Neuroimmune nexus in the pathophysiology and therapy of inflammatory disorders: Role of α7 nicotinic acetylcholine receptors

The secretion of cytokines from macrophages is a highlight of the inflammatory response. The general concept of the α7nAChR-mediated anti-inflammatory pathway was discovered based on the analysis of cytokine expression during α7nAChR activation. As mentioned above, the first discovery was done by Kevin Tracey’s group, which showed the inhibition of TNF-α expression by macrophages after VNS. The critical role of α7nAChR in this mechanism was demonstrated by using α7nAChR-deficient mice where VNS did not affect TNF-α expression [ 3 ]. Since TNF-α expression is regulated via NF-κB transcription factor, it was tempting to hypothesize that NF-κB activation is also affected by α7nAChR-mediated signaling. Indeed, further studies demonstrated that α7nAChR-mediated signaling inhibits NF-kB -dimerization and translocation [ 76 ]. NF-κB is a master transcription factor that regulates more than 200 pro-inflammatory genes including different cytokines (IL-1β, Il-6, IL-8), chemokines (MCP-1, Rantes), and adhesion molecules (VCAM-1, E-selectins). Accordingly, many studies have been focused on the effect of α7nAChR activation on the expression of NF-κB-dependent pro-inflammatory mediators, which are released in pathophysiological conditions or in vitro after NF-κB activation by endotoxin (i.e., LPS). Endogenous α7nAChR ligands and pharmacological agonists have similar effects. Nicotine inhibits the production of the pro-inflammatory cytokines TNF-α, IL-1β and IL-12 in bone marrow monocytes, while stimulating the secretion of IL-10, an anti-inflammatory cytokine [ 77 ]. Acetylcholine and its derivatives inhibit IL-1β secretion from human and rat monocytes in vitro treated with 3’-O-(4-Benzoyl)benzoyl ATP , which a is P2X7 receptor agonist with potent pro-inflammatory functions [ 78 ]. GTS-21 attenuates TNF-α and IL-1β at the transcriptional level in human whole blood activated by exposure to endotoxin [ 79 ]. Notably, other pro-inflammatory cytokines such as IL-6, IL-8, IL-12 were not inhibited by GTS-21 in this study when evaluated 4 h after treatment, however the authors hypothesized that these cytokines may be inhibited with longer treatment. Varenicline decreased cytokine and chemokine expression including TNF-α, IL-6, and IL-1β via α7nAChRs in RAW 264.7 murine macrophage cell line when evaluated at 24 h after LPS-stimulation [ 80 ]. Varenicline, a drug prescribed for smoking cessation, has full agonistic properties on α7nAChRs but is more potent as a partial agonist at α4β2-nAChRs [ 81 ]. Administration of α7nAChR agonists (nicotine, PNU 282987, or PHA 568487) reduced bronchoalveolar lavage MIP-2 (CXCL2) production [ 46 ] in endotoxin and live Escherichia coli -induced acute lung injury (ALI) in mice. MIP-2 assists in the recruitment of neutrophils to sites of injury or infection. Regulation of pro-inflammatory stimulation via α7nAChR occurs naturally during inflammation, and α7nAChR-deficiency increases pro-inflammatory cytokine expression. For example, development of neuroinflammation was compared in wild type (WT) and α7nAChR-deficient mice with intraperitoneally inoculated E.coli. Infected α7nAChR−/− mice showed significantly higher mRNA expression of TNF-α, IL-6, MCP-1, and suppressor of cytokine signaling 1 (SOCS1) in the brain [ 82 ]. Macrophages are the major type of immune cells regulating acute immune response via cytokine secretion. Macrophage functions are regulated by T lymphocytes. The secretions of INFγ from Th1 lymphocytes or IL-4/IL-13 from Th2 lymphocytes promote classical or alternative activation of macrophages, which release different sets of cytokines directed toward pro-inflammatory or remodeling functions, correspondingly. Therefore, the modulation of T cell phenotype by α7nAChR activation is an important regulator of cytokine secretion. For example, during experimental autoimmune encephalomyelitis (EAE), GAT107, an ago-positive allosteric modulator of α7nAChRs, reduced the production of pro-inflammatory cytokines including IL-17, IFNγ, and IL-6, as well as increasing the production of the anti-inflammatory cytokine IL-10 in encephalitogenic T cells [ 41 ]. Accordingly, macrophages will be polarized toward the M2 phenotype (alternative activation), preventing NF-kB activation. Therefore, data regarding the role of α7nAChR in the regulation of IL-10 expression confirms the cholinergic anti-inflammatory mechanism. Taken together, these studies have revealed inhibition of NF-kB-dependent pro-inflammatory cytokines by α7nAChR activation. Apoptosis and proliferation are two opposite mechanisms that can control pathophysiological outcomes by regulating leukocyte accumulation in tissue. It has been demonstrated that the proliferation of macrophages at the site of inflammation is critical for the development of inflammatory responses [ 83 ]. The role of macrophage apoptosis was also recently reconsidered as an important alternative for macrophage efflux during inflammation [ 84 ]. Proliferation and apoptosis may have a protective or pathological effect at different stages of diseases; therefore, it can represent an important part of anti-inflammatory mechanisms that depend on α7nAChR activation. The effect of α7nAChRs on leukocyte proliferation starts with the regulation of hematopoiesis. Recently, it was shown that B-cell derived ACh in bone marrow reduces the proliferation of hematopoietic stem and progenitor cells (HSPCs) [ 4 ]. This leads to decreased proliferation of common myeloid progenitor cells, and thus monocytes, in circulation. Notably, deletion of α7nAChRs leads to higher levels of circulating Ly6C hi monocytes, Ly6G+ neutrophils and CD71+Ter119+ erythrocytes but unchanged platelet counts compared to C57BL/6 WT controls. The effects are mediated via increased proliferation of HSPCs and myeloid progenitors, demonstrating the critical role of α7nAChRs in progenitor proliferation [ 4 ]. α7nAChR activation can also directly regulate proliferation in circulation or tissue. For example, the effect GAT107 on development of neuroinflammation by regulating cell proliferation was demonstrated [ 41 ]. Administration of GAT107 during experimental autoimmune encephalomyelitis (EAE) reduced encephalitogenic T cell proliferation and the production of pro-inflammatory cytokines, as was shown above. Another example comes from study of nicotine effects in rats with cerebral ischemia. It has been shown that nicotine inhibits microglial proliferation. Notably, blockade of α7nAChRs with α-BTX could prevent the inhibitory effects of nicotine on cultured microglial proliferation, suggesting that nicotine inhibits microglial proliferation in an α7nAChR-dependent fashion [ 85 ]. Studying bone-marrow-derived monocytes, St-Pierre and colleagues demonstrated that nicotine reduces M1 monocyte proportions in bone marrow cells ex vivo by increasing cell death and inhibiting their proliferation, but not by preventing their polarization [ 77 ]. Interestingly, in another study nicotine did not effectively reduce cell proliferation of Jurkat T cells or IL-2-dependent Kit-225 T cells over a period of 30 days in culture at concentrations ranging from 10 nM to 100 μM [ 86 ]. These results echo the recent finding that T cell number is not affected in blood after acetylcholinesterase inhibition or ACh inhibition [ 4 ]. For more detail please see section 4.4 Hematopoiesis. At the same time, in circulation and tissue, nicotine may act both as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. Based on α7nAChR knock-down with siRNA, the effect of nicotine is, at least partially, regulated via α7nAChRs [ 86 ]. In several non-neuronal cells, activation of the α7nAChR promotes cell survival and protects cells from apoptosis. It was suggested that nicotine has an important role in promoting cellular survival in macrophages during Mycobacterium avium paratuberculosis infection [ 87 ]. The effect is related to increased expression of the anti-apoptotic protein Bcl2. Another example was obtained using ischemia-reperfusion injury model where vagal stimulation decreases infarct size and inflammatory markers due to antiapoptotic properties of the nicotinic pathway [ 88 ]. The mechanism of cell survival was linked to STAT3 pro-survival pathway, which was activated via α7nAChR and generated a protection of macrophages from endoplasmic reticulum (ER) stress-induced apoptosis [ 89 ]. Interestingly, it works specifically for M2 macrophages, while the apoptosis of M1 macrophages was not significantly affected. Remarkably, this mechanism is completely lost in α7nAChR-deficient M2 macrophages [ 89 ]. Notably, apoptosis has a protective effect during many pathological conditions, therefore a pro-survival role of α7nAChR cannot explain all potential anti-inflammatory outcomes. NF-kB regulates different pro-inflammatory factors including the expression of adhesion molecules and chemokine receptors. Therefore, α7nAChR-mediated inhibition of NF-kB pathway may regulate monocyte/macrophage migration during inflammation. Notably, the presence of leukocytes at the site of inflammation may have protective or pathological outcomes depending on the type and stage of inflammation. The potential role of α7nAChR in the regulation of macrophage migration was considered in a few publications without detailed analysis. First, the effect of α7nAChR stimulation or α7nAChR deficiency on the accumulation of macrophages in tissue was detected. Particularly, GTS-21 treatment in mice with LPS-induced acute lung injury reduced the number of macrophages in the bronchoalveolar lavage fluid [ 90 ]. Nicotine reduced the accumulation of CD11b-positive macrophages in the synovium and spleen during arthritis. However, tissue immunostaining for CD11b can also label neutrophils and other myeloid cells. Activation of α7nAChRs by agonists AZ6983 [ 91 ] or GTS-21 [ 92 ] reduces atherosclerosis development and the number of lesion macrophages in ApoE−/− mice. These results were obtained by immunostaining aortic roots with macrophage specific anti-CD68 antibodies. Surprisingly, another