Search

1. Structure?Activity Relationships (SAR) of Melanocortin and Agouti?Related (AGRP) Peptides Anamika Singh, Erica M. Haslach and Carrie Haskell?Luevano Abstract Introduction Structure and Chemistry of Melanocortin Peptides Structure?Activity Relationships (SAR) Agouti?Related Protein (AGRP) Development of Chimeric Peptide Analogues Conclusion 2. Melanocortin Signalling Mechanisms Paula C. Eves and John W. Haycock Abstract Introduction Melanocortin Receptors Melanocortins and Pigmentation Melanocortin Anti?Inflammatory and Immunomodulatory Roles Intracellular Signalling of Melanocortin Peptides Melanocortin Receptor Antagonists and Agonists Conclusion 3. Distribution and Function of Melanocortin Receptors within the Brain Kathleen G. Mountjoy Abstract Introduction MC1R Is Expressed in the Periaqueductal Gray and the MC1R Regulates Pain and Analgesia MC2R Is Expressed in Teleost Fish Brain and Fetal Mouse Brain, but Not in Adult Mammalian Brain MC3R Is Specifically Expressed in Hypothalamus and the Limbic System Where Its Functions Are Not Clearly Understood MC4R Is Expressed in Distinct Nuclei in Every Region of the Brain Reflecting It's Many Diverse Functions in the Brain MC5R Expression and Function in the Brain Is Unclear Melanocortin Receptor Accessory Proteins (MRAPs) Conclusion 4. Drugs, Exercise, and the Melanocortin?4 Receptor-Different Means, Same Ends: Treating Obesity Jay W. Schaub, Erin B. Bruce, and Carrie Haskell?Luevano Abstract Introduction Melanocortins Exercise Conclusion 5. Melanocortins in Brain Inflamation: The Role of Melanocortin Receptor Subtypes Ruta Muceniece and Maija Dambrova Abstract Introduction Distribution of MC Expressing Neurons and MC Receptors in the Brain MCR Subtypes and Brain Inflammation Main Intracellular Signalling Pathways of MC Receptors Potential Therapeutic Applications of MC Receptor Subtype Selective Compounds Conclusion 6. Melanocortins and the Cholinergic Anti?Inflamatory Pathway DanielaGiuliani, Alessandra Ottani, Domenica Altavilla, Carla Bazzani, Francesco Squadrito and Salvatore Guarini Abstract Introduction The Inflammatory Reflex Antishock Effects of Melanocortins Melanocortins Reverse Circulatory Shock through Activation of the Cholinergic Anti?Inflammatory Pathway Protective Effect of Melanocortins in Myocardial Ischemia/Reperfusion Melanocortins Prevent Myocardial Ischemia/Reperfusion?Induced Damage through Activation of the Cholinergic Anti?Inflammatory Pathway Neuroprotection by Melanocortins in Ischemic Stroke Melanocortins Produce Neuroprotection Against Ischemic Stroke by Activating the Cholinergic Anti?Inflammatory Pathway Conclusion 7. Melanocortin Control of Cell Traficking in Vascular Inflamation Hetal B. Patel, Giovanna Leoni, Trinidad Montero Melendez, Andre L.F. Sampaio and Mauro Perretti Abstract Introduction Inflammation: Cell Trafficking and Microcirculation Melancortins in Experimental Inflammation Effects of Melanocortins on Inflammatory Cells In Vitro Lessons from Melanocortin Knockout Mice Drug Discovery Melanocortin Peptide Pharmacology Conclusion 8. Terminal Signal: Anti?Inflamatory Efects of ??Melanocyte?Stimulating Hormone Related Peptides Beyond the Pharmacophore Thomas Brzoska, Markus Bohm, Andreas Lugering, Karin Loser and Thomas A. Luger Abstract Introduction ??MSH Related Peptides and Melanocortin Receptors-Mediation of Anti?Inflammatory Effects? Anti?Inflammatory Effects of ??MSH Related Tripeptides In Vitro and Vivo Therapeutic Potential of ??MSH?Related Tripeptides in Human Immune?Mediated Inflammatory Diseases Conclusion 9. Protective Efects of Melanocortins in Systemic Host Reactions Stefano Gatti, Caterina Lonati, Andrea Sordi and Anna Catania Abstract Introduction Fever Endotoxemia and Septic Shock Acute Respiratory Distress Syndrome (ARDS) Ischemia and Reperfusion Injury Organ Transplantation Conclusion 10. Development of ??Melanocortin Analogs for Melanoma

Publisher Summary Inflammation is a process of interactions among soluble factors and different cells that can arise in any tissue in response to traumatic, infectious, postischemic, toxic, or autoimmune injury. A number of inflammatory stimuli have been identified. These stimuli can interact with cell receptors created after a sensitizing exposure to trigger an inflammatory cascade that induces host defense. In this scenario, neurogenic inflammation occurs: this is defined as a process by which inflammation is triggered by the nervous system. This means that nerve cells recognize a stimulus and initiate the inflammatory response independent from the immune system. This is called the inflammatory spinal reflex. catecholamines and acetylcholine may control local and systemic immune response during all phases of inflammation. In detail, afferent somatosensory nerves, including the vagus, are able to recognize various signals of things that could threaten the organism. The resulting information may initiate an inflammatory response (neurogenic inflammation) via a spinal reflex. The released pro-inflammatory cytokines may again affect somatosensory nerves and signal the brain that a local inflammatory reaction has occurred. This may activate inhibitory neuroimmune pathways such as the HPA axis, the SNS, and the vagus to support a local anti-inflammatory response. if the inflammatory response dominates, chronic inflammatory diseases may be supported; if the anti-inflammatory response is outbalanced, subsequent immunodepression with risk of infectious complication may occur.

