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Expression of α7 nicotinic acetylcholine receptors (nAChRs) on antigen presenting cells (APCs), such as macrophages and dendritic cells, is now well established. We have shown that GTS-21, a selective α7 nAChR agonist, downregulates APC-dependent CD4+ T cell differentiation into regulatory T cells (Tregs) and effector Th1, Th2 and Th17 cells by inhibiting antigen processing, thereby interfering with antigen presentation. α7 nAChRs on Jurkat human leukemic T cells require functional T cell receptors (TCRs)/CD3 and leukocyte-specific tyrosine kinase to mediate nicotine-induced Ca2+-signaling via Ca2+ release from intracellular stores, and are insensitive to two conventional α7 nAChR antagonists, α-bungarotoxin (α-BTX) and methyllycaconitine (MLA). We investigated the effects of GTS-21, α-BTX and MLA on ovalbumin (OVA)-induced Th cytokine release from spleen cells isolated from OVA-specific TCR transgenic DO11.10 mice. We found that: (1) GTS-21 dose-dependently suppresses OVA-induced IFN-γ, IL-4 and IL-17 release, but neither α-BTX nor MLA alone affected the OVA-induced cytokine release. (2) Neither α-BTX nor MLA abolished the suppressive effects of GTS-21 on IFN-γ and IL-17 release from OVA-activated DO11.10 spleen cells. (3) GTS-21 significantly suppressed OVA-induced APC-dependent CD4+ T cell differentiation into Tregs. Neither MLA nor mecamylamine, a non-specific nAChR antagonist, abolished the suppressive effect of GTS-21 on Treg differentiation. These results suggest that α7 nAChRs on APCs involved in cytokine synthesis and T cell differentiation are insensitive to the conventional α7 nAChR antagonists, α-BTX and MLA, and that α7 nAChRs on APCs differ pharmacologically from those in neurons.
Expression of a non-neuronal cholinergic system in immune cells, including T and B cells as well as antigen presenting cells (APCs), such as macrophages and dendritic cells, has been extensively studied from both genetic and functional viewpoints , . The finding that α7 nicotinic acetylcholine receptor (nAChR) stimulation suppresses the synthesis and release of tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-activated mouse macrophages drew attention to the role of α7 nAChRs in the regulation of inflammatory responses . Later, Fujii et al showed that α7 nAChR gene-deficient (α7-KO) mice immunized with ovalbumin (OVA) exhibit significantly higher serum antigen-specific IgG1 concentrations than wild-type C57BL/6J mice . Moreover, they also found that, in the presence of OVA, OVA-immunized spleen cells from α7-KO mice produce greater amounts of TNF-α, interferon (IFN)-γ and interleukin (IL)-6 than do those from the wild-type mice. All of these findings suggest a role for α7 nAChRs in the regulation of immune function.
Nicotine, acetylcholine (ACh), and α7 selective nAChR agonists, such as 3-[(2,4-dimethoxy)benzylidene]-anabaseine (GTS-21), A-833834 and A-585539, all consistently attenuate LPS-induced TNF-α release from mouse macrophages, RAW 264.7 cells, and human monocytes , , , , , . Several lines of evidence indicate that activation of α7 nAChRs elicits an increase of intracellular Ca2+ concentrations via ionotropic and/or metabotropic signaling pathways in not only neuronal cells but also non-neuronal cells , , , , , , , , , . The results observed in immune cells suggest that suppression of pro-inflammatory cytokine release elicited by α7 nAChR is mediated most likely via metabotropic rather than ionotropic signaling pathways , , . Metabotropic functions elicited by α7 nAChR agonists in neuronal cells such as PC12 cells are blocked by selective antagonists, such as methyllycaconitine (MLA) and α-bungarotoxin (α-BTX) , . On the other hand, there have been conflicting observations as to the sensitivity of α7 nAChRs expressed in macrophages and monocytes to MLA and α-BTX. Whereas MLA or α-BTX reversed most α7 nAChR agonist-induced suppression of cytokine release from activated macrophages and monocytes , , , they did not attenuate α7 nAChR agonist-induced suppression of cytokine release from activated human monocytes or mouse macrophages , , .
Given those findings, we endeavored to clarify the sensitivity of α7 nAChRs on APCs to conventional nAChR antagonists by investigating (1) the effects of GTS-21, α-BTX and MLA on Th cytokine (IFN-γ, IL-4 and IL-17) production in OVA-activated spleen cells from OVA-specific T cell receptor (TCR) transgenic DO11.10 mice; (2) the effects of α-BTX and MLA on the release of IFN-γ and IL-4 from the OVA-activated spleen cells from DO11.10 mice in the presence of GTS-21; and (3) the effects of MLA and mecamylamine (MEC), a nonspecific nAChR antagonist, on OVA-induced, APC-dependent CD4+ T cell differentiation into regulatory T cells (Tregs) in the presence of GTS-21.
OVA-specific TCR transgenic DO11.10 (H-2d) mice on a BALB/c background were purchased from The Jackson Laboratory.
The protocols used in this study were approved by the Animal Care and Use Committee of Osaka Ohtani University and the Ethical Committees of Doshisha Women’s College of Liberal Arts (Nos. Y15012, Y15028, Y16002, Y17010, Y17024, Y18001, Y18010).
