| Background Toxoplasma gondii is an intracellular parasite capable of infecting a broadspectrum of hosts including up to30%of the world’s human population. Infectionoccurs when individual ingest undercooked meat containing cysts of the parasite orconsume food contaminated with oocysts. Although most infections are mild in healthyadults, life-threatening consequences may develop in immunocompromised patients andin utero infection can lead to major defects in the fetus.During acute infection, tachyzoite, the rapidly replicative form of the parasiteelicits an extremely strong type1immune response, characterized by proinflammatorycytokine production such as IL-6, IFN-γ and TNF-α. Macrophages can provide a nichepermissive for parasite replication and are the most abundant cell type infected byToxoplasma. It has been clarified that macrophages can be phenotypically polarized bythe micro-environment to mount specific functional programs. Polarized macrophageshave been classified in two main groups: classically activated macrophages (or M1)whose typical activating stimuli are IFN-γ and LPS, and alternatively activatedmacrophages (or M2) induced by exposure to IL-4and IL-13. M1exhibits antimicrobialfunctions against intracellular pathogens which is conducted by the production ofreactive oxygen and nitrogen intermediates such as NO and promote strong Th1 responses, while M2is accompanied by diminished proinflammatory cytokine secretionand shares high expression of scavenger, mannose (CD206) and galactose receptors.Recent work on T. gondii polymorphisms show that mouse macrophages infectedwith type I and III strains are polarized toward an M2activation state through activationof STAT6, while type II infected macrophages are M1-like cells elicited by activationof NF-kB. Type I and type III rhoptry kinase ROP16, inferred as a virulencedeterminant, can constitutively activate STAT6, and type II dense granule proteinGRA15is responsible for strain-specific NF-kB activation.The majority of T. gondii strains isolated from humans and animals in NorthAmerica and Europe have been grouped into three predominant clonal lineages (types I,II and III) that differ genetically less than1%. Regardless of the genetic background ofmice, type I strains are highly virulent with an LD100=1, whereas type II or type IIIstrains associate with host genetic background and display lower virulence with anLD50≈102and≈105, respectively. Recently there were several studies revealingthat a few major clonal lineages of T.gondii dominate in different geographical regions.For example, the type12lineage is most common in wildlife in North America, and theAfrica1and3are among the major types in Africa. Our previous study showed that thegenotype Chinese1(known as ToxoDB#9) is dominantly prevalent in China.Interestingly, although belonging to the same genotype Chinese1, isolates of TgCtwh3and TgCtwh6have remarkably different virulence to mice, in which TgCtwh3is highlyvirulent and cause lethal infection prior to encystation, whereas TgCtwh6is mildlyvirulent and able to establish latent infection.Objective To compare the difference in the acute immune response of macrophagesinfected with TgCtwh3or TgCtwh6tachyzoites in vitro and in vivo; to observe thephenotypical changes on activated macrophages; to evaluate the activation state ofmacrophages and determine modulation of the signaling pathway elicited by TgCtwh3 or TgCtwh6; to briefly explore the mechanism of human toxoplasmosis caused byisolates of Chinese1genotype.Methods (1) Purification and cell passages of TgCtwh3and TgCtwh6tachyzoites.Tachyzoites of mouse-virulent TgCtwh3isolates were maintained in vitro by serialpassages in L929fibroblasts monolayers cultured in RPMI1640. The TgCtwh6tachyzoites were initially obtained by inoculating brain homogenate containingTgCtwh6cysts from infected mice and then cultured with L929fibroblasts. For initialpassages, fibroblast monolayers were detached by scraping, and cells were forcedthrough a27-gauge needle to release the intracellular parasites. About4weeks laterTgCtwh6were maintained by continual cell passages.