The body's first defense against invading pathogens or tissue injury is the innate immune system. Since excessive immune responses can be damaging, anti-inflammatory mechanisms function to control the pro-inflammatory response and prevent injury. The cholinergic anti-inflammatory pathway is a neural mechanism that suppresses the innate inflammatory response. Knowledge concerning innervation of the immune system offers a unique opportunity to explore previously unrecognized techniques to treat disease. It also enables consideration of the neurological basis of complementary and alternative medical therapies, such as meditation and acupuncture. This evolving area of research has implications for the pathogenesis of chronic inflammatory conditions including inflammatory bowel disease, rheumatoid arthritis, type 2 diabetes, and other conditions of excessive cytokine release.

Inflammation can cause damage and even death. What controls this primitive and potentially lethal innate immune response to injury and infection? Molecular and neurophysiological studies during the past decade have revealed a pivotal answer: immunity is coordinated by neural circuits that operate reflexively. The afferent arc of the reflex consists of nerves that sense injury and infection. This activates efferent neural circuits, including the cholinergic anti-inflammatory pathway, that modulate immune responses and the progression of inflammatory diseases. It might be possible to develop therapeutics that target neural networks for the treatment of inflammatory disorders.

Inhibition of sympathoexcitatory circuits is influenced by cerebral structures and mediated via vagal mechanisms. Studies of pharmacologic blockade of the prefrontal cortex together with neuroimaging studies support the role of the right hemisphere in parasympathetic control of the heart via its connection with the right vagus nerve. Neural mechanisms also regulate inflammation; vagus nerve activity inhibits macrophage activation and the synthesis of tumor necrosis factor in the reticuloendothelial system through the release of acetylcholine. Data suggest an association between heart rate variability and inflammation that may support the concept of a cholinergic anti-inflammatory pathway.

The autonomic nervous system maintains homeostasis through its sympathetic and parasympathetic divisions. During infection, cells of the immune system release cytokines and other mediators that cause fever, hypotension, and tissue injury. Although the effect of cytokines on the nervous system has been known for decades, only recently has it become evident that the autonomic nervous system, in turn, regulates cytokine production through neural pathways. We have previously shown that efferent vagus nerve signals regulate cytokine production through the nicotinic acetylcholine receptor subunit α7, a mechanism termed “the cholinergic antiinflammatory pathway.” Here, we show that vagus nerve stimulation during endotoxemia specifically attenuates TNF production by spleen macrophages in the red pulp and the marginal zone. Administration of nicotine, a pharmacological agonist of α7, attenuated TNF immunoreactivity in these specific macrophage subpopulations. Synaptophysin-positive nerve endings were observed in close apposition to red pulp macrophages, but they do not express choline acetyltransferase or vesicular acetylcholine transporter. Surgical ablation of the splenic nerve and catecholamine depletion by reserpine indicate that these nerves are catecholaminergic and are required for functional inhibition of TNF production by vagus nerve stimulation. Thus, the cholinergic antiinflammatory pathway regulates TNF production in discrete macrophage populations via two serially connected neurons: one preganglionic, originating in the dorsal motor nucleus of the vagus nerve, and the second postganglionic, originating in the celiac-superior mesenteric plexus, and projecting in the splenic nerve.

Introduction The interaction of the CNS and the immune system is well known. A parasympathetic anti-inflammatory pathway has recently been described. Both electrical and pharmacological parasympathetic stimulation attenuate proinflammatory mediator generation. Burn induces abacterial cytokine generation and we sought to evaluate whether parasympathetic stimulation after experimental burn decreases cardiodepressive mediator generation. Material and methods A 30% TBSA full-thickness rat burn model was used. After microsurgical preparation of the cervical portion of the vagus nerve, we performed electric vagus nerve stimulation. Serum was harvested and organ samples of heart and liver were homogenized. Samples were subjected to sandwich-ELISA specific for TNF-α, IL-1β and IL-6. Heart rate measurements were done using left ventricular microcatheterization. Statistical analysis was done using Student's t-tests and analysis of variance (ANOVA). Results Burn induced a significant rise of TNF-α, IL-1β and IL-6 in organ homogenates and serum. After cervical vagal electrostimulation, serum and organ homogenate levels of proinflammatory cytokines were markedly reduced compared to burn controls. Left ventricular microcatheter assessment demonstrated no cardiodepressive effect of the vagal stimulation itself. Conclusion Our results encourage further research regarding the neuroimmunologic background of burn, possibly leading to the development of a novel therapeutic approach to burn-induced organ dysfunction and immunodysregulation.

Objective: Electrical vagus nerve stimulation inhibits proinflammatory cytokine production and prevents shock during lethal systemic inflammation through an 7 nicotinic acetylcholine receptor (7nAChR)-dependent pathway to the spleen, termed the cholinergic anti-inflammatory pathway. Pharmacologic 7nAChR agonists inhibit production of the critical proinflammatory mediator high mobility group box 1 (HMGB1) and rescue mice from lethal polymicrobial sepsis. Here we developed a method of transcutaneous mechanical vagus nerve stimulation and then investigated whether this therapy can protect mice against sepsis lethality. Design: Prospective, randomized study. Setting: Institute-based research laboratory. Subjects: Male BALB/c mice. Interventions: Mice received lipopolysaccharide to induce lethal endotoxemia or underwent cecal ligation and puncture to induce polymicrobial sepsis. Mice were then randomized to receive electrical, transcutaneous, or sham vagus nerve stimulation and were followed for survival or euthanized at predetermined time points for cytokine analysis. Measurements and Main Results: Transcutaneous vagus nerve stimulation dose-dependently reduced systemic tumor necrosis factor levels during lethal endotoxemia. Treatment with transcutaneous vagus nerve stimulation inhibited HMGB1 levels and improved survival in mice with polymicrobial sepsis, even when administered 24 hrs after the onset of disease. Conclusions: Transcutaneous vagus nerve stimulation is an efficacious treatment for mice with lethal endotoxemia or polymicrobial sepsis. (Crit Care Med 2007; 35:2762‐2768)

Cytokine production by the immune system contributes importantly to both health and disease. The nervous system, via an inflammatory reflex of the vagus nerve, can inhibit cytokine release and thereby prevent tissue injury and death. The efferent neural signaling pathway is termed the cholinergic antiinflammatory pathway. Cholinergic agonists inhibit cytokine synthesis and protect against cytokine-mediated diseases. Stimulation of the vagus nerve prevents the damaging effects of cytokine release in experimental sepsis, endotoxemia, ischemia/reperfusion injury, hemorrhagic shock, arthritis, and other inflammatory syndromes. Herein is a review of this physiological, functional anatomical mechanism for neurological regulation of cytokine-dependent disease that begins to define an immunological homunculus.

