Figure 1.
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.
Figure 2.
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.
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 Material and Methods. *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; A: Macroscopic score and B: colon length were evaluated. Values are shown as means ± SEM. Samples were collected on day 3 post-DNBS induction; mice per group ≥8. aP<0.05 as compared to sham-saline-DNBS-treated group, bP<0.05 as compared to VXP-DNBS-treated group or NRX-DNBS-treated group respectively, cP<0.05 as compared to sham-McN-A-343-DNBS-treated group.
Table 1.
Central administration of a M1mAchR agonist alleviates the severity of Dextran sulfate sodium (DSS 5%)–induced colitis.
Figure 3.
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.
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 Material and Methods. *Sham represents data obtained in sham SPX mice, because no significant differences were determined between this group and any other sham group of animals; A: Serum amyloid protein (SAP); B: Colonic interferon-gamma (IFN-γ); C: Colonic interleukin (IL)-17; D: Colonic IL-12p70; E: Colonic IL-23 and F: Colonic IL-4. Values are shown as means ± SEM. Samples were collected on day 3 post-DNBS induction; mice per group ≥8. aP<0.05 as compared to sham-saline-DNBS-treated group, bP<0.05 as compared to VXP-DNBS-treated group or NRX-DNBS-treated group respectively, cP<0.05 as compared to sham-McN-A-343-DNBS-treated group.
Figure 4.
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.
A: IL-12p70 and 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 ex vivo 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. aP<0.05 as compared to DNBS-control group, bP<0.05.
Figure 5.
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.
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 ex vivo or not with GTS-21 (a specific α7nAChR agonist, 100 µM) or with A, B: betulinic acid (a specific NF-κB activator, 10 µM) or C, D: 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. aP<0.05 as compared to DNBS-control group, *P<0.05.
Figure 6.
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.
Effect of McN-A343 (in vivo) and GTS-21 (ex vivo) 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 A: Interferon-gamma (IFN-γ); B: Interleukin (IL)-17 and C: IL-4 were measured in media at 24 hrs; D: CD4+CD25− T cells proliferation. aP<0.05 as compared to DNBS-control group, bP<0.05, n = 8, data are representative of 3 independent experiments with quadruplicated cultures, mean ± SEM.
Figure 7.
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.
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 A, B: anti-p19 mAb (10 ug/ml−1) or anti-p35 mAb (10 ug/ml−1); C, D: 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. *P<0.05, data are representative of 3 independent experiments with quadruplicated cultures, mean ± SEM.