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Functions of acetylcholine-producing lymphocytes in immunobiology
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ScienceDirect Functions of acetylcholine-producing lymphocytes in immunobiology Stephen G Malin, Vladmir S Shavva, Laura Tarnawski and Peder S Olofsson Recent advances in neuroscience and immunology have shown that cholinergic signals are vital in the regulation of inflammation and immunity. Choline acetyltransferase+ (ChAT+) lymphocytes have the capacity to biosynthesize and release acetylcholine, the cognate ligand for cholinergic receptors. Acetylcholine-producing T cells relay neural signals in the ‘inflammatory reflex’ that regulate cytokine release in spleen. Mice deficient in acetylcholine-producing T cells have increased blood pressure, show reduced local vasodilatation and viral control in lymphocytic choriomeningitis virus infection, and display changes in gut microbiota compared with littermates. These observations indicate that ChAT+ lymphocytes play physiologically important roles in regulation of inflammation and anti-microbial defense. However, the full scope and importance of ChAT+ lymphocytes in immunity and vascular biology remains to be elucidated. Here, we review key findings in this emerging area. Address Laboratory of Immunobiology, Center for Bioelectronic Medicine, Department of Medicine, Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Corresponding author: Olofsson, Peder S ([email protected])
Current Opinion in Neurobiology 2020, 62:115–121 This review comes from a themed issue on Brain, gut, and immune system interactions Edited by Asya Rolls and Isaac Chiu
actions of acetylcholine are relatively well understood in the context of nervous and musculoskeletal systems, but our mechanistic understanding of extra-neuronal cholinergic signals is much more limited. Recent developments in neuroscience and immunology show that cholinergic signals are vital in the regulation of inflammation and immunity. Landmark discoveries by the Watkins, Niijima, and Tracey labs revealed that elevated cytokine levels in the periphery cause signals in the cholinergic vagus nerve that promote fever, and elicit motor signals to the spleen that can regulate cytokine release in inflammation [5–7]. These insights led to the definition of the ‘inflammatory reflex’, a concept that has propelled research on neural control of inflammation [8,9]. Subsequent work showed that the cholinergic a7 nicotinic acetylcholine receptor subunit (Chrna7) in immune cells, previously primarily studied in the nervous system, is essential for neural reflex regulation of cytokines in systemic inflammation, for example release of pro-inflammatory cytokines from macrophages [10–18]. Other cholinergic receptors also regulate immune responses. For example, activation of nicotinic or muscarinic acetylcholine receptors on T cells was reported to promote Th1 differentiation or Th2/Th17 differentiation, respectively [19]. The discovery that lymphocytes have the capacity to provide the cognate ligand for cholinergic receptors by biosynthesizing and releasing acetylcholine have revealed additional modes of immune cell communication, but much of this work is only in its infancy. Here, we review known functions of acetylcholineproducing lymphocytes.
https://doi.org/10.1016/j.conb.2020.01.017 0959-4388/ã 2020 Elsevier Ltd. All rights reserved.
Lymphocyte biosynthesis and release of acetylcholine
Introduction Cholinergic regulation of inflammation
Acetylcholine is a key neurotransmitter of cholinergic nerves in animals, but acetylcholine is found across many systems, including in unicellular organisms, bacteria and plants. Measurable levels of acetylcholine have been reported in a range of different tissues, and cognate cholinergic receptors for acetylcholine are expressed by many cell types and across organs inside and outside of the nervous system [1–4]. The cellular and synaptic www.sciencedirect.com
Acetylcholine is most well known as a principal neurotransmitter released in quanta under the control of action potentials in cholinergic nerves. Most of our understanding of acetylcholine biosynthesis and release is derived from studies of neurons, and knowledge of the implications of lymphocyte acetylcholine synthesis is limited. Choline acetyltransferase (ChAT) catalyzes acetylcholine biosynthesis from the precursors acetyl-coenzyme A (acetyl-CoA) and choline. Choline can be synthesized or sourced from the extracellular space through uptake by selective or non-selective transporters, and the rate limiting step for acetylcholine biosynthesis in neurons appears to be choline uptake [20–22]. Acetyl-CoA can originate either from the Current Opinion in Neurobiology 2020, 62:115–121
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oxidative metabolism of glucose through glycolysis or from fatty acids via beta-oxidation. The sources of ChAT substrates in lymphocytes are not well defined, but it is interesting to consider that as T cell metabolic states change with activation and differentiation, availability of choline and acetyl-CoA may shift considerably [23–25], and ChAT biosynthesis of acetylcholine likely changes accordingly. The cellular localization of ChAT may provide clues to the modes of storage and release of acetylcholine [26–29]. Both soluble and membrane-bound ChAT have been isolated from murine brain [30]. However, the cellular localization of active lymphocyte ChAT is unclear. Experimental observations suggest that lymphocytes can release acetylcholine in a non-quantal manner independent of vesicles, but the mechanism of lymphocyte acetylcholine release requires further investigation. Potentially lymphocyte-derived ChAT could also signal to neurons themselves, both in the periphery and CNS, but such mechanisms remain purely speculative at the moment.
