Cells Expressing Choline Acetyltransferase Research Articles

B cells modulate lung antiviral inflammatory responses via the neurotransmitter acetylcholine.

The rapid onset of innate immune defenses is critical for early control of viral replication in an infected host, yet it can also lead to irreversible tissue damage, especially in the respiratory tract. Intricate regulatory mechanisms must exist that modulate inflammation, while controlling the infection. Here, B cells expressing choline acetyl transferase (ChAT), an enzyme required for production of the metabolite and neurotransmitter acetylcholine (ACh) are identified as such regulators of the immediate early response to influenza A virus. Lung tissue ChAT + B cells are shown to interact with a7 nicotinic Ach receptor-expressing lung interstitial macrophages in mice within 24h of infection to control their production of TNFa, shifting the balance towards reduced inflammation at the cost of enhanced viral replication. Thus, innate-stimulated B cells are key participants of an immediate-early regulatory cascade that controls lung tissue damage after viral infection.

M1 cholinergic signaling in the brain modulates cytokine levels and splenic cell sub-phenotypes following cecal ligation and puncture.

The contribution of the central nervous system to sepsis pathobiology is incompletely understood. In previous studies, administration of endotoxin to mice decreased activity of the vagus anti-inflammatory reflex. Treatment with the centrally-acting M1 muscarinic acetylcholine (ACh) receptor (M1AChR) attenuated this endotoxin-mediated change. We hypothesize that decreased M1AChR-mediated activity contributes to inflammation following cecal ligation and puncture (CLP), a mouse model of sepsis. In male C57Bl/6 mice, we quantified basal forebrain cholinergic activity (immunostaining), hippocampal neuronal activity, serum cytokine/chemokine levels (ELISA) and splenic cell subtypes (flow cytometry) at baseline, following CLP and following CLP in mice also treated with the M1AChR agonist xanomeline. At 48h. post-CLP, activity in basal forebrain cells expressing choline acetyltransferase (ChAT) was half of that observed at baseline. Lower activity was also noted in the hippocampus, which contains projections from ChAT-expressing basal forebrain neurons. Serum levels of TNFα, IL-1β, MIP-1α, IL-6, KC and G-CSF were higher post-CLP than at baseline. Post-CLP numbers of splenic macrophages and inflammatory monocytes, TNFα+ and ILβ+ neutrophils and ILβ+ monocytes were higher than baseline while numbers of central Dendritic Cells (cDCs), CD4+ and CD8+ T cells were lower. When, following CLP, mice were treated with xanomeline activity in basal forebrain ChAT-expressing neurons and in the hippocampus was significantly higher than in untreated animals. Post-CLP serum concentrations of TNFα, IL-1β, and MIP-1α, but not of IL-6, KC and G-CSF, were significantly lower in xanomeline-treated mice than in untreated mice. Post-CLP numbers of splenic neutrophils, macrophages, inflammatory monocytes and TNFα+ neutrophils also were lower in xanomeline-treated mice than in untreated animals. Percentages of IL-1β+ neutrophils, IL-1β+ monocytes, cDCs, CD4+ T cells and CD8+ T cells were similar in xanomeline-treated and untreated post-CLP mice. Our findings indicate that M1AChR-mediated responses modulate CLP-induced alterations in serum levels of some, but not all, cytokines/chemokines and affected splenic immune response phenotypes.

Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma.

Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca2+-NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

