Consolidating roles of neuroimmune reflexes: specificity of afferent, central, and efferent signals in homeostatic immune networks.

Neural reflexes occupy a central role in physiological homeostasis. The vagus nerve is a major conduit for transmitting afferent and efferent signals in homeostatic reflex arcs between the body and the brain. Recent advances in neuroscience, immunology, and physiology have revealed important vagus nerve mechanisms in suppressing inflammation and treating rheumatoid arthritis and other autoimmune conditions. Numerous clinical trials indicate that there is significant benefit to vagus nerve stimulation therapy. Although many questions are still unanswered, it will be important, even necessary, to pursue answers that will be useful in guiding interventions to modulate immunological and physiological homeostasis.

Vagal Regulation of Group 3 Innate Lymphoid Cells and the Immunoresolvent PCTR1 Controls Infection Resolution.

Uncovering mechanisms that control immune responses in the resolution of bacterial infections is critical for the development of new therapeutic strategies that resolve infectious inflammation without unwanted side effects. We found that disruption of the vagal system in mice delayed resolution of Escherichia coli infection. Dissection of the right vagus decreased peritoneal group 3 innate lymphoid cell (ILC3) numbers and altered peritoneal macrophage responses. Vagotomy resulted in an inflammatory peritoneal lipid mediator profile characterized by reduced concentrations of pro-resolving mediators, including the protective immunoresolvent PCTR1, along with elevated inflammation-initiating eicosanoids. We found that acetylcholine upregulated the PCTR biosynthetic pathway in ILC3s. Administration of PCTR1 or ILC3s to vagotomized mice restored tissue resolution tone and host responses to E. coli infections. Together these findings elucidate a host protective mechanism mediated by ILC3-derived pro-resolving circuit, including PCTR1, that is controlled by local neuronal output to regulate tissue resolution tone and myeloid cell responses.

Human-relevant near-organ neuromodulation of the immune system via the splenic nerve.

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve-induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

The impact of influenza pulmonary infection and inflammation on vagal bronchopulmonary sensory neurons.

Influenza A virus (IAV) is rapidly detected in the airways by the immune system, with resident parenchymal cells and leukocytes orchestrating viral sensing and the induction of antiviral inflammatory responses. The airways are innervated by heterogeneous populations of vagal sensory neurons which also play an important role in pulmonary defense. How these neurons respond to IAV respiratory infection remains unclear. Here, we use a murine model to provide the first evidence that vagal sensory neurons undergo significant transcriptional changes following a respiratory IAV infection. RNA sequencing on vagal sensory ganglia showed that IAV infection induced the expression of many genes associated with an antiviral and pro-inflammatory response and this was accompanied by a significant increase in inflammatory cell recruitment into the vagal ganglia. Assessment of gene expression in single-vagal sensory neurons confirmed that IAV infection induced a neuronal inflammatory phenotype, which was most prominent in bronchopulmonary neurons, and also evident in some neurons innervating other organs. The altered transcriptome could be mimicked by intranasal treatment with cytokines and the lung homogenates of infected mice, in the absence of infectious virus. These data argue that IAV pulmonary infection and subsequent inflammation induces vagal sensory ganglia neuroinflammation and this may have important implications for IAV-induced morbidity.

The role of the cholinergic anti-inflammatory pathway in autoimmune rheumatic diseases.

The cholinergic anti-inflammatory pathway (CAP) is a classic neuroimmune pathway, consisting of the vagus nerve, acetylcholine (ACh)-the pivotal neurotransmitter of the vagus nerve-and its receptors. This pathway can activate and regulate the activities of immune cells, inhibit cell proliferation and differentiation, as well as suppress cytokine release, thereby playing an anti-inflammatory role, and widely involved in the occurrence and development of various diseases; recent studies have demonstrated that the CAP may be a new target for the treatment of autoimmune rheumatic diseases. In this review, we will summarize the latest progress with the view of figuring out the role of the cholinergic pathway and how it interacts with inflammatory reactions in several autoimmune rheumatic diseases, and many advances are results from a wide range of experiments performed in vitro and in vivo.

