Distinct Roles of α4β2 and α7 Nicotinic Receptors in the Modulation of Inflammatory Responses in TNBS-Induced Colitis

Background and aim: Accumulating evidence indicates that vagus nerve signaling through nicotinic receptors (nAChRs) negatively modulates the inflammatory and immune responses in various clinical and experimental conditions, such as Inflammatory Bowel Disease (IBD), purportedly through a spleen-dependent pathway. Our aims were: 1)to pharmacologically investigate the role played by α4β2 and α7 nAChRs in the modulation of local and systemic inflammatory responses in 2,4,6-TriNitroBenzene Sulfonic acid (TNBS)-colitis in mice; 2)to assess the involvement of the spleen in nicotinic responses. Methods: Colitis was induced in Swiss mice by enema (i.r.) administration of 5mg/mouse TNBS 6 days after skin sensitiza- tion. Mice were randomly assigned to: CTR: saline (0.9% NaCl) 10 ml/kg TC: α4β2 agonist TC2403 5mg/kg DBE: α4β2 antagonist Dihydro-βErythroidine 1.5mg/kg AR-R: α7 agonist AR-R17779 1.5mg/kg MLA: α7 antagonist MethylLycAconitine 1mg/kg Pharmacological treatments started 8h after TNBS enema and were administered subcutaneously (s.c.) twice daily for 3 days. Normal mice (N) received saline 50 μL i.r. and 10 ml/kg s.c.. In a second series of experiments, mice subjected to splenectomy (SPX), performed 14 days before colitis induction, were administered with saline (SPX-CTR) or with AR-R17779 1.5mg/kg (SPX- AR). We determined clinical outcome as Disease Activity Index (DAI), colonic damage as Macroscopic Score (MS) and thickness and colonic and lung myeloperoxidase (MPO) activity. + Splenic and mesenteric lymph nodes (MLN) CD3 T cells were counted by flow cytometry. All animal experiments were performed according to the guidelines for the use and care of laboratory animals (DL 26/2014). Results: Compared to N, CTR mice showed markedly higher DAI (P<0.001), MS (p<0.001), colonic thickness (p<0.01), colonic (P<0.001) and + lungMPO(p<0.01);CD3 Tcellswereincreasedinthespleen(P<0.01)andslightlydecreased in MLN. Compared to CTR, TC improved only DAI (p<0.01), while DBE significantly reduced colonic MPO and DAI (p<0.05). AR-R significantly ameliorated MS (p<0.05), colonic MPO and thickness (p<0.05), while MLA slightly augmented MS and lung MPO but did not affect the other markers. In SPX-AR mice, α7 agonist lost efficacy in decreasing MS and MPO and worsened DAI but reduced colonic thickness (p<0.01); no effects were produced on T cells, either with or without SPX.Conclusions: The weak and seemingly contradictory effects shown by α4β2 agents indicated a controversial role of this nAChR subtype in TNBS colitis, thus requiring further investigations. On the contrary,α7 nAChRs, either tonically activated by the cholinergic anti-inflammatory reflex or exogenously stimu- lated, play a protective role against the inflammatory responses triggered by TNBS: this effect, not involving changes in T cells trafficking, does not rely exclusively on the spleen