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

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

IL-7 gene expression in the thymus and lymph nodes of naive and MOG-injected WT and PACAP KO mice.

<p>The expressions of IL-7 mRNA in the thymus (A) and lymph nodes (LN) (B) of WT and PACAP KO mice were determined by real time RT-PCR on days 0, 14 and 20 after EAE immunization (n = 6 for each group). Arbitrary units were calculated using the 2^−ΔΔCt formula as described in Material and Methods and the means ± SEM are shown. All WT inductions were significant compared to basal levels. Significance of comparison between WT and PACAP KO at each time point analyzed is shown in the Fig., with **<i>P</i><0.01; ***<i>P</i><0.001 by Student’s <i>t-test.</i></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

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

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

Micrographs of main olfactory tissue from Chat-GFP mice show diffuse labeling throughout the bulb with some regions of high labeling in the GL.

<p><b><i>A–C</i></b><b>.</b> High-resolution composite micrographs (shown for visualization only) of MOB tissue. Plane orientation: Sg - sagittal, Cr - coronal, Hz - horizontal; a = anterior, p = posterior, d = dorsal, v = ventral, l = lateral, m = medial. Layer labels: <i>nl -</i> nerve; <i>gl -</i> glomerular; <i>epl</i> - external plexiform; <i>gr</i> - granule cell; <i>ml</i> - mitral cell; <i>ipl</i> - internal plexiform. Scale bar indicates 500 µm. <b><i>A.</i></b> Parasagittal section. <i>Arrow</i> points to region of relatively heavy GFP labeling in the anterior region of the bulb. <i>Arrow head</i> indicates region where relatively little labeling is found. <b><i>B.</i></b> Coronal section. <i>oc -</i> orbital cortex. <i>aob</i> - accessory olfactory bulb. <i>aon -</i> anterior olfactory nucleus. <b><i>C.</i></b> Micrograph of a coronal cross-section of the MOB and AOB. Arrow points to heavily staining glomeruli shown in inset. <b><i>C inset</i></b><b>.</b> High-resolution micrograph of glomeruli with a relatively high amount of GFP staining. <b><i>D</i></b><b>.</b> High-resolution micrograph of the AOB showing that the relatively light GFP staining in the anterior portion of the structure did not co-label with G_oα labeled in red. <b><i>E–F</i></b><b>.</b> Heavily-stained GFP glomeruli do not co-label with PDE 2A (<b>E</b>) or PDE 4A (<b>F</b>). Red = PDE#A, Green = GFP. Scale bar = 100 µm.</p

The pattern and intensity of GFP staining in the GL and GCL of the MOB across different developmental stages in ChAT-GFP mice.

<p><b>A–D.</b> Representative cross-sections of MOB cut horizontally from four different age groups: Post-natal day (PD) 02, 06, 12, and adult. Scale bar indicates 100 µm. <b>E–H</b> Average intensity maps of GFP-staining across the glomerular surface of each MOB. <i>Abscissa</i>: rostro-caudal position realigned to the standard bulb where 0 µm corresponds to the anterior end of the bulb. <i>Ordinate:</i> cylindrical coordinates realigned so that 0° corresponds to dorsal, 90° to lateral, 180° to ventral, 270° to medial surfaces of the bulb. The color bar indicates the level of corrected green intensity in the GL of the MOB with warm colors indicating higher levels of green intensity. <b>I.</b> Intensity map from H overlaid on a standardized, three-dimensional representation of the inner GL of the MOB showing the heterogenous staining pattern on the lateral surface of the bulb. Axes range: d – dorsal, v- ventral; r – rostral, c – caudal; m – medial, l – lateral. <b>J–L.</b> Changes in GFP intensity in the glomerular (GL) and granule cell (GC) layers of the bulb across age groups. <b>J.</b> The level of GFP intensity in the <b>GL layer</b> significantly increases up to post-natal day (PD) 12 and then decreases in adult animals (Anova p-value: 3×10⁻³⁶). A multiple comparison test using Tukey's HSD criterion shows: ** PD12 mean GL intensity is significantly greater than all other age groups. * PD06 and Adult mean GL intensities are significantly greater than PD02, but significantly lower than PD12. They are not significantly different from each other. <b>K.</b> The level of GFP intensity in the <b>GC layer</b> significantly levels out at PD6 and then decreases in adult animals (Anova p-value: 3×10⁻³⁶). L. GC intensity subtracted from GL intensity highlighting the change in relative intensity between GC and GL during development.</p

Standardized and normalized GFP intensity maps show significant differences in the distribution of GFP intensity between PD12 and adult age MOBs.

<p><b>A–B.</b> GFP intensity maps realigned to standardized rostro-caudal and dorso-ventral positions. <b>C.</b> A map of p-values generated from a bin-wise Mann-Whitney U test comparing bins with identical cylindrical coordinates across the PD 12 and adult GFP intensity maps. Each p-value is calculated from a <i>U</i>-test comparing bins from intensity maps across both occluded and non-occluded bulbs that have identical cylindrical coordinates. p-values that fall below the false discovery rate (0.014, hatch on the color bar) are circumscribed in black and indicate regions on the map that exhibit a statistically significant change in the level of GFP intensity <b>D–F.</b> Probability map from <b>A–C</b> overlaid on a 3D reconstruction of the olfactory bulb (standard bulb). Wire mesh in F indicates the bins compared in C.</p

ChAT-GFP colabels with anti-ChAT antibody and distinctly labels cholinergic fibers with a greater signal to noise ratio so that finer details of the cholinergic fibers can be resolved.

<p><b>A.</b> Single color channel (A; GFP & B; ChAT) and merged (C) confocal images of a glomerulus (dashed line) in the main olfactory bulb.</p