report detected the increased size of atherosclerotic lesions and enhanced intraplaque macrophage content in ApoE−/− mice [ 93 ]. However, all these studies indicate the role of α7nAChRs in regulation of macrophage accumulation in tissue that can be mediated either by alteration of monocyte recruitment or macrophage efflux. As discussed above, in addition to migration, macrophage accumulation may also depend on proliferation and apoptosis. However, several other publications made the effort to measure direct migration. The ability of α7nAChR agonists to decrease migration of RAW264.7 cells was shown by simple testing of cell transmigration through uncoated trans-well membranes (Boyden chambers) without a chemokine gradient. Although this setup is simplified, the authors confirmed the well-defined fact that LPS treatment significantly promoted the migration of RAW 264.7 cells. Most importantly, they showed that the number of migrated cells was significantly less in the group treated with LPS and ACh compared to LPS alone [ 94 ]. A similar result was obtained by others who demonstrated that ACh can inhibit LPS-Induced RAW264.7 cell migration [ 95 ]. It was suggested that inhibition of migration was due to blocking MMP-9 or MMP-2 expression. MMPs contribute to 3D macrophage migration through the extracellular matrix (ECM) by degrading ECM proteins and generating space for cell movement. Although the concept is interesting, the presented experiments do not verify this hypothesis. Since this mechanism is not involved in any way in the proposed model of cell migration, where macrophages transmigrate via non-coated 8-μm pore-size membrane without a chemokine gradient. In such a setting, only gravity and diffusion regulate cell motility. Treatment with an α7nAChR agonist, varenicline, generates a similar effect. The rate of LPS-induced cell migration was decreased with varenicline in the Boyden chamber toward serum gradient without any chemokine or post-coat [ 80 ]. As a separate line of evidence, it has been shown that the unique human variant of α7nAChR, CHRFAM7A alters monocyte transmigration in Boyden chamber toward MCP-1 gradient, demonstrating the regulatory role of this receptor variant [ 96 ]. Notably, CHRFAM7A mice presented more anti-inflammatory phenotype and had improved survival compared to WT after a lethal dose of LPS [ 97 ]. Another potential mechanism for α7nAChR-dependent leukocyte migration was suggested by Saeed et al [ 98 ]. They showed that nicotine and cholinergic agent CAP55 inhibit expression of endothelial cell adhesion molecules (VCAM-1 and E-selectin) and significantly block leukocyte transmigration through endothelial monolayer [ 98 ]. Since expression of VCAM-1 and E-selectin is regulated by NF-kB activation, these data correspond to the accepted model that α7nAChR activation prevents NF-kB-mediated pro-inflammatory gene expression. These data agree with recent results of Nahrendorf’s group (discussed below) that detected a reduced number of myeloid leukocytes in the circulation of mice after α7nAChR stimulation due to reduced proliferation of leukocyte progenitors [ 4 ]. Taken together, these observations suggest that α7nAChR activation reduced the number of monocytes and prevented their recruitment, while the α7nAChR-non-treated group may have a higher number of pro-inflammatory monocytes in circulation and increased monocyte transmigration/recruitment due to augmented levels of endothelial adhesive receptors. Thus, current evidence points to the potential role of α7nAChRs in the regulation of macrophage migration. However, critical aspects such as the contribution to particular step(s) of leukocyte recruitment (adhesion to endothelium, transmigration, 3D migration in tissue), type of cell motility (amoeboid or mesenchymal) and important migratory receptors (chemokines or adhesion molecules) are not fully understood. Hematopoiesis occurs in bone marrow and spleen. In a comprehensive paper by Nahrendorf’s group, the critical role of ACh in regulation of hematopoiesis was shown [ 4 ]. It is known that sympathetic (noradrenergic) nerves promote hematopoiesis in the bone marrow. In this paper, the authors showed, that cholinergic stimulation reduces hematopoiesis [ 4 ]. The primary source of ACh in bone marrow is B lymphocytes, which secrete 3-5-fold more ACh compared to other leukocytes [ 4 ] Using the ChEI pyridostigmine to amplify effects of endogenous ACh, the authors demonstrated that increased concentration of ACh reduced the number of CD11b+Ly6C hi monocytes and CD19+ B cells in blood, but did not change Ly6G+ neutrophils, CD3+T cells, CD41+CD61+ platelets and CD71+Ter119+ erythrocytes. These changes depend on hematopoiesis since pyridostigmine treatment reduces the number of common myeloid progenitors, but common lymphocyte progenitors were not affected. A similar result was obtained in human patients with Alzheimer’s disease who were treated with the ChEI donepezil. This treatment led to a significant dose-dependent decrease in the number of leukocytes in blood compared to control samples obtained without treatment. To verify these results, they generated Cd19CreChat fl/fl mice, in which ChAT, essential for producing ACh, was conditionally deleted in B cells. The level of ACh in bone marrow of these mice was reduced 3.6-fold. This reduction leads to an increased amount of SLAM LSK cells (most upstream hematopoietic progenitors), and common myeloid progenitors due to increased proliferation. According to this result, the number of Ly6C hi and Ly6C lo monocytes, Ly6G+ neutrophils and B220+ B cells was increased in the blood. The number of T cells, erythrocytes and platelets was similar in Cd19 CreChatfl/fl and Chat fl/fl mice. The critical role of α7nAChRs in this mechanism was shown by using knockout mice. Analysis of α7nAChR−/− mice demonstrated higher bone marrow numbers of common progenitors and myeloid progenitors as well as higher levels of circulating Ly6C hi monocytes, Ly6G+ neutrophils and CD71+Ter119+ erythrocytes but unchanged platelet counts compared to C57BL/6 WT controls. Further, experiments with BrdU demonstrated increased proliferation in the bone marrow of α7nAChR−/− mice. Most interestingly, these investigators detected that different stromal cells express α7nAChRs and stromal niche cells in bone marrow appear to sense ACh via α7nAChRs. Therefore, ACh released from B cells regulates steady-state hematopoiesis in bone marrow via α7nAChRs. Blocking of ACh or α7nAChR-deficiency leads to the upregulation of pro-inflammatory monocytes (Ly6C hi -positive) and neutrophils in the blood and common hematopoietic progenitors and myeloid progenitors in the bone marrow. Reduced hematopoiesis and leukocyte number in the circulation occur after pharmacological stimulation of the cholinergic system. Thus, these data provide the molecular mechanism for reduced leukocyte number after α7nAChR-mediated treatment ( Fig. 3 ). Notably, the ability of B cells to express ACh was shown previously when Reardon et al [ 99 ]. used ChAT-GFP mice to detect ACh expression in different organs. They found ChAT-GFP positive cells in the spleen, lymph nodes and peripheral blood. In the bone marrow, GFP expression was not detected in immature B cells, but was observed in mature recirculating B cells (B220 hi ). Based on this information, they concluded that ChAT-GFP expression is induced in response to specific signals received after B-cell development in the bone marrow [ 99 ]. This result is mostly consistent with the Nahrendorf study that, in addition, detected ChAT-GFP activity in immature B cells, albeit 5-folds less compared to mature B cells [ 4 ]. The data of Nahrendorf’s group are in agreement with previous findings by Constantini et al. [ 100 ]. They tested CHRFAM7A-transgenic mice. As we discussed above (2.2.1) CHRFAM7A is a unique human gene, which encodes a protein with structural similarity to α7nAChR. However, it has been shown that CHRFAM7A serves as an inhibitor of ligand binding to α7nAChR [ 101 , 102 ]. The authors found that CHRFAM7A increased the hematopoietic stem cell reservoir in the bone marrow and increased their differentiation to the monocyte lineage in vitro [ 100 ]. They also demonstrated that while the hematopoietic stem cell reservoir was depleted during Systemic inflammatory response syndrome, hematopoietic stem cells were spared in CHRFAM7A-transgenic mice and that these mice also had increased immune cell mobilization and myeloid cell differentiation. Interestingly, authors suggest that CHRFAM7A, as a unique human gene may contribute to discrepancies between the effectiveness of α7nAChR agonists in animal models and human clinical trials for inflammatory and neurodegenerative diseases [ 100 ]. Recently, Fielding et al provided another line of evidence that demonstrates the importance of α7nAChR-dependent cholinergic signaling for the regulation of hematopoiesis [ 103 ]. They demonstrated that the activation of α7nAChR on mesenchymal stromal cells in the bone marrow hematopoietic stem cell niche leads to increased CXCL12 expression that preserves hematopoietic stem cell quiescence, thereby helping protect stem cells from exhaustion during proliferative stress. Accordingly, the regulation of hematopoiesis depends on cholinergic signaling on immune progenitors and bone marrow niche cells. The contribution of α7nAChR to the regulation of hematopoiesis in the spleen was described in several papers and demonstrated a similar outcome. Particularly, the administration of α7nAChR partial-agonist GTS-21 leads to significantly reduced spleen size and splenic leukocyte numbers. The effect was most apparent for the Ly6-C hi monocyte population. According to these data, reduced number of hematopoietic stem cells (HSPCs) and myeloid progenitor cells in the spleens of the treated mice was observed [ 92 ]. Using α7nAChR Cre:YFP mice, it has been shown that all hematopoietic organs including bone marrow, spleen, thymus and lymph nodes contain α7nAChR positive cells. However, only 15-30% of hematopoietic cells express α7nAChRs and these cells are CD11b+-myeloid cells and B lymphocytes [ 104 ].

