Splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis.

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 alpha7 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 alpha7 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 alpha7 agonists to inhibit proinflammatory cytokine production during infection and tissue injury.

Splenectomy protects against sepsis lethality and reduces serum HMGB1 levels.

High mobility group box 1 (HMGB1) is a critical mediator of lethal sepsis. Previously, we showed that apoptotic cells can activate macrophages to release HMGB1. During sepsis, apoptosis occurs primarily in lymphoid organs, including the spleen and thymus. Currently, it is unclear whether this accelerated lymphoid organ apoptosis contributes to systemic release of HMGB1 in sepsis. In this study, we report that splenectomy significantly reduces systemic HMGB1 release and improves survival in mice with polymicrobial sepsis. Treatment with a broad-spectrum caspase inhibitor reduces systemic lymphocyte apoptosis, suppresses circulating HMGB1 concentrations, and improves survival during polymicrobial sepsis, but fails to protect septic mice following splenectomy. These findings indicate that apoptosis in the spleen is essential to the pathogenesis of HMGB1-mediated sepsis lethality.

The selective alpha7 agonist GTS-21 attenuates cytokine production in human whole blood and human monocytes activated by ligands for TLR2, TLR3, TLR4, TLR9, and RAGE.

The cholinergic antiinflammatory pathway modulates inflammatory cytokine production through a mechanism dependent on the vagus nerve and the alpha7 subunit of the nicotinic acetylcholine receptor. GTS-21 [3-(2,4-dimethoxybenzylidene) anabaseine], a selective alpha7 agonist, inhibits inflammatory cytokine production in murine and human macrophages and in several models of inflammatory disease in vivo, but to date its antiinflammatory efficacy in human monocytes has not been characterized. We report here our findings that GTS-21 attenuates tumor necrosis factor (TNF) and interleukin 1beta levels in human whole blood activated by exposure to endotoxin. GTS-21 inhibited TNF production in endotoxin-stimulated primary human monocytes in vitro at the transcriptional level. The suppressive effect of GTS-21 was more potent than nicotine in whole blood and monocytes. Furthermore, GTS-21 attenuated TNF production in monocytes stimulated with peptidoglycan, polyinosinic-polycytidylic acid, CpG, HMGB1 (high-mobility group box 1 protein), and advanced glycation end product-modified albumin. GTS-21 decreased TNF levels in endotoxin-stimulated whole blood obtained from patients with severe sepsis. These findings establish the immunoregulatory effect of GTS-21 on human monocytes, and indicate the potential benefits of further exploration of GTS-21's therapeutic uses in human inflammatory disease.

Modulation of TNF release by choline requires alpha7 subunit nicotinic acetylcholine receptor-mediated signaling.

The alpha7 subunit-containing nicotinic acetylcholine receptor (alpha7nAChR) is an essential component in the vagus nerve-based cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines during inflammation. Choline is an essential nutrient, a cell membrane constituent, a precursor in the biosynthesis of acetylcholine, and a selective natural alpha7nAChR agonist. Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and sepsis, and the role of the alpha7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1-50 mM) dose-dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with significant inhibition of NF-kappaB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.) failed to inhibit systemic TNF levels in alpha7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release from endotoxin-activated peritoneal macrophages isolated from alpha7nAChR knockout mice. Choline treatment prior to endotoxin resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed HMGB1 release in vitro and in vivo, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human whole blood and macrophages. Collectively, these data characterize the anti-inflammatory efficacy of choline and demonstrate that the modulation of TNF release by choline requires alpha7nAChR-mediated signaling.

Neuro-immune interactions via the cholinergic anti-inflammatory pathway.

The overproduction of TNF and other cytokines is associated with 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.

Elevated high-mobility group box 1 levels in patients with cerebral and myocardial ischemia.

