PMC

(A) Gene cluster analysis of uninfected and infected BMDMs with the indicated type II toxoplasma strains for markers of alternative activation. For a list of genes that are regulated by GRA15 and a TFBS analysis of this gene set see , respectively. For a list of genes that are co-regulated by ROP16 and GRA15 see .
(B) Arginase activity of wild type (dark bars) or Stat6−/− BMDMs (light bars) infected for 20 h with the indicated parasite strains. Error bars = + SD. (Student t *P value < 0.03, indicated sample vs. all other infected wild type BMDMs; ^#P value < 0.01, type III vs. all other infected wild type BMDMs; ^%P value = 0.06, type II Δgra15 + ROP16_I vs. type II + ROP16_I; ^$P value < 0.03 uninfected Stat6−/− vs. all other infected Stat6−/− macrophages; Infected wild type vs. Stat6−/− BMDMs was significantly different for each parasite strain, P value < 0.02, not shown).
(C) IL-12p70, IL-23 (p40/p19), and IL-10 cytokine secretion by parasite infected BMDMs was determined by ELISA 20 hours after infection (Student t *P value < 0.03, type II vs. all other samples). Error bars = + SD. For a further analysis of the ability of GRA15_II and ROP16_I to induce M1 and M2 markers in the context of polarizing environments in vitro and in vivo see .

(A) RAW264.7 macrophages were infected with the type I (RH), II (Pru) or III (CEP) Toxoplasma strains (MOI=5) and 24 h after infection arginase activity was measured in the lysates of infected and uninfected macrophages by determining the conversion of L-arginine to urea in one hour (Student t *P value < 0.006, type I or III vs. type II). Similar results were obtained with other strains of the three clonal lineages (GT1, ME49, and VEG, not shown). Error bars represent a standard deviation (SD). The relative percentage of infected macrophages following i.p. infection with the three strain types can be seen in .
(B) RAW264.7 macrophages were infected with either the type I, II or III Toxoplasma strains that expressed GFP (MOI=0.5) and one day after infection macrophages were fixed, permeabilized and stained with antibodies against either the macrophage mannose receptor (CD206 also called Mrc1, stained red) or the galactose/N-acetylgalactosamine specific lectin (mMgl/Mgl2, stained red). Nuclei were stained with Hoechst (blue). These results are representative of at least three experiments. Error bars = + SD.
(C) As in A, but DC2.4 dendritic cells were assayed (Student t *P value < 0.02, type I or III vs. type II; ^#P value < 0.002, type II vs. uninfected control). Error bars = + SD.
(D) IL-23 (p40/p19) and IL-12 (p40/p35 or ‘IL-12p70’) cytokine production by type I, II or III infected BMDMs were determined by ELISA 24 h after infection (Student t *P value < 0.03 type II vs. type I or III). These results are representative of at least three experiments. Error bars = + SD.

(A) RAW264.7 macrophages were stimulated with LPS (20 ng/ml) and IFN-γ (100U/ml) in medium containing different concentrations of L-arginine and NO production was measured by determining the concentration of nitrite (NO₂−) in the medium.
(B) RAW264.7 macrophages cultured in medium with either 15 or 40 mg/L L-arginine were mock-infected (control) or infected (MOI=5 or 10) with type I or type I Δrop16 parasites and subsequently stimulated with LPS (20 ng/ml) and IFN-γ (100 U/ml). One day after infection NO production was measured by determining the concentration of nitrite in the medium; and,
(C) arginase activity was determined. Error bars = + SD.
(D) DC2.4 cells were infected with either type I or type I Δrop16 at an MOI of 1 in medium supplemented with 35 mg/L L-arginine and stimulated with 50 ng/mL of IL-4 and/or treated with 100 μM of the arginase inhibitor nor-NOHA. Twenty-four h later the number of parasites per vacuole was quantified by immunofluorescence microscopy. The bars represent the number of vacuoles containing 1 or 2 parasites (dark bars), or ≥ 3 parasites (light bars). Fisher’s exact test one-tailed probabilities ≤ 0.03 are indicated.
(E) As in D, but type II (PruA7 5-8b+, HXGPT+) and type II +ROP16_I (2C4) parasites were assayed after 48 h of infection. The bars depict the number of vacuoles containing 1 to 3 parasites (dark bars), or ≥ 4 parasites (light bars). Fisher’s exact test one-tailed probabilities ≤ 0.03 are indicated (Fisher exact test P value ≤ 0.002, NOHA vs. IL-4 for all infections in D and E, not shown).

