Nicotinic modulation in TNBS-induced colitis: focus on spleen and T-cells

Abstract

Background and aim: Increasing evidence indicates that vagus nerve signalling can modulate inflammation through the activation of nicotinic receptors (nAChRs) in different inflammatory conditions, like Inflammatory Bowel Disease (IBD). However, the kind of nAchRs subtypes and spleen involvement in such “Cholinergic Anti-inflammatory Pathway” (CAP) remain to be elucidated. Our aim was to pharmacologically investigate the role played by α7 and α4β2 nAChRs in 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-colitis by administering different doses of selective α7 and α4β2 ligands in normal and splenectomised mice. Given the crucial role played by T-cells in colitis and CAP, we analyzed T-cells and relative subsets in the spleen and mesenteric lymph nodes (MLN). In particular, we focussed on CD4+CD25+FoxP3+ cells (T-regs), a crucial population in resolving inflammation, to investigate their role in the nicotinic modulation of the inflammatory response, in both normal and splenectomised (SPX) mice. Methods: Colitis was induced in female CD/1 mice (7-12 weeks old) by enema (i.r.) administration of 5mg/mouse TNBS, 6 days after skin sensitization. Mice were randomly assigned to: TNBS: saline 10ml/kg subcutaneously (s.c.); SULF: sulfasalazine (50 mg/kg) per os (p.o.); AR: α7 agonist AR-R 17779 (0.5; 1.5; 5 mg/kg) s.c.; MLA: α7 antagonist methyllycaconitine (0.1; 0.5; 1 mg/kg) s.c; TC: α4β2 agonist TC 2403 (2; 5 mg/kg) s.c.; DBE: α4β2 antagonist Dihydro-βerythroidine (0.5; 1.5; 5 mg/kg) s.c.. Pharmacological treatments started 8h after TNBS enema and were administered twice daily for 3 days. SHAM mice received saline 50 µL i.r. and 10 mL/kg s.c.. In a second series of experiments, colitic SPX mice were similarly administered with saline (SPX/TNBS) or with AR-R17779 1.5 mg/kg (SPX/TNBS+AR), while SPX/SHAM received saline i.r. and s.c.. We determined Disease Activity Index (DAI), colonic Macroscopic Score (MS), colon length and thickness, colonic and lung myeloperoxidase (MPO) activity. Splenic and MLN T-cells and relative subpopulations were analysed by flow cytometry. Colonic levels of IL-10 were determined through ELISA assay. All animal experiments were authorised and performed according to the guidelines for the use and care of laboratory animals (DL 26/2014). Results: TNBS mice, compared to SHAM, showed higher DAI, MS, colonic thickness, colonic and pulmonary MPO levels (p<0.001), whilst colonic length was lower (p<0.001). In colitic mice only treatment with 1.5 mg/kg AR-R showed protective effects, comparable to those elicited by SULF, as it reduced DAI (P<0.01), MS, colonic thickness and colonic MPO and increased colonic length (p<0.05). MLA was ineffective on most of the evaluated parameters. α4β2 agents did not impact the severity of colitis, with the exception of DAI (p<0.01), ameliorated by both TC and DBE, and colonic MPO, decreased by DBE (p<0.05). After SPX, AR-R lost the protective effects as colonic length, thickness and MPO were not significantly different between SPX/TNBS and SPX/TNBS+AR groups, whilst DAI and MS were higher in SPX/TNBS+AR than in SPX/TNBS (p<0.05). As regards T-lymphocytes, colitis induction provoked an increase in splenic CD3+ T-cells (p<0.01) and MLN T-regs (p<0.05), and a mild reduction of splenic T-regs and colonic IL-10 levels. AR treatment reduced splenic T-cells and MLN T-regs (p<0.05) and moderately raised splenic T-regs and colonic IL-10. In SPX mice, MLN T-regs and colonic IL-10 were not different among groups. Conclusions: α7 nAChRs agonist AR-R17779 showed beneficial effects against inflammatory responses elicited by TNBS and these effects were spleen-dependent. Induction of colitis, treatment with AR and splenectomy evoked changes in T-cells and relative subpopulations, resulting in a complex profile. The relationship between these alterations, disease severity and the α7-mediated modulation of the immune response remains to be clarified.