Pituitary adenylate cyclase activating peptide deficient mice exhibit impaired thymic and extrathymic regulatory T cell proliferation during EAE

Abstract

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 do novo 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.

Figure 1. PACAP gene expression is induced in the spinal cord and the lymph nodes of WT mice during EAE.

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 t-test *P<0.05; **P<0.01; ***P<0.001.

Figure 2. 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.

Panel A 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 reports the ratio Teff/Treg (calculated as the % of CD4⁺CD25⁺ cells that are Fox3^−/% of CD4⁺CD25⁺ cells that are Foxp3⁺). Panel C 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 t-test *P<0.05; **P<0.01; ***P<0.001 (*for comparison between values of WT and PACAP KO) and ^# P<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 Fig. S4 for representative FACS plots and Fig. S5 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.

Figure 3. The relative abundance and proliferation of Tregs is diminished in the CNS of PACAP KO and the ratio of Teff to Tregs is enhanced.

Panel A 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 reports the ratio Teff/Treg (calculated as the % of CD4⁺CD25⁺ cells that are Fox3^−/% of CD4⁺CD25⁺ cells that are Foxp3⁺). Panel C 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 t-test *P<0.05; **P<0.01. See Fig. S6 for representative FACS plots and Fig. S7 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.

Figure 4. The expansion of thymic Tregs is impaired in PACAP KO mice.

We determined the proportions (A, B, E) and total numbers (F) of Tregs in the thymus in mice with no EAE or mice with EAE on day 14 and 20 post-immunization by using CD4, CD8 and Foxp3 antibodies and proliferation (C, D) by using an anti-Ki67 antibody. A and C are representative FACS plots for each group, and B and D are the mean values ± SEM with Student’s t-test *P<0.05; **P<0.01; ***P<0.001 (*for comparison between values of WT and PACAP KO) and ^## P<0.01; ^### P<0.001 (^#for comparison between values on 0, 14 or 20 days within WT or PACAP KO mice strains). Data shown are representative of three experiments. The Y axis in panel B represents the percentage of CD4⁺CD8⁻ cells that are Foxp3⁺. Panel D, the Y axis indicates the percentage of CD4⁺CD8⁻Foxp3⁺ cells that are Ki67⁺. See Fig. S8 for determinations of total number of cells during the course of EAE in the thymus of PACAP KO vs. WT mice.

Figure 5. IL-7 gene expression in the thymus and lymph nodes of naive and MOG-injected WT and PACAP KO mice.

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 **P<0.01; ***P<0.001 by Student’s t-test.

Figure 6. Th profile analysis in PACAP KO vs WT mice on days 0, 14, and 20 after EAE induction.

Th profiles were determined in cell suspensions from the lymph nodes (A) and the CNS (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 t-test *P<0.05; **P<0.01, ***P<0.001 (*for comparison between values of WT and PACAP KO) and ^# P<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).