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

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

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 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

VIP KO mice exhibit reduced mortality and lung histopathology in response to LPS injection.

<p>Female WT (C57BL6) and VIP KO mice were injected i.p. with LPS (40 mg/Kg). A, Kaplan Meier curve analysis of survival cumulative of four experiments (total WT n = 29; VIP KO n = 28) (Curve comparison Logrank test **p<0.01). B, Representative sections of lungs from control (noninjected) or LPS-injected WT and VIP KO mice (24 hours post injection) stained with H&E. C, Histological scores of LPS-injected WT vs. VIP KO mice (mean of two experiments; total WT n = 7; VIP KO n = 9), 24 hours after LPS injection, scored from 0 to 3 according to the level of lung inflammation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036922#s2" target="_blank"><i>Materials and Methods</i></a>. (Student's <i>t</i>-test *p<0.05).</p

Peritoneal cells from VIP KO mice exhibit an intrinsic defect in cytokine response to LPS- administration.

<p>Peritoneal cells were collected from WT (n = 3) and VIP KO mice (n = 3), and cultured in complete RPMI in triplicate in the presence or absence of LPS (10 ng/ml). Supernatants were collected 2 (A) and 16 h (B) later, and stored at −20°C for analysis of TNFα and IL-6 levels by ELISA. Student's <i>t</i>-test *p<0.05; **p<0.01; ***p<0.001. Representative data are shown of four independent experiments.</p

VIP KO mice exhibit reduced levels of proinflammatory cytokines in the peritoneal fluid and serum.

<p>Female WT (C57BL6) (n = 6) and VIP KO mice (n = 6) were injected i.p. with LPS (40 mg/Kg), and serum and peritoneal suspensions were collected 0, 3 and 6 (and also 24 for IL-10) hours post-injection. The levels of TNFα, IL-6, IL-12p40 and IL-10 were assessed by sandwich ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036922#s2" target="_blank"><i>Materials and Methods</i></a>. Student's <i>t</i>-test *p<0.05; **p<0.01. One of three representative experiments is shown.</p

Influence of dietary alteration and TBI on molecules associated with anxiety-like behavior.

<p>Protein levels of (<b>A</b>) NPY1R and (<b>B</b>) correlation analysis of NPY1R with anxiety-like behavior in groups fed with omega-3 (n-3 diet) or omega-3 deficient (n-3 def) diet, transitioned to western diet (n-3 diet/WD and n-3 def/WD) and subjected to fluid percussion injury after diet transition (n-3 diet/WD/FPI and n-3 def/WD/FPI). Values are expressed as mean ± SEM. ^#p<0.05 Vs n-3 def, ^@p<0.05 Vs n-3 diet, ^*p<0.05 Vs their respective n-3 diet group by ANOVA (two-way) and Newman–Keuls post-hoc test.</p