Cerebral and myocardial ischemia, two of the leading causes of morbidity and mortality worldwide, are associated with inflammation that can lead to multiple organ failure and death. High-mobility group box 1(HMGB1), a recently described mediator of lethal systemic inflammation, has been detected in individuals with severe sepsis and hemorrhagic shock, but its role during ischemic injury in humans is unknown. To determine whether systemic HMGB1 levels are elevated after ischemic injury, a prospective observational study was performed in subjects with a diagnosis of either Acute Coronary Syndrome (ACS) or cerebral vascular ischemia (transient ischemic attack or cerebral vascular accident). Subjects (n, 16; age [mean], 67+/-16.3 years) were enrolled in the North Shore-LIJ emergency department within 24 h of symptom onset. Blood samples were collected, and HMGB1 levels analyzed by Western blot analysis using previously described methods (Wang et al. Science. 1999). Control samples were obtained from healthy age- and sex-matched volunteers (n, 16; age [mean], 68+/-15.8 years). Here, we report that serum HMGB1 levels were significantly elevated in both myocardial ischemia subjects (myocardial control serum HMGB1, 1.94+/-2.05 ng/mL, vs. myocardial ischemia serum HMGB1, 159+/-54.3 ng/mL; P<0.001); and in cerebral ischemia subjects (cerebral control serum HMGB1, 16.8+/-10.9 ng/mL, vs. cerebral ischemia serum HMGB1, 218+/-18.8 ng/mL; P<0.001). These results suggest that systemic HMGB1 levels are elevated in human ischemic disease.

The effects of CpG DNA on HMGB1 release by murine macrophage cell lines.

DNA containing cytosine-guanine dinucleotide (CpG) motifs (CpG DNA) has potent immunostimulatory activities that resemble those of lipopolysaccharide (LPS) in its effects on the innate immune system. Among its activities, LPS can induce the release of high mobility group protein (HMGB1) by macrophages, a dual function molecule that can mediate the late effects of LPS. To determine whether CpG DNA can also induce HMGB1 release, the effects of a synthetic CpG oligonucleotide (ODN) on HMGB1 release from RAW 264.7 and J774A.1 cells were assessed by Western blotting of culture supernatants. Under conditions in which the CpG ODN activated the cell lines, as assessed by stimulation of tumor necrosis factor alpha and interleukin-12, it failed to cause HMGB1 release into the media. Although unable to induce HMGB1 release by itself, the CpG ODN nevertheless potentiated the action of LPS. With RAW 264.7 cells, lipoteichoic acid and polyinosinic-polycytidylic acid, like LPS, stimulated HMGB1 release as well as cytokine production. These results indicate that the effects of CpG DNA on macrophages differ from other ligands of Toll-like receptors and may lead to a distinct pattern of immune cell activation in the context of infection or its use as an immunomodulatory agent.

Immunologic role of the cholinergic anti-inflammatory pathway and the nicotinic acetylcholine alpha 7 receptor.

Cytokines are small protein molecules that facilitate communication between cells of the immune system and other tissues. Their messenger function elicits responses in other cells, ranging from DNA binding and tissue remodeling to coordinating the local cellular response to inflammation. The magnitude of the cytokine response is regulated closely, because an over- or underabundance of cytokine activity can impair organ function and cause shock and tissue injury. Counterregulatory molecular and humoral mechanisms protect the host from cytokine excess, including the pituitary-adrenal-glucocorticoid system, and the anti-inflammatory cytokine system. These humoral systems are protective, but they are relatively slow-acting, concentration-gradient dependent, and not integrated. We recently discovered that the "cholinergic anti-inflammatory pathway," a nervous system-based, rapid, and locally acting mechanism, can inhibit the cytokine response. Signals transmitted via the vagus nerve converge on cytokine-producing cells that express the nicotinic acetylcholine receptor alpha 7 (nAChR alpha7). The alpha7 receptor is an essential component of the cholinergic anti-inflammatory pathway, because activation of this receptor prevents cytokine release. Advances in understanding the molecular structure and function of alpha7 have begun to provide a better understanding of potential mechanisms regarding its assembly, expression, and cytokine-inhibiting functions. It may be possible to exploit this pathway to therapeutic advantage for diseases caused by excessive cytokine activity.

Experimental therapeutic strategies for severe sepsis: mediators and mechanisms.