(A) Susceptible C57BL/6 mice were orally infected by gavage with 800 cysts of the type II, type II +ROP16_I, type II +ROP16_III (Pru background) or type III (CEP) strains and survival was monitored. The combined results of 2 experiments are shown (n=10).
(B) On day 8 of infection, the entire length of the small intestine was fixed, sectioned and stained with hematoxylin and eosin dyes (top panels), or with antibodies to Gr-1 (Ly6C/G) (RB6-8C5, brown staining) and hematoxylin (lower panels). Representative pictures of the villi (top panels) or Peyer’s patches (lower panels) from the intestines of mice infected with either the type II or type II +ROP16_I strains are shown at 20x magnification. The border of the Peyer’s patch is outlined in white.
(C) On day 8 of infection lymphocytes were harvested from the Peyer’s patch or lamina propria and stimulated for 20 h in vitro with plate bound anti-CD3ε and anti-CD28 antibodies. IFN-γ and IL-22 was detected in the supernatant by ELISA. The average of 3 biological replicates and the standard deviation is plotted. Student t test P values ≤ 0.06 are indicated.
(D) For each mouse intestine (n=3) severe inflammation was quantified along the entire length of the intestine by microscopy of the tissue sections. If a region met the following criteria: 1) increased cellular influx, 2) villi necrosis or villi blunting and 3) mucosal thickening, then that region was considered severely inflamed and the length of that region was measured. The sum of all regions was tallied per mouse intestine and the average of the biological replicates and standard deviation is plotted. The student t test P value is indicated.
(E) For each intestine, the number of villi or villi remnant that stained positive for iNOS (NOS2), Gr-1 (Ly6C/G) or myeloperoxidase (MPO) was quantified and the average of the biological replicates and standard deviation is plotted. Student t test P values ≤ 0.05 are indicated.

(A) RAW264.7 macrophages were mock-infected (control) or infected (MOI=5) with type I, type I Δrop16 or type II parasites and 24 h later arginase activity was determined (Student t *P value < 0.05, type I vs. type I Δrop16 or type II). Error bars = + SD.
(B) As in A, except the macrophages were fixed and stained with antibodies to the parasite surface antigen SAG-1 (green) and the M2 markers CD206 or mMgl. Hoechst dye was used to stain nuclei (blue).
(C) Western blot using antibodies against mMgl1/2, tyrosine phosphorylated STAT6 (pSTAT6) or total STAT6. Antibodies against GAPDH and SAG-1 were used for host cell and parasite loading controls, respectively.
(D) One day after infection with the indicated strain types, macrophages were fixed, permeabilized and stained with antibodies against pSTAT6 and SAG-1. Nuclei were stained with Hoechst (blue).
(E) BMDMs were infected with GFP expressing type I or type I Δrop16 parasites and stained with PE labeled antibodies to the surface receptors B7-H1 (PD-L1), B7-DC (PD-L2), Dectin-1 (Clec7a) or CD86 (which is not regulated by ROP16). Histogram plots depicting the relative surface expression of these markers on infected GFP⁺ (black lines) and non-infected GFP^neg (blue lines) BMDMs in the same well. Isotype staining with Rat IgG2a antibodies is also shown (shaded histogram).
(F) BALB/c mice were infected i.p. with type I, type I Δrop16 or type II parasites. Twenty-one h post-infection, PECs were harvested and stained for CD11b and the SAG-1 parasite surface antigen. Infected (toxo⁺) and uninfected (toxo^neg) CD11b⁺ PECs were analyzed for their expression of pSTAT6 or CD206 by histogram (gray = ‘minus one’ staining control where cells are stained with all staining reagents except the anti-STAT6 or CD206 primary antibodies; red = type I; blue = type II; green = type I Δrop16; black = PECs from an uninfected mouse stimulated with recombinant murine IL-4 for 10 minutes at 37°C). Data are representative of 2 independent experiments (n=3).
(G) Bar graphs depict the percentage of positively staining CD11b⁺ PECs as analyzed in F (‘KO’= type I Δrop16). N.D.= not detected. Error bars = + SD. ANOVA one-way analysis of variance was used to determine statistical significance. The percentage of infected CD11b⁺ cells was similar in type I, type II, and type I Δrop16 infected mice (data not shown).
See for a list of genes regulated by ROP16, as well as for a TFBS analysis of this gene set.