Fig 1.
Schematic experimental design of OVA-induced lung inflammation murine model.
Our experiments involved the use of 4 different mice groups (4 mice/group), as described in Methods. In all cases, mice were sacrificed at day 14 and the lungs were isolated for analysis.
Table 1.
Histological scoring system for inflammation in lungs of mice treated with DC and ACh-DC.
Fig 2.
ACh increases the expression of MHC class II and the stimulatory ability of murine DC.
(A) Phenotype of immature DC differentiated from bone marrow precursors from BALB/c mice was analyzed by flow cytometry. A representative experiment is shown. (B-C) DC (1x106 cells/ml) were treated or not with different concentrations of ACh or LPS 1μg/ml for 18h and the expression of MHC class II (anti-IAd) was evaluated by flow cytometry (B), while CD86 expression (C) was evaluated on ACh 10−11 M-treated DC. Representative histograms of median intensity of fluorescence (MIF) are shown and bars represent the mean ± SEM of 5–7 experiments (2 replicates/experiment). *p<0.05, **p<0.01 vs untreated DC, nonparametric Friedman test. (D) ACh 10−11 M-treated-DC and DC (5x104 cells) were washed and co-cultured with C57BL/6 splenocytes (1:5 ratio) for 5 days. The proliferation was determined by thymidine incorporation and data are expressed as the mean ± SEM of [3H] thymidine incorporation (n = 5 experiments, 2 replicates/experiment). *p<0.05, nonparametric Wilcoxon test.
Fig 3.
ACh enhances the production of pro-inflammatory cytokines on murine DC.
(A-D) DC (1x106 cells/ml) were incubated with or without ACh 10−11 M for 18 h. TNF-α (A), MCP-1 (B), IL-12p70 (C) and IL-10 (D) secretion was measured by ELISA (mean ± SEM, n = 4 experiments). *p<0.05; **p<0.01 nonparametric Wilcoxon test.
Fig 4.
ACh-induced activation of DC is mediated by mAChR.
(A-C) DC (1x106 cells/ml) were exposed or not to cholinergic receptors antagonists (AT and MM, 10-9M for 30 m) and then were incubated with or without ACh 10−11 M for 18 h. MHC Class II IAd expression (A) was measured by flow cytometry and a representative histogram of MIF of n = 6 experiments is shown (mean ± SEM, 2 replicates/experiment). (B-C) TNF-α (B) and MCP-1 (C) secretion was measured by ELISA (mean ± SEM, n = 4 experiments). *p<0.05, nonparametric Kruskal-Wallis for multiple comparisons with Dunn’s post-test. A. U.: arbitrary units.
Fig 5.
M3 mAChR is involved in ACh-induced activation of DC.
(A-C) DC (1x106 cells/ml) were exposed or not to selective M3 receptor antagonist (Tio 30nM for 30 m) and then were incubated with or without ACh 10−11 M for 18 h. M3 (A) and MHC II IAd (B) expression was determined by flow cytometry and representative histograms of MIF of 5 experiments are shown (mean ± SEM, 2 replicates/experiment). TNF-α production (C) was measured by ELISA (mean ± SEM, n = 4 experiments). *p<0.05, nonparametric Wilcoxon test.
Fig 6.
ACh-DC facilitates the development of inflammatory process in onset of OVA-induced lung inflammation murine model.
(A) H&E (upper and middle panels) and PAS staining (lower panel) of fixed lungs from mice of groups I-III are shown. In the upper panel, the full arrows indicate the alveolar septum (x50 magnification). In the middle panel the ellipses indicate peribronchiolar mononuclear infiltrate, the black arrows indicate epithelial damage and the insert in group II shows the recruitment of mononuclear cells into blood vessel (x400 magnification). In the lower panel, the full arrows indicate PAS positive cells, the black arrows indicate mucin secretion while the orange arrows indicate epithelial desquamation (x400 magnification). Al: Alveolus, V: Blood vessel, Br: Bronchus, Brl: Bronchiole. A representative experiment is shown (n = 4). (B) Peribronchial mononuclear infiltrate was quantified at x40 magnification in at least ten fields on sections stained with H&E and results are expressed as mean ± SEM of fold increase (B, left panel) while the alveolar involvement was evaluated according to a scoring system described in Methods (B, right panel). (C) Lung sections stained with PAS were examined at x10 magnification and the area proportion of mucus in % (mean ± SEM) was determinate as described in Methods. *p<0.05; ***p<0.001 nonparametric Wilcoxon test, n = 4 experiments.
Fig 7.
ACh-DC induces the recruitment of CD11b+CD11c- cells in the onset of OVA-induced lung inflammation accompanied by increased TNF-α production.
(A-B) Cells isolated from lungs were stained with mAb directed to CD11c and CD11b and analyzed by flow cytometry, gating mononuclear cells and excluding for the analysis the High Autofluorescence Cells. A representative experiment (A) and the quantification of CD11b+CD11c- and CD11b-CD11c+ cells (B) are shown (mean % ± SEM, n = 4 experiments). (C-D) Level of circulating anti-OVA IgE antibodies (C) and TNF-α (D) was measured at the end of the in vivo model in mice serum samples of day 14 by ELISA (mean ± SEM, n = 4 experiments). (C) A representative experiment is shown (OD media ± SEM, n = 4 animals/group). *p<0.05 nonparametric Wilcoxon test.