Severe sepsis is the leading cause of mortality in intensive care units. The limited ability of current therapies to reduce sepsis mortality rates has fueled research efforts for the development of novel treatment strategies. Through the close collaboration between clinicians and scientists, progress can be seen in the struggle to develop effective therapeutic approaches for the treatment of sepsis and other immune and inflammatory disorders. Indeed, significant advances in intensive care, such as lung protective mechanical ventilation, improved antibiotics, and superior monitoring of systemic perfusion, are improving patient survival. Nonetheless, specific strategies that target the pathophysiological disorders in sepsis patients are essential to further improve clinical outcomes. This article reviews current clinical management approaches and experimental interventions that target pleiotropic or late-acting inflammatory mediators like caspases, C5a, MIF, and HMGB1, or the body's endogenous inflammatory control mechanisms such as the cholinergic anti-inflammatory pathway. These inflammatory mediators and anti-inflammatory mechanisms, respectively, show significant potential for the development of new experimental therapies for the treatment of severe sepsis and other infectious and inflammatory disorders.

Cholinergic anti-inflammatory pathway activity and High Mobility Group Box-1 (HMGB1) serum levels in patients with rheumatoid arthritis.

High Mobility Group Box-1 (HMGB1) is a cytokine implicated in the pathogenesis of rheumatoid arthritis (RA) and other inflammatory diseases. The cholinergic anti-inflammatory pathway, a vagus nerve-dependent mechanism, inhibits HMGB1 release in experimental disease models. Here, we examine the relationship between vagus nerve activity and HMGB1 in patients with RA. We compared RR interval variability, an index of cardiac vagal modulation, HMGB1 and hsCRP serum levels, and disease activity scores in thirteen RA patients and eleven age- and sex-matched controls. In RA patients, serum levels of HMGB1 and hsCRP were elevated as compared with controls (HMGB1=71 ng/mL [45-99] vs. 18 ng/mL [0-40], P<0.0001; hsCRP=14.5 mg/L [0.7-59] vs. 1 mg/L [0.4-2.9], P<0.001). RR interval variability in RA patients was significantly decreased as compared with controls (HF=38 msec2 [14-80] vs. 288 msec2 [38-364], P<0.0001; rMSSD=20.9+/-9.79 msec, 52.6+/-35.3 msec, P<0.01). HMGB1 levels and RR interval variability were significantly related (rho=-0.49, P<0.01). HMGB1 serum levels significantly correlated with disease activity scores (DAS-28) in patients with RA (P=0.004). The study design does not enable a determination of causality, but the results are consistent with the hypothesis that decreased cholinergic anti-inflammatory pathway activity is associated with increased HMGB1 levels in patients with RA.

Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis.

OBJECTIVE: Tumor necrosis factor and high mobility group box 1 are critical cytokine mediators of inflammation. The efferent vagus nerve inhibits cytokine release through alpha7-nicotinic acetylcholine receptor-mediated cholinergic signaling. Here we studied whether GTS-21, a selective alpha7-nicotinic acetylcholine receptor agonist, inhibits proinflammatory cytokines in vitro and in vivo and improves survival in murine endotoxemia and severe sepsis. DESIGN: Randomized and controlled in vitro and in vivo study. SETTINGS: Research laboratory and animal facility rooms. SUBJECTS: RAW 264.7 cells and BALB/c mice treated with endotoxin or subjected to cecal ligation and puncture (CLP). INTERVENTIONS: RAW 264.7 cells were exposed to endotoxin (4 ng/mL or 10 ng/mL) in the presence or absence of GTS-21 (1-100 muM), and tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation were analyzed. Mice were treated with GTS-21 (0.4 mg/kg or 4 mg/kg, intraperitoneally) or saline 30 mins before endotoxin (6 mg/kg, intraperitoneally), and serum tumor necrosis factor was analyzed 1.5 hrs after the onset of endotoxemia. In survival experiments, mice were treated with GTS-21 (0.4 or 4.0 mg/kg, intraperitoneally) or saline 30 mins before and 6 hrs after endotoxin and then twice daily for 3 days. Severe sepsis was induced by CLP. Mice were treated with GTS-21 (4 mg/kg) or saline immediately and 6 hrs and 24 hrs after CLP, and serum high mobility group box 1 was analyzed 30 hrs after CLP. In survival experiments, GTS-21 (0.4 or 4 mg/kg) treatment was initiated 24 hrs after CLP and continued twice daily for 3 days. MEASUREMENTS AND MAIN RESULTS: GTS-21 dose-dependently inhibited tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation in vitro. GTS-21 (4 mg/kg) significantly inhibited serum tumor necrosis factor during endotoxemia and improved survival (p < .0001). GTS-21 (4 mg/kg) significantly inhibited serum high mobility group box 1 levels in CLP mice and improved survival (p < .0006). CONCLUSION: These findings are of interest for the development of alpha7-nicotinic acetylcholine receptor agonists as a new class of anti-inflammatory therapeutics.

