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The overproduction of TNF and other cytokines can cause the pathophysiology of numerous diseases. Controlling cytokine synthesis and release is critical for preventing unrestrained inflammation and maintaining health. Recent studies identified an efferent vagus nerve-based mechanism termed “the cholinergic anti-inflammatory pathway” that controls cytokine production and inflammation. Here we review current advances related to the role of this pathway in neuro-immune interactions that prevent excessive inflammation. Experimental evidence indicates that vagus nerve cholinergic anti-inflammatory signaling requires alpha7 nicotinic acetylcholine receptors expressed on non-neuronal cytokine producing cells. Alpha7 nicotinic acetylcholine receptor agonists inhibit cytokine release and protect animals in a variety of experimental lethal inflammatory models. Knowledge related to the cholinergic anti-inflammatory pathway can be exploited in therapeutic approaches directed towards counteracting abnormal chronic and hyper-activated inflammatory responses.
Nervous system interaction with the immune system is vital for modulating innate immune responses and controlling inflammation (Tracey, 2002; Pavlov et al., 2003; Pavlov and Tracey, 2004; Czura and Tracey, 2005). Inflammation, a highly regulated response to infection and injury, has evolved as a beneficial component of the physiological defense systems of the host organism. Inflammation is critically mediated by tumor necrosis factor (TNF) and other pro- and anti-inflammatory cytokines, which are produced by activated macrophages and other innate immune cells (Tracey et al., 1986; Tracey, 2002). The production and release of cytokines is part of the advantageous response of the host innate immune system towards neutralizing the invading pathogen and promoting wound healing. These benefits are an asset to host survival; however, the innate immune mechanisms underlying inflammatory responses must be extremely well balanced in order to prevent the deleterious effects of over-production of TNF and other pro-inflammatory cytokines that can result in systemic inflammation and secondary tissue injury (Tracey, 2002). Abnormal systemic inflammation is a characteristic event associated with the pathology of rheumatoid arthritis, inflammatory bowel diseases, sepsis and other disorders (Tracey, 2002). Systemic inflammation can also be experimentally induced by administering lipopolysaccharide (LPS, endotoxin), an active major component of the outer membranes of Gram-negative bacteria and prototypical activator of innate immune responses. Recombinant human TNF (cachectin) elicits the same pathophysiological consequences that are caused by high dose endotoxin administration in rats including hypotension, metabolic acidosis, hemoconcentration and death occurring within hours after TNF administration (Tracey et al., 1986). Animals receiving neutralizing anti-TNF monoclonal antibody fragments prior to bacterial challenge are completely protected against shock, organ dysfunction, and death (Tracey et al., 1987). The discovery that administration of TNF, in doses similar to the levels produced by the host in response to endotoxin, evoked the symptoms caused by endotoxin administration was paramount. This scientific breakthrough identified TNF as a necessary and sufficient mediator of systemic inflammation. These findings indicated the possibility that anti-TNF strategies could be used in the treatment of life threatening diseases characterized by abnormally elevated TNF levels (Tracey et al., 1987). The overabundance of TNF and other pro-inflammatory cytokines may be lethal and conserved physiological mechanisms have evolved to counteract pro-inflammatory cytokine excess. Anti-inflammatory mechanisms include the release of glucocorticoids, anti-inflammatory cytokines such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β), and soluble receptors which neutralize the activity of cytokines. Recently, a neural efferent vagus nerve-mediated mechanism that can suppress the overproduction of TNF and other pro-inflammatory cytokines was described (Borovikova, et al. 2000). This pathway, termed the “cholinergic anti-inflammatory pathway” represents a physiological mechanism by which the nervous system interacts with the innate immune system to restrain systemic inflammatory responses.
