IL-17A expression during experimental autoimmune encephalomyelitis.

<p>Wild-type or Smart-17A mice were immunized with MOG-CFA to induce EAE. (A) At the indicated time point, cells were harvested from the draining axial, brachial and inguinal lymph nodes (LN) at day 6 or spinal cords and cerebellums (CNS) at day 12 and numbers of cells were enumerated. (B) Cells were assayed for hNGFR expression. (C) The total numbers of hNGFR⁺ cells were calculated and the percentage attributable to each cell population is shown in a pie graph. The percentage of background staining seen in a wild-type mouse under identical conditions was subtracted before performing all calculations to control for nonspecific staining. (D) Cells were isolated from the LN or CNS and immediately stained for surface markers or restimulated for 5 hr with PMA/ionomycin and then stained for surface markers and/or intracellular expression of IL-17A. The experiments in A–C were repeated 3 times with n>2 mice at each time point. The experiment in D was repeated 2 times with n>5 mice at each time point. For bar and pie graphs, data from independent experiments were compiled. For flow cytometry, representative plots are shown. For the CNS data, mice were excluded that did not display symptoms of paralysis.</p

IL-17A expression during infection with <i>Klebsiella pneumoniae.</i>

<p>Wild-type or Smart-17A mice were infected with 500–1000 <i>K. pneumoniae</i>. (A) At the indicated time points, cells were harvested from lungs and numbers of cells were enumerated. (B) Cells were isolated from the lungs of mice 2 days after infection and assayed for hNGFR expression. (C) The total number of hNGFR⁺ and YFP⁺ cells on day 2 post-infection were calculated and the percentage attributable to each cell population is shown in a pie graph. The percentage of background staining seen in a wild-type mouse under identical conditions was subtracted before performing all calculations to control for nonspecific staining. This experiment was repeated 3 times with n >3 mice at each time point. For bar and pie graphs, data from independent experiments were compiled. For flow cytometry, representative plots are shown.</p

Expression of IL-17A from innate-like T lymphocytes cells can be induced by IL-1β and/or IL-23.

<p>(A) Smart-17A mice were inoculated intranasally with PBS or with 500 ng IL-1β, IL-23 or both cytokines. Lungs were harvested 8 hr later and cells were analyzed for hNGFR expression. Gates were set using a wild-type control. The experiment was repeated 2 times and representative data are shown. (B) Wild-type mice were infected with <i>K. pneumoniae</i> and levels of IL-1β and the IL-23 subunit p19 mRNA in whole lung homogenate were measured using quantitative PCR. Expression of GAPDH was used as a reference to define relative expression. The experiment was done twice and a representative experiment is shown, n = 3 for all groups.</p

Generation of Smart-17A mice.

<p>(A) Targeting strategy for the <i>il17a</i> locus. For detailed description, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039750#s4" target="_blank">Materials and Methods</a>. (B) CD4⁺ T cells were isolated from wild-type or Smart-17A mice and polarized under Th17 conditions for 4 days. hNGFR was detected using a surface antibody and IL-17A was assayed using intracellular cytokine staining after restimulation. A representative flow cytometry plot is shown from >5 comparable experiments.</p