Abstract P112: Evidence Of Renin Expression And Function In The Brainstem

The role of the brain renin-angiotensin system (RAS) in blood pressure (BP) and autonomic control isrecognized. However, the lack of the precise source of renin in the brain is one of the main barrierstowards a complete understanding of how the brain RAS operates. Here, we hypothesized that renin isexpressed in brain regions involved in sympathetic control such as the rostroventral lateral medulla(RVLM). We employed RNAscope, a highly sensitive and specific in situ hybridization technology.Surprisingly, the renin positive signals were absent in the RVLM, but we identified renin transcripts inthe nucleus ambiguus (NA), a site in the vicinity of the RVLM that contains cardiac vagal motoneurons.There was no signal in this region with the negative control scrambled probe. The signal strength wasgreater than that observed in other regions of the brain classically associated with the brain RAS. Wealso observed that the NA contains other necessary RAS components to produce angiotensin.Interestingly, renin was colocalized with cells expressing choline acetyltransferase (ChAT), suggesting itspossible role in cholinergic neurotransmission. Therefore, we generated mice lacking renin specifically incholinergic neurons (Ren-a ChAT-null ). Because we previously identified an alternative brain-specific reninisoform (ren-b) that elicits cardioprotective roles, we also generated mice lacking ren-b in cholinergicneurons (Ren-b ChAT-null ). At baseline, Ren-a ChAT-null exhibited a trend towards decreased BP, while Ren-b ChAT-null exhibited higher BP, although these differences were not statistically significant. Importantly,Ren-a ChAT-null exhibited an attenuated tachycardiac and pressor responses to atropine (p<0.005 andp<0.05, respectively; n=4-7), indicating a lower parasympathetic tone in these mice. Conversely, Ren-b ChAT-null exhibited an augmented pressor response to atropine (p<0.005; n=7) suggesting an elevatedparasympathetic tone. These data suggest that the activation of the brain RAS in the NA might play arole in cholinergic neurotransmission, and it defines the NA as a potential source of endogenous renin inthe brainstem. Studies to understand the differential contribution of Ren-a and Ren-b in the NA to BPare in progress.

Coronary vasodilation mediated by T cells expressing choline acetyltransferase

CD4+ T cells expressing choline acetyltransferase (ChAT) have recently been shown to cause a drop in systemic blood pressure when infused into mice. The aim of this study was to determine if ChAT-expressing T cells could regulate coronary vascular reactivity. Preconstricted segments of epicardial and intramyocardial porcine coronary arteries relaxed in response to Jurkat T cells (JT) that overexpressed ChAT (JTChAT cells). The efficacy of the JTChAT cells was similar in epicardial and intramyocardial vessels with a maximum dilator response to 3 × 105 cells/mL of 38.0 ± 6.7% and 38.7 ± 7.25%, respectively. In contrast, nontransfected JT cells elicited a weak dilator response, followed by a weak contraction. The response of JTChAT cells was dependent on the presence of the endothelial cells. In addition, the response could be significantly reduced by Nω-nitro-l-arginine methyl ester (l-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the presence of indomethacin. JTChAT cells, but not JT cells, increased the expression of phosphorylated endothelial nitric oxide synthase (eNOS). JTChAT cells contained significantly greater levels of acetylcholine compared with JT cells; however, the nonselective muscarinic antagonist atropine and the M1 receptor antagonist pirenzepine both failed to block the dilator effect of JTChAT cells. Exogenously added acetylcholine induced only a weak relaxation (∼10%) at low concentrations, which became a contractile response at higher concentrations. These data illustrate the capacity for cells that express ChAT to regulate coronary vascular reactivity, via mechanisms that are dependent on interaction with the endothelium and in part mediated by the release of nitric oxide.NEW & NOTEWORTHY This study shows ChAT-expressing T cells can induce vasodilation of the blood vessel in the coronary circulation and that this effect relies on a direct interaction between T cells and the coronary vascular endothelium. The study establishes a potential immunomodulatory role for T cells in the coronary circulation. The present findings offer an additional possibility that a deficiency of ChAT-expressing T cells could contribute to reduced coronary blood flow and ischemic events in the myocardium.

Multichannel polymer scaffold seeded with activated Schwann cells and bone mesenchymal stem cells improves axonal regeneration and functional recovery after rat spinal cord injury.

The adult mammalian CNS has a limited capacity to regenerate after traumatic injury. In this study, a combinatorial strategy to promote axonal regeneration and functional recovery after spinal cord injury (SCI) was evaluated in adult rats. The rats were subjected to a complete transection in the thoracic spinal cord, and multichannel scaffolds seeded with activated Schwann cells (ASCs) and/or rat bone marrow-derived mesenchymal stem cells (MSCs) were acutely grafted into the 3-mm-wide transection gap. At 4 weeks post-transplantation and thereafter, the rats receiving scaffolds seeded with ASCs and MSCs exhibited significant recovery of nerve function as shown by the Basso, Beattie and Bresnahan (BBB) score and electrophysiological test results. Immunohistochemical analyses at 4 and 8 weeks after transplantation revealed that the implanted MSCs at the lesion/graft site survived and differentiated into neuron-like cells and co-localized with host neurons. Robust bundles of regenerated fibers were identified in the lesion/graft site in the ASC and MSC co-transplantation rats, and neurofilament 200 (NF) staining confirmed that these fibers were axons. Furthermore, myelin basic protein (MBP)-positive myelin sheaths were also identified at the lesion/graft site and confirmed via electron microscopy. In addition to expressing mature neuronal markers, sparse MSC-derived neuron-like cells expressed choline acetyltransferase (ChAT) at the injury site of the ASC and MSC co-transplantation rats. These findings suggest that co-transplantation of ASCs and MSCs in a multichannel polymer scaffold may represent a novel combinatorial strategy for the treatment of spinal cord injury.

Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the urotensin II-related peptide (ust2b) gene as a driver.

Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP+ cells in the larvae. More than 98% of the GFP+ cells expressed choline acetyltransferase, while their projections were co-localized with α-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP+ cells were observed. In addition, intracellular electrophysiological recordings of GFP+ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord.

Treatment of inflammatory bowel disease with neural stem cells expressing choline acetyltransferase

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder, with increasing incidence and prevalence in the last one-half century. IBD patients suffer from autonomic vagal neuropathy and nerve dysfunction, with deficiency of acetylcholine in inflamed mucosa. Recent studies showed that death of enteric neuron in local tissue was induced during the process of IBD, which also played a crucial role in the pathogenesis of disease. Stem cells have been demonstrated a new biological treatment for IBD, therefore, we proposal that neural stem cells expressing choline acetyltransferase may enhance enteric neural cell regeneration, restore acetylcholine production in intestinal mucosa, and thus inhibit immune responses of IBD.

The nestin-expressing and non-expressing neurons in rat basal forebrain display different electrophysiological properties and project to hippocampus

BackgroundNestin-immunoreactive (nestin-ir) neurons have been identified in the medial septal/diagonal band complex (MS/DBB) of adult rat and human, but the significance of nestin expression in functional neurons is not clear. This study investigated electrophysiological properties and neurochemical phenotypes of nestin-expressing (nestin+) neurons using whole-cell recording combined with single-cell RT-PCR to explore the significance of nestin expression in functional MS/DBB neurons. The retrograde labelling and immunofluorescence were used to investigate the nestin+ neuron related circuit in the septo-hippocampal pathway.ResultsThe results of single-cell RT-PCR showed that 87.5% (35/40) of nestin+ cells expressed choline acetyltransferase mRNA (ChAT+), only 44.3% (35/79) of ChAT+ cells expressed nestin mRNA. Furthermore, none of the nestin+ cells expressed glutamic acid decarboxylases 67 (GAD67) or vesicular glutamate transporters (VGLUT) mRNA. All of the recorded nestin+ cells were excitable and demonstrated slow-firing properties, which were distinctive from those of GAD67 or VGLUT mRNA-positive neurons. These results show that the MS/DBB cholinergic neurons could be divided into nestin-expressing cholinergic neurons (NEChs) and nestin non-expressing cholinergic neurons (NNChs). Interestingly, NEChs had higher excitability and received stronger spontaneous excitatory synaptic inputs than NNChs. Retrograde labelling combined with choline acetyltransferase and nestin immunofluorescence showed that both of the NEChs and NNChs projected to hippocampus.ConclusionsThese results suggest that there are two parallel cholinergic septo-hippocampal pathways that may have different functions. The significance of nestin expressing in functional neurons has been discussed.

Developmental Changes in Brainstem Neurons Regulating Lower Airway Caliber

Premature infants are at risk for lower airway obstruction; however, maturation of reflex pathways regulating lower airway patency is inadequately studied. We hypothesized that postnatal maturation causes developmental change in brainstem efferent airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) that project to the airways and in pulmonary afferent fibers that terminate in the nucleus tractus solitarius (NTS). Ferrets aged 7, 14, 21, and 42 d received intrapulmonary injection of cholera toxin (CT)-beta subunit, a transganglionic retrograde tracer. Five days later, their brainstem was processed for dual immunolabeling of CT-beta and the cholinergic marker, choline acetyl transferase. CT-beta-labeled AVPNs and CT-beta-labeled afferent fiber optical density (OD) were analyzed. There was a significantly higher CT-beta-labeled cell number within the rNA at the youngest compared with older ages. All efferent CT-beta-labeled cells expressed choline acetyl transferase. OD of CT-beta-labeled afferent fibers was also higher at 7 d compared with 14 d. We conclude that the number of efferent AVPNs and afferent fiber OD both diminish over the second postnatal week. We speculate that exposure to injurious agents in early postnatal life may inhibit natural remodeling and thereby enhance later vulnerability to airway hyperreactivity.