The potential link between the parasympathetic nervous system and immune response was discovered by Kevin Tracey’s group when they showed the inhibition of TNF-α synthesis by VNS [ 3 ], as discussed earlier. In a further study, Ulloa and Tracey demonstrated that nicotine improves the survival of mice in LPS-induced endotoxemia and cecal-ligation and puncture (CLP) sepsis [ 76 ]. The effect was mediated via nicotinic inhibition of HMGB1 release and blocking of the NF-kB pathway via activation of α7nAChRs. This manuscript initiated the detailed study of the protective effect of α7nAChR stimulation in the treatment of sepsis. The effective contribution of α7nAChRs to the anti-inflammatory response was demonstrated using several α7nAChR agonists. Treatment with choline or GTS-21 improves survival of mice after CLP or LPS-induced endotoxemia [ 105 , 106 ]. Interestingly, choline administration 24 h after CLP-induced polymicrobial sepsis still significantly improved survival in mice, showing high impact of the CAP. In agreement with these results, activation of α7nAChRs by nicotine enhances survival in sepsis-induced ALI induced by instilling E. coli into the airways. The result was verified by using α7nAChR-deficient mice, which had significantly lower survival [ 107 ]. Further studies showed that the α7nAChR protective mechanism is tightly related to splenocyte functions, since VNS fails to inhibit TNF-α in splenectomized animals during lethal endotoxemia. Furthermore, the administration of nicotine actually increases proinflammatory cytokine production and lethality from polymicrobial sepsis (CLP) in splenectomized mice [ 2 ]. Based on these results, it was suggested that VNS activates T cells in the spleen that release ACh for macrophage anti-inflammatory shift, described in detail in introduction section. Notably, recent data demonstrate that B lymphocytes in bone marrow and the circulation are alternative sources of ACh for activation of anti-inflammatory cholinergic response [ 4 ]. The NF-kB pathway can be activated via several pro-inflammatory signaling pathways. Signaling via different TLRs, formation of IL-1-initiated signalsome or TNF-α-mediated stimulation initiate NF-kB release from I-kBα complex and translocation to the nucleus. In several following publications, the effect of α7nAChRs on different NF-kB-mediated signaling pathways was evaluated. Accordingly, it has been shown that α7nAChR stimulation inhibits the expression of TLR4 and CD14 [ 108 ], and this mechanism can occur via α7nAChR/PI3K signaling [ 109 ]. Different endogenous agonists of the α7nAChR, including ACh, choline, and phosphocholine inhibit ATP-mediated IL-1β release in human and rat monocytes [ 78 ]. In agreement with these results, α7nAChR stimulation by GTS-21 inhibits the production of cytokines from monocytes activated by ligands for TLR2, TLR3, TLR4, TLR9 and RAGE [ 79 ]. These results demonstrate a comprehensive effect of α7nAChR activation on the prevention of NF-kB-mediated pro-inflammatory stimulation. Accordingly, the cholinergic anti-inflammatory mechanism may inhibit the development of different inflammatory diseases initiated by different pathogens. Numerous data demonstrate that classical activation of macrophages in vivo or M1 macrophage polarization in vitro leads to NF-kB activation that indicates a direct link between NF-kB and pro-inflammatory macrophage phenotype. It would be logical to expect that α7nAChR stimulation modifies macrophage phenotype. Indeed, GTS-21 [ 90 ] or PNU-282987 [ 110 ] treatment significantly diminished the number of M1-polarized macrophages and increased the number of M2-polarized macrophages in lungs after LPS-mediated acute lung injury (ALI) in mice. Also, stimulation with nicotine decreases M1 and increases M2 macrophages in the decidua of pregnant mice treated with LPS [ 111 ]. In agreement with this result, ACh inhibited LPS-induced IL-1β and IL-6 elevation (M1 phenotype) and promoted IL-4 and IL-10 production (M2 phenotype) in microglia, and knockdown of the α7nAChR abolished these effects of ACh [ 112 ]. Apparently, a shift in macrophage phenotype reduces cytokine storm and regulates the organism response to infection. The generation of α7nAChR-mediated anti-inflammatory response by the inhibition of macrophage pro-inflammatory cytokine release is generally accepted. However, other potential mechanisms are still under consideration. For example, the contribution of α7nAChR to the reduction of inflammatory response in acute lung injury (ALI) by regulating maturation, phenotype, and quantity of DCs was described [ 113 ]. Another non-NF-kB-related mechanism was suggested by testing tissue edema formation. It is well known that sepsis increases microvascular permeability that dysregulates homeostasis. However, microvascular permeability was significantly reduced in animals treated with GTS-21 simultaneously and 1h after induction of endotoxemia [ 114 ]. Sepsis development strongly depends on the recruitment of myeloid immune cells to damaged organs [ 115 ]. Accordingly, the potential effect of α7nAChRs on monocyte and macrophage migration can make an important contribution. More details on macrophages migration are in section 4.3 Migration. Notably, most in vivo data were obtained by using α7nAChR agonists but not by α7nAChR-deficient mice. To the best of our knowledge, only one study demonstrated the increased mortality in α7nAChR-deficient mice after CLP and the results are moderate - 80% survival of α7nAChR−/− mice versus 100% survival of WT [ 116 ]. Another paper demonstrates that a deficiency of α7nAChR worsens the reduced survival in E. coli pneumonia [ 46 ]. However, there is no data that compare the survival rate of α7nAChR−/− versus WT mice in the classical LPS-induced endotoxemia model. Certainly, the exogenous stimulation of α7nAChR via vagus nerve or agonist mediates a protective anti-inflammatory response. However, the level of α7nAChR activation in natural conditions during sepsis development is not fully understood. These questions still require clarification. SARS-CoV-2, the causative agent of the global pandemic beginning in 2019, is a continuing threat with both health and economic implications for healthcare providers and patients. The SARS-CoV-2 virus primarily causes respiratory disease but is known to cause severe inflammation and damage in other organs, with multiple potential extrapulmonary sequelae. While comorbidities and immune status play a major role in determining the severity of disease in each patient, it has been reported that there are potential interactions between the α7nAChR and SARS-CoV-2 that could have immunomodulatory effects via the α7nAChR. Investigations into these interactions stemmed largely from the observation that fewer smokers developed severe SARS-CoV-2 disease compared to non-smokers [ 117 ]. Nicotine, the addictive agent in tobacco products, is a non-specific agonist of the α7nAChR. The observations published as a result of these studies may present opportunities for therapies aimed at preventing severe disease. It has been suggested that SARS-CoV-2 spike protein has the potential to interact with the α7nAChR [ 117 ]. The spike protein contains a motif similar to known nicotinic ACh receptor antagonists, the Y674-R685 region, which is accessible in the fully glycosylated protein. This Y674-R685 region is homologous to the portion of α-BTX that interacts with α7nAChRs. This led to the hypothesis that spike protein interacts with nAChRs, which was investigated with molecular dynamics simulations [ 118 ]. This in silico prediction was validated experimentally using the α7nAChR expressed on human cell lines and mitochondria extracted from mouse brains. In a competitive ELISA, the SARS-CoV-2 spike protein fragment Y674-R685 inhibited the binding of an antibody specific to the α7nAChR region 179–190. It was additionally reported that the SARS-CoV-2 fragment was able to disrupt cytochrome c release from mitochondria, through the interruption of α7-bax complexes in the mitochondrial membrane. Coronaviruses are known to express proteins that either initiate or delay apoptotic processes. This data suggests that spike protein fragment Y674-R685 attenuates apoptosis of the infected cell to support its viability while the virus replicates. Interestingly, this inhibition of apoptosis is only observed when the fragment interacts with mitochondrial α7nAChR, not with the α7nAChR on the plasma membrane [ 119 ]. The α7nAChR has been studied in SARS-CoV-2 disease not only due to potential interaction with the virus’s spike protein, but as a possible therapeutic target to combat the cytokine storm that often proves fatal for patients. Cytokine release syndrome is a characteristic of severe disease, where hyperinflammation of lung and other tissues result from the release of IL-6, IL-8, and TNF-α by both the adaptive and innate immune systems. Because of the well-characterized ability of α7nAChR activation to decrease pro-inflammatory cytokines, it was hypothesized that nicotine may be useful in controlling aberrant cytokine release. Additionally, it has been suggested that SARS-CoV-2 might impair macrophage function and upset M1/M2 balance [ 120 ]. Nicotine’s widespread availability as well as its function as an α7nAChR agonist may prove useful in the treatment of SARS-CoV-2 [ 121 ]. Most recently, treatment with pyridostigmine has been studied in a phase 2/3 clinical trial in SARS-CoV-2 patients, resulting in reduced mortality [ 122 ]. Self-managed tVNS is also being investigated for the treatment of lasting symptoms after convalescence, termed “long COVID”, in a preliminary controlled trial. tVNS was reported to be safe and feasible for participants and resulted in reductions in mental fatigue scores. Mental fatigue, or “brain fog” is one of the most common sequelae of SARS-CoV-2 after convalescence [ 123 ]. VNS is a potential therapy in active disease, as a means to prevent severe illness by reducing inflammatory cytokine production [ 124 ]. Coronary artery disease, cardiac ischemia, myocardial infarction (MI), and heart failure (HF) are related cardiac disorders that are major contributors to global morbidity and mortality. While multiple factors contribute to the underlying pathology, activation of the immune system and recruitment of leukocytes to the heart plays a pivotal role in adverse cardiac remodeling, at least in part due to excessive stimulation of pro-inflammatory pathways. In this regard, several preclinical studies have provided direct and/or indirect evidence for beneficial effects of α7nAChR stimulation on cardiac remodeling and function. Furthermore, preclinical and some clinical studies have established the therapeutic potential of VNS. Occlusion of a coronary artery causes ischemia downstream, which leads to MI and impaired cardiac pump function. Two major responses to this damage are reflex activation of the sympathetic nervous system and stimulation of systemic and local immune responses. In addition to stimulating the heart, sympathetic activation can also stimulate myelopoiesis and trafficking of monocytes from the spleen to the infarct region, where they become macrophages. Balanced activation of pro- and anti-inflammatory macrophage phenotypes is crucial to limiting infarct size and healing. However, excessive activation of the pro-inflammatory phenotype plays a major role in adverse cardiac remodeling. Early work established beneficial effects