release acetylcholine has not been definitively defined. However, considering the observations to date in mice, many different T cell populations are permissive to ChAT upregulation in response to T cell receptor activation within a suitable cytokine environment. There are also several reports on ChAT expression in human mononuclear cells [43,44]. Of note, most experimental work on ChAT+ leukocytes was performed on cell lines and in vitro [44]. Regrettably, many of the available anti-ChAT antibodies show insufficient specificity when used with non-neural tissue. Considering this and to the best of our knowledge, it is unknown whether primary human T cells have the capacity to express functional ChAT protein and biosynthesize acetylcholine under physiological conditions. Addressing the existence of human ChAT+ lymphocytes, if such a population exists, is a priority for future research in the field. Regulation of blood pressure
In lymphocytes, ChAT is induced by cellular activation, including ligation of pattern recognition receptors and T cell receptors [31–33], administration of /7nAChR ligand SLURP-1 and possibly by autocrine upregulation by acetylcholine [34]. However, details of the involved pathways and regulatory elements are sparse, particularly for primary lymphocytes [35]. In mouse T cells, interleukin 21 exposure strongly promotes ChAT expression [33,36]. Release of acetylcholine by T cells is promoted by noradrenalin exposure and T cell receptor engagement [32,33,37] and cholecystokinin exposure promotes B cell acetylcholine release [31].
Acetylcholine-producing ChAT+ T cells Discoveries over the last decade have revealed unexpected roles for ChAT+ T cells, including the regulation of cytokine release, blood pressure and in regulating leukocyte tissue entry in infection. Appreciating the local and systemic effects of leukocyte cholinergic signaling in infection and autoimmunity could perhaps help identify new treatment targets in inflammatory diseases [38–40]. Phenotype
At the initial discovery that ChAT+ T cells relay neural signals in the mouse inflammatory reflex, these T cells were defined as CD3+CD4+CD44hiCD62Llow, that is of an effector/memory phenotype [32]. Subsequent reports show that several different phenotypes of T cells can express ChAT. Studies using ChAT-eGFP reporter mice [41] revealed that both CD4+ and CD8+ T cells express ChAT in Lymphocytic choriomeningitis virus (LCMV) infection [33] and that ChAT-expression was found in several lineages, including in effector, memory, and follicular helper CD4+ T cells. In the mouse intestine, ChAT+ T cells expressed markers of the Th17 lineage [42]. The exact nature of which T cell lineages have the capacity to upregulate ChAT and biosynthesize and Current Opinion in Neurobiology 2020, 62:115–121
It has long been known that acetylcholine is abundant in spleen [45] and that cholinergic agonists regulate cytokine release [46], but the spleen lacks cholinergic innervation and is instead supplied by the adrenergic splenic nerve. The discovery that ChAT+ T cells relay neural signals to immune cells in spleen by releasing acetylcholine [32] showed that T lymphocytes are an integral part of the inflammatory reflex capable of supplying the appropriate ligand to cholinergic receptors (Figure 1a). It turned out that ChAT+ T cells may perform a similar function also in other organs, for example the vasculature. Importantly, cholinergic receptors are abundant on vascular endothelial cells. It is well established that activation of these receptors by acetylcholine promotes phosphorylation of endothelial nitric oxide synthase (eNOS), thereby leading to endothelial production of nitric oxide (NO). Diffusion of NO to outer layers of the vascular wall induces relaxation of vascular smooth muscle cells [47]. However, the role of acetylcholine in the regulation of vascular contractility in vivo and its importance under physiological conditions are not well understood. Furthermore, the source of acetylcholine for these vascular endothelial cholinergic receptors has not been determined. The vasculature largely lacks cholinergic motor innervation, and free acetylcholine cannot be transported far in blood because it is quickly degraded by acetylcholinesterases with high catalytic activity [48,49]. Interestingly, T cells are involved in development of hypertension in rodents, and mice deficient in acetylcholine-producing ChAT+ T cells show elevated blood pressure and signs of increased cardiac afterload [50,51]. Conversely, Jurkat T cells engineered to overexpress ChAT and release acetylcholine promote eNOS phosphorylation in vitro, and reduction of blood pressure when injected in vivo, a capacity that is lost by blocking cholinergic receptors [51]. Hence, it appears that acetylcholine-producing T cells have the capacity to provide the cognate ligand for endothelial cell cholinergic receptors www.sciencedirect.com
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Figure 1
(b)
(a)
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ACh Current Opinion in Neurobiology
Schematic representation of proposed physiological effects of ChAT+ lymphocytes. (a) ChAT+ T cells respond to adrenergic signals with increased release of acetylcholine (ACh), a ligand also for alpha7 nicotinic acetylcholine receptor subunit containing receptors. Ligands for alpha7 nicotinic acetylcholine receptor subunit containing receptors can activate Jak2/STAT3 and/or cAMP signaling in macrophage-like cells and reduce release of pro-inflammatory cytokines. (b) ChAT+ T cells release acetylcholine (ACh), the cognate ligand for muscarinic acetylcholine receptors on vascular endothelial cells. Acetylcholine exposure leads to endothelial production of nitric oxide, which promotes smooth muscle cell relaxation and vascular dilatation. (c) Mice deficient in ChAT+ T cells show reduced expression of intestinal antimicrobial peptides and different diversity of the jejunal microbiome. Additionally, Ach-mediated iNOS activation in intestinal endothelial cells may induce production of NO, which potentially has anti-microbial effects (d) B cells can express ChAT and release ACh which may prevent neutrophil recruitment to the peritoneum.