Recent data suggest remarkable effects of vagus stimulation (reduction) and vagotomy (exacerbation) on acute inflammation in rats, the so-called "inflammatory reflex". Its role in humans remains unknown. Therefore, the aim was to explore whether surgical vagotomy in humans would affect the risk of a prototype inflammatory disease, rheumatoid arthritis. This was a case-control study. Assessment of the relative risk (RR) of developing rheumatoid arthritis after surgical vagotomy during 1964-2001 in 63,092 prevalent rheumatoid arthritis cases versus 125,404 matched controls from the general population and in 2548 incident rheumatoid arthritis cases versus 24,357 matched controls from the general population, respectively, was done. For comparison, we assessed RRs for hospitalisation for gastric disorders not including vagotomy. Data on exposures and rheumatoid arthritis were retrieved from population-based and prospectively recorded Swedish registers. A pre-rheumatoid arthritis vagotomy was not significantly associated with an increased risk for rheumatoid arthritis (RR = 1.17, 95% CI 0.97 to 1.40). RRs in the same range were observed for several other pre-rheumatoid arthritis gastric conditions that do not include vagotomy (eg, gastric ulcer RR = 1.21, 95% 1.11 to 1.33). Vagotomy has no specific effect on the risk of developing rheumatoid arthritis in humans. Gastroduodenal ulcers occur more often than expected even before the occurrence of rheumatoid arthritis.

The innate immune system protects against infection and tissue injury through the specialized organs of the reticuloendothelial system, including the lungs, liver, and spleen. The central nervous system regulates innate immune responses via the vagus nerve, a mechanism termed the cholinergic antiinflammatory pathway. Vagus nerve stimulation inhibits proinflammatory cytokine production by signaling through the α7 nicotinic acetylcholine receptor subunit. Previously, the functional relationship between the cholinergic antiinflammatory pathway and the reticuloendothelial system was unknown. Here we show that vagus nerve stimulation fails to inhibit tumor necrosis factor (TNF) production in splenectomized animals during lethal endotoxemia. Selective lesioning of the common celiac nerve abolishes TNF suppression by vagus nerve stimulation, suggesting that the cholinergic pathway is functionally hard wired to the spleen via this branch of the vagus nerve. Administration of nicotine, an α7 agonist that mimics vagus nerve stimulation, increases proinflammatory cytokine production and lethality from polymicrobial sepsis in splenectomized mice, indicating that the spleen is critical to the protective response of the cholinergic pathway. These results reveal a specific, physiological connection between the nervous and innate immune systems that may be exploited through either electrical vagus nerve stimulation or administration of α7 agonists to inhibit proinflammatory cytokine production during infection and tissue injury.

Background & Aims: The recently proposed Inflammatory Reflex describes an interaction between the vagus nerve and peripheral macrophages, resulting in attenuation of proinflammatory cytokine release in response to systemic exposure to bacterial endotoxin. The purpose of this study was to determine whether a similar vagus/macrophage axis modulates the inflammatory responses in the colon in mice. Methods: We assessed the Disease Activity Index (DAI), macroscopic and histologic damage, serum amyloid-P level, and myeloperoxidase activity in colitis induced by administration of dextran sodium sulfate (DSS) in healthy and vagotomized C57BL/6 and in mice deficient in macrophage-colony stimulating factor (M-CSF)–induced and in hapten-induced colitis. A pyloroplasty was performed in vagotomized mice. Results: DAI, macroscopic and histologic scores, myeloperoxidase activity, levels of serum amyloid-P, and colonic tissue levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were increased significantly in vagotomized mice 5 days post-DSS and 3 days after hapten-induced colitis compared with sham-operated mice that received DSS or the hapten. Pretreatment with nicotine significantly decreased each of these markers in vagotomized mice with DSS colitis, and all markers except DAI and IL-6 in sham-operated DSS-treated mice. Conversely, hexamethonium treatment significantly increased each of these markers in the sham-operated DSS-treated mice. Vagotomy had no effect on the colitis in M-CSF–deficient mice. Conclusions: The vagus nerve plays a counterinflammatory role in acute colitis via a macrophage-dependent mechanism, involving hexamethonium-sensitive nicotinic receptors. The identification of a counterinflammatory neural pathway would open new therapeutic avenues for treating acute exacerbations of inflammatory bowel disease.

Endothelial cell activation plays a critical role in regulating leukocyte recruitment during inflammation and infection. Based on recent studies showing that acetylcholine and other cholinergic mediators suppress the production of proinflammatory cytokines via the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expressed by macrophages and our observations that human microvascular endothelial cells express the alpha7 nAChR, we examined the effect of cholinergic stimulation on endothelial cell activation in vitro and in vivo. Using the Shwartzman reaction, we observed that nicotine (2 mg/kg) and the novel cholinergic agent CAP55 (12 mg/kg) inhibit endothelial cell adhesion molecule expression. Using endothelial cell cultures, we observed the direct inhibitory effects of acetylcholine and cholinergic agents on tumor necrosis factor (TNF)-induced endothelial cell activation. Mecamylamine, an nAChR antagonist, reversed the inhibition of endothelial cell activation by both cholinergic agonists, confirming the antiinflammatory role of the nAChR cholinergic pathway. In vitro mechanistic studies revealed that nicotine blocked TNF-induced nuclear factor-kappaB nuclear entry in an inhibitor kappaB (IkappaB)alpha- and IkappaBepsilon-dependent manner. Finally, with the carrageenan air pouch model, both vagus nerve stimulation and cholinergic agonists significantly blocked leukocyte migration in vivo. These findings identify the endothelium, a key regulator of leukocyte trafficking during inflammation, as a target of anti-inflammatory cholinergic mediators.