of VNS in a rat ischemia/reperfusion model of MI [ 88 ]. For these experiments, ischemia was induced by ligating the left anterior descending (LAD) coronary artery for 30 min, and the ligature was then removed for reperfusion. Stimulation of the right cervical vagus nerve, starting 5 min before ischemia and ending 5 min after reperfusion, decreased infarct size and recruitment of macrophages to the area at risk when evaluated at 24 h. Beneficial effects of VNS were blocked by local cardiac administration of the non-selective nicotinic antagonist mecamylamine but remained when the left atrium was paced during VNS to prevent bradycardia. Thus, the beneficial effects of VNS were mediated by nicotinic receptors, most likely within the heart, but were not related to parasympathetic slowing of the heart rate and the resulting decreased oxygen demand. Double staining for CD68 and α7nAChR suggested the presence of α7nAChRs on macrophages, however, the specificity of nicotinic receptor antibodies has been questioned [ 22 , 125 ]. α7nAChRs have also been implicated in the beneficial effects of VNS in a mouse model of acute MI [ 126 ]. VNS delivered separately during ischemia or reperfusion reduced infarct size, determined 2 h after reperfusion, by two different cholinergic mechanisms. Atropine and wortmannin blocked the protective effect of VNS given during ischemia, implicating muscarinic receptors and the Akt/GSK-3β pathway, which was confirmed by Western blotting. In contrast, the protection achieved by VNS during reperfusion was blocked by MLA and not atropine, suggesting mediation by α7nAChRs. Further experiments found that effects of VNS during reperfusion were also attenuated by blockade of the JAK2 pathway, which has been implicated in α7 mediated anti-inflammatory effects [ 12 , 22 ]. Interestingly, VNS during reperfusion still reduced infarct size after sectioning the vagus nerve below the diaphragm or splenectomy, suggesting mediation by α7nAChRs intrinsic to the heart rather than the classical CAP. While it is expected that α7nAChRs receptors are present on resident cardiac macrophages and monocytes-derived macrophages, this has not been demonstrated directly for cardiac tissue. In another study using rats, administration of the α7nAChR agonist, PNU-282987, just before reperfusion, reduced infarct size, serum markers of cardiac injury, and serum levels of the pro-inflammatory cytokines TNF-α, IL-6, and HMGB1 when measured after 2 h of reperfusion [ 127 ]. Levels of the anti-inflammatory cytokine IL-10 were unaffected in this study. Interestingly, postconditioning by brief hindlimb ischemia had similar but smaller effects compared to PNU-282987, and inhibition of cardiac NF-κBp65 signaling via GSK-3β was implicated for both. While it is not expected that PNU-282987 would affect hearts rate, cardiac function was not evaluated. These findings agree with earlier work that found similar beneficial effects in the same model after pretreatment with PNU-120596, a positive allosteric modulator of α7nAChRs [ 38 ]. Effects of PNU-120596, which require some source of endogenous ACh, were blocked by α-BTX. Unfortunately, effects of PNU-120596 on heart rate were not evaluated. Recent experiments with another mouse model of MI support the idea that intrinsic activation of α7nAChRs can have a cardioprotective influence. These investigators found that permanent occlusion of the LAD coronary artery in α7nAChR deficient mice produced larger infarcts and impaired cardiac function more than occurred in WT control mice when evaluated at 24 h post-occlusion [ 128 ]. Likewise, MI caused larger systemic inflammatory responses in knockouts compared to controls. The latter was evidenced by serum pro-inflammatory cytokine levels and the expression of genes for these cytokines in the spleen. Inflammation also plays a major role in adverse remodeling associated with heart failure (HF), so targeting this pathology through cholinergic anti-inflammatory mechanisms makes sense. Furthermore, autonomic imbalance, with enhanced sympathetic and reduced vagal efferent tone, occurs in HF, as in other cardiovascular diseases, providing another rationale for VNS. Several studies have evaluated effects of cholinergic pharmacotherapy and VNS in experimental models of HF. Daily treatment of rats with PNU-282987 for 4 weeks after ligation of the LAD, reduced adverse cardiac remodeling, caused a modest improvement of ventricular function, and reduced inducibility of ventricular arrhythmias [ 129 ]. Treatment with PNU-282987 also reduced proinflammatory cytokine levels in the peri-infarct region. Beneficial effects were attenuated by an AMPK inhibitor in this model and in isolated LPS stimulated macrophages, which favor the M2 phenotype in the presence of PNU-282987 alone. Several animal studies have demonstrated that chronic activation of cholinergic efferent pathways by treatment with cholinesterase inhibitors or by VNS can have beneficial effects in HF models. Many of the pharmacological studies have utilized cholinesterase inhibitors like donepezil, which are known to activate the cholinergic anti-inflammatory pathway by a centrally mediated effect [ 58 , 130 – 132 ]. Recent work, with a rat model of MI-induced chronic heart failure, provided convincing evidence that cardioprotective effects of donepezil are mediated by peripheral α7nAChRs [ 130 ]. For this work, donepezil was administered in drinking water, beginning 2 weeks after MI was induced by permanent ligation of the LAD and continued for 4 weeks, when terminal studies were performed. Despite the delayed initiation of donepezil therapy, it caused significant improvement of all cardiac function parameters, decreased cardiac fibrosis, and increased capillary density without decreasing heart rate. Furthermore, donepezil blunted sympathetic activation, as indicated by substantial drops in plasma catecholamines, decreased plasma BNP, and reduced inflammatory markers in the heart. All these beneficial effects of donepezil were blocked by peripheral but not central infusion of MLA, suggesting a major role of peripheral α7nAChRs. Since donepezil and similar drugs are already widely used in the treatment of Alzheimer’s disease, such agents might have therapeutic applications for diseases having inflammatory components such as HF. This concept is supported by results from a retrospective study, which demonstrated that Alzheimer’s patients treated with ChEIs had a reduced incidence of new-onset HF and cardiovascular death compared to patients not treated with these drugs [ 133 ]. Other studies have shown that peripheral inhibition of cholinesterase with pyridostigmine, given by gavage [ 134 ] or included in the drinking water [ 61 ], can attenuate the early development of HF when assessed one week after coronary ligation in rat models. In both cases, pyridostigmine also had anti-inflammatory effects in the infarct and peri-infarct region of the left ventricles. MI also increased TNF-α in the spleen, and this response was likewise attenuated by pyridostigmine [ 61 ]. Immunohistochemical evidence was presented for increased trafficking of leukocytes to the infarct after pyridostigmine treatment, but there was also a switch to higher proportion of the M2 anti-inflammatory macrophage phenotype. Vagal stimulation is another approach being explored as a potential therapy for HF in animal studies as well as clinical studies [ 135 – 137 ]. Efficacy has been evaluated in HF with reduced ejection fraction (HFrEF) and HF with preserved EF (HFpEF), however, only a few studies have examined the role of α;7nAChRs. While most studies of VNS have used direct stimulation of the cervical vagus, a few recent investigations used tVNS, which has the advantage of not requiring surgery. In the Dahl salt-sensitive model, rats treated with high salt diet develop hypertension and HFpEF. Four weeks of tVNS lowered blood pressure, improved cardiac function, and decreased inflammatory cytokines in serum, macrophage infiltration into the left ventricle, and cardiac fibrosis [ 138 ]. Survival was likewise improved, and all these beneficial effects of tVNS were blocked by MLA, suggesting mediation by α7nAChRs. Interestingly, treatment with the AT1 receptor blocker, olmesartan, also lowered blood pressure but did not improve cardiac function or reduce inflammation. Heart rate variability was reduced in this model of HFpEF, and tVNS normalized this parameter. However, this effect of tVNS was not blocked by MLA, suggesting mediation by direct neural mechanisms. Transcriptomic analysis showed that HFpEF caused differential expression of over 500 genes, primarily related to inflammation and fibrosis, and tVNS reversed most of these changes. Such effects were largely blocked by MLA, suggesting a primary involvement of α7nAChRs. Recently, the same group has reported encouraging results in a pilot clinical study of tVNS in patients with HFpEF [ 139 ]. Here, VNS was applied to the tragus, which is innervated by the auricular branch of the vagus, and sham control stimulation was applied to the earlobe. Patients with co-morbidities indicative of a pro-inflammatory state were selected to enhance efficacy based on the known anti-inflammatory effects of VNS. After 3 months of tVNS, patients had reduced global longitudinal strain and levels of serum TNF-α. Furthermore, better quality of life was determined based on a standard questionnaire. To summarize, a growing body of literature supports targeting cholinergic mechanisms for treatment of cardiac disease. Such effects can be mediated two distinct cholinergic systems, which can be activated by pharmacological or bioelectronic approaches ( Fig. 4 ). One approach is restoring balanced autonomic regulation of the heart such that vagal efferent effects mediated by muscarinic receptors are returned toward normal. The other mechanism involves attenuation or reversal of adverse cardiac remodeling by activating α7nAChRs on macrophages that mediate cholinergic anti-inflammatory responses. Macrophages in the heart are most likely the dominant effectors for therapeutic approaches targeting the α7nAChR in cardiac disease, but VNS and treatment with ChEIs also work by improving cholinergic transmission to myocytes. Some benefit might also derive from cholinergic anti-inflammatory effects at the spleen in cardiac diseases (e.g., reduced output of inflammatory cytokines), but the full impact of α7 based therapies on spleen-heart interactions requires much more study. Lipid uptake and deposition by macrophages in the artery intima leads directly to the formation of atherosclerotic plaques. The formation of these plaques not only decreases the size of the artery lumen, but their rupture can have catastrophic consequences such as heart attack and stroke. Among the anti-inflammatory effects of α7nAChR, it has been demonstrated to attenuate the development of atherosclerosis. Multiple mouse studies, in both Ldlr−/− and ApoE−/− models, have demonstrated that the activation of α7nAChRs with a specific agonist attenuates atherosclerosis development. Infusion of AZ6983 to ApoE−/− mice through osmotic mini-pumps decreased lesion area by 37% in the aortic root and lipid accumulation in plaques by 48% when compared with controls [ 91 ]. Decreased plaque size and lipid content is corroborated using other agonists: namely AR- R17779 [ 140 ] and GTS-21 [ 92 ]. As a critical addition to this experimental data in mice, it has been demonstrated