and, in this way, reduce vascular contractility and blood pressure in mice (Figure 1b). Whether ChAT+ T cells play a role in human blood pressure regulation has not yet been established. Facilitation of T cell tissue entry in infection
Regulation of blood vessel contractility is important in the inflammatory response to infection and tissue injury, but in itself is not sufficient to accomplish leukocyte extravasation [52]. Surprisingly, genetic deletion of Chat in the T cell compartment significantly reduced local vasodilatation, tissue entry of cytotoxic T cells specific for LCMV viral antigen, and virus clearance in LCMV-infected mice [33]. Treatment of infected mice deficient in ChAT+ T cells with a vasodilator improved capacity for virus clearance, while inhibiting nitric oxide synthesis in wild type mice reduced virus clearance [33]. In light of these findings, acetylcholine-mediated vasodilatation may be an additional mechanism important for tissue entry of www.sciencedirect.com
blood-borne lymphocytes in infection and clearance of virus [53]. The mechanism by which ChAT+ T cells facilitate tissue entry of antigen-specific cytotoxic T cells in infected tissues has not been characterized. It is possible that ChAT+ T cell derived acetylcholine affects a number of mechanisms at the intersection between blood and the vascular wall, including activation of endothelial muscarinic receptors, changes in expression of endothelial adhesion molecules, production of nitric oxide, and changes in vascular wall permeability and smooth muscle cell contractility (Figure 1b). At present, mechanistic details of the interactions of acetylcholineproducing T cells in blood and the vascular wall are incompletely understood. Gut homeostasis and host defense
A number of reports have linked ChAT+ T cells to the response to microorganisms. Dhawan et al. investigated mice deficient in ChAT+ T cells and found that Current Opinion in Neurobiology 2020, 62:115–121
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expression of a number of antimicrobial peptides was reduced in jejunal mucosa, and in mice deficient in ChAT+ T cells, the microbial diversity was increased compared with littermates [42] (Figure 1c). Another study from the same group concluded that ChAT+ T cells worsen the local intestinal innate immune response in experimental induced colitis, whereas ChAT+ T promote resolution of inflammation in induced colitis [54]. Hence, it is possible that the presence of ChAT+ lymphocytes may affect the mucosal environment in the intestine and potentially the composition of the intestinal microflora and impact mucosal inflammation. The percentage and number of splenic ChAT+ T cells were increased in mice after experimental polymicrobial sepsis of intestinal origin, and the authors propose that ChAT+ T cells play a role in the immunosuppression phenotype observed in survivors of experimental sepsis [55]. Ramires et al. observed recruitment of ChAT+ T cells upon Citrobacter rodentium infection in mice and conditional T cell ChAT knockout lead to increased infection and local inflammation. However, antimicrobial peptide production was unaffected. In the absence of ChAT+ T cells, iNos expression in intestinal epithelial cells decreased while the cholinomimetic carbachol together with INFg promoted iNos expression in murine colonic epithelial cell line CMT-93 [56]. Given the established antimicrobial properties of NO [57], these data support a role for ChAT+ T cells in host defence, potentially both in regulation of antimicrobial molecule production and in controlling inflammation at the site of infection.
