Central Muscarinic Cholinergic Activation Alters Interaction between Splenic Dendritic Cell and CD4⁺CD25^- T Cells in Experimental Colitis

<div><p>Background</p><p>The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4⁺/CD25⁻T cell activation in the context of experimental colitis.</p><p>Methods</p><p>The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4⁺CD25⁻T cell co-culture were determined.</p><p>Results</p><p>McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4⁺CD25⁻T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy.</p><p>Conclusions</p><p>Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.</p></div

Central administration of a M1mAchR agonist alleviates the severity of 2, 4-dinitrobenzene sulfonic acid (DNBS)–induced colitis through vagus nerve and splenic nerve signaling to the spleen.

<p>Vagotomy (VXP) and/or splenectomy (SPX), splenic neurectomy (NRX) and/or splenectomy (SPX) were performed 10 days prior to initiating McN-A-343 (5 ng/kg/day, i.c.v.) treatment and/or colitis induction as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109272#s2" target="_blank">Material and Methods</a>. *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; <b><i>A</i></b>: Serum amyloid protein (SAP); <b><i>B</i></b>: Colonic interferon-gamma (IFN-γ); <b><i>C</i></b>: Colonic interleukin (IL)-17; <b><i>D</i></b>: Colonic IL-12p70; <b><i>E</i></b>: Colonic IL-23 and <b><i>F</i></b>: Colonic IL-4. Values are shown as means ± SEM. Samples were collected on day 3 post-DNBS induction; mice per group ≥8. ^a<i>P</i><0.05 as compared to sham-saline-DNBS-treated group, ^b<i>P</i><0.05 as compared to VXP-DNBS-treated group or NRX-DNBS-treated group respectively, ^c<i>P</i><0.05 as compared to sham-McN-A-343-DNBS-treated group.</p

Central administration of a M1mAChR agonist alleviates splenic CD11c⁺ dendritic cells (DCs) interleukin (IL)-12p70 and IL-23 release in the context of 2, 4-dinitrobenzene sulfonic acid (DNBS)–induced colitis through vagus nerve and splenic nerve signaling to the spleen.

<p><b><i>A</i></b>: IL-12p70 and <b><i>B</i></b>: IL-23 production from splenic CD11c⁺ DCs. Splenic CD11c⁺ DCs were isolated from control and McN-A-343 (5 ng/kg/day, i.c.v. for 4 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 3 post-DNBS induction incubated <i>ex vivo</i> or not with GTS-21 (a specific α7nAChR agonist, 100 µM). IL-12p70 and IL-23 were measured in media at 24 hrs following treatments. Values are shown as means ± SEM, 3 independent experiments with 4 mice per group. ^a<i>P</i><0.05 as compared to DNBS-control group, ^b<i>P</i><0.05.</p

Central administration of a M1mAchR agonist alleviates macroscopic markers of 2, 4-dinitrobenzene sulfonic acid (DNBS)–induced colitis through vagus nerve and splenic nerve signaling to the spleen.

<p>Vagotomy (VXP) and/or splenectomy (SPX), splenic neurectomy (NRX) and/or splenectomy (SPX) were performed 10 days prior to initiating McN-A-343 (5 ng/kg/day, i.c.v.) treatment and/or colitis induction as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109272#s2" target="_blank">Material and Methods</a>. *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; <b><i>A</i></b>: Macroscopic score and <b><i>B</i></b>: colon length were evaluated. Values are shown as means ± SEM. Samples were collected on day 3 post-DNBS induction; mice per group ≥8. ^a<i>P</i><0.05 as compared to sham-saline-DNBS-treated group, ^b<i>P</i><0.05 as compared to VXP-DNBS-treated group or NRX-DNBS-treated group respectively, ^c<i>P</i><0.05 as compared to sham-McN-A-343-DNBS-treated group.</p

Central administration of a M1mAchR agonist alleviates the severity of Dextran sulfate sodium (DSS 5%)–induced colitis.

<p>Vagotomy (VXP) or splenic neurectomy (NRX) were performed 10 days prior to initiating McN-A-343 (5 ng/kg/day, i.c.v.) treatment and/or colitis induction as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109272#s2" target="_blank">Material and Methods</a>. Macroscopic score, colon length, serum amyloid protein (SAP), colonic interferon-gamma (IFN-γ), interleukin (IL)-17, IL-12p70, IL-23 and IL-4. IL-12p70 and IL-23 release from splenic CD11c⁺ dendritic cells (DC). Splenic CD11c⁺ DCs were isolated from McN-A-343 (5 ng/kg/day, i.c.v. for 6 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 5 post-colitis induction. Effect of McN-A343 (<i>in vivo</i>) treatment on splenic CD11c⁺ DCs function and sequential CD4⁺ CD25⁻T cell activation. Splenic CD11c⁺ DCs isolated from different colitic group were cultured in for 24 h before being co-cultured with CD4⁺CD25⁻T cells isolated from naïve mice. The levels of IFN-γ and IL-17 and IL-4 were measured in media at 24 hrs.</p>a<p><i>P</i><0.05 as compared to sham-saline-DSS-treated group,</p>b<p><i>P</i><0.05 as compared to VXP-DSS-treated group or NRX-DSS-treated group.</p>c<p><i>P</i><0.05 as compared to NRX-DSS-treated group.</p><p>Central administration of a M1mAchR agonist alleviates the severity of Dextran sulfate sodium (DSS 5%)–induced colitis.</p

Experimental protocol.