The 'cytokine theory of disease' states that an overproduction of cytokines can cause the clinical manifestations of disease. Much effort has been expended to determine how cytokines are regulated in normal health. Transcriptional, translational and other molecular control mechanisms protect the host from excessive cytokine production. A recent discovery revealed an unexpected pathway that inhibits macrophage cytokine production. The inflammatory reflex is a physiological pathway in which the autonomic nervous system detects the presence of inflammatory stimuli and modulates cytokine production. Afferent signals to the brain are transmitted via the vagus nerve, which activates a reflex response that culminates in efferent vagus nerve signalling. Termed the 'cholinergic anti-inflammatory pathway', efferent activity in the vagus nerve releases acetylcholine (ACh) in the vicinity of macrophages within the reticuloendothelial system. ACh can interact specifically with macrophage alpha7 subunits of nicotinic ACh receptors, leading to cellular deactivation and inhibition of cytokine release. This 'hard-wired' connection between the nervous and immune systems can be harnessed therapeutically in animal models of inflammatory disease, via direct electrical stimulation of the vagus nerve, or through the use of cholinergic agonists that specifically activate the macrophage alpha7 subunit of the ACh receptor. Autonomic dysfunction has been associated with human inflammatory diseases including rheumatoid arthritis, diabetes and sepsis; whether this dysfunction results from the inflammatory component of these diseases, or is actually an underlying cause, is now less clear. The description of the cholinergic anti-inflammatory now brings to the fore several new therapeutic strategies for inflammatory disease, and suggests that many of these diseases may actually be diseases of autonomic dysfunction.

This review outlines the mechanisms underlying the interaction between the nervous and immune systems of the host in response to an immune challenge. The main focus is the cholinergic anti-inflammatory pathway, which we recently described as a novel function of the efferent vagus nerve. This pathway plays a critical role in controlling the inflammatory response through interaction with peripheral α7 subunit-containing nicotinic acetylcholine receptors expressed on macrophages. We describe the modulation of systemic and local inflammation by the cholinergic anti-inflammatory pathway and its function as an interface between the brain and the immune system. The clinical implications of this novel mechanism also are discussed.

Background— We investigated whether electrical stimulation (STIM) of efferent vagus nerves may suppress nuclear factor (NF)-κB activation and the inflammatory cascade in hemorrhagic (Hem) shock. Methods and Results— Rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Hem shock was induced by intermittent withdrawing of blood until mean arterial pressure stabilized within the range of 35 to 40 mm Hg. Application of constant voltage pulses to the caudal vagus ends (STIM; 5 V, 2 ms, 1 Hz for 12 minutes, 5 minutes after mean arterial pressure stabilization) increased survival time (VGX+Hem+Sham STIM=38±3 minutes; VGX+Hem+STIM >180 minutes), reverted the marked hypotension (VGX+Hem+Sham STIM=33±3 mm Hg; VGX+Hem+STIM=66±5 mm Hg), inhibited IκBα liver loss, and blunted the augmented NF-κB activity, decreased hepatic tumor necrosis factor (TNF)-α mRNA (VGX+Hem+Sham STIM=1.42±0.5 amount of TNF-α m-RNA; VGX+Hem+STIM=0.51±0.2 amount of TNF-α mRNA), and reduced plasma TNF-α (VGX+Hem+Sham STIM=190±24 pg/mL; VGX+Hem+STIM=87±15 pg/mL). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Conclusions— Our results show a parasympathetic inhibition of NF-κB by which the brain opposes NF-κB activation in the liver and modulates the inflammatory response during acute hypovolemic hemorrhagic shock.