Transcutaneous vagus nerve stimulation reduces serum high mobility group box 1 levels and improves survival in murine sepsis.

OBJECTIVE: Electrical vagus nerve stimulation inhibits proinflammatory cytokine production and prevents shock during lethal systemic inflammation through an alpha7 nicotinic acetylcholine receptor (alpha7nAChR)-dependent pathway to the spleen, termed the cholinergic anti-inflammatory pathway. Pharmacologic alpha7nAChR 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.

Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia.

TNF has a critical mediator role in inflammation and is an important therapeutic target. We recently discovered that TNF production is regulated by neural signals through the vagus nerve. Activation of this "cholinergic antiinflammatory pathway" inhibits the production of TNF and other cytokines and protects animals from the inflammatory damage caused by endotoxemia and severe sepsis. Here, we describe a role for central muscarinic acetylcholine receptors in the activation of the cholinergic antiinflammatory pathway. Central muscarinic cholinergic activation by muscarine, the M1 receptor agonist McN-A-343, and the M2 receptor antagonist methoctramine inhibited serum TNF levels significantly during endotoxemia. Centrally administered methoctramine stimulated vagus-nerve activity measured by changes in instantaneous heart-rate variability. Blockade of peripheral muscarinic receptors did not abolish antiinflammatory signaling through the vagus nerve, indicating that peripheral muscarinic receptors on immune cells are not required for the cytokine-regulating activities of the cholinergic antiinflammatory pathway. The role of central muscarinic receptors in activating the cholinergic antiinflammatory pathway is of interest for the use of centrally acting muscarinic cholinergic enhancers as antiinflammatory agents.

Anti-HMGB1 neutralizing antibody ameliorates gut barrier dysfunction and improves survival after hemorrhagic shock.

Intestinal barrier dysfunction occurs following hemorrhagic shock and resuscitation (HS/R). High-mobility group B1 (HMGB1) has been shown to increase the permeability of Caco-2 human enterocyte-like epithelial monolayers in vitro. In this study, we found that serum concentrations of HMGB1 were higher in blood samples obtained from 25 trauma victims with hemorrhagic shock than in 9 normal volunteers. We also studied whether treatment with anti-HMGB1 antibody can ameliorate HS/R-induced gut barrier dysfunction in mice. Animals were shocked by withdrawal of blood to maintain mean arterial pressure at 25 to 30 mmHg for 2 h. After resuscitation with shed blood plus Ringer's lactate solution, the mice were treated with either anti-HMGB1 antibody or nonimmune rabbit IgG. Serum HMGB1 concentrations were significantly higher in trauma victims than control mice. Treatment with anti-HMGB1 antibody improved survival at 24 h and ameliorated the development of ileal mucosal hyperpermeability to FITC-labeled dextran. At 24 h after HS/R, treatment with anti-HMGB1 antibody decreased bacterial translocation to mesenteric lymph nodes and was associated with lower circulating concentrations of IL-6 and IL-10. These data support the notion that HMGB1 is a mediator of HS/R-induced gut barrier dysfunction and suggest that anti-HMGB1 antibodies warrant further evaluation as a therapeutic to ameliorate the morbidity of HS/R in trauma patients.