Cholinergic regulation of vascular endothelial function by human ChAT+ T cells.

Endothelial dysfunction and impaired vasodilation are linked with adverse cardiovascular events. T lymphocytes expressing choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of the vasorelaxant acetylcholine (ACh), regulate vasodilation and are integral to the cholinergic antiinflammatory pathway in an inflammatory reflex in mice. Here, we found that human T cell ChAT mRNA expression was induced by T cell activation involving the PI3K signaling cascade. Mechanistically, we identified that ChAT mRNA expression was induced following the attenuation of RE-1 Silencing Transcription factor REST-mediated methylation of the ChAT promoter, and that ChAT mRNA expression levels were up-regulated by GATA3 in human T cells. In functional experiments, T cell-derived ACh increased endothelial nitric oxide-synthase activity, promoted vasorelaxation, and reduced vascular endothelial activation and promoted barrier integrity by a cholinergic mechanism. Further, we observed that survival in a cohort of patients with severe circulatory failure correlated with their relative frequency of ChAT +CD4+ T cells in blood. These findings on ChAT+ human T cells provide a mechanism for cholinergic immune regulation of vascular endothelial function in human inflammation.

Vagus nerve stimulation promotes resolution of inflammation by a mechanism that involves Alox15 and requires the α7nAChR subunit.

Nonresolving inflammation underlies a range of chronic inflammatory diseases, and therapeutic acceleration of resolution of inflammation may improve outcomes. Neural reflexes regulate the intensity of inflammation (for example, through signals in the vagus nerve), but whether activation of the vagus nerve promotes the resolution of inflammation in vivo has been unknown. To investigate this, mice were subjected to electrical vagus nerve stimulation (VNS) or sham surgery at the cervical level followed by zymosan-induced peritonitis. The duration of inflammation resolution was significantly reduced and efferocytosis was significantly increased in mice treated with VNS as compared with sham. Lipid mediator (LM) metabololipidomics revealed that mice treated with VNS had higher levels of specialized proresolving mediators (SPMs), particularly from the omega-3 docosahexaenoic (DHA) and docosapentaenoic (n-3 DPA) metabolomes, in peritoneal exudates. VNS also shifted the ratio between proinflammatory and proresolving LMs toward a proresolving profile, but this effect by VNS was inverted in mice deficient in 12/15-lipoxgenase (Alox15), a key enzyme in this SPM biosynthesis. The significant VNS-mediated reduction of neutrophil numbers in peritoneal exudates was absent in mice deficient in the cholinergic α7-nicotinic acetylcholine receptor subunit (α7nAChR), an essential component of the inflammatory reflex. Thus, VNS increased local levels of SPM and accelerated resolution of inflammation in zymosan-induced peritonitis by a mechanism that involves Alox15 and requires the α7nAChR.

Inflammation neuroscience: neuro-immune crosstalk and interfaces.

Inflammation is a key process in antimicrobial defence and tissue repair, and failure to properly regulate inflammation can result in tissue damage and death. Neural circuits play important roles throughout the course of an inflammatory response, and the neurophysiological and molecular mechanisms are only partly understood. Here, we review key evidence for the neural regulation of inflammation and discuss emerging technologies to further map and harness this neurophysiology, a cornerstone in the rapidly evolving field of inflammation neuroscience.

The resolvin D1 receptor GPR32 transduces inflammation resolution and atheroprotection.

Chronic inflammation is a hallmark of atherosclerosis and results from an imbalance between proinflammatory and proresolving signaling. The human GPR32 receptor, together with the ALX/FPR2 receptor, transduces biological actions of several proresolving mediators that stimulate resolution of inflammation. However, since no murine homologs of the human GPR32 receptor exist, comprehensive in vivo studies are lacking. Using human atherosclerotic lesions from carotid endarterectomies and creating a transgenic mouse model expressing human GPR32 on a Fpr2×ApoE double-KO background (hGPR32myc×Fpr2-/-×Apoe-/-), we investigated the role of GPR32 in atherosclerosis and self-limiting acute inflammation. GPR32 mRNA was reduced in human atherosclerotic lesions and correlated with the immune cell markers ARG1, NOS2, and FOXP3. Atherosclerotic lesions, necrotic core, and aortic inflammation were reduced in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice as compared with Fpr2-/-×Apoe-/- nontransgenic littermates. In a zymosan-induced peritonitis model, the hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice had reduced inflammation at 4 hours and enhanced proresolving macrophage responses at 24 hours compared with nontransgenic littermates. The GPR32 agonist aspirin-triggered resolvin D1 (AT-RvD1) regulated leukocyte responses, including enhancing macrophage phagocytosis and intracellular signaling in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice, but not in Fpr2-/-×Apoe-/- nontransgenic littermates. Together, these results provide evidence that GPR32 regulates resolution of inflammation and is atheroprotective in vivo.