Expression of natriuretic peptide-activated guanylate cyclases by cholinergic and dopaminergic amacrine cells of the rat retina

Recently, the natriuretic peptides were detected in the cholinergic and dopaminergic amacrine cells of the retina. We performed immunofluorescence labeling of rat retinal sections to examine the immunoreactivity of natriuretic peptide-activated guanylate cyclases (NPR-A and NPR-B) in the rat retina, in particular whether they were localized to dopaminergic and cholinergic amacrine cells. NPR-A and NPR-B immunoreactivity was detected in several layers of the retina including amacrine cells. In amacrine cells, both NPR-A and NPR-B were co-localized with tyrosine hydroxylase, a marker of dopaminergic cells. NPR-B, but not NPR-A, was localized to amacrine cells expressing choline acetyltransferase (ChAT), a marker of cholinergic cells. These findings suggest that natriuretic peptides have different regulatory systems in dopaminergic and cholinergic amacrine cells in rat retina.

Regionally specific induction of BDNF and truncated trkB.T1 receptors in the hippocampal formation after intraseptal injection of kainic acid

The septo-hippocampal cholinergic and GABAergic systems were lesioned with single unilateral injections of kainic acid (KA) into the septum to further characterize the role of these afferents in the regulation of hippocampal brain-derived neurotrophic factor (BDNF) expression. Nearly all cells expressing choline acetyltransferase, trkA or glutamic acid decarboxylase mRNA disappeared in the medial septum 7 days after the neurotoxin administration. The lesion resulted in a complete loss of CA3 pyramidal cells, and robust increases in BDNF mRNA levels in hippocampal granular dentate cells and in the amygdala. There were rapid transient increases of BDNF mRNA levels in the hippocampal formation and cortex. In addition, we found a strong induction of truncated trkB.T1 mRNA receptors in the stratum radiatum and stratum oriens of the CA3 subfield. The prolonged induction of BDNF mRNA levels suggests an important role of this neurotrophin, possibly mediated by truncated trkB receptors, in the regulation of hippocampal plasticity following injury.

Embryonic Precursor Cells That Express Trk Receptors: Induction of Different Cell Fates by NGF, BDNF, NT-3, and CNTF

Epidermal growth factor (EGF)-treated neurosphere cultures from embryonal striatum contain multipotential cells capable of neuronal, astrocytic, and oligodendroglial differentiation. In this study, we tested whether these neural precursor cells differentiate in the presence of neurotrophic factors. We first assayed neurosphere cells for expression of neurotrophin receptors. TrkA, TrkB, TrkC, and gp75 were detected by immunofluorescence microscopy in 60–80% of cells. In addition, the ciliary neurotrophic factor receptor α was expressed in 50–60% of cells. In the presence of the mitogen, EGF, treatment of stem cells with neurotrophic factors had no apparent effect. Removal of EGF from cells resulted in cessation of cell proliferation and pronounced astrocytic (glial fibrillary acidic protein+) differentiation. Neuronal (neurofilament+) and oligodendroglial (galactocerebroside+) cells appeared in cultures treated with neurotrophic factors. Nerve growth factor (NGF) resulted in bipolar neuronal cells, and brain-derived neurotrophic factor led to multipolar neuronal cells. Treatment with neurotrophin-3 or ciliary neurotrophic factor resulted in bipolar neuronal cells and oligodendrocytes. Neuronal differentiation in the presence of NGF was enhanced by extracellular matrix, and the resulting neuronal cells expressed choline acetyltransferase and, to a lesser degree, tyrosine hydroxylase. These studies demonstrate that neurotrophic factors influence the fates of these multipotential precursor cells. Indeed, the true utility of multipotential precursor cells is the production of different types of cells in different situations. Local cues, such as neurotrophic factors and extracellular matrix, may regulate production of different types of neural cells during development or in response to other stimuli, such as injury.

Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion.

Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.