj or determinant of the outcome of infection and the ability of ROP 16 to activate the host transcription factor STAT6 is considered an important determinant of virulence.Here,parasites that inj ect Cre-recombinase(CEP-Cre)with their rhoptry proteins were used to dissect macrophage-parasite interactions.Comparison of the transcriptional profile of macrophages challenged in vitro revealed that injection of ROP 16 was sufficient to induce a STAT6 dependent M2 phenotype.Similar results were observed in vivo but,productive infection was associated with reduced expression of anti-microbial genes and antigen presentation pathways.In vivo,ROP 16 was important for early parasite replication in macrophages but STAT6 was not a major determinant of virulence.Rather,the loss of ROP16 was associated with heightened parasite specifie T cell response.Together,these studies indicate a STAT6-independent role for Rop16 in limiting the magnitude of parasite specific T cell response.