The vagus nerve is the tenth cranial nerve and major constituent of the parasympathetic part of the autonomic nervous system. It contains sensory (afferent) and motor (efferent) fibers. The efferent vagus nerve arises in the brainstem medulla oblongata and innervates visceral organs. The vagus nerve is traditionally associated with the regulation of vital physiological functions including heart rate, bronchoconstriction, and gastrointestinal function through its principal neurotransmitter, acetylcholine as well as other neuronal and humoral substances (Pavlov and Tracey, 2004). Research in our laboratory and others has shown that the efferent vagus nerve inhibits pro-inflammatory cytokine production and systemic inflammation, thus identifying a non-classical immunoregulatory and anti-inflammatory function of the efferent vagus nerve (Borovikova et al., 2000; Wang et al., 2003; De Jonge et al., 2005; Huston et al., 2006; Ghia et al., 2006) (Fig.1). Accumulating evidence indicates that this regulation requires interaction between efferent vagus nerve signaling and nicotinic acetylcholine receptors expressed on macrophages and other non-neuronal cytokine producing cells that reside in organs of the reticuloendothelial system. These findings, together with the major neurotransmitter function of acetylcholine in pre- and post-ganglionic efferent vagal neurons as well as its anti-inflammatory role in vitro (Borovikova et al., 2000), have given rise to the concept of the efferent vagus nerve driven cholinergic anti-inflammatory pathway. Animals receiving direct electrical stimulation of the vagus nerve exhibit significantly reduced systemic levels of TNF and other pro-inflammatory cytokines during endotoxemia (Borovikova et al., 2000; Wang et al., 2003; Pavlov et al., 2006). Interestingly, vagus nerve stimulation does not change systemic anti-inflammatory cytokine levels (Borovikova et al., 2000). Pro-inflammatory cytokine levels are higher in animals subjected to vagotomy during endotoxemia (Borovikova et al., 2000) and intestinal inflammation (Ghia et al., 2006), which indicates a tonic inhibitory effect of the vagus nerve on pro-inflammatory cytokine production. Vagus nerve stimulation also attenuates the development of lethal shock in endotoxemic rats (Borovikova et al., 2000). Moreover, vagus nerve stimulation significantly inhibits serum, cardiac and hepatic TNF levels as well as attenuates the development of shock in animal models of ischemia reperfusion as a result of aortic occlusion (Bernik et al., 2000) and hypovolemic hemorrhagic shock (Guarini et al., 2003). The development of acute colitis in mice also is dependent on the anti-inflammatory function of the vagus nerve (Ghia et al., 2006). These findings have indicated the profound beneficial effect of the efferent vagus nerve in several experimental models of diseases as shown in Table 1.
Current knowledge indicates that the vagus nerve provides an important bi-directional communication circuit by which the brain modulates inflammation (Tracey 2002; Pavlov et al., 2003). The presence of inflammation can be detected by the sensory (afferent) vagus nerve and communicated to the nucleus tractus solitarus in the brainstem medulla oblongata. Neural communication between this, other brainstem nuclei and “higher” brain structures including the hypothalamus are associated with the generation of brain-derived anti-inflammatory output through the efferent vagus nerve-mediated cholinergic anti-inflammatory pathway (Tracey 2002; Pavlov et al., 2003; Pavlov and Tracey, 2005). Knowledge related to this vagus nerve dominated “inflammatory reflex” and the mechanisms of its regulation contribute to a more detailed understanding of the neuro-immune interactions which regulate innate immune responses and inflammation. A recent study demonstrates this reflex through a dietary fat-induced vago-vagal mechanism that controls inflammation (Luyer et al., 2005). Dietary fat intake causes the release of cholecystokinin (CCK), activation of CCK receptors and consequent stimulation of afferent and efferent vagus nerve activity which results in the inhibition of pro-inflammatory cytokines in rats during hemorrhagic shock (Luyer et al., 2005). This study also sheds new light on the role that nutrition may have in the cholinergic anti-inflammatory pathway (Luyer et al., 2005). In addition to the vago-vagal neural inflammatory reflex, humoral pathways also are stimulated, and activation of the hypothalamo-pituitary-adrenal axis results in the release of glucocorticoids producing an anti-inflammatory signal. Activation of the sympathetic division of the autonomic nervous system may also be induced during inflammation resulting in the release of epinephrine (adrenaline) and norepinephrine (noradrenaline), which are involved in the complex receptor-dependent regulation of inflammatory responses (Pavlov and Tracey, 2004). Thus, the host organism mobilizes neural and neurohumoral mechanisms to control inflammation during an immune challenge.