ChAT+ B cell biology Acquired immunity to foreign pathogens critically depends on functional B cells that develop in the bone marrow, in contrast to T lymphopoiesis that is largely restricted to the thymus in mammals. Although T cells have been the most widely studied lymphocyte population with regards to ChAT expression, ChAT+ B cells have been detected in several studies. There is limited understanding of regulation of ChAT expression and activity in B cells, but there are reports that TLR ligands (CpG, TLR2 ligand; LPS, TLR4 ligand; R848, TLR7 ligand; Pam3Cys TLR9 ligand) promote ChAT expression in murine B2 and B1b cells in a MyD88-dependent manner [31]. Phenotype
The ChAT-eGFP transgenic reporter line has again been instrumental in isolating and assigning functional roles for ChAT B cells. This reporter mouse line is controlled by the ChAT promoter elements that drive ChAT expression in cholinergic neurons [41]. Although this strain is thought to correctly mirror expression of ChAT, interpretations of results obtained with this strain must be tempered with the probable extended Current Opinion in Neurobiology 2020, 62:115–121
of half-life of GFP versus ChAT and also that the transgenic construct faithfully recapitulates lymphocyte ChAT expression. Examination of the thymus in ChATeGFP reporter mice failed to detect GFP within the predominant CD4-expressing and CD8-expressing populations [31]. It remains possible that the minor so-called double negative population, where correct rearrangement of the alpha/beta T cell receptor is completed, may contain ChAT populations that have been herewith overlooked. Commitment to the B cell lineage in the bone marrow occurs at the pro-B cell stage, which is also characterized by completion of V-DJ recombination of the immunoglobulin heavy chain. Rearrangement of the light chain in the pre-B cell stage allows for production of a functional B cell receptor. However, robust GFP expression seemed to be restricted to the later mature B cell subsets that can recirculate back to the bone marrow, rather than these earlier progenitors [31]. A more refined characterization of earlier B lymphopoiesis may still reveal ChAT-expressing subsets. However, available data would imply that ChAT expression is not a characteristic of lymphocytes undergoing V-DJ recombination and by inference central tolerance mechanisms during B-lymphopoiesis and T-lymphopoiesis. Interestingly, the IMMGEN database [58] indicates that none of the major immune cell populations express ChAT. The reason for this discrepancy remains unclear. Function
ChAT-expressing B cells are relatively abundant in the peripheral lymphoid organs, and may even be more numerous than T cells [31,59]. Reardon et al. detected robust expression of ChAT in subsets of the marginal zone (MZ) and B1 cell subsets. Interestingly, follicular B cells also seem to express ChAT in the spleen and also presumably in the lymph nodes, but the identity and functional importance of this putative subset has not been determined. Addressing this question will be of considerable interest, as follicular B cells are the major B cell subset responsible for the extra-follicular and germinal center responses that are critical for host defense and autoimmunity. Whether ChAT expression is dynamic within B cells, delineates a separate subset, or rather that ChAT-expressing B cells shares markers with other common B cell populations has not been fully determined. Notably however, fate-mapping approaches would seem to indicate that ChAT expression is transient within B cells, indicating that production of acetylcholine could be an acquired function within a given lymphocyte population [31]. Conceptually, it is the expression of defined receptors that respond to B cell mitogens and stimulants that would engender a B cell permissive to ChAT expression. A number of recent reports have implicated neural control in the regulation of adaptive and innate immune www.sciencedirect.com
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responses of B cells. The expression of ChAT within MZ and B1 cells would imply a role for ChAT in the antibacterial responses that characterize MZ and B1 cells [60] and would be consistent with other known functions of the inflammatory reflex in systemic inflammation. Indeed, ChAT B cells played an important role in limiting the recruitment of neutrophils into the peritoneum following LPS administration and furthermore ChAT expression in B cells can be dynamically regulated through Toll-like receptor ligands (Figure 1d). Potential neuronal control of B cell-mediated antibacterial responses have stimulated efforts to understand how innervation of the spleen could regulate this process, given the critical function of the spleen in hosting humoral immunity to gram-positive bacteria typified by Streptococcus pneumoniae. Stimulation of the vagus nerve was demonstrated to prevent accumulation of CD11b cells within the marginal zone following immunization with S. pneumoniae, and that antibody secreting cells seemed to colocalize with nerve fibers [61]. It could be speculated that communication between neurons and B cells may be involved in the T-cell independent responses that dominate in the B cell responses to gram-positive bacteria. Notably however, vagal nerve stimulation (VNS) did not affect antibody secretion, indicating that either the inflammatory reflex is dispensable or that the VNS protocol has not yet been optimized for these readouts. As well as producing acetylcholine, B cells may also be responsive to acetylcholine as well. Loss of Chrna7 leads to an increase in humoral immune responses and increases in CD40 expression. Expression of the Chrna7 also seems to be upregulated as B cell mature, indicative of possible autocrine mechanisms [62,63]. Of note, the IMMGEN database fails to show any robust expression of Chrna7 in immune cells.
Conclusion In summary, the capacity of some lymphocyte subpopulations to express ChAT and biosynthesize acetylcholine has been known for decades. Only recently have distinct functional capacities of acetylcholine-producing primary lymphocytes been defined. Although available data are limited, ChAT+ T cells are essential for regulation of systemic release of pro-inflammatory cytokines by the inflammatory reflex [32], involved in blood pressure regulation [51], required for optimal anti-viral defense in mice [33] and implicated in gut microbial homeostasis [42]. ChAT-expressing B cells are relatively abundant in peripheral lymphoid organs and might function to regulate T-cell independent immune responses. Mapping the mechanisms of ChAT regulation, acetylcholine transport and release in lymphocytes, as well as molecular interactions with other immune cells and the vasculature, will be important to undertake in the immediate future. www.sciencedirect.com
Conflict of interest statement SM, LT, and VSS have no conflicts of interest to declare relative to this manuscript. PSO is a co-founder and shareholder of Emune AB and ChAT Therapeutics.
Acknowledgements The work was funded by grants to PSO from the Knut and Alice Wallenberg Foundation, the Swedish Medical Council and the Stockholm Region (ALF).