In this issue of Experimental Physiology, Bratton et al. (2012) report negative results. The authors have failed to find a synaptic connection from vagal preganglionic neurons to splenic sympathetic postganglionic neurons in the rat. Furthermore, the authors have shown that vagal efferent neurons do not drive the ongoing activity of splenic neurons. Although negative results are a common subject of jokes among scientists, they can be significant. Identification of the limitations of a current model (and this is what good negative results often do) is as necessary for improving our understanding as proposing a new model (which is often based on positive results). Bratton et al. (2012) tested the current model of the neuroanatomical substrate of the inflammatory reflex and, more specifically, the efferent arm of the reflex. What is the inflammatory reflex? In the past, the terms neurogenic inflammation, reflex inflammation and, occasionally, inflammatory reflex were used to refer to axon reflexes and some multineuronal spinal reflexes that mediate local inflammatory reactions. These reflexes have been implicated in inflammatory vasodilatation and in the production of multiple ingredients of what is commonly known as the inflammatory soup. A new meaning of the term inflammatory reflex has emerged during the last decade (Tracey, 2002; Rosas-Ballina & Tracey, 2009). Although it is still a work in progress, the principal feature of the new meaning is that it refers to systemic inflammation. In systemic inflammation, the inflammatory soup (vaguely similar to what is found at local inflammation sites) is cooked in the spleen, liver and perhaps other organs for remote consumption; it circulates throughout the body and exerts profound systemic effects, both adaptive (combating an infection) and detrimental (causing multiple organ dysfunction and failure). According to the new thinking, systemic levels of inflammatory mediators in systemic inflammation are controlled by the nervous system, and this control is realized via the inflammatory reflex. In contrast to reflexes in local inflammation, the arc of the proposed reflex is more complex; it involves neurons located in the brain and possibly travels through several brain structures. This reflex is thought to constitute the basis for homeostatic regulation of inflammation, similar to the regulation of heart rate or body temperature (Tracey, 2002; Rosas-Ballina & Tracey, 2009). The proposed regulation is thought to involve integration of inflammatory signals in the brain and co-ordination of effector responses. However, several elements of this innovative model may require further experimental testing and perhaps conceptual modifications. The afferent arm of what later became known as the inflammatory reflex attracted attention first. In the late 1980s and early 1990s, several groups proposed that peripheral-to-brain signalling by nerves is important for triggering various components of the systemic inflammatory response (for review, see Romanovsky et al. 2005). The component with which I am most familiar is fever. Two procedures were used to block the transduction of peripheral pyrogenic signals by nerves: surgical vagotomy and capsaicininduced inactivation (desensitization) of abdominal sensory nerves expressing the transient receptor potential vanilloid-1 (TRPV1) channel. Either procedure attenuated the febrile response to bacterial lipopolysaccharide, thus seeming to demonstrate the importance of signals conveyed to the brain by nerves. However, the majority of early studies ignored the fact that surgical vagotomy caused severe complications, including thermogenic deficiency. In later studies, when caution was exercised to prevent these complications, most – if not all – effects of vagotomy on fever disappeared. Several of these later studies were especially convincing, because they were conducted by the same authors who reported an attenuation of fever by vagotomy in their earlier papers. As for desensitization by capsaicin, studies from my laboratory have shown that it blocks the first phase of lipopolysaccharide-induced fever via a non-neural, non-TRPV1-mediated mechanism (Romanovsky et al. 2005). Hence, even though there is still some evidence (not reviewed here) suggesting that the immune system may send signals to the brain via nerves, it is hard to find experimental support for the proposition that signals conveyed by nerves are crucial (or even important) for driving effector responses, at least in the case of fever. Is a reflex an adequate model for afferent signals reaching the brain by non-neural (humoral) routes? The efferent arm of the inflammatory reflex is a newer construction. To explain the exaggeration of hypothermia and other symptoms of lipopolysaccharideinduced shock by vagotomy, Borovikova et al. (2000) proposed that efferent vagal activity, via a nicotinic receptor-mediated mechanism, inhibited the production of some components of the inflammatory soup, such as tumour necrosis factorα. This cholinergic anti-inflammatory pathway became the efferent arm of the inflammatory reflex. Initially, effector processes controlled by this pathway were thought to be localized in the liver. Subsequently, the focus shifted to the spleen; activation of splenic sympathetic nerves and the resultant suppression of the cytokine response in the spleen were shown to play dominant roles (Nance & Sanders, 2007; Rosas-Ballina & Tracey, 2009). To explain how the vagal anti-inflammatory pathway activates sympathetic suppression of splenic cytokine production, a synaptic connection has been proposed between vagal preganglionic neurons and sympathetic postganglionic neurons that innervate the spleen (RosasBallina & Tracey, 2009). By using neuroanatomical and electrophysiological approaches, Bratton et al. (2012) attempted to identify the proposed vago-splenic synapses. The anatomical approach encompassed injecting an anterograde tracer into the dorsal motor nucleus of the vagus and a retrograde tracer into the spleen and then searching for synaptic connections between anterogradely and retrogradely labelled

Inflammation is a complex set of interactions among soluble factors and cells that can arise in any tissue in response to traumatic, infectious, post-ischaemic, toxic or autoimmune injury. The process normally leads to recovery from infection and to healing, However, if targeted destruction and assisted repair are not properly phased, inflammation can lead to persistent tissue damage by leukocytes, lymphocytes or collagen. Inflammation may be considered in terms of its checkpoints, where binary or higher-order signals drive each commitment to escalate, go signals trigger stop signals, and molecules responsible for mediating the inflammatory response also suppress it, depending on timing and context. The non-inflammatory state does not arise passively from an absence of inflammatory stimuli; rather, maintenance of health requires the positive actions of specific gene products to suppress reactions to potentially inflammatory stimuli that do not warrant a full response.

Inflammation is a local, protective response to microbial invasion or injury. It must be fine-tuned and regulated precisely, because deficiencies or excesses of the inflammatory response cause morbidity and shorten lifespan. The discovery that cholinergic neurons inhibit acute inflammation has qualitatively expanded our understanding of how the nervous system modulates immune responses. The nervous system reflexively regulates the inflammatory response in real time, just as it controls heart rate and other vital functions. The opportunity now exists to apply this insight to the treatment of inflammation through selective and reversible 'hard-wired' neural systems.

Efferent activity in the vagus nerve can prevent endotoxin-induced shock by attenuating tumor necrosis factor (TNF) synthesis. Termed the “cholinergic antiinflammatory pathway,” inhibition of TNF synthesis is dependent on nicotinic α-bungarotoxin-sensitive acetylcholine receptors on macrophages. Vagus nerve firing is also stimulated by CNI-1493, a tetravalent guanylhydrazone molecule that inhibits systemic inflammation. Here, we studied the effects of pharmacological and electrical stimulation of the intact vagus nerve in adult male Lewis rats subjected to endotoxin-induced shock to determine whether intact vagus nerve signaling is required for the antiinflammatory action of CNI-1493. CNI-1493 administered via the intracerebroventricular route was 100,000-fold more effective in suppressing endotoxin-induced TNF release and shock as compared with intravenous dosing. Surgical or chemical vagotomy rendered animals sensitive to TNF release and shock, despite treatment with CNI-1493, indicating that an intact cholinergic antiinflammatory pathway is required for antiinflammatory efficacy in vivo. Electrical stimulation of either the right or left intact vagus nerve conferred significant protection against endotoxin-induced shock, and specifically attenuated serum and myocardial TNF, but not pulmonary TNF synthesis, as compared with sham-operated animals. Together, these results indicate that stimulation of the cholinergic antiinflammatory pathway by either pharmacological or electrical methods can attenuate the systemic inflammatory response to endotoxin-induced shock.