Role of Neuroendocrine, Immune, and Autonomic Nervous System in Anorexia Nervosa-Linked Cardiovascular Diseases.

Anorexia nervosa represents a severe mental disorder associated with food avoidance and malnutrition. In patients suffering from anorexia nervosa, cardiovascular complications are the main reason leading to morbidity and mortality. However, the origin and pathological mechanisms leading to higher cardiovascular risk in anorexia nervosa are still unclear. In this aspect, the issue of exact pathological mechanisms as well as sensitive biomarkers for detection of anorexia nervosa-linked cardiovascular risk are discussed. Therefore, this review synthesised recent evidence of dysfunction in multiple neuroendocrine axes and alterations in the immune system that may represent anorexia nervosa-linked pathological mechanisms contributing to complex cardiovascular dysregulation. Further, this review is focused on identification of non-invasive biomarkers for the assessment of increased cardiovascular risk in anorexia nervosa that can be linked to a clinical application. Complex non-invasive assessment of cardiovascular autonomic regulation-cardiac vagal control (heart rate variability), sympathetic vascular activity (blood pressure variability), and cardiovascular reflex control (baroreflex sensitivity)-could represent a promising tool for early diagnosis, personalized therapy, and monitoring of therapeutic interventions in anorexia nervosa particularly at a vulnerable adolescent age.

Apical splenic nerve electrical stimulation discloses an anti-inflammatory pathway relying on adrenergic and nicotinic receptors in myeloid cells.

The autonomic nervous system innervates all lymphoid tissues including the spleen therefore providing a link between the central nervous system and the immune system. The only known mechanism of neural inhibition of inflammation in the spleen relies on the production of norepinephrine by splenic catecholaminergic fibers which binds to ß2-adrenergic receptors (ß 2-ARs) of CD4+ T cells. These CD4+ T cells trigger the release of acetylcholine that inhibits the secretion of inflammatory cytokines by macrophages through α7 nicotinic acetylcholine receptor (α7nAchRs) signaling. While the vagal anti-inflammatory pathway has been extensively studied in rodents, it remains to be determined whether it coexists with other neural pathways. Here, we have found that three nerve branches project to the spleen in mice. While two of these nerves are associated with an artery and contain catecholaminergic fibers, the third is located at the apex of the spleen and contain both catecholaminergic and cholinergic fibers. We found that electrical stimulation of the apical nerve, but not the arterial nerves, inhibited inflammation independently of lymphocytes. In striking contrast to the anti-inflammatory pathway mechanism described so far, we also found that the inhibition of inflammation by apical nerve electrical stimulation relied on signaling by both ß 2-ARs and α7nAchRs in myeloid cells, with these two signaling pathways acting in parallel. Most importantly, apical splenic nerve electrical stimulation mitigated clinical symptoms in a mouse model of rheumatoid arthritis further providing the proof-of-concept that such an approach could be beneficial in patients with Immune-mediated inflammatory diseases.

Essential Neuroscience in Immunology.

The field of immunology is principally focused on the molecular mechanisms by which hematopoietic cells initiate and maintain innate and adaptive immunity. That cornerstone of attention has been expanded by recent discoveries that neuronal signals occupy a critical regulatory niche in immunity. The discovery is that neuronal circuits operating reflexively regulate innate and adaptive immunity. One particularly well-characterized circuit regulating innate immunity, the inflammatory reflex, is dependent upon action potentials transmitted to the reticuloendothelial system via the vagus and splenic nerves. This field has grown significantly with the identification of several other reflexes regulating discrete immune functions. As outlined in this review, the delineation of these mechanisms revealed a new understanding of immunity, enabled a first-in-class clinical trial using bioelectronic devices to inhibit cytokines and inflammation in rheumatoid arthritis patients, and provided a mosaic view of immunity as the integration of hematopoietic and neural responses to infection and injury.