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Current Opinion in Neurobiology 2020, 62:115–121
ScienceDirect Functions of acetylcholine-producing lymphocytes in immunobiology Stephen G Malin, Vladmir S Shavva, Laura Tarnawski and Peder S Olofsson Recent advances in neuroscience and immunology have shown that cholinergic signals are vital in the regulation of inflammation and immunity. Choline acetyltransferase+ (ChAT+) lymphocytes have the capacity to biosynthesize and release acetylcholine, the cognate ligand for cholinergic receptors. Acetylcholine-producing T cells relay neural signals in the ‘inflammatory reflex’ that regulate cytokine release in spleen. Mice deficient in acetylcholine-producing T cells have increased blood pressure, show reduced local vasodilatation and viral control in lymphocytic choriomeningitis virus infection, and display changes in gut microbiota compared with littermates. These observations indicate that ChAT+ lymphocytes play physiologically important roles in regulation of inflammation and anti-microbial defense. However, the full scope and importance of ChAT+ lymphocytes in immunity and vascular biology remains to be elucidated. Here, we review key findings in this emerging area. Address Laboratory of Immunobiology, Center for Bioelectronic Medicine, Department of Medicine, Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Corresponding author: Olofsson, Peder S ([email protected])
Current Opinion in Neurobiology 2020, 62:115–121 This review comes from a themed issue on Brain, gut, and immune system interactions Edited by Asya Rolls and Isaac Chiu
actions of acetylcholine are relatively well understood in the context of nervous and musculoskeletal systems, but our mechanistic understanding of extra-neuronal cholinergic signals is much more limited. Recent developments in neuroscience and immunology show that cholinergic signals are vital in the regulation of inflammation and immunity. Landmark discoveries by the Watkins, Niijima, and Tracey labs revealed that elevated cytokine levels in the periphery cause signals in the cholinergic vagus nerve that promote fever, and elicit motor signals to the spleen that can regulate cytokine release in inflammation [5–7]. These insights led to the definition of the ‘inflammatory reflex’, a concept that has propelled research on neural control of inflammation [8,9]. Subsequent work showed that the cholinergic a7 nicotinic acetylcholine receptor subunit (Chrna7) in immune cells, previously primarily studied in the nervous system, is essential for neural reflex regulation of cytokines in systemic inflammation, for example release of pro-inflammatory cytokines from macrophages [10–18]. Other cholinergic receptors also regulate immune responses. For example, activation of nicotinic or muscarinic acetylcholine receptors on T cells was reported to promote Th1 differentiation or Th2/Th17 differentiation, respectively [19]. The discovery that lymphocytes have the capacity to provide the cognate ligand for cholinergic receptors by biosynthesizing and releasing acetylcholine have revealed additional modes of immune cell communication, but much of this work is only in its infancy. Here, we review known functions of acetylcholineproducing lymphocytes.
https://doi.org/10.1016/j.conb.2020.01.017 0959-4388/ã 2020 Elsevier Ltd. All rights reserved.
Lymphocyte biosynthesis and release of acetylcholine
Introduction Cholinergic regulation of inflammation
Acetylcholine is a key neurotransmitter of cholinergic nerves in animals, but acetylcholine is found across many systems, including in unicellular organisms, bacteria and plants. Measurable levels of acetylcholine have been reported in a range of different tissues, and cognate cholinergic receptors for acetylcholine are expressed by many cell types and across organs inside and outside of the nervous system [1–4]. The cellular and synaptic www.sciencedirect.com
Acetylcholine is most well known as a principal neurotransmitter released in quanta under the control of action potentials in cholinergic nerves. Most of our understanding of acetylcholine biosynthesis and release is derived from studies of neurons, and knowledge of the implications of lymphocyte acetylcholine synthesis is limited. Choline acetyltransferase (ChAT) catalyzes acetylcholine biosynthesis from the precursors acetyl-coenzyme A (acetyl-CoA) and choline. Choline can be synthesized or sourced from the extracellular space through uptake by selective or non-selective transporters, and the rate limiting step for acetylcholine biosynthesis in neurons appears to be choline uptake [20–22]. Acetyl-CoA can originate either from the Current Opinion in Neurobiology 2020, 62:115–121
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oxidative metabolism of glucose through glycolysis or from fatty acids via beta-oxidation. The sources of ChAT substrates in lymphocytes are not well defined, but it is interesting to consider that as T cell metabolic states change with activation and differentiation, availability of choline and acetyl-CoA may shift considerably [23–25], and ChAT biosynthesis of acetylcholine likely changes accordingly. The cellular localization of ChAT may provide clues to the modes of storage and release of acetylcholine [26–29]. Both soluble and membrane-bound ChAT have been isolated from murine brain [30]. However, the cellular localization of active lymphocyte ChAT is unclear. Experimental observations suggest that lymphocytes can release acetylcholine in a non-quantal manner independent of vesicles, but the mechanism of lymphocyte acetylcholine release requires further investigation. Potentially lymphocyte-derived ChAT could also signal to neurons themselves, both in the periphery and CNS, but such mechanisms remain purely speculative at the moment.