that α7nAChR is expressed in human atherosclerotic plaques. Human carotid endarterectomy specimens were obtained and analyzed for α7nAChR expression. Most lesions, 7 out of 10 samples, were positive for α7nAChR immunoperoxidase staining. Additionally, α7nAChR staining coincided with staining for CD68 and CD163 positive macrophages [ 141 ]. In the same study, α7nAChR deficiency was evaluated in atherosclerosis using Ldlr−/− mice reconstituted with α7nAChR−/− or WT bone marrow. The Ldlr−/− mice receiving α7nAChR−/− bone marrow showed a 72% increase in atherosclerosis at the aortic root and more advanced plaques with larger lipid deposits than those that received WT bone marrow [ 141 ]. Activation of α7nAChR is a potential approach to accompany current treatments of atherosclerotic disease. Not only does the activation decrease inflammatory cytokines, but it has also been reported to reduce the lipid content of atherosclerotic lesions. Conversely, α7nAChR deficiency increases atherosclerosis severity and was reported to increase cholesterol accumulation in macrophages. Deficiency of α7nAChR, regardless of ApoE status, increases lipid uptake. In ApoE +/+ /α7nAChR −/− mice, oxidized LDL uptake by peritoneal macrophages increased by 35% when compared to ApoE +/+ /α7nAChR +/+ control mice. In ApoE/α7nAChR double knockouts, cholesterol mass of peritoneal macrophages increased by 29% compared to the control mice [ 142 ]. The same study also examined mRNA levels of CD36, a class B scavenger receptor participating in lipid uptake by macrophages. They found that CD36 mRNA levels were significantly increased in ApoE +/+ /α7nAChR −/− mice when compared to control mice, suggesting that α7nAChRs may regulate macrophage lipid uptake, and therefore plaque formation, through altering expression of scavenger receptors [ 142 ]. While most published studies support an anti-atherogenic role for α7nAChR, there are some controversial data showing α7nAChR exacerbates atherosclerosis. In Ldlr−/− mice reconstituted with either control or α7nAChR−/− bone marrow, advanced lesions in the mice with α7nAChR deficient bone marrow had reduced size and macrophage content [ 143 ]. It was also reported that a common smoking cessation drug, varenicline, exacerbated atherosclerosis by activating α7nAChR on macrophages. Plaques in the whole aorta of ApoE−/− mice were increased 1.5-fold after daily treatment with 0.5 mg/kg varenicline for three weeks. This effect was validated by using an antagonist, MLA, in conjunction with varenicline [ 144 ]. The same research group published data demonstrating that varenicline also increases lipid uptake of macrophages by up regulating expression of CD36 and LOX-1, two scavenger receptors heavily involved in the progression of atherosclerosis [ 145 ]. While MLA was used to confirm the involvement of α7nAChR in the macrophage response to varenicline, it is crucial to note that varenicline was designed to be a high specificity α4ß2 nAChR antagonist [ 146 ]. Nonetheless, studies to clarify the effects of α7nAChR stimulation in atherosclerotic disease are invaluable to the advancement of patient treatment options. Metabolic dysfunction is a complex process that depends on many body functions including pancreatic β-cells, adipocytes, and macrophages in fat tissue. The important role of macrophages in this process points to a potential mechanism, which resembles other chronic inflammatory diseases like atherosclerosis. Accumulation of pro-inflammatory macrophages and secretion of pro-inflammatory cytokines contributes to disease progression. This common element makes it possible that activation of α7nAChR anti-inflammatory pathway has a protective mechanism for the regulation of insulin secretion and blood glucose during metabolic dysfunction. Indeed, oral administration of the α7nAChR agonist TC-7020 to homozygous leptin-resistant diabetic (db/db) obese mice reduced elevated glucose and glycated hemoglobin levels. In addition, it decreased weight gain, food intake, and lowered serum TNF-α. Changes were reversed by the α7nAChR-selective antagonist MLA, verifying α7nAChR-mediated effects [ 147 ]. Unfortunately, only fasting glucose was evaluated, and a glucose tolerance test may provide much more detailed information. This problem was resolved in the following papers that showed that α7nAChR stimulation in diet-induced obese mice by the agonist ICH3 improved glucose tolerance and insulin sensitivity [ 148 ]. In agreement with this result, GTS-21 or PNU-282987 stimulation of db/db mice lower levels of blood glucose in an oral glucose tolerance test [ 149 ]. Critical evidence was obtained by Gausseres and co-authors who tested metabolic parameters in α7nAChR-knockout mice fed a standard chow diet. 12 weeks old α7nAChR −/− mice display chronic mild hyperglycemia combined with impaired glucose tolerance and a marked deficit in β-cell mass. Moreover, 24-week-old mice demonstrated both glucose intolerance and insulin resistance, as well as adipose tissue inflammation and late-onset excessive gain in body weight due to increased fat mass [ 150 ]. These results clearly demonstrate the physiological protective effect of α7nAChR in the development of type 2 diabetes. Notably, the mice were kept on a normal chow diet. It is tempting to speculate that a similar experiment set up on a high-fat diet would demonstrate a much stronger difference in metabolic parameters of α7nAChR−/− and WT mice. Interestingly, the development of diabetes affects α7nAChR expression in different cells. It has been shown that streptozotocin-induced diabetes in Wistar rats upregulates α7nAChR gene expression in the cerebellum [ 151 , 152 ]. In contrast, the expression of α7nAChR in obese diabetic human subjects demonstrated reduced α7nAChR expression by adipocytes compared to normal weight controls. The expression of α7nAChR was partially re-established by diet and physical activity. In addition, in vitro treatment of human adipocytes in the presence of α7nAChR agonists PNU282987 or genistein increases α7nAChR expression [ 153 ]. In contrast to these data, another model of diabetes, non-obese type 1 insulin-dependent diabetes (NOD) mice demonstrated different results. Treatment of mice with AR- R17779 or nicotine did not affect overall inflammation and development of diabetes [ 154 ]. Probably, it can be explained by the different metabolic mechanism in type 1 diabetes; namely, a reduced level of adipose tissue inflammation and related signaling in NOD model. The protective mechanism of α7nAChR function during metabolic disorder is activated via different cell types. In addition to adipocytes and adipose macrophages, it has been shown that VNS suppresses hepatic IL-6/STAT3 signaling via α7-nAChRs on Kupffer cells. Hepatic IL-6 expression was suppressed by PNU-282987 administration in high-fat diet-induced obese mice, suggesting that cholinergic action suppresses IL-6 expression even in obese and insulin-resistant mice [ 155 ]. In agreement with the protective effect of α7nAChR in metabolic regulation, several independent reports demonstrated that activation of α7nAChR by PNU282987 [ 156 ] or nicotine [ 157 ] promotes diabetic wound healing. The effect is mediated via suppression of TNF-α production in streptozotocin (STZ)-induced diabetic mice [ 156 ] and by blocking TLR2 signaling in leptin-resistant db/db obese mice [ 157 ]. To summarize, the protective role of α7nAChRs in metabolic diseases was demonstrated in several independent studies. The mechanism depends on the reduced expression of pro-inflammatory cytokines and improved function of adipocytes and pancreatic β-cells, but the direct role of macrophages in the regulation of diabetes by α7nAChR was not shown yet. The convincing results in animal studies were supported by evidence of vagus nerve contribution to the regulation of metabolic parameters in humans. The studies were performed for different pathophysiological conditions but have interesting implications regarding metabolic regulation. The Netherlands Study of Depression and Anxiety performed on 1,883 individuals demonstrated that a decreased vagus nerve activity and increased sympathetic activity were associated with metabolic syndrome [ 158 ]. The application of device-generated VNS in patients with epilepsy is associated with significant weight loss [ 159 ]. Cervical VNS treatment of patients with severe treatment-resistant depression also results in significant weight loss without additional dieting or exercising [ 160 ]. Specific clinical trials to assess metabolic parameters were performed using galantamine, a centrally acting ChEI with anti-inflammatory properties. The subjects received oral galantamine for 12 weeks or placebo (n=30 individuals per group). Galantamine significantly reduced plasma levels of TNF-α and leptin and increased the level of IL-10 and adiponectin [ 161 ]. In the following study with the same parameters (n = 22 individuals per group), galantamine treatment significantly increased antioxidant enzyme activities and decreased lipid peroxidation, which results in reduced oxidative stress [ 162 ]. Based on these clinical studies, a low dose of galantamine alleviates inflammation and insulin resistance in metabolic syndrome subjects. These data demonstrate that stimulation of the CAP has a high therapeutic potential for the treatment of obesity and metabolic syndrome. Endometriosis is a chronic inflammatory disease characterized by ectopic endometrial tissue implanting in extra-uterine sites. It affects 176 million women, presenting with chronic pain, dysmenorrhea, and infertility. Available treatments are limited, typically target hormones, and are not consistently tolerated or effective. As not all patients desire treatments that affect hormones, there remains an opportunity to develop treatments for reducing inflammation underlying endometriosis symptoms. Recent studies demonstrate that targeting α7nAChR may be promising for reducing inflammation, therefore pain, in endometriosis. Multiple changes occur in the endometrial cells that implant at distal sites, including increased NF-kB expression and activation. In a study utilizing endometriosis patient biopsies, NF-kB activity was increased in highly inflammatory (“red”) lesions. Red lesions also had a higher expression of ICAM-1 [ 163 ]. Peritoneal macrophages isolated from endometriosis patients were also shown to have increased NF-kB expression, a key observation, as the condition of the peritoneal environment is hypothesized to be involved in the pathogenesis of the disease [ 164 ]. NF-kB is known to activate many pro-inflammatory genes, such as cytokines associated with inflammation like TNF-α, therefore its increased expression and activity in endometriosis lesions supports interest in α7nAChR as a putative treatment target. Crucially, Hao et al [ 165 ] reported that human endometrial tissue is positive for α7nAChR. Endometrial tissue samples were collected from female patients categorized as having either ovarian or deep (peritoneal) lesions, and age-matched controls were recruited from women undergoing colposcopy or loop electrosurgical excision procedures. In these samples, endometriosis patients in either category had significantly reduced α7nAChR staining in the glandular epithelium when compared to endometrium of control patients, which was associated with a higher extent of fibrosis and greater severity of dysmenorrhea. Cholinergic agonists were next tested in an