Vagus nerve cholinergic circuitry to the liver and the gastrointestinal tract in the neuroimmune communicatome.

Improved understanding of neuroimmune communication and the neural regulation of immunity and inflammation has recently led to proposing the concept of the "neuroimmune communicatome." This advance is based on experimental evidence for an organized and brain-integrated reflex-like relationship and dialogue between the nervous and the immune systems. A key circuitry in this communicatome is provided by efferent vagus nerve fibers and cholinergic signaling. Inflammation and metabolic alterations coexist in many disorders affecting the liver and the gastrointestinal (GI) tract, including obesity, metabolic syndrome, fatty liver disease, liver injury, and liver failure, as well as inflammatory bowel disease. Here, we outline mechanistic insights regarding the role of the vagus nerve and cholinergic signaling in the regulation of inflammation linked to metabolic derangements and the pathogenesis of these disorders in preclinical settings. Recent clinical advances using this knowledge in novel therapeutic neuromodulatory approaches within the field of bioelectronic medicine are also briefly summarized.

Vernet syndrome resulting from varicella zoster virus infection-a very rare clinical presentation of a common viral infection.

Vernet syndrome is a unilateral palsy of glossopharyngeal, vagus, and accessory nerves. Varicella zoster virus (VZV) infection has rarely been described as a possible cause. A 76-year-old man presented with 1-week-long symptoms of dysphonia, dysphagia, and weakness of the right shoulder elevation, accompanied by a mild right temporal parietal headache with radiation to the ipsilateral ear. Physical examination showed signs compatible with a right XI, X, and XI cranial nerves involvement and also several vesicular lesions in the right ear's concha. He had a personal history of poliomyelitis and chickenpox. Laringoscopy demonstrated right vocal cord palsy. Brain MRI showed thickening and enhancement of right lower cranial nerves and an enhancing nodular lesion in the ipsilateral jugular foramen, in T1 weighted images with gadolinium. Cerebrospinal fluid (CSF) analysis disclosed a mild lymphocytic pleocytosis and absence of VZV-DNA by PCR analysis. Serum VZV IgM and IgG antibodies were positive. The patient had a noticeable clinical improvement after initiation of acyclovir and prednisolone therapy. The presentation of a VZV infection with isolated IX, X, and XI cranial nerves palsy is extremely rare. In our case, the diagnosis of Vernet syndrome as a result of VZV infection was made essentially from clinical findings and supported by analytical and imaging data.

Vagal afferent activation suppresses systemic inflammation via the splanchnic anti-inflammatory pathway.

Electrical stimulation of the vagus nerve (VNS) is a novel strategy used to treat inflammatory conditions. Therapeutic VNS activates both efferent and afferent fibers; however, the effects attributable to vagal afferent stimulation are unclear. Here, we tested if selective activation of afferent fibers in the abdominal vagus suppresses systemic inflammation. In urethane-anesthetized rats challenged with lipopolysaccharide (LPS, 60 µg/kg, i.v.), abdominal afferent VNS (2 Hz for 20 min) reduced plasma tumor necrosis factor alpha (TNF) levels 90 min later by 88% compared with unmanipulated animals. Pre-cutting the cervical vagi blocked this anti-inflammatory action. Interestingly, the surgical procedure to expose and prepare the abdominal vagus for afferent stimulation ('vagal manipulation') also had an anti-inflammatory action. Levels of the anti-inflammatory cytokine IL-10 were inversely related to those of TNF. Prior bilateral section of the splanchnic sympathetic nerves reversed the anti-inflammatory actions of afferent VNS and vagal manipulation. Sympathetic efferent activity in the splanchnic nerve was shown to respond reflexly to abdominal vagal afferent stimulation. These data demonstrate that experimentally activating abdominal vagal afferent fibers suppresses systemic inflammation, and that the efferent neural pathway for this action is in the splanchnic sympathetic nerves.