release acetylcholine has not been definitively defined. However, considering the observations to date in mice, many different T cell populations are permissive to ChAT upregulation in response to T cell receptor activation within a suitable cytokine environment. There are also several reports on ChAT expression in human mononuclear cells [43,44]. Of note, most experimental work on ChAT+ leukocytes was performed on cell lines and in vitro [44]. Regrettably, many of the available anti-ChAT antibodies show insufficient specificity when used with non-neural tissue. Considering this and to the best of our knowledge, it is unknown whether primary human T cells have the capacity to express functional ChAT protein and biosynthesize acetylcholine under physiological conditions. Addressing the existence of human ChAT+ lymphocytes, if such a population exists, is a priority for future research in the field. Regulation of blood pressure
In lymphocytes, ChAT is induced by cellular activation, including ligation of pattern recognition receptors and T cell receptors [31–33], administration of /7nAChR ligand SLURP-1 and possibly by autocrine upregulation by acetylcholine [34]. However, details of the involved pathways and regulatory elements are sparse, particularly for primary lymphocytes [35]. In mouse T cells, interleukin 21 exposure strongly promotes ChAT expression [33,36]. Release of acetylcholine by T cells is promoted by noradrenalin exposure and T cell receptor engagement [32,33,37] and cholecystokinin exposure promotes B cell acetylcholine release [31].
Acetylcholine-producing ChAT+ T cells Discoveries over the last decade have revealed unexpected roles for ChAT+ T cells, including the regulation of cytokine release, blood pressure and in regulating leukocyte tissue entry in infection. Appreciating the local and systemic effects of leukocyte cholinergic signaling in infection and autoimmunity could perhaps help identify new treatment targets in inflammatory diseases [38–40]. Phenotype
At the initial discovery that ChAT+ T cells relay neural signals in the mouse inflammatory reflex, these T cells were defined as CD3+CD4+CD44hiCD62Llow, that is of an effector/memory phenotype [32]. Subsequent reports show that several different phenotypes of T cells can express ChAT. Studies using ChAT-eGFP reporter mice [41] revealed that both CD4+ and CD8+ T cells express ChAT in Lymphocytic choriomeningitis virus (LCMV) infection [33] and that ChAT-expression was found in several lineages, including in effector, memory, and follicular helper CD4+ T cells. In the mouse intestine, ChAT+ T cells expressed markers of the Th17 lineage [42]. The exact nature of which T cell lineages have the capacity to upregulate ChAT and biosynthesize and Current Opinion in Neurobiology 2020, 62:115–121
It has long been known that acetylcholine is abundant in spleen [45] and that cholinergic agonists regulate cytokine release [46], but the spleen lacks cholinergic innervation and is instead supplied by the adrenergic splenic nerve. The discovery that ChAT+ T cells relay neural signals to immune cells in spleen by releasing acetylcholine [32] showed that T lymphocytes are an integral part of the inflammatory reflex capable of supplying the appropriate ligand to cholinergic receptors (Figure 1a). It turned out that ChAT+ T cells may perform a similar function also in other organs, for example the vasculature. Importantly, cholinergic receptors are abundant on vascular endothelial cells. It is well established that activation of these receptors by acetylcholine promotes phosphorylation of endothelial nitric oxide synthase (eNOS), thereby leading to endothelial production of nitric oxide (NO). Diffusion of NO to outer layers of the vascular wall induces relaxation of vascular smooth muscle cells [47]. However, the role of acetylcholine in the regulation of vascular contractility in vivo and its importance under physiological conditions are not well understood. Furthermore, the source of acetylcholine for these vascular endothelial cholinergic receptors has not been determined. The vasculature largely lacks cholinergic motor innervation, and free acetylcholine cannot be transported far in blood because it is quickly degraded by acetylcholinesterases with high catalytic activity [48,49]. Interestingly, T cells are involved in development of hypertension in rodents, and mice deficient in acetylcholine-producing ChAT+ T cells show elevated blood pressure and signs of increased cardiac afterload [50,51]. Conversely, Jurkat T cells engineered to overexpress ChAT and release acetylcholine promote eNOS phosphorylation in vitro, and reduction of blood pressure when injected in vivo, a capacity that is lost by blocking cholinergic receptors [51]. Hence, it appears that acetylcholine-producing T cells have the capacity to provide the cognate ligand for endothelial cell cholinergic receptors www.sciencedirect.com
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Figure 1
(b)
(a)
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Schematic representation of proposed physiological effects of ChAT+ lymphocytes. (a) ChAT+ T cells respond to adrenergic signals with increased release of acetylcholine (ACh), a ligand also for alpha7 nicotinic acetylcholine receptor subunit containing receptors. Ligands for alpha7 nicotinic acetylcholine receptor subunit containing receptors can activate Jak2/STAT3 and/or cAMP signaling in macrophage-like cells and reduce release of pro-inflammatory cytokines. (b) ChAT+ T cells release acetylcholine (ACh), the cognate ligand for muscarinic acetylcholine receptors on vascular endothelial cells. Acetylcholine exposure leads to endothelial production of nitric oxide, which promotes smooth muscle cell relaxation and vascular dilatation. (c) Mice deficient in ChAT+ T cells show reduced expression of intestinal antimicrobial peptides and different diversity of the jejunal microbiome. Additionally, Ach-mediated iNOS activation in intestinal endothelial cells may induce production of NO, which potentially has anti-microbial effects (d) B cells can express ChAT and release ACh which may prevent neutrophil recruitment to the peritoneum.
