FIG 4.

Pneumocystis infection induces an AAM phenotype in vivo in an IL-4Rα-dependent manner. Wildtype BALB/c and IL-4Rα^−/− mice were infected with 1x10⁷ Pneumocystis (PC) nuclei by i.t. instillation and euthanized at 7 days post-infection. Cells were isolated from the alveolar space via lung lavage, and subsequently whole lungs were digested to create single cell suspensions. (A) Arginase activity in lung digest was determined by measurement of the conversion of arginine into urea and standardized to total protein. (B) mRNA was isolated from lung digest cells and reverse transcribed to cDNA. Gene expression was quantified using qPCR to determine gene expression levels of Arg1, Fizz1, and Ym1 genes. qPCR data was normalized to values generated in wildtype uninfected animals and expressed as fold increase over controls. (C) Cells isolated from lung lavage and lung digest samples were stained for surface expression of CD11b, CD11c, Gr1, MR, and CCR7 and analyzed by flow cytometry. Cells that were CD11b⁺Gr1⁻ and CD11c⁺Gr1⁻ were gated upon, and the numbers of cells in each subset expressing CCR7 or MR were determined in both the lung lavage and lung digest samples. (D) The number of CD11c⁺Gr1⁻ (left) and CD11b⁺Gr1⁻ (right) cells producing IL-10, TGFβ, and TNFα were measured by intracellular cytokine staining and flow cytometry. Lung digest preparations were incubated with PMA, ionomycin, and brefeldin A as described and surface stained for CD11b, CD11c, and Gr1. Following fixation they were subsequently stained for intracellular up-regulation of TNFα, TGFβ, and IL-10. (E) Kinetics of PC clearance over time post infection, expressed as PC nuclei microscopically counted in lung digest samples. Mean ± standard deviations are reported for groups of 4 mice per strain, and are representative of 3 experimental replicates. Statistically significant differences between treatment groups at each timepoint are denoted as follows: *, P < 0.05 by one-way ANOVA and Bonferroni post test. Asterisk above bar represent difference compared to uninfected controls for each strain, and asterisk above connecting line represents difference between knockout and WT strains.