AMPA-Type Glutamate Receptors Associated With Vascular Smooth Muscle Cell Subpopulations in Atherosclerosis and Vascular Injury.

Objectives and Aims: Vascular smooth muscle cells (VSMCs) are key constituents of both normal arteries and atherosclerotic plaques. They have an ability to adapt to changes in the local environment by undergoing phenotypic modulation. An improved understanding of the mechanisms that regulate VSMC phenotypic changes may provide insights that suggest new therapeutic targets in treatment of cardiovascular disease (CVD). The amino-acid glutamate has been associated with CVD risk and VSMCs metabolism in experimental models, and glutamate receptors regulate VSMC biology and promote pulmonary vascular remodeling. However, glutamate-signaling in human atherosclerosis has not been explored. Methods and Results: We identified glutamate receptors and glutamate metabolism-related enzymes in VSMCs from human atherosclerotic lesions, as determined by single cell RNA sequencing and microarray analysis. Expression of the receptor subunits glutamate receptor, ionotropic, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA)-type subunit 1 (GRIA1) and 2 (GRIA2) was restricted to cells of mesenchymal origin, primarily VSMCs, as confirmed by immunostaining. In a rat model of arterial injury and repair, changes of GRIA1 and GRIA2 mRNA level were most pronounced at time points associated with VSMC proliferation, migration, and phenotypic modulation. In vitro, human carotid artery SMCs expressed GRIA1, and selective AMPA-type receptor blocking inhibited expression of typical contractile markers and promoted pathways associated with VSMC phenotypic modulation. In our biobank of human carotid endarterectomies, low expression of AMPA-type receptor subunits was associated with higher content of inflammatory cells and a higher frequency of adverse clinical events such as stroke. Conclusion: AMPA-type glutamate receptors are expressed in VSMCs and are associated with phenotypic modulation. Patients suffering from adverse clinical events showed significantly lower mRNA level of GRIA1 and GRIA2 in their atherosclerotic lesions compared to asymptomatic patients. These results warrant further mapping of neurotransmitter signaling in the pathogenesis of human atherosclerosis.

The brain-gut axis, inflammatory bowel disease and bioelectronic medicine.

The hallmark of inflammatory bowel diseases (IBD) is chronic intestinal inflammation with typical onset in adolescents and young adults. An abundance of neutrophils is seen in the inflammatory lesions, but adaptive immunity is also an important player in the chronicity of the disease. There is an unmet need for new treatment options since modern medicines such as biological therapy with anti-cytokine antibodies still leave a substantial number of patients with persisting disease activity. The role of the central nervous system and its interaction with the gut in the pathophysiology of IBD have been brought to attention both in animal models and in humans after the discovery of the inflammatory reflex. The suggested control of gut immunity by the brain-gut axis represents a novel therapeutic target suitable for bioelectronic intervention. In this review, we discuss the role of the inflammatory reflex in gut inflammation and the recent advances in the treatment of IBD by intervening with the brain-gut axis through bioelectronic devices.

Molecular Imaging of Inflammation in a Mouse Model of Atherosclerosis Using a Zirconium-89-Labeled Probe.

