Tuft cell acetylcholine is released into the gut lumen to promote anti-helminth immunity.

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.

Characterization of social behavior in young and middle-aged ChAT-IRES-Cre mouse.

The cholinergic system is an important modulator of brain processes. It contributes to the regulation of several cognitive functions and emotional states, hence altering behaviors. Previous works showed that cholinergic (nicotinic) receptors of the prefrontal cortex are needed for adapted social behaviors. However, these data were obtained in mutant mice that also present alterations of several neurotransmitter systems, in addition to the cholinergic system. ChAT-IRES-Cre mice, that express the Cre recombinase specifically in cholinergic neurons, are useful tools to investigate the role of the cholinergic circuits in behavior. However, their own behavioral phenotype has not yet been fully characterized, in particular social behavior. In addition, the consequences of aging on the cholinergic system of ChAT-IRES-Cre mice has never been studied, despite the fact that aging is known to compromise the cholinergic system efficiency. The aim of the current study was thus to characterize the social phenotype of ChAT-IRES-Cre mice both at young (2-3 months) and middle (10-11 months) ages. Our results reveal an alteration of the cholinergic system, evidenced by a decrease of ChAT, CHT and VAChT gene expression in the striatum of the mice, that was accompanied by mild social disturbances and a tendency towards anxiety. Aging decreased social dominance, without being amplified by the cholinergic alterations. Altogether, this study shows that ChAT-IRES-Cre mice are useful models for studying the cholinergic system's role in social behavior using appropriate modulating technics (optogenetic or DREADD).

Cancer Induces a Stress Ileopathy Depending on ß-Adrenergic Receptors and Promoting Dysbiosis that Contributes to Carcinogenesis.

Gut dysbiosis has been associated with intestinal and extraintestinal malignancies, but whether and how carcinogenesis drives compositional shifts of the microbiome to its own benefit remains an open conundrum. Here, we show that malignant processes can cause ileal mucosa atrophy, with villous microvascular constriction associated with dominance of sympathetic over cholinergic signaling. The rapid onset of tumorigenesis induced a burst of REG3γ release by ileal cells, and transient epithelial barrier permeability that culminated in overt and long-lasting dysbiosis dominated by Gram-positive Clostridium species. Pharmacologic blockade of ß-adrenergic receptors or genetic deficiency in Adrb2 gene, vancomycin, or cohousing of tumor bearers with tumor-free littermates prevented cancer-induced ileopathy, eventually slowing tumor growth kinetics. Patients with cancer harbor distinct hallmarks of this stress ileopathy dominated by Clostridium species. Hence, stress ileopathy is a corollary disease of extraintestinal malignancies requiring specific therapies. SIGNIFICANCE: Whether gut dysbiosis promotes tumorigenesis and how it controls tumor progression remain open questions. We show that 50% of transplantable extraintestinal malignancies triggered a ß-adrenergic receptor-dependent ileal mucosa atrophy, associated with increased gut permeability, sustained Clostridium spp.-related dysbiosis, and cancer growth. Vancomycin or propranolol prevented cancer-associated stress ileopathy. This article is highlighted in the In This Issue feature, p. 873.

Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths.

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.

Acetylcholine Modulates the Hormones of the Growth Hormone/Insulinlike Growth Factor-1 Axis During Development in Mice.

Pituitary growth hormone (GH) and insulinlike growth factor (IGF)-1 are anabolic hormones whose physiological roles are particularly important during development. The activity of the GH/IGF-1 axis is controlled by complex neuroendocrine systems including two hypothalamic neuropeptides, GH-releasing hormone (GHRH) and somatostatin (SRIF), and a gastrointestinal hormone, ghrelin. The neurotransmitter acetylcholine (ACh) is involved in tuning GH secretion, and its GH-stimulatory action has mainly been shown in adults but is not clearly documented during development. ACh, together with these hormones and their receptors, is expressed before birth, and somatotroph cells are already responsive to GHRH, SRIF, and ghrelin. We thus hypothesized that ACh could contribute to the modulation of the main components of the somatotropic axis during development. In this study, we generated a choline acetyltransferase knockout mouse line and showed that heterozygous mice display a transient deficit in ACh from embryonic day 18.5 to postnatal day 10, and they recover normal ACh levels from the second postnatal week. This developmental ACh deficiency had no major impact on weight gain and cardiorespiratory status of newborn mice. Using this mouse model, we found that endogenous ACh levels determined the concentrations of circulating GH and IGF-1 at embryonic and postnatal stages. In particular, serum GH level was correlated with brain ACh content. ACh also modulated the levels of GHRH and SRIF in the hypothalamus and ghrelin in the stomach, and it affected the levels of these hormones in the circulation. This study identifies ACh as a potential regulator of the somatotropic axis during the developmental period.

Selective disruption of acetylcholine synthesis in subsets of motor neurons: a new model of late-onset motor neuron disease.

Motor neuron diseases are characterized by the selective chronic dysfunction of a subset of motor neurons and the subsequent impairment of neuromuscular function. To reproduce in the mouse these hallmarks of diseases affecting motor neurons, we generated a mouse line in which ~40% of motor neurons in the spinal cord and the brainstem become unable to sustain neuromuscular transmission. These mice were obtained by conditional knockout of the gene encoding choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine. The mutant mice are viable and spontaneously display abnormal phenotypes that worsen with age including hunched back, reduced lifespan, weight loss, as well as striking deficits in muscle strength and motor function. This slowly progressive neuromuscular dysfunction is accompanied by muscle fiber histopathological features characteristic of neurogenic diseases. Unexpectedly, most changes appeared with a 6-month delay relative to the onset of reduction in ChAT levels, suggesting that compensatory mechanisms preserve muscular function for several months and then are overwhelmed. Deterioration of mouse phenotype after ChAT gene disruption is a specific aging process reminiscent of human pathological situations, particularly among survivors of paralytic poliomyelitis. These mutant mice may represent an invaluable tool to determine the sequence of events that follow the loss of function of a motor neuron subset as the disease progresses, and to evaluate therapeutic strategies. They also offer the opportunity to explore fundamental issues of motor neuron biology.