Research in our laboratory utilizing antisense and knockout approaches identified the critical importance of the α7 nicotinic acetylcholine receptor (α7nAChR) in mediating cholinergic anti-inflammatory signaling (Wang et al., 2003). Experiments with α7nAChR knockout mice revealed that in the absence of the α7nAChR, vagus nerve stimulation was ineffective at preventing TNF release indicating that the α7nAChR is essential for the effectiveness of the cholinergic anti-inflammatory pathway (Wang et al., 2003). Recently, a functional connection between the vagus nerve anti-inflammatory activity and the spleen was identified (Huston et al., 2006). The spleen was shown to be a major producer of TNF during endotoxemia, and vagus nerve stimulation significantly suppressed splenic TNF through an α7nAChR mediated mechanism (Huston et al., 2006). Further studies are needed to elucidate the mechanisms involved and the importance of this regulation under different inflammatory conditions.
The α7nAChR is expressed on non-neuronal cells including macrophages, endothelial cells, dendritic cells, keratinocytes and lymphocytes (Grando et al., 2003; Saeed et al., 2005; Kawashima et al., in press). In addition, these cells express other markers of the cholinergic system, including acetylcholine release, a variety of other nicotinic and muscarinic acetylcholine receptors, and the enzymes choline acetyltransferase and acetylcholinesterase, thus forming a non-neuronal cholinergic system (Grando et al., 2003, Kawashima et al., in press). While macrophages have been identified as the major source of TNF during endotoxemia, dendritic cells, endothelial cells and lymphocytes also synthesize and release pro-inflammatory cytokines and are substantial contributors to the innate immune activation underlying inflammatory responses. Macrophages also have a prominent role in mediating intestinal inflammation, and a recent study has identified these cells as the main target of the anti-inflammatory function of the vagus nerve in a murine model of inflammatory bowel disease (Ghia et al., 2006).
Although muscarinic acetylcholine receptors are expressed on macrophages and other cytokine producing cells they do not seem to play a critical role in transmitting vagal anti-inflammatory output during endotoxemia (Pavlov et al., 2006). Administration of atropine methyl nitrate, which blocks peripheral muscarinic receptors including those expressed on cytokine-producing cells, does not interrupt the TNF suppressing effect of vagus nerve stimulation in endotoxemic rats (Pavlov et al., 2006). These findings highlight a major difference between classical vagal cholinergic regulation of physiological functions such as heart rate, etc. that are predominantly mediated by muscarinic receptors and the immunomodulatory function of the vagus nerve, mediated by the α7nAchR. Moreover, we have recently demonstrated a role for muscarinic receptors in the central nervous system (CNS) in inhibiting systemic inflammation in endotoxemic rats (Pavlov et al., 2006). We have shown that activation of muscarinic cholinergic transmission in the CNS by muscarinic receptor ligands lowers serum TNF levels (Pavlov et al., 2006). We have also shown that central muscarinic cholinergic activation results in higher efferent vagus nerve activity (as demonstrated by an increase in the high frequency power component of heart rate variability), thus indicating a role for the efferent vagus nerve in conveying the central cholinergic signal to the periphery, which leads to inhibition of systemic TNF levels. Our study, together with observations that muscarinic receptors in the CNS are involved in controlling the vagus nerve anti-inflammatory function during hemorrhagic shock in rats (Guarini et al., 2004), indicates a role for central muscarinic receptor mechanisms in controlling the cholinergic anti-inflammatory pathway in rats. The central (brain) neuronal circuits underlying this regulation remain enigmatic.