endometriosis mouse model, using Balb/C mice, where PNU-282987 significantly reduced total endometriosis lesion weight and fibrosis when compared to controls. It was verified by treatment using the α7 antagonist MLA, which ablated any beneficial effects of PNU-282987 treatment [ 165 ]. The same research group evaluated VNS in the Balb/C endometriosis model, which also reduced overall lesion weight [ 166 ]. In a separate study, the same endometriosis mouse model demonstrated significantly reduced lesion weight when treated with the specific agonist PHA-543,613 [ 167 ]. Overall, these studies demonstrate that activation of the CAP by VNS may be utilized to reduce lesion size and combat inflammation associated with endometriosis, although they lack detail on specific mechanisms. As of writing this review, there is an ongoing clinical trial of tVNS for endometriosis pain at Hospital Foch, Suresnes, France [ 168 ]. Arthritis, in general, is simultaneously a disease of chronic inflammation and autoimmunity that results in degeneration of joints such as the hips, fingers, and knees. There are multiple clinical classifications under the umbrella of arthritis: osteoarthritis, rheumatoid, and psoriatic arthritis. All arthritic conditions result in greatly reduced quality of life. Treatment focuses on mitigation of symptoms by decreasing inflammation, relieving pain, and preventing further loss of joint mobility. However, these treatment strategies do not always mitigate disease progression for all patients, especially in osteoarthritis cases. Additional safe and effective early treatments are desperately needed. Rheumatoid arthritis (RA) is characterized by autoantibodies to IgG FcR bound by rheumatoid factor and anti-citrullinated protein antibodies. The pathophysiology of rheumatoid arthritis involves, among other factors, infiltration of activated leukocytes and inflammatory cytokine production within the synovial tissues [ 169 ]. In a collagen-induced RA model using DBA/1 mice, activation of α7nAChRs was reported to modulate the infiltration of macrophages into the synovium. Mice given intraperitoneal treatment with nicotine had significant decreases in CD11b positive cells in the synovium compared with control mice and the group receiving vagotomy. In addition, mice receiving nicotine treatment had reduced arthritis scores, synovial inflammation, and expression of ICAM-1 and CCR2 in synovial tissue. While this study relied heavily on immunohistochemistry in the absence of other methods, it illustrates the impact of leukocyte infiltration, and the CAP, on RA severity [ 170 ]. Dendritic cells (DCs) are among the activated leukocytes infiltrating the synovium and contributing to disease progression in RA, thus targeting α7nAChR on DCs may be effective in treatment. Using the collagen-induced arthritis model, treatment with GTS-21 was reported to significantly decrease arthritis scores and DC infiltration into the synovium when compared to controls [ 74 ]. Overall, reduction of leukocyte infiltration and pro-inflammatory mediators within the synovium via α7nAChR activation could be a strategy to mitigate disease. Osteoarthritis is primarily a disease of articular cartilage degradation and remodeling of synovial joints. Specific mechanisms underlying the pathology of osteoarthritis are not well understood, but it is suggested that synovial chondrocytes may overexpress inflammatory mediators as well as matrix metalloproteinases. In a monosodium iodoacetate-induced model of osteoarthritis using Wistar rats, treatment with the α7nAChR agonist PNU-282987 reduced cartilage degeneration and MMP-1 and MMP-13 expression. Additionally, activating α7nAChRs in this osteoarthritis model promoted autophagy in chondrocytes and mitigated disease progression [ 171 ]. While this result is promising, further study is desperately needed to confirm α7nAChR as a treatment target in osteoarthritis. Psoriatic arthritis typically features not only synovial inflammation and damage, but the same skin lesions associated with psoriasis, an autoimmune disease resulting in a high amount of inflammation and over-proliferation of skin cells. Using synovial joint samples from nine patients with a confirmed diagnosis of psoriatic arthritis, expression of α7nAChR was confirmed within these joints using immunoperoxidase staining. Double immunofluorescence staining revealed that α7nAChR staining coincided with CD68/CD163+ macrophages and fibroblasts [ 172 ]. While it is crucial to report that α7nAChR is expressed in human joints, translational studies in mice examining the effects of its activation are lacking. Inflammatory bowel diseases, namely Crohn’s disease and ulcerative colitis, have a complex etiology that combines genetic, immunologic, and environmental factors. Patients with inflammatory bowel diseases have a wide range of gastrointestinal symptoms, stemming from aberrant inflammation in the intestines. Paradoxically, it was noted by clinicians that cigarette smoking reduced symptoms in ulcerative colitis patients, but exacerbated disease in Crohn’s disease patients. Subsequently, the CAP became of major interest in inflammatory bowel diseases, as nicotine is an agonist of the α7nAChR. Decreasing pain, ulceration, and other debilitating symptoms by ameliorating inflammation through activating the α7nAChR is a promising therapeutic strategy. Crohn’s disease can occur anywhere along the digestive tract, presenting with swelling, abdominal pain, fatigue, and malnutrition. Triggers for the onset of disease are being heavily studied, and leading hypotheses suggest an initial viral or bacterial infection [ 173 ]. Management plans for patients typically include drugs like corticosteroids, immunosuppressants, and biologics, which must be taken long term. Because treatments often extend throughout a patien’s lifespan, non-drug alternatives are sought out by many. The vagus nerve is a crucial part of the axis connecting the gut, brain, and intestinal microbiota. Vagotomy in patients is associated with the development of inflammatory bowel disease, particularly Crohn’s disease [ 174 ]. For these reasons, VNS has been evaluated clinically to activate the CAP and elicit a therapeutic response. Nine Crohn’s disease patients with moderate active disease were selected to participate in a year-long study on the efficacy and safety of VNS for Crohn’s disease. Vagal nerve stimulators were surgically implanted around the left vagus nerve. After one year of consistent vagus nerve stimulation, five patients were in clinical remission and six were in endoscopic remission. Patients also had restored vagal tone, reduced digestive pain scores, and lowered serum levels of inflammatory cytokines [ 175 ]. Immune cells within the gastrointestinal tract are thought to be influential in this effect of the vagus nerve on Crohn’s disease, namely macrophages, dendritic cells, and mast cells. Confirmation for this hypothesized mechanism behind the success of VNS in Crohn’s disease patients is needed. Ulcerative colitis (UC) is a primarily colorectal inflammatory bowel disease, which not only causes intestinal sores, pain, and bloody stool in patients but also increases their risk of developing colon cancer. Colitis can be induced in BALB/c mice using dextran sulfate sodium (DSS), resulting in not only bloody stools but also colon shortening. In this colitis model, treatment with GTS-21 reduced colon shortening and immune cell infiltration in mice [ 176 ]. Paracellular invasion of intestinal bacteria into the mucosa is another concern with UC, usually resulting from the destruction of tight junctions due to inflammation and NF-kB activation. It was demonstrated that the number of tight junctions in the mucosa was improved by GTS-21 treatment, with increases in zonula occludens-1, claudin-1, occludin, and JAM-1. Leakage of FITC-dextran from the intestinal pouch was reduced in GTS-21 treated mice with DSS induced colitis, which was further confirmed by using the probe EUB338 to track bacterial translocation. GTS-21 treated mice showed attenuated bacterial translocation [ 176 ]. Treatment with PNU-282987, another α7nAChR agonist, combined with an SHP2 (a tyrosine phosphatase) inhibitor, gave similar results: reducing colon shortening and histological changes in the colon such as damage to intestinal crypts [ 177 ].

The α7nAChR was originally cloned from DNA libraries created from brain tissue, but subsequent work has shown that it is expressed by numerous additional cell types, including those of the immune system, where it has a major role in regulating inflammation. Expression of α7nAChR on leukocytes has been demonstrated in both RNA and cell-surface studies. The presence of α7nAChR is also indirectly identified when leukocytes respond to stimulation with nicotinic agonists, like ACh and nicotine, and such responses are blocked by MLA or α-BTX. Expression of α7nAChR is well documented in monocytes and macrophages. Human donor monocytes were shown to express α7nAChR at the mRNA level [ 68 ]. Macrophages retain α7nAChR expression after their differentiation from monocytes. Wang et al used FITC-labeled α-BTX to show α7nAChRs studding the surface of macrophages in fluorescent microscopy images [ 3 ]. For details on the cellular functions altered by α7nAChR on monocytes/macrophages, see Section 4.0 Cellular Functions Altered by α7nAChR activation. Monocytes and macrophages are not the only myeloid cells that express a functional α7nAChR, as the receptor is also expressed by neutrophils. Neutrophils not only express α7nAChR at the mRNA level, but they also respond to α7nAChR agonists. Stimulation of neutrophils with ACh and nicotine altered both adhesion and respiratory burst [ 69 ]. In addition to cells of myeloid lineage, both T-cells and B-cells express α7nAChR. Expression of nicotinic ACh receptors on T-cells was first investigated by examining the binding of labeled agonists to the receptor, such as radiolabeled ACh, which showed their presence on the surface of murine lymphocytes [ 70 ]. The expression of specific subunits was confirmed by measuring mRNAs. Sato et al [ 68 ] demonstrated the expression of α7nAChR in human donor T and B lymphocytes. T-cells themselves are critical for the function of the CAP, but their expression of α7nAChR is not [ 71 ]. However, activating the α7nAChR on T-cells by nicotine results in reduced antigen-mediated signaling [ 72 ]. B lymphocytic cell lines were used by Arredondo et al [ 73 ] to study both the expression of α7nAChR and effects of activation. B-lymphocytic cell lines showed the highest expression of α7nAChR in the lymphoblast stage (Daudi cells), with significantly lower expression at the mature stage (Ramos cells). Stimulation of the α7nAChR resulted in an increase in Bcl-6 and CD138 expression. Other effects of α7nAChR activation include many shared with macrophages, like decrease in expression and secretion of cytokines and inflammatory markers [ 72 , 73 ]. Dendritic cells also express α7nAChRs. Activation of α7nAChR on dendritic cells by agonists, like GTS-21 or ACh, can affect the way they interact with other immune cells. Co-stimulatory molecule CD80 as well as MHCII expression were down regulated after treatment of dendritic cells in a collagen induced arthritis model, possibly impacting the downstream activation of T and B lymphocytes [ 74 ]. Activation of α7nAChR can also result in decreased secretion of inflammatory cytokines, like IL-23 [ 75 ].

