br Materials and methods br Results br Discussion The data

Materials and methods
Results
Discussion The data presented here demonstrate the important role of B lymphocyte-expressed α7 nAChRs in regulating the antibody immune response. It is shown that, in addition to ACh produced by activated T lymphocytes (Fujii et al., 2012), activated B lymphocytes can be affected by endogenous, self-produced ACh, which is a negative regulator of their proliferation. The anti-proliferative effect of ACh is mediated by α7 nAChRs, which are the main nAChR subtype of mature B lymphocytes (Koval et al., 2011). This conclusion is in accord with our previously published data showing increased proliferative response in B lymphocytes of α7−/− mice and in the presence of MLA (Koval et al., 2009, Koval et al., 2011). However, it argues against the previously published evidence that α7-positive B lymphocytes propagated in the spleen of α7-negative hosts only if the hosts were chronically treated with nicotine (Skok et al., 2006). Here, we observed proliferation of non-stimulated B lymphocytes in the presence of either MLA or α7-specific antibody. This data suggests that B lymphocyte-expressed α7 nAChRs are connected to intercellular mitogenic pathway, which can be triggered in ion channel-independent manner and, therefore, the stimulating effect of nicotine may be not related to the nAChR ion channel function. This suggestion may help explain contradictory results obtained concerning the pro- or anti-proliferative effects of nicotine in different experimental models (Ishizuka et al., 2012, Cardinale et al., 2012, Medjber et al., 2015, Zeng et al., 2014). In contrast, MLA inhibited LPS-induced IgM production by B lymphocytes suggesting that accelerated proliferation was disadvantageous for the antibody synthesis. In one of our early papers, we have already shown that blocking hybridoma cell proliferation stimulated its antibody production, illustrating the reciprocal relation of mitogenic and biosynthetic activity of B lymphocyte-derived BMS-387032 (Skok et al., 2003). The data presented here indicate that ion-independent signaling through α7 nAChR favors B lymphocyte proliferation but prevents the antibody production. It is important that, after being activated, B lymphocytes down-regulate α7 nAChR expression and, therefore, become less sensitive to cholinergic regulation through this nAChR subtype. Previously we reported that α7 nAChRs are equally important for the formation of follicular, transitional and marginal zone splenic B lymphocyte subpopulations (Skok et al., 2006). Here we show that both CD5+ and Foxp3+ B lymphocytes are α7high and α7 nAChRs are necessary for the formation, induction and functioning of regulatory B lymphocytes: B cells from α7−/− mice formed less Foxp3+ cells in response to LPI activation and their induction in WT mice was inhibited by both PNU282987 and MLA. IL-10 production stimulated by LPI was also inhibited by α7 nAChR ligands. Therefore, signaling through α7 nAChR is required for activation/induction of both regulatory B cell subsets: Foxp3 expressing and IL-10 producing ones; it can be impaired by either lack of α7 or its inhibition (MLA), desensitization by chronic presence of agonist (PNU282987) or blocking by the antibody. Expression and importance of α7 nAChRs in regulatory T lymphocytes has been documented (Galitovskiy et al., 2011). In that paper, stimulation of CD4+ colonic T lymphocytes by nicotine in vivo favored Foxp3 expression and formation of IL-10-producing Tregs that correlates with our data. Taken together, this data indicate that regulatory branch of immunity (Tregs and Bregs) is subjected to cholinergic modulation via α7 nAChRs. CD5+ B lymphocytes also include a B1 subset, which belongs to the evolutionary ancient branch of immunity (Janeway Jr et al., 2001). Cholinergic regulation by non-neuronal ACh is also an ancient mechanism influencing cell survival and proliferation (Wessler et al., 1999) and α7 subunits are encoded by one of the most ancient members of the nAChR genes superfamily (Ortells and Lunt, 1995). Therefore, high level of α7 nAChR expression in B1 cells may be important for supporting their survival and self-renewal.