A novel flexible cuff-like microelectrode for dual purpose, acute and chronic electrical interfacing with the mouse cervical vagus nerve.

OBJECTIVE: Neural reflexes regulate immune responses and homeostasis. Advances in bioelectronic medicine indicate that electrical stimulation of the vagus nerve can be used to treat inflammatory disease, yet the understanding of neural signals that regulate inflammation is incomplete. Current interfaces with the vagus nerve do not permit effective chronic stimulation or recording in mouse models, which is vital to studying the molecular and neurophysiological mechanisms that control inflammation homeostasis in health and disease. We developed an implantable, dual purpose, multi-channel, flexible 'microelectrode' array, for recording and stimulation of the mouse vagus nerve. APPROACH: The array was microfabricated on an 8 µm layer of highly biocompatible parylene configured with 16 sites. The microelectrode was evaluated by studying the recording and stimulation performance. Mice were chronically implanted with devices for up to 12 weeks. MAIN RESULTS: Using the microelectrode in vivo, high fidelity signals were recorded during physiological challenges (e.g potassium chloride and interleukin-1ß), and electrical stimulation of the vagus nerve produced the expected significant reduction of blood levels of tumor necrosis factor (TNF) in endotoxemia. Inflammatory cell infiltration at the microelectrode 12 weeks of implantation was limited according to radial distribution analysis of inflammatory cells. SIGNIFICANCE: This novel device provides an important step towards a viable chronic interface for cervical vagus nerve stimulation and recording in mice.

The haptoglobin beta subunit sequesters HMGB1 toxicity in sterile and infectious inflammation.

BACKGROUND: Extra-corpuscular haemoglobin is an endogenous factor enhancing inflammatory tissue damage, a process counteracted by the haemoglobin-binding plasma protein haptoglobin composed of alpha and beta subunits connected by disulfide bridges. Recent studies established that haptoglobin also binds and sequesters another pro-inflammatory mediator, HMGB1, via triggering CD163 receptor-mediated anti-inflammatory responses involving heme oxygenase-1 expression and IL-10 release. The molecular mechanism underlying haptoglobin-HMGB1 interaction remains poorly elucidated. METHODS: Haptoglobin ß subunits were tested for HMGB1-binding properties, as well as efficacy in animal models of sterile liver injury (induced by intraperitoneal acetaminophen administration) or infectious peritonitis (induced by cecal ligation and puncture, CLP, surgery) using wild-type (C57BL/6) or haptoglobin gene-deficient mice. RESULTS: Structural-functional analysis demonstrated that the haptoglobin ß subunit recapitulates the HMGB1-binding properties of full-length haptoglobin. Similar to HMGB1-haptoglobin complexes, the HMGB1-haptoglobin ß complexes also elicited anti-inflammatory effects via CD163-mediated IL-10 release and heme oxygenase-1 expression. Treatment with haptoglobin ß protein conferred significant protection in mouse models of polymicrobial sepsis as well as acetaminophen-induced liver injury, two HMGB1-dependent inflammatory conditions. CONCLUSIONS: Haptoglobin ß protein offers a novel therapeutic approach to fight against various inflammatory diseases caused by excessive HMGB1 release.

Novel strategies for targeting innate immune responses to influenza.

We previously reported that TLR4(-/-) mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist Eritoran blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post infection. Herein we extend these findings: anti-TLR4- or -TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h before PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4(-/-) mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks high-mobility group B1 (HMGB1)-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI; (iii) interleukin (IL)-1ß contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the antiviral drug oseltamivir were administered starting 4 days post infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.

Central cholinergic activation of a vagus nerve-to-spleen circuit alleviates experimental colitis.

The cholinergic anti-inflammatory pathway is an efferent vagus nerve-based mechanism that regulates immune responses and cytokine production through α7 nicotinic acetylcholine receptor (α7nAChR) signaling. Decreased efferent vagus nerve activity is observed in inflammatory bowel disease. We determined whether central activation of this pathway alters inflammation in mice with colitis and the mediating role of a vagus nerve-to-spleen circuit and α7nAChR signaling. Two experimental models of colitis were used in C57BL/6 mice. Central cholinergic activation induced by the acetylcholinesterase inhibitor galantamine or a muscarinic acetylcholine receptor agonist treatments resulted in reduced mucosal inflammation associated with decreased major histocompatibility complex II level and pro-inflammatory cytokine secretion by splenic CD11c⁺ cells mediated by α7nAChR signaling. The cholinergic anti-inflammatory efficacy was abolished in mice with vagotomy, splenic neurectomy, or splenectomy. In conclusion, central cholinergic activation of a vagus nerve-to-spleen circuit controls intestinal inflammation and this regulation can be explored to develop novel therapeutic strategies.

