Serum withdrawal induces tPA expression in RT4-D6P2T cells.

<p>Photomicrographs (<b>a</b>) and relative quantifications of tPA-like immunoreactivity in RT4-D6P2T cells (<b>a’</b>) cultured either in the presence (Control) or absence of serum (Serum Starved) after 24h. Immunofluorescent images were captured using an Axiovert 40 fluorescence microscope (Carl Zeiss Inc.). Each condition was reproduced using at least three dishes <i>per</i> experiment. Representative photomicrographs were taken from at least three fields <i>per</i> dish in a fixed pattern. Original magnification 40X. Scale bar = 30μm. Details on the procedure can be found in MATERIALS AND METHODS section. (<b>a’</b>) Bar graph depicts the relative fluorescence ± SEM, expressed as arbitrary units. <i>**p<0.01 Vs Control</i>, as determined using the unpaired two-tailed Student <i>t</i>-test. (<b>b</b>) Representative immunoblot and related quantifications of bands obtained by resolving cell lysates from cultures treated as indicated above. Normalized tPA expression levels were calculated using β-tubulin as the loading control. Each result displayed in the bar graph represents the mean ± SEM from three independent experiments. *<i>*p<0.01 Vs Control</i>; Unpaired two-tailed Student <i>t</i>-test. Ctrl = 10% FBS cultured RT4-D6P2T cells after 24h. SS = Serum starved cells.</p

BDNF boosts endogenous PACAP/PAC₁ receptor signaling to induce tPA expression in RT4-D6P2T cells.

<p>Western blots and related quantifications (<b>a</b> and <b>b</b>) showing the effect of 2x10⁻⁹M BDNF treatment on PACAP peptide and PAC₁ receptor expression at different exposure times (0, 6, 12 and 24h). For experimental procedures please refer to the appropriate section in MATERIALS AND METHODS. Results in (<b>b</b>) are the mean ± SEM of normalized values obtained from at least three separate experiments. β-tubulin was used as the loading control. <i>*p<0.05 or **p<0.01 Vs Control</i>; ANOVA followed by Tukey <i>post-hoc</i> test. Inhibitory effects of the non-specific PAC₁/VPAC type receptor antagonist PACAP6-38 (10μM) on tPA protein expression were determined by Western blots (<b>c</b>). Cells were either left untreated (control) or pretreated with PACAP6-38 in the absence or presence of PACAP or BDNF and tPA expression was evaluated. Quantification of bands was performed by normalizing tPA band density to that of β-tubulin, which was used as loading control. Each experiment was reproduced three times with similar results. Depicted in the bar graph are the average ratios ± SEM. <i>*p<0.05 or ***p<0.001 Vs Control</i>; ANOVA followed by Tukey <i>post-hoc</i> test.</p

Localization of tPA-like immunoreactivity in RT4-D6P2T cells stimulated with PACAP, maxadilan, VIP or brain-derived neutrophic factor (BDNF).

<p>Depicted are representative immunocytofluorescence photomicrographs (<b>a</b>) and bar graphs showing semi-quantification of fluorescent intensity (<b>b</b>) in cells in which 1° Ab was omitted (Neg. Control), were left untreated (Control) or were treated with the indicated concentrations of PACAP, the PAC₁ agonist (maxadilan), VIP or BDNF for 24h. Briefly, RT4-D6P2T cells cultured on glass cover slips were fixed in 4% para-formaldehyde, permeabilized with 0.2% Triton X-100, blocked with 0.1% BSA in PBS and then probed with the tPA antibody (1:50 dilution). Detection was then performed using an Alexa fluor 488-conjugated secondary antibody. Nuclei were counterstained with DAPI (#940110 Vector Laboratories). Immunofluorescent images were captured using an Axiovert 40 fluorescence microscope (Carl Zeiss Inc.). Each condition was reproduced in three dishes <i>per</i> experiment. Representative photomicrographs were taken from at least three fields <i>per</i> dish in a fixed pattern. Original magnification 40X. Scale bar = 30μm.</p

Pituitary Adenylate Cyclase Activating Peptide Deficient Mice Exhibit Impaired Thymic and Extrathymic Regulatory T Cell Proliferation during EAE