Spleen cells were prepared from DO11.10 mice , , ,  as described previously . Briefly, to examine the effects of GTS-21,
In DO11.10 spleen cells, OVA is taken up into endosomes within APCs and processed to OVA peptide326-339 (OVAp), the antigenic epitope of OVA, by cathepsins. After conjugation of OVAp with MHC class II molecules (MHC II), the OVAp-MHC II conjugates are translocated to the surface of the APCs. Subsequent recognition of OVAp presented on MHC II by OVAp-specific TCRs on naïve DO.11.10 CD4+ T cells triggers a series of T cell activation reactions leading to differentiation into Tregs and effector T
As described above, it is well established that ɑ7 nAChR mRNA is expressed in immune cells , . In addition, using DO11.10 spleen cells, we recently showed that GTS-21 downregulates OVA-induced naïve CD4+ T cell differentiation into Tregs and effector T cells by inhibiting antigen processing, thereby interfering with antigen presentation . This suppressive effect of GTS-21 on naïve CD4+ T cell differentiation was not observed in the co-cultures with α7-KO APCs , suggesting GTS-21
This work was supported in part by Grants-in-Aid for Scientific Research (18K06903) from the Ministry of Education, Science, Sports and Culture (C) of Japan (MM) and funding from the Smoking Research Foundation (MM, SO, KK, TF), Tokyo, Japan.
CRediT authorship contribution statement
Masato Mashimo: Investigation, Writing - original draft, Data curation, Visualization. Shiori Takeshima: Investigation. Hiromi Okuyama: Investigation. Ayako Matsurida: Investigation. Mami Murase: Investigation. Shiro Ono: Conceptualization, Methodology, Writing - review & editing, Supervision. Koichiro Kawashima: Conceptualization, Writing - review & editing, Supervision, Funding acquisition. Takeshi Fujii: Writing - review & editing, Investigation, Formal analysis, Project administration,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
- E.P. van der Zanden et al.
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- M. Mashimo et al.
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- J. Li et al.
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- R.L. Papke
Merging old and new perspectives on nicotinic acetylcholine receptors
- N. Kabbani et al.
Beyond the channel: metabotropic signaling by nicotinic receptors
Trends Pharmacol. Sci.
- J.R. King et al.
Identification and characterization of a G protein-binding cluster in alpha7 nicotinic acetylcholine receptors
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- M. Kox et al.
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- Y.X. Fujii et al.
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Citation Excerpt :
As previously mentioned, the cholinergic system affects the secretion of cytokines and antigen presentation by macrophages and participates in the regulation of the M1/M2 functional state, CCR2+ /CCR2- macrophage phenotype transition, and chemotaxis recruitment. GTS-21 interferes with APC antigen processing, thus inhibiting antigen-specific and antigen processing-dependent (OVA) CD4+ T cells from differentiating into regulatory T cells and effector T cells (Th1, Th2, and Th17) as well as the production of Th cytokines; in contrast, GTS-21 activates α7nAChRs expressed on CD4+ T cells and promotes development into all linkages under the condition that naïve T cells are induced without depending on antigen specificity and antigen processing (OVAp) or induction by a nonantigen (anti-CD3/CD28 monoclonal induction), suggesting that α7nAChRs expressed on T cells and APCs have different effects [98,223,224] Recent studies have shown that myocardial nonspecific effector T cells become the main cardiac-infiltrating CD4+ T-cell subset in the later stage of myocarditis, and immune adoption of these cells has no effect on the severity of inflammation at the peak of the disease; however, it can protect the heart from fibrosis remodeling and cardiac insufficiency after inflammation . Therefore, it has become a new therapeutic direction of the cholinergic pathway: the α7nAChR on APCs can regulate the differentiation and development of T-helper cell subsets and inhibit antigen processing to regulate the proportion and immunity state of autoreactive CD4+ T cells/non-specific Teff cells.
The immune system and the nervous system depend on each other for their fine tuning and working, thus cooperating to maintain physiological homeostasis and prevent infections. The cholinergic system regulates the mobilization, differentiation, secretion, and antigen presentation of adaptive and innate immune cells mainly through α7 nicotinic acetylcholine receptors (α7nAChRs). The neuro-immune interactions are established and maintained by the following mechanisms: colocalization of immune and neuronal cells at defined anatomical sites, expression of the non-neuronal cholinergic system by immune cells, and the acetylcholine receptor-mediated activation of intracellular signaling pathways. Based on these immunological mechanisms, the protective effects of cholinergic system in animal models of diseases were summarized in this paper, such as myocardial infarction/ischemia–reperfusion, viral myocarditis, and endotoxin-induced myocardial damage. In addition to maintaining hemodynamic stability and improving the energy metabolism of the heart, both non-neuronal acetylcholine and neuronal acetylcholine in the heart can alleviate myocardial inflammation and remodeling to exert a significant cardioprotective effect. The new findings on the role of cholinergic agonists and vagus nerve stimulation in immune regulation are updated, so as to develop improved approaches to treat inflammatory heart disease.
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