We have presented basic information on the presence and function of α7nAChRs in the immune system and reviewed evidence showing that they are relevant targets for treating a wide range of diseases that have inflammation as a key contributor to pathophysiology. The preclinical worked presented in this review was selected to be representative rather than all inclusive, so potential applications for α7 based therapy are enormous. Much of this review has focused on the CAP and its anti-inflammatory effect on splenic monocytes/macrophages. While this is clearly an important therapeutic target, there is still much to learn about α7 contributions to other aspects of immune regulation. For example, what effect does α7 activation have on trafficking of leukocytes, hematopoiesis, and the functions of B and T lymphocytes? Recent work suggests a major role for α7nAChRs in the bone marrow where they affect hematopoiesis and trafficking of inflammatory myeloid cells to other tissues [ 4 ]. Importantly, activation of a7nAChRs in bone marrow occurs by a unique mechanism that is independent of the CAP. To broaden our understanding of α7 functions in immune regulation, future studies will need to consider the broad cellular expression of a7nAChRs, the localization and mobility of these cells, and regional sources of ACh (neurons and/or non-neuronal). While a wealth of preclinical work has established the potential value of α7 based therapies (drugs and VNS) in a wide range of inflammatory disease models, translation of this work to patients has been limited. Neuromodulation therapy with VNS has gained the most traction, especially for the treatment of heart failure, but this approach is limited by the need for surgery. Recent success with tVNS suggests that this non-invasive approach could have broader applications. Pharmacological activation of the CAP has been limited to clinical studies using ChEIs approved for other indications. Despite intensive work by pharmaceutical companies to develop new drugs that activate α7nAChRs, this work has targeted central disorder. While some of these drugs have been used to activate the CAP in preclinical studies, this work has not been moved to the clinics. Perhaps broader use and success of tVNS and ChEIs for treating inflammatory disorders will be a catalyst for similar clinical investigation of α7 active drugs.

Stimulation of the vagus nerve by means of electrodes applied directly to the cervical vagus has been instrumental in the discovery and characterization of the CAP in preclinical studies [ 1 – 3 , 5 , 6 ]. Furthermore, potential translation of this approach to patients to achieve anti-inflammatory responses should be facilitated by decades of experience gained from the use of VNS to treat patients with drug-resistant epilepsy or depression [ 7 – 9 ]. The neuroanatomical circuitry that mediates activation of the CAP in the spleen after direct stimulation of the cervical vagus has been subject to debate, the main point of contention being the role of vagal efferent input to the celiac/superior mesenteric ganglia. There is much functional evidence supporting the idea that excitatory cholinergic input to these ganglia is activated directly by VNS. However, preganglionic cholinergic input that drives sympathetic ganglia normally comes from the spinal cord, and there is solid anatomical evidence for spinal projections to the celiac/superior mesenteric ganglia via the splanchnic nerve [ 10 ]. The latter pathway requires vagal afferent input to the brain and activation of central circuity to increase splanchnic nerve activity. Most evidence links the vagal efferent circuit to activation of the spleen CAP, while the splanchnic circuit evokes an α7 independent anti-inflammatory response [ 10 , 11 ]. The latter is mediated by noradrenergic stimulation of β 2 receptors on macrophages. Given the broad distribution of vagal efferent nerves ( Fig. 1 ), it is not surprising that cholinergic anti-inflammatory effects can also occur in other tissues, such as the gut, via direct effects of neuronal ACh on α7nAChR expressing macrophages [ 12 ]. Two factors that limit the utility of invasive VNS clinically are the need for surgery to implant the electrode and stimulator and the occurrence of off-target effects such as coughing [ 13 ]. There is also the potential for surgical complications and infection. This has led to the development of non-invasive approaches such as transcutaneous VNS (tVNS) and peripheral focused ultrasound stimulation (pFUS). As the name implies, this approach involves passing current through the skin to activate vagal nerve fibers [ 14 ]. Some studies have used this approach in acute experiments to stimulate in the cervical vagus region, and others have targeted the auricular branch of the vagus nerve by applying transcutaneous stimulation of the concha and inner tragus of the outer ear [ 13 ]. Precise neuroanatomical mechanisms by which these approaches modulate autonomic efferent tone and the full extent of their therapeutic spectrum for treating peripheral disease and suppression of inflammation are topics of intense investigation [ 13 , 15 , 16 ]. While there is some evidence that tVNS can suppress inflammatory responses [ 16 ], the ability to recruit the CAP and α7nAChRs in the spleen has not been well tested. Focused low intensity ultrasound stimulation is an emerging technique that can safely modulate neuronal activity deep within the brain [ 17 ], and recent work aims to apply this promising technology to alleviate disease in peripheral tissues by neuromodulation [ 18 ]. One obvious target for this approach is the CAP in the spleen. Here the goal is to stimulate release of NE from sympathetic nerves by pFUS. In theory, this should allow activation of the CAP with no off-target effects. Recent preclinical studies using rodent models have provided extensive evidence that FUS applied to the spleen can activate the CAP by stimulating sympathetic nerves in the tissue, bypassing the need for upstream VNS [ 19 – 21 ]. Application of FUS to the spleen produced equivalent inhibition of LPS-evoke inflammation to that achieved by invasive VNS and did so without producing bradycardia or altering companion LPS-evoked hyperglycemia [ 20 ]. The anti-inflammatory response to FUS was absent in mice lacking α7nAChRs (knockouts) or cholinergic T cells (genetic ablation) and after treatment with reserpine to deplete catecholamines. In contrast, FUS applied to a specific hepatic site, blocked LPS-induced hyperglycemia by a non-CAP mechanism involving hepatic sensory fibers but did not alter the inflammatory response to LPS [ 20 ]. Anti-inflammatory effects of FUS have also been demonstrated in mouse models of arthritis [ 21 ] and pneumonia [ 19 ]. There is substantial interest in developing drugs that can stimulate α7nAChR selectively for potential use in treating inflammatory conditions as well as diseases of the central nervous system (CNS) such as depression, schizophrenia, and Alzheimer’s disease. Interestingly, the later CNS diseases are now recognized to also have inflammatory components. Work to develop α7nAChR active drugs and characterize their pharmacology at the cellular level has contributed to improved understanding of the structure and function of this unique receptor [ 22 ]. Nicotinic receptors are pentamers comprising specific subunits, which give each variant its distinctive properties. While most nicotinic receptors are heteromeric, having different combinations of specific α and β subunits, α7nAChRs are unique in being homomers containing five α7 subunits. All nicotinic receptors function as ligand-gated cation channels when activated by agonists such as ACh, but α7nAChRs differ in having a preferential permeability to calcium compared to sodium. They also undergo rapid and reversible desensitization to a non-conducting state when stimulated by ACh. Non-neuronal localization of α7nAChRs is common in the periphery, where their presence on macrophages is central to this review because they mediate anti-inflammatory effects. Accumulating evidence gained from biochemical studies of macrophage responses to α7 receptor activation suggests that signaling in these cells is not mediated exclusively by channel activity. Rather, such responses have characteristics typical of metabotropic signaling [ 22 ]. Specifically, stimulation of α7 receptors in these cells stimulates the Jak2/STAT3 pathway, which inhibits NFκB activation and thereby decreases production of inflammatory cytokines while increasing anti-inflammatory cytokines. How α7 receptors connect to this pathway remains unknown, but it is thought that such signaling is associated with the desensitized, non-conductive state [ 22 ]. Signaling through α7nAChRs in humans also involves complications introduced by CHRFAM7A, a human-specific duplication of the α7nAChR gene, consisting of exons A, B, C, and E of the ULK4 gene and exons 5-10 of the CHRNA7 gene. It is located on chromosome 15 in either a direct or inverted orientation to CHRNA7 [ 23 ]. In addition to these two orientations, a 2-bp deletion and variable copy numbers have been identified [ 24 ]. The CHRFAM7A gene encodes a functionally altered α7 subunit, dup-α7, which is thought to be regulated at the transcriptional level [ 25 ]. Dup-α7 can form heteropentamers with functional α7 subunits in varying proportions, requiring at least two α7 subunits for the ion channel to retain function [ 23 , 26 ]. Data show that dup-α7 may negatively modulate α7nAChR activity as part of a heteropentamer, hypothetically through the reduction of ligand binding sites, which are located in the N-terminal region (exons 1-7) of the subunits, and partially lost in the CHRFAM7A chimera [ 23 ]. Its expression during inflammatory disease is related to the presence of inflammatory stimuli, which down regulate CHRFAM7A expression and increase CHRNA7 expression in an NF-κB dependent manner. It was suggested that this method of regulation blocks unneeded CAP activation in homeostatic state and promotes CAP activation in the presence of inflammatory mediators [ 23 ]. The proportions of CHRFAM7A:CHRNA7 vary between inflammatory diseases, and further study regarding the role of CHRFAM7A is warranted, as this duplicate gene may be a translational gap between laboratory animal and human studies. Many drugs have been synthesized and tested with the goal of identifying agents that can elicit α7 mediated responses with high selectivity and potency [ 22 ]. One limitation of this work has been the need to measure α7 channel activity as a functional assay. Nevertheless, this