<div><p>We have shown that mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) manifest enhanced sensitivity to experimental autoimmune encephalomyelitis (EAE), supporting the anti-inflammatory actions described for this neuropeptide. In addition to an increased proinflammatory cytokine response in these mice, a reduction in regulatory T cell (Treg) abundance in the lymph nodes (LN) was observed, suggesting altered Treg kinetics. In the present study, we compared in PACAP deficient (KO) vs. wild type mice the abundances and rates of proliferation FoxP3⁺ Tregs in three sites, the LN, central nervous system (CNS) and thymus and the relative proportions of Th1, Th2, and Th17 effector subsets in the LN and CNS. Flow cytometry analyses revealed a decrease in Treg proliferation and an increased T effector/Tregs ratio in the LN and CNS of PACAP KO mice. In the thymus, the primary site of <i>do novo</i> natural Treg production, the total numbers and proliferative rates of FoxP3⁺ Tregs were significantly reduced. Moreover, the expression of IL-7, a cytokine implicated in thymic Treg expansion during EAE, failed to increase at the peak of the disease in the thymus and LN of PACAP KO mice. In addition to these Treg alterations, a specific reduction of Th2 cells (about 4-fold) was observed in the lymph nodes in PACAP KO mice, with no effects on Th1 and Th17 subsets, whereas in the CNS, Th1 and Th17 cells were increased and Th2 decreased. Our results suggest that endogenous production of the neuropeptide PACAP protects against EAE by modulating Treg expansion and Th subsets at multiple sites.</p> </div

Dose- and time-dependent effects of PACAP on tissue plasminogen activator (tPA) mRNA and protein expression.

<p>Representative immunoblots and densitometric analyses (<b>a – b</b>) showing the effects of increasing concentrations of PACAP38 (10⁻¹⁰M to 10⁻⁶M, respectively) or (<b>a</b>) of a single concentration of the peptide (10⁻⁷M) on tPA protein expression at different time points (0, 3, 6, 12, 24, 48h). Protein extracts (20μg) obtained from rat RT4-D6P2T cultures grown as described were separated by SDS-PAGE and transferred to nitrocellulose membranes. Then, membranes were incubated using a rabbit anti-tPA (1:300, sc-15346, Santa Cruz Biotechnology) and a rabbit anti-β-tubulin antibody (H-235, cat n. sc-9104, Santa Cruz Biotechnology; 1:500) and scanned with an Odyssey Infrared Imaging System, as described in Materials and Methods section. Densitometric analyses were performed using the ImageJ software and values obtained were normalized to β-tubulin, which was used as loading control. Results are expressed as the average ratios ± S.E.M. from at least three independent determinations. <i>*p<0.05 or **p<0.01 Vs untreated control or t0</i>, as determined by One-way ANOVA followed by Dunnett’s <i>post-hoc</i> test. Kinetics of tPA mRNA expression in untreated cells (Ctrl) or following treatment with 10⁻⁷M PACAP38 at different times (0, 1, 2, 4, 6, 8, 10, 12, 24 and 48h, respectively) as determined by quantitative real time PCR analyses (<b>c</b>). Results are presented as mean fold changes with respect to Ctrl at the same experimental time ± S.E.M. Fold changes of tPA gene expression were obtained after normalization to the endogenous ribosomal protein S18 (housekeeping gene) and then calculated using the comparative ΔCt method. Baseline expression levels of the control group (Ctrl) were set to 1. Experiments were performed three times independently. <i>*p<0.05 or ***p<0.001 vs Ctrl at the same experimental time</i>, as determined by ANOVA followed by Dunnett’s <i>post-hoc</i> comparison.</p

PACAP- and BDNF-mediated induction of tPA expression and activity involves both the MAPK^Erk1/2 and Akt/cAMP-responsive element binding protein (CREB) signaling pathways.

<p>Representative zymographic (<b>a</b>) and Western blot analyses (<b>b</b>) demonstrating the effects of 10⁻⁷M PACAP or 2x10⁻⁹M BDNF treatment on tPA and uPA activity (<b>a</b>) and tPA protein expression (<b>b</b>) in RT4-D6P2T cells after 24h. tPA and uPA activities were determined by zymography as detailed in MATERIALS AND METHODS. The representative picture shows lytic areas (white bands) on a Coomassie-stained gel (blue background) that correspond to the predicted molecular weight for tPA (64-70kDa) and uPA (~50kDa). Bar graphs depicted below the gel indicate the relative activity of each plasminogen activator, measured as the mean band intensity obtained from at least three separate determinations and calculated using the ImageJ software. <i>*p<0.05 or **p<0.01 Vs Control</i>; ANOVA followed by Dunnett’s <i>post-hoc</i> test. (<b>c</b> and <b>c’</b>) Activation of the MAPK^Erk1/2 and Akt/CREB signalling cascades was assessed by measuring Erk1/2, Akt and CREB phosphorylation at the indicated phospho-sites using the same procedures described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117799#pone.0117799.g001" target="_blank">Fig. 1</a>. Relative activation was calculated by normalizing each phospho-protein level over the corresponding unphosphorylated protein, which also served as loading control. Each plotted result represents the mean ± SEM from three separate experiments. <i>**p<0.01 Vs Control</i>; ANOVA followed by Dunnett’s <i>post-hoc</i> test. n.s. = not significant.</p

PACAP gene expression is induced in the spinal cord and the lymph nodes of WT mice during EAE.