(2) For in vivo infection,1×105tachyzoites of TgCtwh3or1×106tachyzoites of TgCtwh6were inoculated i.p. into six-to eight-week old BALB/C mice. At2-6d postinfection mice were euthanized andperitoneal exudates cells (PECs) were harvested by washing of the peritoneal cavitywith10ml of ice-cold D-Hanks solution. PECs were cultured in12-well plates (1×106cells per well) in RPMI1640, and three hours later nonadherent cells were washed offand the remaining adherent Mφ (termed Wh3-Mφ and Wh6-Mφ, respectively) were leftuntreated12h. Supernatants were collected for cytokine measurement, NO productionand arginase activity. The simultaneous detection of IL-6, IFN-γ, TNF, IL-4and IL-10was performed by Cytometric Bead Array Cytokine Kit, and IL-12p40was detected byMouse IL-12/IL-23p40Flex Set according to manufacturer’s instructions. Samples wereacquired on a cytometer and data were analyzed using FCAP Array Software. NOproduction was assessed by nitrite accumulation using the Griess Reagent System andarginase activity was measured according to previously published protocols.(3) For invitro infection assays, maintained in6-well plates (2×106cells per well), BMMφ andRAW264.7cells were pre-stimulated for24h with rMuIFN-γ plus LPS, or withrMuIL-13plus IL-4, or left unstimulated, then infected with freshly lysed T. gondii tachyzoites at a parasite to host cell ratio of2:1. Cells were incubated for an additional24h. Supernatants were collected for detection of arginase activity.(4) After beingwashed twice with PBS containing3%FCS, Wh3-Mφ and Wh6-Mφ were labeled withthe antibodies of interest and appropriate isotypes at the appropriate dilution for20minat4°C in the dark. The antibodies include FITC-conjugated anti-F4/80,PE-Cy5-conjugated anti-MHC Ⅱ, PE-conjugated PD-L2, PE-conjugated PD-L1,PE-Cy5-conjugated anti-CD80, APC-conjugated anti-CD86as well as Alexa Fluor647-conjugated anti-CD206. Noninfected macrophages were performed in parallel.Cells were washed three times in FACS buffer and fixed in2%paraformaldehydebefore FACS analysis.(5) By resuspension in TRIzol reagent RNA was prepared fromWh3-Mφ or Wh6-Mφ after being cultured for12h, or TgCwh3or TgCwh6-infectedBMMφ infected at24h. Total RNA was extracted and cDNA was synthesized using theRevertAid First Strand cDNA Synthesis Kit. The mRNA levels of Th1/Th2cytokineand M1/M2markers were detected by real-time RT-PCR.(6) BMMφ grown in a6-wellplate were infected with TgCtwh3and TgCtwh6parasites respectively (2:1ratio ofparasites to Mφ) for24h. After being washed with ice-cold PBS, in the presence ofProtease/Phosphatase Inhibitor Cocktail infected cells were lysed by addition of lysisbuffer, or nuclear proteins were extracted according to manufacturer’s instructions.Total cell lysates were subjected to SDS-PAGE and immunoblotting using several Abs,including anti-iNOS, anti-Ym1, anti-arginase1, anti-Stat6, anti-pY641-Stat6, anti-IκBα,anti-p-IκBα, anti-T.gondii SAG1. Nuclear proteins were subjected to SDS-PAGE fordetecting anti-NF-κB p65.(7) TgCtwh3or TgCtWh6parasites were allowed to invadecells on coverslips and incubated for different time points. Then coverslips were fixed,permeabilized and blocked in goat serum. To determine the nuclear or cytoplasmicNF-κB p65expression, HeLa cells were simultaneously incubated with anti-NF-κB p65along with anti-T.gondii p30-FITC overnight at4°C. Coverslips were incubated for1hat room temperature with tetramethylrhodamine goat-anti-rabbit IgG (H+L), and then incubated with Hoechst dye for5min for DNA visualization. Cell preparations wereexamined by inverted fluorescence microscope.Results (1) Both TgCtwh3and TgCtwh6triggered Th1immune response with distinctcytokines profile. Compared with non-infected Mφ, remarkedly elevated IL-6, IL-10,IL-12p40, IFN-γ and TNF were found in Wh6-Mφ, whereas modest increases of thesecytokines were displayed in Wh3-Mφ except for IL-10. In concert with protein levels,Wh6-Mφ also showed elevated mRNA expression of IL-10, IL-12p40, IFN-γ andTNF-α. Unexpectedly, although IL-4concentration was below the level of detectionincreased transcription of IL-4mRNAs was exhibited on Wh3-Mφ.