The discovery of the critical role for the α7nAChR in mediating cholinergic anti-inflammatory signaling led to the utilization of nicotine and other α7nAChR agonists in mechanistic studies. Nicotine has been shown to inhibit nuclear factor kappa B (NF-κB) translocation to the nucleus in endotoxin-stimulated RAW 264.7 macrophages (Wang et al., 2004) (Fig.1). NF-κB is a key transcription factor for the synthesis of TNF and other cytokines and preventing its nuclear translocation in response to endotoxin and other immunogenic stimuli is critical for decreasing pro-inflammatory cytokine production. Vagus nerve stimulation causes nicotinic receptor-dependent suppression of hepatic NF-κB activation during hemorrhagic shock (Guarini et al., 2003). This data combined with the finding of lower hepatic TNF mRNA and serum protein levels as a result of vagus nerve stimulation provide additional evidence that cholinergic anti-inflammatory signaling is associated with suppression of NF-κB activation in vivo (Guarini et al., 2003). Nicotine has also been demonstrated to inhibit resident peritoneal macrophage activation ex vivo, attenuating pro-inflammatory cytokine release through α7nAChR - mediated activation of the janus kinase (JAK)/ signal transducer and activator of transcription (STAT) pathway (De Jonge et al., 2005). Binding of nicotine to the α7nAChR triggers activation (phosphorylation) of JAK2 and subsequent phosphorylation of effectors such as STAT3. Phosphorylated STAT3 translocates to the nucleus and is correlated downstream with decreased levels of TNF, macrophage inflammatory protein 2 (MIP-2), and interleukin 6 (IL-6) (De Jonge et al., 2005; Gallowitsch-Puerta and Tracey, 2005) (Fig.1). The importance of cholinergic anti-inflammatory signaling through the JAK/STAT pathway also is supported by the finding that the anti-inflammatory function of vagus nerve stimulation is abolished in STAT-3 deficient mice (De Jonge et al., 2005). The involvement of other alternative intracellular pathways in mediating cholinergic anti-inflammatory signaling remains to be evaluated; although some of these mechanisms such as calcium signaling have been extensively studied in neurons, their mediating role in macrophages and other non-excitable cells is still enigmatic.
In addition to inhibiting TNF and other “early” pro-inflammatory cytokines, α7nAChR-dependent cholinergic signaling also is implicated in suppressing the release of high mobility group box 1 (HMGB1), a “late” cytokine mediator of systemic inflammation (Wang et al., 2004). Studies have shown that HMGB1 plays important functions in mediating the pathology of experimental severe sepsis and other inflammatory disorders (Wang et al., 1999; Tracey, 2005). Analysis of patients with sepsis and cerebral and myocardial ischemia has shown elevated systemic levels of HMGB1 (Wang et al, 1999; Goldstein et al., 2006), indicating the potential for this protein as a therapeutic target. In fact anti-HMGB1 antibodies have been shown to improve survival in experimental models of severe sepsis (Tracey, 2005). Treatment with nicotine results in lower systemic levels of HMGB1 in endotoxemic and septic mice and significantly increases survival rates when compared with vehicle administered controls (Wang et al., 2004).
In addition to the vagus nerve, the anti-inflammatory function of nicotine has been explored in various experimental models of diseases as summarized in Table 1. Future studies will contribute to validating the efficacy of these approaches in the clinical management of inflammatory diseases.
The cholinergic anti-inflammatory pathway is a physiological neuro-immune mechanism that regulates innate immune function and controls inflammation. Current knowledge indicates that the functional activity of this pathway can be modulated through its neuronal (efferent vagal neurons and higher brain structures) and non-neuronal (α7nAChR on cytokine-producing cells) cholinergic components. Future studies on the neuro-immune interactions through the cholinergic anti-inflammatory pathway in rodents and humans will contribute to unraveling the immunoregulatory mechanisms and therapeutic potential of this pathway.
This work was supported by the Feinstein Institute for Medical Research Reward Program, North Shore-Long Island Jewish GCRC (General Clinical Research Center), MO1 RR018535, and the NIGMS (National Institute of General Medical Sciences). We would like to thank Kevin J. Tracey and William R. Parrish for critically reading this manuscript. We apologize to those authors whose work could not be cited because of the format (reference limitations) of this article.
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