<p>PACAP mRNA levels were determined by real time RT-PCR in the spinal cord (A) and the lymph nodes (B) of C57BL/6 mice on days 0, 14 and 30 post-disease induction. The expression of PACAP was elevated in both tissues on day 14, and maintained on day 30. Bar charts, mean ± SEM of six individual mice. Student’s <i>t</i>-test *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

The relative abundance and proliferation of Tregs is diminished in the CNS of PACAP KO and the ratio of Teff to Tregs is enhanced.

<p>Panel <b>A</b> indicates the percentages of CD4⁺ cells in the CNS that were Tregs (CD4⁺CD25⁺Foxp3⁺) in WT and PACAP KO mice on days 0 (ND = non detectable), 14 and 20 days after EAE induction. Panel <b>B</b> reports the ratio Teff/Treg (calculated as the % of CD4⁺CD25⁺ cells that are Fox3^−/% of CD4⁺CD25⁺ cells that are Foxp3⁺). Panel <b>C</b> indicates, the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs that are proliferating as determined by co-expression with the Ki67 antibody (C). Values represented are mean ± SEM. One experiment representative of three (n = 6 each) is shown. Student’s <i>t</i>-test *<i>P</i><0.05; **<i>P</i><0.01. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061200#pone.0061200.s006" target="_blank">Fig. S6</a> for representative FACS plots and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061200#pone.0061200.s007" target="_blank">Fig. S7</a> for determinations of total number of cells, % of total cells that are Tregs, and total numbers of Tregs during the course of EAE in the CNS of PACAP KO vs. WT mice.</p

The relative abundance and proliferation of Tregs is diminished in the lymph nodes of PACAP KO and the ratio of Teff to Tregs is enhanced.

<p>Panel <b>A</b> indicates the percentages of CD4⁺ cells in the lymph nodes (LN) that were Tregs (CD4⁺CD25⁺Foxp3⁺) in WT and PACAP KO mice on days 0, 14 and 20 days after EAE induction. Panel <b>B</b> reports the ratio Teff/Treg (calculated as the % of CD4⁺CD25⁺ cells that are Fox3^−/% of CD4⁺CD25⁺ cells that are Foxp3⁺). Panel <b>C</b> indicates, the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs that are proliferating as determined by co-expression with the Ki67 antibody (C). Values represented are mean ± SEM. One experiment representative of three (n = 6 each) is shown. Student’s <i>t</i>-test *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 (*for comparison between values of WT and PACAP KO) and ^#<i>P</i><0.05; ^##P<0.01; ^###P<0.001 (^#for comparison between values on 0, 14 or 20 days within WT or PACAP KO mice strains). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061200#pone.0061200.s004" target="_blank">Fig. S4</a> for representative FACS plots and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061200#pone.0061200.s005" target="_blank">Fig. S5</a> for determinations of total number of cells, % of total cells that are Tregs, and total numbers of Tregs during the course of EAE in the lymph nodes of PACAP KO vs. WT mice.</p

Th profile analysis in PACAP KO vs WT mice on days 0, 14, and 20 after EAE induction.

<p>Th profiles were determined in cell suspensions from the lymph nodes <b>(A)</b> and the CNS <b>(B),</b> by intracellular flow cytometry staining of IFNγ (Th1), IL-17 (Th17) and IL-4 (Th2) after 4 h incubation with PMA/ionomycin/monensin. The graphs, where Y axis represents the percentage of CD4⁺ cells that are IFNγ⁺, IL-17⁺ or IL-4⁺, respectively, are accompanied by representative FACS plots corresponding to EAE day 14. Bars represent the mean ± SEM of six individual mice. A representative experiment out of three is shown. Student’s <i>t</i>-test *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001 (*for comparison between values of WT and PACAP KO) and ^#<i>P</i><0.05; ^##<i>P</i><0.01, ^###<i>P</i><0.001 (^#for comparison between values on 0, 14 or 20 days within WT or PACAP KO mice strains).</p