<p>On day 1, mice were anaesthetized using ketamine (150 mg/kg, i.p) and xylazine (10 mg/kg, i.p) and i.c.v. Placement of the cannula, splenic neurectomy (NRX), subdiaphragmatic bilateral vagotomy (VXP), or splenectomy (SPX) were performed. Mice were allowed to recover for 10 days. Completeness of vagotomy was verified using the CCK-8 satiety test starting on day 8. On day 11, pharmacological treatment started: micro-osmotic pumps were connected to cannula and filled with vehicle or the McN-A-343. On day 12, colitis was induced using two specific models of gut inflammation: 2,4-dinitrobenzene sulfonic acid (DNBS) or dextran sulfate sodium (DSS) models. Colitis was induced by intra-rectal administration of 100 µl of 4 mg of DNBS solution in 30% ethanol and the mice left for 3 days. Alternatively, DSS was added to the drinking water in a final concentration of 5% (wt/vol) for 5 days. Controls were all time-matched and consisted of mice that received normal drinking water only. Colonic samples were collected 3 and 5 days post-DNBS and DSS activation respectively.</p

Role of the CD11c⁺ dendritic cells (DCs) in CD4⁺CD25⁻ T cells priming in the context of 2, 4-dinitrobenzene sulfonic acid (DNBS)–induced colitis.

<p>Effect of McN-A343 (<i>in vivo</i>) and GTS-21 (<i>ex vivo</i>) treatments on splenic CD11c⁺ DCs function and sequential CD4⁺CD25⁻T cell activation. Splenic CD11c⁺ DCs were isolated from control and McN-A-343 (5 ng/kg/day, i.c.v. for 4 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 3 post-DNBS induction and were cultured in the presence or absence of GTS-21 (a specific α7nAChR agonist, 100 µM) for 24 hrs before medium was washed and co-cultured with CD4⁺/CD25⁻ T cells isolated from naïve mice. The level of <b><i>A</i></b>: Interferon-gamma (IFN-γ); <b><i>B</i></b>: Interleukin (IL)-17 and <b><i>C</i></b>: IL-4 were measured in media at 24 hrs; <b><i>D</i></b>: CD4⁺CD25⁻ T cells proliferation. ^a<i>P</i><0.05 as compared to DNBS-control group, ^b<i>P</i><0.05, n = 8, data are representative of 3 independent experiments with quadruplicated cultures, mean ± SEM.</p

Implication of the interleukin (IL)-12p70 and IL-23 pathways during CD4⁺CD25⁻ T cells priming by splenic CD11c⁺ dendritic cells (DCs) in the context of 2, 4-dinitrobenzene sulfonic acid (DNBS)–induced colitis.

<p>Splenic CD11c⁺ DCs isolated from different colitic group were cultured in for 24 h before being co-cultured with CD4⁺/CD25⁻ T cells isolated from naïve mice in the presence or absence of <b><i>A, B</i></b>: anti-p19 mAb (10 ug/ml⁻¹) or anti-p35 mAb (10 ug/ml⁻¹); <b><i>C, D</i></b>: recombinant (r)IL-12p70 (25 ng/ml⁻¹) or rIL-23 (25 ng/ml⁻¹) protein. Supernatant were collected after 24 h. The levels of interferon-gamma (IFN-γ) and IL-17 in the culture supernatant were investigated at 24 hrs. *<i>P</i><0.05, data are representative of 3 independent experiments with quadruplicated cultures, mean ± SEM.</p

Implication of the NF-kB pathway in splenic CD11c⁺ dendritic cells (DCs) cytokine release in the context of 2, 4-dinitrobenzene sulfonic acid (DNBS)–induced colitis.

<p>Interleukin (IL)-12p70 and IL-23 production from splenic CD11c⁺ DCs. Splenic CD11c⁺ DCs were isolated from control and McN-A-343 (5 ng/kg/day, i.c.v. for 4 days)-treated groups of colitic mice subjected to sham-operation, vagotomy (VXP) or splenic neurectomy (NRX) on day 3 post-DNBS induction incubated <i>ex vivo</i> or not with GTS-21 (a specific α7nAChR agonist, 100 µM) or with <b><i>A, B</i></b>: betulinic acid (a specific NF-κB activator, 10 µM) or <b><i>C, D</i></b>: BAY 11-7082 (a specific NF-κB inhibitor, 10 µM). IL-12p70 and IL-23 were measured in media at 24 hrs following treatments. Values are shown as means ± SEM, 3 independent experiments with 4 mice per group. ^a<i>P</i><0.05 as compared to DNBS-control group, ^*<i>P</i><0.05.</p

GTS-21 attenuates cisplatin-induced renal cell apoptosis.

<p>Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. Renal cell apoptosis was measured by TUNEL staining and representative photomicrographs are shown (at 400x magnification; scale bar, 50 μm) in (A). Apoptosis was determined by counting the number of TUNEL positive cells per high power field (HPF) using random sections and the mean apoptosis scores (±SEM) are shown (B). *** = p<0.001 vs. CTRL, ⁺⁺ = p<0.01 vs. CIS.</p