and, in this way, reduce vascular contractility and blood pressure in mice (Figure 1b). Whether ChAT+ T cells play a role in human blood pressure regulation has not yet been established. Facilitation of T cell tissue entry in infection
Regulation of blood vessel contractility is important in the inflammatory response to infection and tissue injury, but in itself is not sufficient to accomplish leukocyte extravasation [52]. Surprisingly, genetic deletion of Chat in the T cell compartment significantly reduced local vasodilatation, tissue entry of cytotoxic T cells specific for LCMV viral antigen, and virus clearance in LCMV-infected mice [33]. Treatment of infected mice deficient in ChAT+ T cells with a vasodilator improved capacity for virus clearance, while inhibiting nitric oxide synthesis in wild type mice reduced virus clearance [33]. In light of these findings, acetylcholine-mediated vasodilatation may be an additional mechanism important for tissue entry of www.sciencedirect.com
blood-borne lymphocytes in infection and clearance of virus [53]. The mechanism by which ChAT+ T cells facilitate tissue entry of antigen-specific cytotoxic T cells in infected tissues has not been characterized. It is possible that ChAT+ T cell derived acetylcholine affects a number of mechanisms at the intersection between blood and the vascular wall, including activation of endothelial muscarinic receptors, changes in expression of endothelial adhesion molecules, production of nitric oxide, and changes in vascular wall permeability and smooth muscle cell contractility (Figure 1b). At present, mechanistic details of the interactions of acetylcholineproducing T cells in blood and the vascular wall are incompletely understood. Gut homeostasis and host defense
A number of reports have linked ChAT+ T cells to the response to microorganisms. Dhawan et al. investigated mice deficient in ChAT+ T cells and found that Current Opinion in Neurobiology 2020, 62:115–121
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expression of a number of antimicrobial peptides was reduced in jejunal mucosa, and in mice deficient in ChAT+ T cells, the microbial diversity was increased compared with littermates [42] (Figure 1c). Another study from the same group concluded that ChAT+ T cells worsen the local intestinal innate immune response in experimental induced colitis, whereas ChAT+ T promote resolution of inflammation in induced colitis [54]. Hence, it is possible that the presence of ChAT+ lymphocytes may affect the mucosal environment in the intestine and potentially the composition of the intestinal microflora and impact mucosal inflammation. The percentage and number of splenic ChAT+ T cells were increased in mice after experimental polymicrobial sepsis of intestinal origin, and the authors propose that ChAT+ T cells play a role in the immunosuppression phenotype observed in survivors of experimental sepsis [55]. Ramires et al. observed recruitment of ChAT+ T cells upon Citrobacter rodentium infection in mice and conditional T cell ChAT knockout lead to increased infection and local inflammation. However, antimicrobial peptide production was unaffected. In the absence of ChAT+ T cells, iNos expression in intestinal epithelial cells decreased while the cholinomimetic carbachol together with INFg promoted iNos expression in murine colonic epithelial cell line CMT-93 [56]. Given the established antimicrobial properties of NO [57], these data support a role for ChAT+ T cells in host defence, potentially both in regulation of antimicrobial molecule production and in controlling inflammation at the site of infection.