HMGB1 mediates splenomegaly and expansion of splenic CD11b+ Ly-6C(high) inflammatory monocytes in murine sepsis survivors.

BACKGROUND: More than 500,000 hospitalized patients survive severe sepsis annually in the USA. Recent epidemiological evidence, however, demonstrated that these survivors have significant morbidity and mortality, with 3-year fatality rates higher than 70%. To investigate the mechanisms underlying persistent functional impairment in sepsis survivors, here we developed a model to study severe sepsis survivors following cecal ligation and puncture (CLP). METHODS: Sepsis was induced in mice by CLP and survivors were followed for twelve weeks. Spleen and blood were collected and analyzed at different time points post-sepsis. RESULTS: We observed that sepsis survivors developed significant splenomegaly. Analysis of the splenic cellular compartments revealed a major expansion of the inflammatory CD11b+ Ly-6CHigh pool. Serum high-mobility group box 1 (HMGB1) levels in the sepsis surviving mice were significantly elevated for 4-6 weeks after post-sepsis, and administration of an anti-HMGB1 monoclonal antibody significantly attenuated splenomegaly as well as splenocyte priming. Administration of recombinant HMGB1 to naive mice induced similar splenomegaly, leukocytosis and splenocyte priming as observed in sepsis survivors. Interestingly analysis of circulating HMGB1 from sepsis survivors by mass spectroscopy demonstrated a stepwise increase of reduced form of HMGB1 (with known chemo-attractant properties) during the first 3 weeks, followed by disulphide form (with known inflammatory properties) 4-8 weeks after CLP. DISCUSSION: Our results indicate that prolonged elevation of HMGB1 is a necessary and sufficient mediator of splenomegaly and splenocyte expansion, as well as splenocyte inflammatory priming in murine severe sepsis survivors.

The pulse of inflammation: heart rate variability, the cholinergic anti-inflammatory pathway and implications for therapy.

Biological therapeutics targeting TNF, IL-1 and IL-6 are widely used for treatment of rheumatoid arthritis, inflammatory bowel disease and a growing list of other syndromes, often with remarkable success. Now advances in neuroscience have collided with this therapeutic approach, perhaps rendering possible the development of nerve stimulators to inhibit cytokines. Action potentials transmitted in the vagus nerve culminate in the release of acetylcholine that blocks cytokine production by cells expressing acetylcholine receptors. The molecular mechanism of this cholinergic anti-inflammatory pathway is attributable to signal transduction by the nicotinic alpha 7 acetylcholine receptor subunit, a regulator of the intracellular signals that control cytokine transcription and translation. Favourable preclinical data support the possibility that nerve stimulators may be added to the future therapeutic armamentarium, possibly replacing some drugs to inhibit cytokines.

HMGB1 release and redox regulates autophagy and apoptosis in cancer cells.

The functional relationship and cross-regulation between autophagy and apoptosis is complex. In this study we show that the high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anticancer agents enhanced autophagy and apoptosis, as well as HMGB1 release. HMGB1 release may be a prosurvival signal for residual cells after various cytotoxic cancer treatments. Diminished HMGB1 by short hairpin RNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led predominantly to apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGEs), but not to Toll-like receptor 4, induces Beclin1-dependent autophagy and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA intercalators (oxaliplatin or adriamycin). On the contrary, oxidized HMGB1 increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release, as well as its redox state, thus links autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.

Whole blood cytokine attenuation by cholinergic agonists ex vivo and relationship to vagus nerve activity in rheumatoid arthritis.

