Neutralization of IL-17 inhibits the progression of tumors in DMBA/TPA induced cutaneous carcinogenesis.

<p>Wild type C57Bl/6 mice were topically treated once with DMBA and then twice weekly with TPA. All mice developed cutaneous tumors at 12 weeks. The mice were divided (10 mice/group) and treated intravenously with adenovirus encoding GFP (Ad-GFP), IL-17R:Fc (Ad-IL-17R:Fc) or left untreated (Ctrl) at week 12 and 16. All mice were continuously treated with TPA twice a week throughout the experiments. The tumor growth was monitored weekly. Bar graph shows the mean tumor number per mouse +/− SEM at the end of experiments (* P<0.05). Data are representative of 2 independent experiments.</p

TPA induced promotion of tumor growth is abrogated in IL-17R-/- mice.

<p>A). TPA induced inflammation enhances tumor growth. Wild type C57Bl/6 mice were treated epicutaneously with TPA or acetone (vehicle controls) every other day for a total of 5 applications. The mice were then inoculated subcutaneously with EG7 tumor cells. Tumor growth was monitored (n = 5). B). TPA induced inflammation increases tumor specific IL-17 producing cells. At the end of experiments, the draining lymph node lymphocytes were collected from EG7 tumor bearing mice that were treated with TPA or acetone. The cells stimulated with OVA pulsed BM-DC for 4 days. Concentrations of cytokines in supernatants were measured by ELISA (n = 4). Naïve T cells with OVA pulsed BM-DC (Naïve) and immune T cells with BM-DC without OVA (Neg) served as controls in the cultures. C). TPA induced tumor promotion is abrogated in IL-17R-/- mice. Wild type or IL-17R-/- were epicutaneously treated with TPA or acetone every other day for a total of 5 applications. The mice were then inoculated with EG7 tumor cells and tumor growth was monitored (n = 5). Data are representative of 2–3 independent experiments (mean +/− SEM, * P<0.05).</p

IL-17 has opposing effects on the infiltration of CD11b+ macrophages and CD8+ T cells in DMBA/TPA treated skin.

<p>Mice were treated with DMBA and TPA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032126#pone-0032126-g001" target="_blank">figure 1</a> and skin tissues were harvested at the end of the experiment (26 weeks). A). Frozen tissue sections were stained with CD11b antibodies (brown) and counterstained with hematoxylin (blue, upper panels) or with anti-CD8 antibodies (green) and counterstained with a fluorescence dye DAPI (blue, lower panel). Photos were taken microscopically (20× objective). Numbers of positive cells were counted microscopically. Islets show the enlarged areas of circled squares with positive cells. Graphs show the mean number of positive cells per field +/− SEM, <i>n</i> = 10, * p<0.05, ** P<0.01. B). Gr-1 and CD11b double positive cells in spleens were stained with fluorescence labeled antibodies and positive cells were analyzed in a flow cytometer. The numbers in the right upper quadrant indicate the percent of positive cells in whole spleen cells. C). Statistical analysis of GR-1/CD11b+ (myeloid derived suppressor cells), CD19/B220+ (B cells), CD8+ and CD4+ T cells in spleens (mean +/− SEM, n = 8, ** P<0.01). Data are representative of two independent experiments.</p

The deficiency in IL-17 receptor inhibits TPA induced tumor promoting inflammation in the skin.

<p>Mice were treated topically with TPA every other day for a total of three applications. Skin tissues were harvested 24 hours after the last treatment. A). Paraffin embedded tissue sections were stained with hematoxylin. Skin samples from naïve untreated mice served as controls. Dashed lines indicate the border between the epidermis and dermis. The thickness of epidermis was measured microscopically (n = 10). B). Frozen tissue sections were stained immunohistochemcially with antibodies. CD11b+ (monocytes/macrophages) and Gr-1+ (granulocytes) cells were counted microscopically (n = 6). C) Concentrations of cytokines in the skin lysates were measured by ELISA (n = 4). D). S100A8 and S100A9 proteins in the skin lysates were detected by Western blots using specific antibodies. E). Concentrations of PGE2 in the skin lysates were measured by a PGE2 EIA kit (n = 3). F). Expression levels of mRNA for chemokines and cytokines in the skin lysates were quantified by real time RT-PCR (n = 3). Data are representative of 2–3 independent experiments (mean +/− SEM, * P<0.05: ** P<0.01).</p