Autonomic regulation of the immune system in cardiovascular diseases.

The autonomic nervous system is a powerful regulator of circulatory adjustments to acute hemodynamic stresses. Here we focus on new concepts that emphasize the chronic influence of the sympathetic and parasympathetic systems on cardiovascular pathology. The autonomic neurohumoral system can dramatically influence morbidity and mortality from cardiovascular disease through newly discovered influences on the innate and adaptive immune systems. Specifically, the end-organ damage in heart failure or hypertension may be worsened or alleviated by pro- or anti-inflammatory pathways of the immune system, respectively, that are activated through neurohumoral transmitters. These concepts provide a major new perspective on potentially life-saving therapeutic interventions in the deadliest of diseases.

Rethinking inflammation: neural circuits in the regulation of immunity.

Neural reflex circuits regulate cytokine release to prevent potentially damaging inflammation and maintain homeostasis. In the inflammatory reflex, sensory input elicited by infection or injury travels through the afferent vagus nerve to integrative regions in the brainstem, and efferent nerves carry outbound signals that terminate in the spleen and other tissues. Neurotransmitters from peripheral autonomic nerves subsequently promote acetylcholine-release from a subset of CD4(+) T cells that relay the neural signal to other immune cells, e.g. through activation of α7 nicotinic acetylcholine receptors on macrophages. Here, we review recent progress in the understanding of the inflammatory reflex and discuss potential therapeutic implications of current findings in this evolving field.

The influence of sensitization on mechanisms of organophosphorus pesticide-induced airway hyperreactivity.

We previously demonstrated that antigen sensitization increases vulnerability to airway hyperreactivity induced by the organophosphorus pesticide (OP) parathion. Sensitization also changes the mechanism of parathion-induced airway hyperreactivity to one that is dependent on IL-5. To determine whether this effect can be generalized to other OPs, and to other classes of pesticides, we measured airway responsiveness to vagal stimulation or intravenous acetylcholine in nonsensitized and ovalbumin-sensitized guinea pigs 24 hours after a single subcutaneous injection of the OPs diazinon or chlorpyrifos, or the pyrethroid permethrin. Sensitization exacerbated the effects of chlorpyrifos on bronchoconstriction in response to vagal stimulation or intravenous acetylcholine. Pretreatment with function-blocking IL-5 antibody prevented chlorpyrifos-induced airway hyperreactivity in sensitized, but not in nonsensitized, guinea pigs. In sensitized guinea pigs, blocking IL-5 decreased eosinophil activation, as measured by decreased eosinophil major basic protein in the trachea. In contrast, sensitization did not alter diazinon-induced airway hyperreactivity, and permethrin did not cause airway hyperreactivity in either nonsensitized or sensitized guinea pigs. None of the pesticides affected inflammatory cells in the bronchoalveolar lavage fluid or blood. We have previously shown that three different OPs cause airway hyperreactivity via loss of neuronal M2 muscarinic receptor function. Similar to parathion, but unlike diazinon, the mechanism of chlorpyrifos-induced airway hyperreactivity is changed by sensitization. Thus, OP-induced airway hyperreactivity is dependent on sensitization status and on the OP used, which may influence therapeutic approaches.

Allergen challenge sensitizes TRPA1 in vagal sensory neurons and afferent C-fiber subtypes in guinea pig esophagus.

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE.

Toll-like receptor 9 plays a key role in the autonomic cardiac and baroreflex control of arterial pressure.