Authors Helt, Kelley, Boorstein, Pandey, and Fein, askedauthors Kinsbourne and Herbert to respond to the Goodletter because of their expertise in this area. Following isDr. Kinsbourne’s response.Curran et al. (2007) confirmed that children with autismbehaveless aberrantly when they are febrile due to infections,a phenomenon that might offer insight into the cause andtreatment of autistic symptoms. Good (2011) attributes theimprovement to an increase in cerebral blood flow. Somebrain regions related to autistic symptoms are indeed under-perfused (Ohnishi et al. 2000). However, because regionalcerebral blood flow is proportionate to metabolic need,diminished perfusion reflects diminished metabolism ratherthan cause it. Evidence is lacking that fever increases theperfusion of these regions, or that they would function betterif it did. Indeed, Curran et al. (2007) found no relationshipbetween degree of fever and degree of improvement.Fever is the presenting symptom of inflammatoryreactions to a pathogen. Absent inflammation, heat stressdoes not improve behavior in autism (Miles 2010, cited byGood). Therefore, the benefits do not accrue from the risingtemperature as such. I suggest that they derive from theimmune system’s efforts to limit the inflammatory reaction.The febrile inflammatory response to pathogens ismediated by proinflammatory cytokines, notably IL-1 betaand TNF-alpha (Mackowiak 1998) and promoted by thesympathetic nervous system. In excess, these cytokineswould be toxic. The proinflammatory cytokine response israpidly curtailed by IL-1ra, a specific IL-1 antagonist, andby anti-inflammatory cytokines, such as IL-10, TNF-binding protein and transforming growth factor-b (TGF-b)(Czura and Tracey 2005). Moreover, the inflammatoryreaction with its sympathetic activation is buffered by thevagal cholinergic anti-inflammatory system (CAIS) (Tracey2007; Thayer 2009).The inflammatory reflex of the vagus nerve preventsexcessive release of cytokines from macrophages. Whenthe CAIS of mice was inactivated, the cytokine response toendotoxin was excessive (Borovikova et al. 2000). Electri-cal vagal stimulation is used to treat inflammatory diseases,including depression, which features neuroinflammation(Pucak et al. 2007). Vagal stimulation also increases heartrate variability, as autonomic tone veers from sympathetictoward parasympathetic dominance.In controlling inflammation and sympathetic predomi-nance, the CAIS may lower arousal level. Much autisticbehavior appears to be a compensatory reaction to chronicpathologically heightened arousal (Kinsbourne 2011).Vargas et al. (2005)discoveredneuroinflammation in peoplewith autism, both in vivo and at autopsy. An outpouring ofcytokines from activated microglia, the CNS counterparts ofmacrophages, involves the excessive release of glutamate,the preponderant excitatory neurotransmitter, increasing thebrain activation toward hyperarousal. By subduing neuro-inflammation (Nizri et al. 2009), the CAIS would down-regulate arousal and enable adaptive behavior to emerge.Sympathetic hyperarousal in autism is also well docu-mented. By fostering parasympathetic (vagal) predominance,the CAIS might ameliorate the fearful sensory rejection andurge toward isolation and inward shift of attention thatcharacterizes the autistic state (Kinsbourne 2011).That autistic behavior can improve during fever demon-strates that it is not necessarily fixed but can constitute areaction to potentially remediable factors. If the suggested

CNI-1493 is a potent anti-inflammatory agent, which deactivates macrophages and inhibits the synthesis of proinflammatory mediators. The objective of the present study was to identify the role of the central nervous system (CNS) and efferent vagus nerve signaling in CNI-1493-mediated modulation of acute inflammation in the periphery. CNI-1493 was administered either intracerebroventricularly (i.c.v., 0.1-1,000 ng/kg) or intravenously (i.v., 5 mg/kg) in anesthetized rats subjected to a standard model of acute inflammation (subcutaneous (s.c.) injection of carrageenan). I.c.v. CNI-1493 significantly suppressed carrageenan-induced paw edema, even in doses at least 6-logs lower than those required for a systemic effect. Bilateral cervical vagotomy or atropine blockade (1 mg/kg/h) abrogated the anti-inflammatory effects of CNI-1493 (1 microg/kg, i.c.v. or 5 mg/kg, i.v.), indicating that the intact vagus nerve is required for CNI-1493 activity. Recording of the efferent vagus nerve activity revealed an increase in discharge rate starting at 3-4 min after CNI-1493 administration (5 mg/kg, i.v.) and lasting for 10-14 min (control activity=87+/-5.4 impulses/s versus CNI-1493-induced activity= 229+/-6.7 impulses/s). Modulation of efferent vagus nerve activity by electrical stimulation (5 V, 2 ms, 1 Hz) of the transected peripheral vagus nerve for 20 min (10 min before carrageenan administration and 10 min after) also prevented the development of acute inflammation. Local administration of the vagus nerve neurotransmitter, acetylcholine (4 microg/kg, s.c.), or cholinergic agonists into the site of carrageenan-injection also inhibited acute inflammation. These results now identify a previously unrecognized role of efferent vagus nerve activity in mediating the central action of an anti-inflammatory agent.

The immune system operates as a diffuse sensory system, detecting the presence of specific chemical constituents associated with dangerous micro-organisms, and then signalling the brain. In this way, immunosensation constitutes a chemosensory system. Several submodalities of this sensory system function as pathways conveying immune-related information, and can be classified as either primarily brain barrier associated or neural. The vagus nerve provides the major neural pathway identified to date. The initial chemosensory transduction events occur in immune cells, which respond to specific chemical components expressed by dangerous micro-organisms. These immune chemosensory cells release mediators, such as cytokines, to activate neural elements, including primary afferent neurons of the vagal sensory ganglia. Primary afferent activation initiates local reflexes (e.g. cardiovascular and gastrointestinal) that support host defense. In addition, at least three parallel pathways of ascending immune-related information activate specific components of the illness response. In this way, immunosensory systems represent highly organized and coherent pathways for activating host defense against infection.

