Figure 3
The generation of adenosine is compromised during the acute phase of toxoplasmosis. C57BL/6 mice were infected orally with 40 cysts of ME-49. Cells were isolated from the spleen, MLN, LP, and IEL, stained for TCRβ, CD4, CD39, CD73, and Foxp3 and analyzed by flow cytometry. (a) Dot blots represent CD73 and Foxp3 expression by CD4+ T lymphocytes at day 0 or at day 9 post infection (p.i.) from spleen and LP compartments. (b) Percentages of TCRβ+ CD4+ Foxp3 negative or positive cells expressing CD73 between days 0 and 9 after infection in spleen, MLN, LP, and IEL compartments. The results are representative of five independent experiments (n=5). (c) At day 0 or at day 9 p.i., TCRβ+ CD4+ lymphocytes from spleen, MLN, LP, or IEL were purified and mRNA extracted. Quantitative RT-PCR of the expression of CD73 mRNA was performed. Data are pooled from four independent experiments. (d) Percentages of TCRβ+ CD4+ cells expressing CD39 between days 0 and 9 after infection in spleen, MLN, LP, and IEL compartments (n=5). (e) TCRβ+ CD4+ lymphocytes from spleen, MLN, LP, or IEL were purified at day 0 or at day 9 p.i. and incubated with AMP. The supernatants were collected and adenosine concentration was analyzed by HPLC. Data are pooled from four independent experiments. (f) Amounts of adenosine were assessed by HPLC in sera from naive or day 9 infected mice. Data are pooled from three independent experiments (n=12). (c, e, f). Graph bars represent the median with interquartile range. The non-parametric Mann–Whitney U-test was used to determine statistical differences (*P<0.05, ***P< 0.001). HPLC, high-performance liquid chromatography; IEL, intraepithelial lymphocyte; LP, lamina propria; MLN, mesenteric lymph node; RT-PCR, reverse transcription-PCR.