The crosstalk between the immune and the autonomic nervous system may impact the cardiovascular function. Toll-like receptors are components of the innate immune system and play developmental and physiological roles. Toll-like receptor 9 (TLR9) is involved in the pathogenesis of cardiovascular diseases, such as hypertension and heart failure. Since such diseases are commonly accompanied by autonomic imbalance and lower baroreflex sensitivity, we hypothesized that TLR9 modulates cardiac autonomic and baroreflex control of arterial pressure (AP). Toll-like receptor 9 knockout (TLR9 KO) and wild-type (WT) mice were implanted with catheters into carotid artery and jugular vein and allowed to recover for 3 days. After basal recording of AP, mice received methyl-atropine or propranolol. AP and pulse interval (PI) variability were evaluated in the time and frequency domain (spectral analysis), as well as by multiscale entropy. Spontaneous baroreflex was studied by sequence technique. Behavioral and cardiovascular responses to fear-conditioning stress were also evaluated. AP was similar between groups, but TLR9 KO mice exhibited lower basal heart rate (HR). AP variability was not different, but PI variability was increased in TLR9 KO mice. The total entropy was higher in TLR9 KO mice. Moreover, baroreflex function was found higher in TLR9 KO mice. Atropine-induced tachycardia was increased in TLR9 KO mice, whereas the propranolol-induced bradycardia was similar to WT mice. TLR9 KO mice exhibit increased behavioral and decreased tachycardia responses to fear-conditioning stress. In conclusion, our findings suggest that TLR9 may negatively modulate cardiac vagal tone and baroreflex in mice.

Impaired vagus-mediated immunosuppression in microsomal prostaglandin E synthase-1 deficient mice.

The cholinergic anti-inflammatory pathway controls innate immune responses and inflammation. The prostaglandin (PG) system is involved in several neuro-processes and associated with inflammatory activation of cells in vagal nuclei. Here we aimed to investigate the potential role of PG in cholinergic neuro-regulation. The effect of vagus nerve stimulation (VNS) has been evaluated in microsomal prostaglandin E synthase-1 (mPGES-1) knockout (-/-) and wild-type (+/+) mice regarding cytokine and PG levels after lipopolysaccharides (LPS) challenge. As expected, VNS decreased the release of pro-inflammatory cytokines both in serum and spleen extracts of mPGES-1 (+/+)animals. However, the immune suppressive effect of VNS was completely abolished in mPGES-1 (-/-) mice. The PG content was not affected by VNS in the spleen of mPGES-1 (+/+) and mPGES-1 (-/-) mice but interestingly, acetylcholine (ACh) release in spleen induced by VNS confirmed an intact cholinergic pathway in mPGES-1 (+/+) whereas no VNS-induced ACh release was found in mPGES-1 (-/-) animals. Our data show that mPGES-1 and consequently PGE2 are crucial in the cholinergic anti-inflammatory pathway. Moreover, the mechanisms involved do not affect PG content in the spleen, but lack of mPGES-1 was found to strongly affect cholinergic mechanisms in the inflamed spleen. These findings illustrate previously unrecognized associations between the cholinergic and prostaglandin systems, and may be of importance for further development of therapeutic strategies directed at modulation of the inflammatory reflex, and immunosuppression in chronic inflammatory diseases.

Alterations in vagal-immune pathway in long-lasting mental stress.

We studied a potential impact of chronic psychosocial load on the allostatic biomarkers--cardiac vagal activity, inflammation, and oxidative stress in healthy undergraduate students. Continuous resting ECG signals were monitored in a group of 16 female healthy students (age: 23.2±0.2 years, BMI: 20.9±0.5 kg/m2) at two time periods: right after holiday (rest period) and a day before final exams (stress period). Vagal activity was quantified by spectral analysis of heart rate variability at high frequency band (HF-HRV). The immune response was assessed from the level of tumor necrosis factor-alpha (TNF-α) in plasma. In addition, mean RR intervals were evaluated. We found that HF-HRV was significantly reduced and the TNF-α was increased in the stress period compared with the rest period. No significant changes were found in the RR interval. In conclusion, allostatic load induced by stress and the accompanying greater immune response decreased cardiovagal regulation in healthy young subjects. These findings may help understand the pathway by which stress can influence health and disease.