The vagus nerve plays an important role in the regulation of homeostasis, and its efferent pathway emit signals able to control immune function, acting on the inflammatory response of lymphocytes and macrophages via the inflammatory reflex. Our hypothesis is that this route may also alter the function of dendritic cells, since these cells are essential for the formation of an adaptive immune response, are sensitive to an increase in proinflammatory cytokines and have cholinergic receptors. Therefore, dendritic cells generated from bone marrow precursor cells of mice subjected to unilateral cervical vagotomy were analyzed for the phenotypic markers CD11c and MHC-II and the co-stimulatory molecules CD80 and CD86, as well as its phagocytic function and antigen presentation to lymphocytes from OT-II mice. The analysis of phenotype and phagocytic function revealed no significant changes between the groups of partially vagotomized animals and control, and data obtained in the OT-II lymphocyte proliferation assay showed that dendritic cells generated from the bone marrow of partially vagotomized animals promote a response similar to non-vagotomized animals. Therefore, the results to date refute the initial hypothesis that the partial vagotomy can alter the phenotype and function of dendritic cells generated from bone marrow precursor cells.

Background The inflammatory reflex regulates innate and adaptive immunity. Activation of its efferent arm (the Cholinergic Anti-inflammatory Pathway (CAP)), by electrical vagus nerve stimulation (VNS) reduces systemic inflammation and ameliorates disease in many acute and chronic animal models 1 . Objectives We determined whether VNS could improve clinical manifestations and biomarkers of inflammation in rheumatoid arthritis (RA). Methods This is an open label study of patients with active RA (>/= 4 tender and 4 swollen joints, and CRP at least 7 mg/L) despite stable methotrexate for 3 months. After a pre-implantation baseline visit, patients were surgically implanted with a Cyberonics VNS system. The device delivered the first VNS during its standard intraoperative diagnostic check sequence. Two weeks after implantation, patients returned for initial in-clinic VNS. Treatment continued through the primary endpoint at Day 42, was withdrawn for a 14-day hiatus, reinitiated at day 56, and then maintained through the final day 84 visit. Biomarker assessments including serum cytokine levels, FACS of circulating cell markers, and whole blood in vitro LPS-stimulated TNF release were performed. Results 8 patients (7/8 RF+, 6/8 ACPA+) were enrolled. Implantation and stimulation were generally well tolerated. Moderate postoperative hoarseness occurred in one patient. ACR 20/50/70 responses at the day 42 primary endpoint were 75, 50, and 25%, respectively. During the 14-day treatment withdrawal period after day 42, the mean DAS28 worsened significantly (+0.95, 95% CI 0.15-1.75). After treatment re-initiation at day 56, the ACR 20/50/70 responses at the day 84 final visit were 62.5, 25, and 12.5%, respectively. Circulating CD25+FoxP3+Tregs were increased by 31%, and whole blood in vitro LPS-stimulated TNF release was reduced by 59% at day 42 compared with pre-implantation baseline values. In patients with a day 42 EULAR response, serum IL-6 was reduced by 56%. Conclusions VNS was generally well tolerated and improved signs and symptoms of RA. Treatment withdrawal was associated with worsening, and re-initiation with improvement in disease activity. Clinical improvement was associated with reduction in important mediators of systemic inflammation, increases in circulating T regulatory cells and decrease in the ability of circulating leukocytes to release TNF in response to endotoxin. These improvements in objective biomarkers were consistent with effects observed in preclinical inflammation models using VNS. This is the first demonstration in humans that stimulation of the CAP can favorably impact clinical manifestations of systemic inflammation. If efficacy and safety are confirmed in larger controlled studies, implantable medical devices may offer a feasible alternative approach to the treatment of RA and other chronic inflammatory diseases. References Andersson U, Tracey K, Annu. Rev. Immunol. 2012; 30:313 Disclosure of Interest F. Koopman: None Declared, S. Miljko Grant/research support from: SetPoint Medical, S. Grazio Grant/research support from: SetPoint Medical, S. Sokolovic Grant/research support from: SetPoint Medical, K. Tracey Shareholder of: SetPoint Medical, Consultant for: SetPoint Medical, Y. Levine Shareholder of: SetPoint Medical, Employee of: SetPoint Medical, R. Zitnik Shareholder of: SetPoint Medical, Employee of: SetPoint Medical, P.-P. Tak Grant/research support from: SetPoint Medical, Consultant for: SetPoint Medical

Introduction The cholinergic anti-inflammatory pathway (ie, inflammatory reflex) comprises an important neuro-immune link whereby inflammation such as arthritis can be tightly controlled by the autonomic nervous system and the vagus nerve (VN). Prostaglandins are known to mediate central effects in neurotransmission, by modulating glial interaction with neurons. Therefore, the authors have investigated the role of prostaglandins, including the major prostaglandin E 2 producing enzyme mPGES-1, in the cholinergic anti-inflammatory pathway. Material and methods Knock-out mice for the mPGES-1 enzyme (-/-) have been used for the study, with comparison to wild-type (+/+) animals. After VN isolation, the authors injected intraperitoneally lipopolysaccharides (LPS, 2 mg/kg). The authors then electrically stimulated the VN for 5 min. After a recovery of 6 h, mice were killed and blood was collected. Tumour necrosis factor α (TNFα), IL-1β and other pro-inflammatory cytokines were measured using the meso scale discovery (MSD) instrument. In addition, the authors evaluated the effect of PGE 2 on the release of cytokines in vitro, using peripheral blood monuclear cells (PBMC) stimulated by LPS. Supernatants were analysed for TNFα concentration with ELISA. Results After vagus nerve stimulation (VNS) and analysis of the serum, wild-type animals showed a decrease of the release of TNFα (62.6+/−8.5 with VNS vs 90.3+/−9.1 pg/ml without VNS; p 2 (1 µM) was shown to inhibit the release of TNFα by PBMC after LPS stimulation from 375.1+/−73.7 to 45.5+/−22.1 pg/ml (p Conclusion Our data indicate that mice deficient in mPGES-1 do not respond to vagus-mediated immunosuppression. These results implicate an involvement of PGE 2 in the cholinergic anti-inflammatory pathway and provide support for further investigations regarding the interaction between cholinergic and prostaglandin systems.

