Temporal control of cellular and molecular inflammatory phenotypes by rapamycin.

<p>(A) Representative Alcian Blue/Periodic Acid Schiff (AB/PAS) stains for inflamed animals treated with vehicle (Veh.) or rapamycin (Rap.) for the indicated periods of time. Intracellular and extracellular mucins are stained dark purple and indicate the appearance of mature goblet cells. (B) Measurement of the proliferative zone before and after rapamycin. The proliferative zone is defined as the ratio of the highest phosphorylated histone H3 (PH3)-positive cell position in a crypt divided by the height of that crypt. For each animal, 12–25 crypts were measured, and bars represent the average proliferative zone for 2–3 animals. Significance was determined by one-way ANOVA with Tukey-Kramer post-test. * represents <i>p</i> ≤ 0.05; ** represents <i>p</i> ≤ 0.01. Bar graphs represent mean +/− standard error. (C) Average crypt height profiles for each treatment group, with the standard error about the mean indicated in shaded regions at points binned in 500-μm increments. <i>N</i> = at least 2 for each group. (D) Flow cytometric measurements of colonic immune infiltrate. Inflamed animals show an even mix of T cells, macrophages, and neutrophils, but rapamycin treatment leads to a loss of macrophages and neutrophils by 48 hours. Significance determined by ANOVA with Tukey-Kramer post-test. * represents <i>p</i> ≤ 0.05; ** represents <i>p</i> ≤ 0.01, both with respect to naïve T, vehicle control. <i>N</i> = 2–3 animals per condition. Bar graphs represent mean +/− standard error. (E) Representative cytokine expression patterns of response to acute rapamycin treatment. Macrophage colony-stimulating factor (M-CSF) drops immediately follow rapamycin exposure, while macrophage inflammatory protein 1-alpha (MIP-1α) declines slowly over 24 hours. Some proteins, like interleukin (IL)-6, transiently increase in expression following rapamycin treatment. <i>N</i> = 2–3 animals per condition. Bar graphs represent mean +/− standard error. Underlying numerical values for panels B–E are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002417#pbio.2002417.s001" target="_blank">S1 Data</a>.