work has resulted in several drugs that evoke anti-inflammatory activity in vitro and in animal models of inflammation. These include agonists , positive allosteric modulators, and silent agonists . There has also been progress in discovery of selective antagonists, which have an important role in mechanistic studies required to establish that anti-inflammatory effects of drugs and vagal stimulation are mediated by the α7nAChR. Table 1 lists major drugs that have been used in preclinical studies of cholinergic anti-inflammatory effects. These drugs evoke channel opening by binding to active orthosteric sites on the receptor, just as ACh. GTS-21 is the most widely used drug in this class and has well-established α7-mediated anti-inflammatory effects. It is a partial agonist that produces long-lasting desensitization, which could be important for metabotropic signaling [ 22 , 27 ]. It also blocks 5-HT 3a serotonin receptors and, at higher concentrations, blocks some other nicotinic receptors [ 22 ]. PNU-282987 and PHA-543,613 are full, rapid desensitizing agonists at the α7nAChRs[ 22 , 28 – 30 ], and both have documented anti-inflammatory activity. Tropisetron is another agent in this class with some properties like GTS-21 [ 31 ]. It is approved for clinical use as an antiemetic, due to its antagonist activity at 5-HT 3a receptors, but it is not available in the United States. Limited evidence from preclinical and clinical studies suggests tropisetron has α7-mediated anti-inflammatory activity [ 32 – 35 ] Nicotinic receptors have both orthosteric and allosteric binding sites, and binding of drugs to allosteric sites can alter the response to orthosteric agonists like ACh. Two types of PAM have been identified, and these potentiate current responses evoked by ACh while not opening channels when applied alone [ 22 ]. Both types increase peak current, but only Type II drugs prolong responses. There is limited evidence that one of these compounds, PNU-120596 [ 36 ], can elicit α7-mediated anti-inflammatory responses [ 37 , 38 ]. Two additional profiles have been identified for novel drugs acting at the α7 receptor. GAT107 appears to activate the receptor directly through a distinct allosteric activation site and have PAM activity as well. Such drugs have been termed “ago-positive allosteric modulators” [ 22 ]. Effects of GAT107 on the CAP have not been evaluated, but there are reports that this agent reverses inflammatory pain by stimulation of α7 receptors [ 39 ] and macrophage dysfunction caused by hyperoxia [ 40 ]. Other studies have shown that GAT107 can cause α7-mediated suppression of neuroinflammation and pathology in a murine model of experimental autoimmune encephalomyelitis [ 41 ]. Interestingly, the latter effects occur at least in part by stimulation of α7 receptors on T cells, although the same study also established that GAT107 reduced the inflammatory response of RAW267.4 cells (macrophage line) to LPS. Lastly, NS6740 is an early representative of the silent agonist class [ 22 , 42 ]. Such drugs have minimal channel activating activity but do induce a long-lasting non-conducting state (desensitization). If this condition is associated with metabotropic signaling, as proposed [ 22 , 42 ], then silent agonists could be especially effective agents for treating chronic inflammatory diseases. A few studies found that NS6740 can attenuate pro-inflammatory effects of LPS on primary microglia from rats [ 43 ] and on a microglia cell line [ 44 ]. While selective antagonists have no clinical applications, such compounds are valuable for mechanistic studies aimed at confirming the role of α7nAChRs in anti-inflammatory effects evoked by nerve stimulation or drugs (e.g., GTS-21). α-Bungarotoxin (α-BTX) is a snake toxin that has a high affinity for blocking α7nAChRs, but it also blocks nicotinic receptors at the neuromuscular junction. This limits its utility for in vivo studies, although it can be given directly into the central nervous system. Nevertheless, it can be useful for in vitro studies to evaluate the role α7 receptors on leukocytes and labeled forms of α-BTX can be used to study the cellular localization of these receptors. Methyllycaconitine (MLA) is the most widely used α7 antagonist, but its selectivity is concentration dependent, and blockade of ganglia can occur at higher doses [ 22 ]. While it is generally regarded as a competitive antagonist, a few studies indicate it can have more complicated actions, at least in some test systems. One cell culture study of microglial cells found that MLA can attenuate LPS-induced release of TNF-α[ 45 ], which is contrary to results from many other studies of macrophages [ 45 – 48 ]. Recent voltage-clamp studies of α7nAChRs expressed in Xenopus oocytes also suggest that MLA might function as an inverse agonist rather than a simple competitive blocker of ACh [ 49 ]. These findings highlight the importance of using complementary approaches (e.g., α7 knockout) to confirm the role of α7nAChRs in neuroimmune mechanisms. Specific cholinergic neurons in the brain have output that stimulates the CAP via the vagal efferent pathway ( Fig. 2 ) [ 50 – 54 ]. Although precise circuitry needs to be defined, recent evidence has implicated cholinergic neurons in the medial septum/diagonal band regions, which have projections to downstream areas where cholinergic transmission occurs by activation of postsynaptic M1 muscarinic receptors [ 51 ]. This pathway can be activated by centrally acting cholinesterase inhibitors (ChEIs) like galantamine, which enhance cholinergic neurotransmission [ 51 , 53 ]. It can also be activated by intracerebral injection of M1 muscarinic agonists, such as McN-A-343 and CNI-1493, as well as the nonselective muscarinic agonist muscarine [ 52 ]. Likewise, peripheral administration of the centrally acting M1 agonist xanomeline attenuated pro-inflammatory cytokine release evoked by LPS and increased survival after LPS administration and in a mouse model of sepsis [ 54 ]. Such effects of M1 agonists are centrally mediated since they were blocked by atropine, which penetrates to the brain, and unaffected by atropine methyl nitrate, which does not cross the blood brain barrier [ 51 , 52 , 54 , 55 ]. Blockade of inhibitory presynaptic M2 muscarinic autoreceptors on cholinergic nerve ending has the same effect by enhancing release of ACh, and thereby potentiating cholinergic neurotransmission [ 52 ]. Central activation of the CAP can also be triggered by central or peripheral administration of BQCA, an allosteric modulator of M1 muscarinic receptors [ 51 ]. This novel drug acts by augmenting the response evoked by endogenous ACh binding to M1 receptors [ 56 , 57 ]. Each of these approaches ultimately elicits an anti-inflammatory effect mediated by the CAP and peripheral α7nAChRs. Drugs that inhibit ChEs in the periphery can amplify and prolong actions mediated by endogenous ACh, whether released from cholinergic nerves or non-neuronal sources such as T and B cells. Therefore, is it logical to expect that peripheral ChEIs might elicit anti-inflammatory effects by this mechanism. Several preclinical studies have evaluated the anti-inflammatory efficacy of pyridostigmine and related quaternary amines in a wide variety of models [ 58 ]. Many of these studies found that selective inhibition of peripheral ChE reduced inflammatory indices and most showed improved outcome [ 59 – 63 ], but others reported lack of anti-inflammatory efficacy [ 64 , 65 ]. Nevertheless, several studies that compared centrally active ChEIs to those that act only in the periphery, showed greater anti-inflammatory efficacy with the drugs that inhibit both central and peripheral ChE [ 58 , 66 , 67 ]. Such superiority of centrally acting ChEIs might be attributed to the fact that they activate the CAP and potentiate its activity by protecting ACh released at peripheral sites.

Interest in the α7 nicotinic acetylcholine receptor (α7nAChR) as a target for suppressing adverse inflammatory responses has its origin in the early 2000’s when research established that stimulation of the vagus nerve suppressed the inflammatory response to injection of lipopolysaccharide (LPS) in rats [ 1 ]. Early work showed that the spleen was required for this vagal anti-inflammatory effect and determined essential roles for nicotinic receptors and sympathetic nerves [ 1 – 3 ]. Ultimately, a series of experiments using α7nAChR deficient mice and cholinergic phenotype reporter mice, along with neuroanatomical techniques and flow cytometry, delineated the current model for the cholinergic anti-inflammatory pathway (CAP), which is illustrated in Figure 1 . This pathway originates in the brainstem with preganglionic cholinergic neurons of the dorsal motor nucleus, which sends long axonal projections to the periphery by mean of the vagus nerve. While many of these axons innervate parasympathetic ganglia in various end organs, some also control a subpopulation of sympathetic neurons in the celiac and superior mesenteric ganglia, which send noradrenergic nerves to the splenic via the splenic nerve. Activation of this circuitry causes the release of norepinephrine (NE) from varicosities of noradrenergic nerves distributed within the splenic white pulp. Next, NE stimulates β 2 adrenergic receptors located on a special population of CD4+ T cells that can synthesize and release ACh. These cells contain the ACh synthetic enzyme, choline acetyltransferase (ChAT), but release of ACh occurs by a transporter mechanism instead of exocytosis as occurs with cholinergic nerves. While close juxtaposition of noradrenergic nerves with cholinergic T cells was considered important initially, it now appears that diffusion of transmitter to the target T cell population is sufficient. For the final step in the pathway, ACh diffuses to monocyte- and macrophage-rich areas of the spleen and stimulates α7nAChR receptors located in their cell membrane. Binding of ACh activates a signaling cascade that inhibits the synthesis and release of pro-inflammatory cytokines such as tissue necrosis factor-α (TNF-α). Given the central role that inflammation plays in many diseases, there has been explosive growth in studies investigating potential therapeutic applications for activation of the CAP in the spleen and similar mechanisms in other tissues by using bioelectronic and pharmacologic approaches. Furthermore, recent work has identified novel α7 dependent mechanisms in the bone marrow that regulate hematopoiesis and leukocyte trafficking [ 4 ], suggesting an additional target for α7 based immunomodulatory therapy.