Background The cholinergic anti-inflammatory pathway is proposed to be part of the so-called vago-vagal ‘inflammatory reflex’. The aim of this study is to provide neuro-anatomical evidence to support the existence of a functional neuronal circuit and its activation in response to intestinal inflammation. Methods The expression of c-fos was evaluated at different levels of the neurocircuitry in the course of postoperative ileus (POI) in a mouse model. Specific activation of the motor neurons innervating the inflamed intestine and the spleen was monitored by retrograde tracing using cholera toxin-b. The role of the vagal afferent pathway nerve was evaluated by selective vagal denervation of the intestine. Key Results Abdominal surgery resulted in subtle inflammation of the manipulated intestine at 24 h (late phase), but not after 2 and 6 h (early) after surgery. This local inflammation was associated with activation of neurons in the nucleus of the solitary tract and in the dorsal nucleus of the vagus. The vagal output mainly targeted the inflamed zone: 42% of motor neurons innervating the intestine expressed c-fos IR in contrast to 7% of those innervating the spleen. Vagal denervation of the intestine abolished c-fos expression in the brain nuclei involved in the neuronal network activated by intestinal inflammation. Conclusions & Inferences Our data demonstrate that intestinal inflammation triggers a vagally mediated circuit leading mainly to activation of vagal motor neurons connected to the inflamed intestine. These findings for the first time provide neuro-anatomical evidence for the existence of the endogenous ‘inflammatory reflex’ and its activation during inflammation.

Inflammation and other immune responses are involved in the variety of diseases and disorders. The acute response to endotoxemia includes activation of innate immune mechanisms as well as changes in autonomic nervous activity. The autonomic nervous system and the inflammatory response are intimately linked and sympathetic and vagal nerves are thought to have anti-inflammation functions. The basic functional circuit between vagus nerve and inflammatory response was identified and the neuroimmunomodulation loop was called cholinergic anti-inflammatory pathway. Unique function of vagus nerve in the anti-inflammatory reflex arc was found in many experimental and pre-clinical studies. They brought evidence on the cholinergic signaling interacting with systemic and local inflammation, particularly suppressing immune cells function. Pharmacolo-gical/electrical modulation of vagal activity suppressed TNF-α and other proinflammatory cytokines production and had beneficial therapeutic effects. Many questions related to mapping, linking and targeting of vagal-immune interactions have been elucidated and brought understanding of its basic physiology and provided the initial support for development of Tracey´s inflammatory reflex. This review summarizes and critically assesses the current knowledge defining cholinergic anti-inflammatory pathway with main focus on studies employing an experimental approach and emphasizes the potential of modulation of vagally-mediated anti-inflammatory pathway in the treatment strategies.

Thoracic traumas with extra-thoracic injuries result in an immediate, complex host response. The immune response requires tight regulation and can be influenced by additional risk factors such as obesity, which is considered a state of chronic inflammation. Utilizing high-dimensional mass and regular flow cytometry, we define key signatures of obesity-related alterations of the immune system during the response to the trauma. In this context, we report a modification in important components of the splenic response to the inflammatory reflex in obese mice. Furthermore, during the response to trauma, obese mice exhibit a prolonged increase of neutrophils and an early accumulation of inflammation associated CCR2+CD62L+Ly6Chimonocytes in the blood, contributing to a persistent inflammatory phase. Moreover, these mice exhibit differences in migration patterns of monocytes to the traumatized lung, resulting in decreased numbers of regenerative macrophages and an impaired M1/M2 switch in traumatized lungs. The findings presented in this study reveal an attenuation of the inflammatory reflex in obese mice, as well as a disturbance of the monocytic compartment contributing to a prolonged inflammation phase resulting in fewer phenotypically regenerative macrophages in the lung of obese mice.

Experimental evidence indicates that small concentrations of inflammatory molecules produced by damaged tissues activate afferent signals through ascending vagus nerve fibers, that act as the sensory arm of an “inflammatory reflex”. The subsequent activation of vagal efferent fibers, which represent the motor arm of the inflammatory reflex, rapidly leads to acetylcholine release in organs of the reticuloendothelial system. Acetylcholine interacts with α7 subunit-containing nicotinic receptors in tissue macrophages and other immune cells and rapidly inhibits the synthesis/release of tumor necrosis factor-α and other inflammatory cytokines. This neural anti-inflammatory response called “cholinergic anti-inflammatory pathway” is fast and integrated through the central nervous system. Preclinical studies are in progress, with the aim to develop therapeutic agents able to activate the cholinergic anti-inflammatory pathway. Melanocortin peptides bearing the adrenocorticotropin/α-melanocyte-stimulating hormone sequences exert a protective and life-saving effect in animals and humans in conditions of circulatory shock. These neuropeptides are likewise protective in other severe hypoxic conditions, such as prolonged respiratory arrest, myocardial ischemia, renal ischemia and ischemic stroke, as well as in experimental heart transplantation. Moreover, experimental evidence indicates that melanocortins reverse circulatory shock, prevent myocardial ischemia/reperfusion damage and exert neuroprotection against ischemic stroke through activation of the cholinergic anti-inflammatory pathway. This action occurs via stimulation of brain melanocortin MC3/MC4 receptors. Investigations that determine the molecular mechanisms of the cholinergic anti-inflammatory pathway activation could help design of superselective activators of this pathway.

Backgrounds Patients with rheumatoid arthritis (RA) have increased risk of sudden cardiac death (SCD), which is two-fold higher than general population. The driving cause of SCD was considered due to lift-threatening arrhythmia where systemic inflammation acts as the pathophysiological basis linking RA to autonomicdysfunction. Methods To assess the sympathetic over-activity of “inflammatory reflex”, we measured heart rate variability (HRV) in a rat collagen-induced arthritis (CIA) model, whose arthritis is induced in Lewis rats by intradermal injection of emulsion of type II collagen. Single-lead electrocardiogram (ECG) was recorded for 30 min every two days. Time and frequency-domain parameters, detrended fluctuation analysis (DFA), deceleration (DC) and acceleration capacity (AC) were analyzed. Results Compared with 9 control rats, many of HRV parameters of 9 CIA rats revealed significant different. At the beginning of arthritis, LF/HF was significant higher than controls (1st week: 2.41 ± 0.7 vs. 1.76 ± 0.6, p