(2) TgCtwh3andTgCtwh6down-regulated CD80, CD86and MHC Ⅱ molecules expression onmacrophages. Our results indicated that both Wh3-Mφ and Wh6-Mφ expressed lowerF4/80, with MFI being40.51±6.22and38.86±5.69respectively, than uninfectedmacrophages with900.6±144.33(both p＜0.001). Additionally, compared withuninfected controls neither Wh3-Mφ nor Wh6-Mφ could up-regulate MHC class IImolecule expression, and both CD80expression in Wh3-Mφ and Wh6-Mφ was less asshown by MFI. Besides, Wh3-Mφ expressed the levels of CD86similar to uninfectedcontrols with MFI being41.3±4.60vs42.0±4.43(p＞0.05), contrary to lower levelsof CD86in Wh6-Mφ with MFI being29.54±6.12(p＜0.01).(3) Wh3-Mφ up-regulatedPD-L2and CD206expression whereas Wh6-Mφ modestly expressed PD-L1. Comparedwith uninfected Mφ TgCtwh3strongly promoted PD-L2expression and stained positivefor CD206, with MFI being112.78±19.91vs5.97±2.96(p＜0.001) and105.32±25.59vs10.28±3.02(p＜0.001) respectively. On the other hand, relative to residentperitoneal Mφ with low PD-L1and no PD-L2expression, Wh6-Mφ modestlyup-regulated the expression of PD-L1and PD-L2with MFI being139.83±20.82vs27.86±4.88(p＜0.001) and37.48±10.34vs5.97±2.96(p＜0.01) respectively.(4)Wh6-Mφ generated high levels of NO whereas Wh3-Mφ up-regulated Ym1and argniase expression. Our result showed that in contrast to low level of NO in Wh3-Mφ(2-fold higher than uninfected Mφ), less virulent TgCtwh6isolates induced robustproduction of NO,14-fold higher than uninfected Mφ. At the same time TgCtwh3infection elicited high arginase activity in Mφ as measured by the production of ureawhen compared with TgCtwh6. Additionally, in vivo experiment also suggested thatTgCtWh6triggered enhanced expression of iNOS on transcript and protein levels,whereas TgCtwh3up-regulated not only argniase activity, but also Ym1and argnise-1mRNA and protein expressions.(5) Highly expression of arginase in M2associatedwith parasites proliferation. To evaluate the effects of M1or M2on parasites,RAW264.7cells, pretreated either with IL-4plus IL-13or with IFN-γ plus LPS for24h,were infected with TgCtwh3and TgCtwh6respectively, and assayed for arginaseactivity and parasites growth. In general, arginase activity was all enhanced in IL-4andIL-13-treated cells, especially in those co-infected with TgCtwh3. Moreover, theproliferation of TgCtwh3in IL-4and IL-13-treated cells was also obvious by countingthe number of parasites per vacuole.(6) By immunofluorescence we found that manyHeLa cells infected with TgCtwh6showed high levels of NF-κB p65in their nucleus,whereas no translocation or only low levels of NF-κB p65to the nucleus induced byTgCtwh3. Furthermore, the results from Western blotting revealed that TgCtwh3induced phospho-STAT6whereas TgCtWh6led to the phosphorylation of IκBα andactivation and nuclear translocation of NF-κBp65.Conclusions (1) Although sharing the Chinese1genotype, different virulent TgCtwh3and TgCtWh6isolates elicited Th1immune response in distinct extent with variantkinds of cytokines.(2) Both TgCtwh3and TgCtWh6tachyzoites down-regulated CD80,CD86and MHCⅡ molecules expression on macrophages.(3) TgCtwh3and TgCtWh6isolates induced macrophage-biased responses in different pathways. Virulent TgCtwh3 whereas cyst-forming isolate of TgCtwh6activated NF-κB pathway and promoteinfected macrophages similar to M1phenotype. |
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