OBJECTIVE: The central nervous system regulates innate immunity in part via the cholinergic anti-inflammatory pathway, a neural circuit that transmits signals in the vagus nerve that suppress pro-inflammatory cytokine production by an alpha-7 nicotinic acetylcholine receptors (alpha7nAChR) dependent mechanism. Vagus nerve activity is significantly suppressed in patients with autoimmune diseases, including rheumatoid arthritis (RA). It has been suggested that stimulating the cholinergic anti-inflammatory pathway may be beneficial to patients, but it remains theoretically possible that chronic deficiencies in this pathway will render these approaches ineffective. METHODS: Here we addressed the hypothesis that inflammatory cells from RA patients can respond to cholinergic agonists with reduced cytokine production in the setting of reduced vagus nerve activity. RESULTS: Measurement of RR interval variability (heart rate variability, HRV), in RA patients (n = 13) and healthy controls (n = 10) revealed that vagus nerve activity was significantly depressed in patients. Whole blood cultures stimulated by exposure to endotoxin produced significantly less tumour necrosis factor in samples from RA patients as compared to healthy controls. Addition of cholinergic agonists (nicotine and GTS-21) to the stimulated whole blood cultures however significantly suppressed cytokine production to a similar extent in patients and healthy controls. CONCLUSION: These findings suggest that it is possible to pharmacologically target the alpha7nAChR dependent control of cytokine release in RA patients with suppressed vagus nerve activity. As alpha7nAChR agonists ameliorate the clinical course of collagen induced arthritis in animals, it may be possible in the future to explore whether alpha7nAChR agonists can improve clinical activity in RA patients.

Cholinergic control of inflammation.

Cytokine production is necessary to protect against pathogens and promote tissue repair, but excessive cytokine release can lead to systemic inflammation, organ failure and death. Inflammatory responses are finely regulated to effectively guard from noxious stimuli. The central nervous system interacts dynamically with the immune system to modulate inflammation through humoral and neural pathways. The effect of glucocorticoids and other humoral mediators on inflammatory responses has been studied extensively in the past decades. In contrast, neural control of inflammation has only been recently described. We summarize autonomic regulation of local and systemic inflammation through the 'cholinergic anti-inflammatory pathway', a mechanism consisting of the vagus nerve and its major neurotransmitter, acetylcholine, a process dependent on the nicotinic acetylcholine receptor alpha7 subunit. We recapitulate additional sources of acetylcholine and their contribution to the inflammatory response, as well as acetylcholine regulation by acetylcholinesterase as a means to attenuate inflammation. We discuss potential therapeutic applications to treat diseases characterized by acute or chronic inflammation, including autoimmune diseases, and propose future research directions.

Cholinergic agonists attenuate renal ischemia-reperfusion injury in rats.

Inflammation plays a significant role in the pathophysiology of renal ischemia-reperfusion injury. Local inflammation is modulated by the brain via the vagus nerve and nicotinic acetylcholine receptors such that electrical or pharmacologic stimulation of this cholinergic anti-inflammatory pathway results in suppression of proinflammatory cytokine production. We examined the effects of cholinergic stimulation using agonists, nicotine or GTS-21, given before or after bilateral renal ischemia-reperfusion injury in rats. Pretreatment of rats with either agonist significantly attenuated renal dysfunction and tubular necrosis induced by renal ischemia. Similarly, tumor necrosis factor-alpha protein expression and leukocyte infiltration of the kidney were markedly reduced following treatment with cholinergic agonists. We found functional nicotinic acetylcholine receptors were present on rat proximal tubule epithelial cells. Cholinergic stimulation significantly decreased tubular necrosis in vagotomized rats after injury, implying an intact vagus nerve is not required for this renoprotective effect.

Controlling inflammation: the cholinergic anti-inflammatory pathway.

Innate immune responses and inflammation are regulated in part by neural mechanisms. In the present paper, we summarize experimental evidence that reveals that innate immunity and inflammation are controlled by the vagus nerve, previously known as a regulator of other vital physiological functions. Activation of vagus nerve cholinergic signalling inhibits TNF (tumour necrosis factor) and other pro-inflammatory cytokine overproduction through 'immune' alpha7 nicotinic receptor-mediated mechanisms. This efferent vagus nerve-based 'cholinergic anti-inflammatory pathway' has been elucidated as a critical regulator of inflammation in several experimental models of diseases. Our recent observations have shown that activation of central (brain) cholinergic transmission by selective muscarinic receptor ligands results in lower systemic TNF levels in rodents and indicate that the efferent vagus nerve may provide a functional brain-to-immune connection. Thus central cholinergic signalling is implicated in the activation of the cholinergic anti-inflammatory pathway. Electrical vagus nerve stimulation is clinically approved for the treatment of epilepsy and depression and current knowledge suggests that it could be utilized to control inflammation. Advances in understanding the receptor and molecular mechanisms of cholinergic anti-inflammatory signalling indicate that selective alpha7 nicotinic receptor agonists and centrally acting cholinergic enhancers can be used in the treatment of pathological conditions characterized by cytokine overproduction.