Hyperfunction of muscarinic receptor maintains long-term memory in 5-HT4 receptor knock-out mice.

Patients suffering from dementia of Alzheimer's type express less serotonin 4 receptors (5-HTR(4)), but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR(4) knock-out (KO) and wild-type (WT) mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR(4) control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR(4). Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg) to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR(4) KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT), the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR(4) KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR(4) KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR(4) to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR(4) aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR(4) mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.

Silencing of choline acetyltransferase expression by lentivirus-mediated RNA interference in cultured cells and in the adult rodent brain.

RNA interference (RNAi) is a potent mechanism for local silencing of gene expression and can be used to study loss-of-function phenotypes in mammalian cells. We used RNAi to knockdown specifically the expression of choline acetyltransferase (ChAT), the enzyme of acetylcholine biosynthesis, both in cultured cells and in the adult brain. We first identified a 19-nucleotide sequence in the coding region of rat and mouse ChAT transcripts that constitutes a target for potent silencing of ChAT expression by RNAi. We generated a lentiviral vector that produces both a small hairpin RNA (shRNA) targeting ChAT mRNAs and the enhanced green fluorescent protein (EGFP) reporter protein to facilitate identification of transduced cells. In the cholinergic cell line NG108-15, there was at least 90% less of the ChAT protein, as measured by assaying its enzymatic activity, 3 days postinfection with this vector than in cells infected with a control vector. The vector was used to transduce cholinergic neurons in vivo and reduced ChAT expression strongly and specifically in the cholinergic neurons of the medial septum in adult rats, without affecting the expression of the vesicular acetylcholine transporter. This lentiviral vector is thus a powerful tool for specific inactivation of cholinergic neurotransmission and can therefore be used to study the role of cholinergic nuclei in the brain. This lentiviral-mediated RNAi approach will also allow the development of new animal models of diseases in which cholinergic neurotransmission is specifically altered.

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia.

Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter.

Quantification of transcriptional activities of reporter gene constructs in primary cultures of sympathetic neurons.

Primary cultures of sympathetic neurons provide an attractive cellular model for investigating the mechanisms of neurotransmitter phenotypic plasticity. However, it has not been possible to transfect these neurons by conventional techniques, and this has been a major impediment to molecular investigations of neuronal gene expression in this system. Here, reporter plasmids were transferred into the nuclei of cultured sympathetic neurons by microinjection. We developed and improved this procedure and were able to measure the transcriptional activities of two coinjected promoters in small groups of neurons, and even from a single neuron. Promoter activities can thus be quantified and normalized relative to that of a constitutively expressed promoter, allowing correction for variability in the injection and assay procedures. High and low promoter activities can be reliably quantified. Importantly, this method can be used not only for reporter plasmids but also for DNA fragments containing only a promoter and reporter gene without any vector sequence that might interfere with promoter. Using this approach, we measured neuronal promoter activities and found that one promoter region of the gene encoding choline acetyltransferase was up-regulated by more than sevenfold by leukemia inhibitory factor. This method thus provides the means to investigate the function of neuronal genes and the mechanisms that regulate their transcription in cultured sympathetic neurons.

More than one way to toy with ChAT and VAChT.

Expression of choline acetyltransferase (ChAT) and of the vesicular acetylcholine transporter (VAChT) is required for the acquisition and the maintenance of the cholinergic phenotype. The ChAT and VAChT genes have been demonstrated to share a common gene locus and this suggests a coordinate regulation of their expression. In the present work, we examined the effects of several differentiating treatments on the modulation of ChAT and VAChT expression at the mRNA and protein levels in growing and differentiating NG108-15 cells. In cells grown in the presence of serum, all the agents tested-retinoic acid, dexamethasone and dibutyrylcyclicAMP (dbcAMP)-induced an increase of ChAT and VAChT mRNA levels but with different efficacy. Treatment with dbcAMP plus dexamethasone resulted in the largest increase of VAChT mRNA amount while retinoic acid mostly enhanced ChAT mRNA level. However, while ChAT activity was increased by all agents, no change in the VAChT protein level was detected. In cells differentiated by serum deprivation, only retinoic acid was effective in inducing an increase of VAChT and ChAT mRNA and ChAT activity, while we observed a downregulation by dbcAMP and dexamethasone. Treatment with the antimitotic agent cytosine arabinoside led to an increase of ChAT activity which was further largely enhanced by the addition of dbcAMP plus dexamethasone, but to only a slight change in VAChT activity. We further showed that complex glycosylation processes which might play a role in targeting and/or stability of the membrane protein VAChT are deficient in these cells. Indeed, in transient transfection assays with the reporter soluble enzyme luciferase placed under regulatory and promoter regions of the VAChT gene, we observed a modulation of luciferase expression by differentiating agents. These data illustrate the complexity of the processes which participate to the expression of the ChAT and VAChT genes, both at the transcriptional and posttranslational levels.