BACKGROUND: Beyond clinical atherosclerosis imaging of vessel stenosis and plaque morphology, early detection of inflamed atherosclerotic lesions by molecular imaging could improve risk assessment and clinical management in high-risk patients. To identify inflamed atherosclerotic lesions by molecular imaging in vivo, we studied the specificity of our radiotracer based on maleylated (Mal) human serum albumin (HSA), which targets key features of unstable atherosclerotic lesions. MATERIALS AND METHODS: Mal-HSA was radiolabeled with a positron-emitting metal ion, zirconium-89 (89Zr4+). The targeting potential of this probe was compared with unspecific 89Zr-HSA and 18F-FDG in an experimental model of atherosclerosis (Apoe-/- mice, n=22), and compared with wild-type (WT) mice (C57BL/6J, n=21) as controls. RESULTS: PET/MRI, gamma counter measurements, and autoradiography showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe-/- mice. The maximum standardized uptake values (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P<0.05) in Apoe-/- mice than in control WT mice, whereas no difference in SUVmax was observed for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta, as evaluated by a gamma counter 48 h postinjection, was 32% higher (P<0.01) for Apoe-/- mice than in WT mice, and the aorta-to-blood ratio was 8-fold higher (P<0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA-based probes were mainly distributed to the liver, spleen, kidneys, bone, and lymph nodes. The phosphor imaging autoradiography (PI-ARG) results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe-/- mice than in WT mice (9.4±1.4 vs 0.8±0.3%; P<0.001). CONCLUSION: 89Zr radiolabeling of Mal-HSA probes resulted in detectable activity in atherosclerotic lesions in aortas of Apoe-/- mice, as demonstrated by quantitative in vivo PET/MRI. 89Zr-Mal-HSA appears to be a promising diagnostic tool for the early identification of macrophage-rich areas of inflammation in atherosclerosis.

Functions of acetylcholine-producing lymphocytes in immunobiology.

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.

Neural Control of Inflammation: Bioelectronic Medicine in Treatment of Chronic Inflammatory Disease.

Inflammation is important for antimicrobial defense and for tissue repair after trauma. The inflammatory response and its resolution are both active processes that must be tightly regulated to maintain homeostasis. Excessive inflammation and nonresolving inflammation cause tissue damage and chronic disease, including autoinflammatory and cardiovascular diseases. An improved understanding of the cellular and molecular mechanisms that regulate inflammation has supported development of novel therapies for several inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. Many of the specific anticytokine therapies carry a risk for excessive immunosuppression and serious side effects. The discovery of the inflammatory reflex and the increasingly detailed understanding of the molecular interactions between homeostatic neural reflexes and the immune system have laid the foundation for bioelectronic medicine in the field of inflammatory diseases. Neural interfaces and nerve stimulators are now being tested in human clinical trials and may, as the technology develops further, have advantages over conventional drugs in terms of better compliance, continuously adaptable control of dosing, better monitoring, and reduced risks for unwanted side effects. Here, we review the current mechanistic understanding of common autoinflammatory conditions, consider available therapies, and discuss the potential use of increasingly capable devices in the treatment of inflammatory disease.

An Effective Method for Acute Vagus Nerve Stimulation in Experimental Inflammation.

Neural reflexes regulate inflammation and electrical activation of the vagus nerve reduces inflammation in models of inflammatory disease. These discoveries have generated an increasing interest in targeted neurostimulation as treatment for chronic inflammatory diseases. Data from the first clinical trials that use vagus nerve stimulation (VNS) in treatment of rheumatoid arthritis and Crohn's disease suggest that there is a therapeutic potential of electrical VNS in diseases characterized by excessive inflammation. Accordingly, there is an interest to further explore the molecular mechanisms and therapeutic potential of electrical VNS in a range of experimental settings and available genetic mouse models of disease. Here, we describe a method for electrical VNS in experimental inflammation in mice.

The Efficacy and Safety of Sendai Viral Reprograming of Mouse Primary Cells Using Human Vectors.

Induced pluripotent stem cells (iPSCs) remain a promising approach to target diseases with a loss of functional parenchyma. This technology comes with a number of concerns for clinical applications, including teratogenic potential and genomic instability. Here we focused on evaluating the safety of cross-species Sendai viral reprogramming, as well as investigating the transcriptional dynamics during reprogramming and differentiation. We established that Sendai viral vectors carrying human Oct4, Sox2, Klf4, and c-Myc (OSKM) could produce mouse iPSCs free of transduced viral materials. Gene expression analysis revealed an efficient silencing of the virally-introduced human pluripotency factors and upregulation of the endogenous pluripotency network over time. In addition, single cell gene expression analysis of proof-of-principle-derived cardiomyocytes revealed distinct expression patterns indicative of subspecialized cardiac cell lineages. Moreover, our results demonstrate the importance of monitoring genomic aberrations before any clinical or preclinical applications, as we detected a high prevalence of chromosomal instability. Taken together, we demonstrated the successful use of a clinically germane method to reprogram terminally differentiated mouse cells and their potential to generate specialized cardiac cell types. Additionally, our results suggest a plasticity of OSKM to reprogram more divergent species and provide a new application of an established reprogramming approach.