ChAT+ B cell biology Acquired immunity to foreign pathogens critically depends on functional B cells that develop in the bone marrow, in contrast to T lymphopoiesis that is largely restricted to the thymus in mammals. Although T cells have been the most widely studied lymphocyte population with regards to ChAT expression, ChAT+ B cells have been detected in several studies. There is limited understanding of regulation of ChAT expression and activity in B cells, but there are reports that TLR ligands (CpG, TLR2 ligand; LPS, TLR4 ligand; R848, TLR7 ligand; Pam3Cys TLR9 ligand) promote ChAT expression in murine B2 and B1b cells in a MyD88-dependent manner [31]. Phenotype
The ChAT-eGFP transgenic reporter line has again been instrumental in isolating and assigning functional roles for ChAT B cells. This reporter mouse line is controlled by the ChAT promoter elements that drive ChAT expression in cholinergic neurons [41]. Although this strain is thought to correctly mirror expression of ChAT, interpretations of results obtained with this strain must be tempered with the probable extended Current Opinion in Neurobiology 2020, 62:115–121
of half-life of GFP versus ChAT and also that the transgenic construct faithfully recapitulates lymphocyte ChAT expression. Examination of the thymus in ChATeGFP reporter mice failed to detect GFP within the predominant CD4-expressing and CD8-expressing populations [31]. It remains possible that the minor so-called double negative population, where correct rearrangement of the alpha/beta T cell receptor is completed, may contain ChAT populations that have been herewith overlooked. Commitment to the B cell lineage in the bone marrow occurs at the pro-B cell stage, which is also characterized by completion of V-DJ recombination of the immunoglobulin heavy chain. Rearrangement of the light chain in the pre-B cell stage allows for production of a functional B cell receptor. However, robust GFP expression seemed to be restricted to the later mature B cell subsets that can recirculate back to the bone marrow, rather than these earlier progenitors [31]. A more refined characterization of earlier B lymphopoiesis may still reveal ChAT-expressing subsets. However, available data would imply that ChAT expression is not a characteristic of lymphocytes undergoing V-DJ recombination and by inference central tolerance mechanisms during B-lymphopoiesis and T-lymphopoiesis. Interestingly, the IMMGEN database [58] indicates that none of the major immune cell populations express ChAT. The reason for this discrepancy remains unclear. Function
ChAT-expressing B cells are relatively abundant in the peripheral lymphoid organs, and may even be more numerous than T cells [31,59]. Reardon et al. detected robust expression of ChAT in subsets of the marginal zone (MZ) and B1 cell subsets. Interestingly, follicular B cells also seem to express ChAT in the spleen and also presumably in the lymph nodes, but the identity and functional importance of this putative subset has not been determined. Addressing this question will be of considerable interest, as follicular B cells are the major B cell subset responsible for the extra-follicular and germinal center responses that are critical for host defense and autoimmunity. Whether ChAT expression is dynamic within B cells, delineates a separate subset, or rather that ChAT-expressing B cells shares markers with other common B cell populations has not been fully determined. Notably however, fate-mapping approaches would seem to indicate that ChAT expression is transient within B cells, indicating that production of acetylcholine could be an acquired function within a given lymphocyte population [31]. Conceptually, it is the expression of defined receptors that respond to B cell mitogens and stimulants that would engender a B cell permissive to ChAT expression. A number of recent reports have implicated neural control in the regulation of adaptive and innate immune www.sciencedirect.com
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responses of B cells. The expression of ChAT within MZ and B1 cells would imply a role for ChAT in the antibacterial responses that characterize MZ and B1 cells [60] and would be consistent with other known functions of the inflammatory reflex in systemic inflammation. Indeed, ChAT B cells played an important role in limiting the recruitment of neutrophils into the peritoneum following LPS administration and furthermore ChAT expression in B cells can be dynamically regulated through Toll-like receptor ligands (Figure 1d). Potential neuronal control of B cell-mediated antibacterial responses have stimulated efforts to understand how innervation of the spleen could regulate this process, given the critical function of the spleen in hosting humoral immunity to gram-positive bacteria typified by Streptococcus pneumoniae. Stimulation of the vagus nerve was demonstrated to prevent accumulation of CD11b cells within the marginal zone following immunization with S. pneumoniae, and that antibody secreting cells seemed to colocalize with nerve fibers [61]. It could be speculated that communication between neurons and B cells may be involved in the T-cell independent responses that dominate in the B cell responses to gram-positive bacteria. Notably however, vagal nerve stimulation (VNS) did not affect antibody secretion, indicating that either the inflammatory reflex is dispensable or that the VNS protocol has not yet been optimized for these readouts. As well as producing acetylcholine, B cells may also be responsive to acetylcholine as well. Loss of Chrna7 leads to an increase in humoral immune responses and increases in CD40 expression. Expression of the Chrna7 also seems to be upregulated as B cell mature, indicative of possible autocrine mechanisms [62,63]. Of note, the IMMGEN database fails to show any robust expression of Chrna7 in immune cells.
Conclusion In summary, the capacity of some lymphocyte subpopulations to express ChAT and biosynthesize acetylcholine has been known for decades. Only recently have distinct functional capacities of acetylcholine-producing primary lymphocytes been defined. Although available data are limited, ChAT+ T cells are essential for regulation of systemic release of pro-inflammatory cytokines by the inflammatory reflex [32], involved in blood pressure regulation [51], required for optimal anti-viral defense in mice [33] and implicated in gut microbial homeostasis [42]. ChAT-expressing B cells are relatively abundant in peripheral lymphoid organs and might function to regulate T-cell independent immune responses. Mapping the mechanisms of ChAT regulation, acetylcholine transport and release in lymphocytes, as well as molecular interactions with other immune cells and the vasculature, will be important to undertake in the immediate future. www.sciencedirect.com
Conflict of interest statement SM, LT, and VSS have no conflicts of interest to declare relative to this manuscript. PSO is a co-founder and shareholder of Emune AB and ChAT Therapeutics.
Acknowledgements The work was funded by grants to PSO from the Knut and Alice Wallenberg Foundation, the Swedish Medical Council and the Stockholm Region (ALF).
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