Autonomic neural regulation of immunity.

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.

High mobility group box 1 (HMGB1) protein: possible amplification signal in the pathogenesis of falciparum malaria.

High mobility group box 1 (HMGB1) protein, a DNA-binding protein that can also act as a pro-inflammatory cytokine if released from cells, is an important amplification signal in various forms of inflammation. The concentration of HMGB1 in serum taken at admission was increased in falciparum malaria in sixteen African children, more so in fatal cases than in those who subsequently recovered (P<0.001). Serum from both non-fatal (P=0.0048) and fatal (P<0.001) cases contained significantly more circulating HMGB1 than did serum from healthy Caucasian adults. These data provide an additional argument that malaria is fundamentally a systemic inflammatory state. In keeping with its developing role in sepsis, HMGB1 may enhance and prolong the inflammatory processes, and thus illness, in malaria.

Activation of human umbilical vein endothelial cells leads to relocation and release of high-mobility group box chromosomal protein 1.

The nuclear protein high-mobility group box chromosomal protein 1 (HMGB1) was recently described to act as a pro-inflammatory cytokine and as a late mediator of severe sepsis and septic shock. The protein is released from monocytes in response to endotoxin and activates monocytes and endothelial cells through nuclear factor kappa B. We have previously demonstrated that the B-box of HMGB1 mediates a pro-inflammatory effect on endothelial cells including the upregulation of cell-adhesion molecules and release of interleukin (IL)-8 and granulocyte colony-stimulating factor. Here, we report that HMGB1 is released from human umbilical vein endothelial cells (HUVEC) in response to lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-alpha. A nuclear relocation of HMGB1 to the cytoplasm was seen at 4 h. Subsequently, high amounts of HMGB1 could be seen in the supernatants from stimulated cells after 16 h. It was also observed that the pro-inflammatory activity of HMGB1 is sensitive to dexamethasone. Interestingly, the HMGB1-induced TNF-alpha release from monocytes could be inhibited by either the A-box of the protein or the p38 inhibitor CNI-1493, but neither had any inhibitory effects on the HMGB1-dependent upregulation of cell-adhesion molecules on HUVEC. Altogether, these results suggest that HUVEC may be an important source of HMGB1 secretion in response to systemic infection and that endothelial cells and monocytes may use different signalling pathways.

Neural regulators of innate immune responses and inflammation.

The nervous system regulates immune function and inflammation. Experimental evidence shows an important role of the autonomic nervous system in the bidirectional communication between the brain and the immune system, underlying the ability of the brain to monitor immune status and control inflammation. Here we review the involvement of the autonomic nervous system in regulating inflammation, with a focus on the vagus nerve. The clinical implications of the recently discovered anti-inflammatory role of the efferent vagus nerve are also discussed.

HMGB1, a pro-inflammatory cytokine of clinical interest: introduction.

Therapeutic intervention against exaggerated cytokine activity has been proved to be clinically successful in several serious, inflammatory disorders [reviewed in 1, 2] in the last decade. Half a million patients with chronic arthritis have shown tremendous improvement with tumour necrosis factor (TNF)- or interleukin (IL)-1-blocking treatment. Similarly anti-TNF therapy is beneficial for chronic inflammatory bowel disorders such as Crohn's disease. The success of this novel strategy has generated a search for additional endogenous mediators suitable for therapeutic targeting. The high mobility group box protein 1 (HMGB1) is a lately discovered candidate molecule identified as an important extracellular mediator in local and systemic inflammation in both human and experimental diseases such as, e.g., arthritis and sepsis [3]. Therapeutic neutralization of HMGB1 has shown encouraging results in experimental disease models, but has not yet reached clinical trials. This volume of the Journal of Internal Medicine contains a collection of four reviews addressing novel aspects of HMGB1 biology of potentially clinical interest. The manuscripts are the product of a recent meeting entitled the 'First HMGB1 Cytokine World Congress' sponsored by the Journal of Internal Medicine.