Towards improved control of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by activation of both the innate and adaptive immune system in genetically susceptible individuals, resulting in chronic intestinal inflammation. The triggers that initiate and perpetuate this continuous inflammation are the subject of much speculation and research, although the central role of the intestinal microbiota is recognized, and is even a target for treatment in some circumstances. The mainstay of modern IBD treatment is suppression of the immune response towards as yet unspecified antigens, and conventional therapy includes corticosteroids, 5-aminosalicylic acid (5-ASA), thiopurines and methotrexate. Reducing activity of specific mediators has proven efficacious, including adhesion molecules, such as the gut-homing integrin α4 ß7 expressed on the surface of circulating immune cells, and cytokines, such as tumour necrosis factor α (TNF-α). This has been achieved using biologic agents including monoclonal antibodies. Recent discoveries in immunology and neuroscience have revealed that signals in the peripheral nervous system regulate inflammation, including levels of TNF-α. The understanding of the mechanisms of the neuro-immune communication involved in inflammation control in the gut is evolving, but is as yet incomplete. Clinical studies using implanted vagus nerve stimulators for treatment of IBD show encouraging results. Accordingly, the neural reflex control of inflammation is emerging as a potential therapeutic target in treatment of IBD. Here, we review current therapeutic options and neural reflex control of gut immunity in the context of intestinal inflammation.

Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex.

Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1-60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.

CD137: A checkpoint regulator involved in atherosclerosis.

Inflammation is associated with atherosclerotic plaque development and precipitation of myocardial infarction and stroke, and anti-inflammatory therapy may reduce disease severity. Costimulatory molecules are key regulators of immune cell activity and inflammation, and are associated with disease development in atherosclerosis. Accumulating evidence indicates that a costimulatory molecule of the Tumor Necrosis Factor Receptor superfamily, the checkpoint regulator CD137, promotes atherosclerosis and vascular inflammation in experimental models. In light of the burgeoning consideration of CD137-targeted therapy in the clinic, it will be important to better understand costimulator immunobiology in development of cardiovascular disease. Here, we review available data on the costimulator CD137 and its potential role in atherosclerosis.

Dietary nitrate attenuates renal ischemia-reperfusion injuries by modulation of immune responses and reduction of oxidative stress.

Ischemia-reperfusion (IR) injury involves complex pathological processes in which reduction of nitric oxide (NO) bioavailability is suggested as a key factor. Inorganic nitrate can form NO in vivo via NO synthase-independent pathways and may thus provide beneficial effects during IR. Herein we evaluated the effects of dietary nitrate supplementation in a renal IR model. Male mice (C57BL/6J) were fed nitrate-supplemented chow (1.0mmol/kg/day) or standard chow for two weeks prior to 30min ischemia and during the reperfusion period. Unilateral renal IR caused profound tubular and glomerular damage in the ischemic kidney. Renal function, assessed by plasma creatinine levels, glomerular filtration rate and renal plasma flow, was also impaired after IR. All these pathologies were significantly improved by nitrate. Mechanistically, nitrate treatment reduced renal superoxide generation, pro-inflammatory cytokines (IL-1ß, IL-6 and IL-12 p70) and macrophage infiltration in the kidney. Moreover, nitrate reduced mRNA expression of pro-inflammatory cytokines and chemo attractors, while increasing anti-inflammatory cytokines in the injured kidney. In another cohort of mice, two weeks of nitrate supplementation lowered superoxide generation and IL-6 expression in bone marrow-derived macrophages. Our study demonstrates protective effect of dietary nitrate in renal IR injury that may be mediated via modulation of oxidative stress and inflammatory responses. These novel findings suggest that nitrate supplementation deserve further exploration as a potential treatment in patients at high risk of renal IR injury.

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.

Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific αMHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo.

FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression.

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.