Vagus nerve stimulation primes platelets and reduces bleeding in hemophilia A male mice.

Deficiency of coagulation factor VIII in hemophilia A disrupts clotting and prolongs bleeding. While the current mainstay of therapy is infusion of factor VIII concentrates, inhibitor antibodies often render these ineffective. Because preclinical evidence shows electrical vagus nerve stimulation accelerates clotting to reduce hemorrhage without precipitating systemic thrombosis, we reasoned it might reduce bleeding in hemophilia A. Using two different male murine hemorrhage and thrombosis models, we show vagus nerve stimulation bypasses the factor VIII deficiency of hemophilia A to decrease bleeding and accelerate clotting. Vagus nerve stimulation targets acetylcholine-producing T lymphocytes in spleen and α7 nicotinic acetylcholine receptors (α7nAChR) on platelets to increase calcium uptake and enhance alpha granule release. Splenectomy or genetic deletion of T cells or α7nAChR abolishes vagal control of platelet activation, thrombus formation, and bleeding in male mice. Vagus nerve stimulation warrants clinical study as a therapy for coagulation disorders and surgical or traumatic bleeding.

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.

Transcutaneous Cervical Vagus Nerve Stimulation Induces Changes in the Electroencephalogram and Heart Rate Variability of Healthy Dogs, a Pilot Study.

Transcutaneous cervical vagus nerve stimulation (tcVNS) has been used to treat epilepsy in people and dogs. Objective electroencephalographic (EEG) and heart rate variability (HRV) data associated with tcVNS have been reported in people. The question remained whether EEG and electrocardiography (ECG) would detect changes in brain activity and HRV, respectively, after tcVNS in dogs. Simultaneous EEG and Holter recordings, from 6 client-owned healthy dogs were compared for differences pre- and post- tcVNS in frequency band power analysis (EEG) and HRV. The feasibility and tolerance of the patients to the tcVNS were also noted. In a general linear mixed model, the average power per channel per frequency band was found to be significantly different pre- and post-stimulation in the theta (p = 0.02) and alpha bands (p = 0.04). The pooled power spectral analysis detected a significant decrease in the alpha (p < 0.01), theta (p = 0.01) and beta (p = 0.035) frequencies post-stimulation. No significant interaction was observed between dog, attitude, and stimulation in the multivariate model, neither within the same dog nor between individuals. There was a significant increase in the HRV measured by the standard deviation of the inter-beat (SDNN) index (p < 0.01) and a decrease in mean heart rate (p < 0.01) after tcVNS. The tcVNS was found to be well-tolerated. The results of this pilot study suggest that EEG and ECG can detect changes in brain activity and HRV associated with tcVNS in healthy dogs. Larger randomized controlled studies are required to confirm the results of this study and to assess tcVNS potential therapeutic value.

Non-invasive Vagus Nerve Stimulation for COVID-19: Results From a Randomized Controlled Trial (SAVIOR I).

Background: Severe coronavirus disease 2019 (COVID-19) is characterized, in part, by an excessive inflammatory response. Evidence from animal and human studies suggests that vagus nerve stimulation can lead to reduced levels of various biomarkers of inflammation. We conducted a prospective randomized controlled study (SAVIOR-I) to assess the feasibility, efficacy, and safety of non-invasive vagus nerve stimulation (nVNS) for the treatment of respiratory symptoms and inflammatory markers among patients who were hospitalized for COVID-19 (ClinicalTrials.gov identifier: NCT04368156). Methods: Participants were randomly assigned in a 1:1 allocation to receive either the standard of care (SoC) alone or nVNS therapy plus the SoC. The nVNS group received 2 consecutive 2-min doses of nVNS 3 times daily as prophylaxis. Efficacy and safety were evaluated via the incidence of specific clinical events, inflammatory biomarker levels, and the occurrence of adverse events. Results: Of the 110 participants who were enrolled and randomly assigned, 97 (nVNS, n = 47; SoC, n = 50) had sufficient available data and comprised the evaluable population. C-reactive protein (CRP) levels decreased from baseline to a significantly greater degree in the nVNS group than in the SoC group at day 5 and overall (i.e., all postbaseline data points collected through day 5, combined). Procalcitonin level also showed significantly greater decreases from baseline to day 5 in the nVNS group than in the SoC group. D-dimer levels were decreased from baseline for the nVNS group and increased from baseline for the SoC group at day 5 and overall, although the difference between the treatment groups did not reach statistical significance. No significant treatment differences were seen for clinical respiratory outcomes or any of the other biochemical markers evaluated. No serious nVNS-related adverse events occurred during the study. Conclusions: nVNS therapy led to significant reductions in levels of inflammatory markers, specifically CRP and procalcitonin. Because nVNS has multiple mechanisms of action that may be relevant to COVID-19, additional research into its potential use earlier in the course of COVID-19 and its potential to mitigate some of the symptoms associated with post-acute sequelae of COVID-19 is warranted.

Neuromodulation Strategies to Reduce Inflammation and Improve Lung Complications in COVID-19 Patients.

Since the outbreak of the COVID-19 pandemic, races across academia and industry have been initiated to identify and develop disease modifying or preventative therapeutic strategies has been initiated. The primary focus has been on pharmacological treatment of the immune and respiratory system and the development of a vaccine. The hyperinflammatory state ("cytokine storm") observed in many cases of COVID-19 indicates a prognostically negative disease progression that may lead to respiratory distress, multiple organ failure, shock, and death. Many critically ill patients continue to be at risk for significant, long-lasting morbidity or mortality. The human immune and respiratory systems are heavily regulated by the central nervous system, and intervention in the signaling of these neural pathways may permit targeted therapeutic control of excessive inflammation and pulmonary bronchoconstriction. Several technologies, both invasive and non-invasive, are available and approved for clinical use, but have not been extensively studied in treatment of the cytokine storm in COVID-19 patients. This manuscript provides an overview of the role of the nervous system in inflammation and respiration, the current understanding of neuromodulatory techniques from preclinical and clinical studies and provides a rationale for testing non-invasive neuromodulation to modulate acute systemic inflammation and respiratory dysfunction caused by SARS-CoV-2 and potentially other pathogens. The authors of this manuscript have co-founded the International Consortium on Neuromodulation for COVID-19 to advocate for and support studies of these technologies in the current coronavirus pandemic.

Pharmacological stimulation of the cholinergic antiinflammatory pathway.

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 alpha-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.

Cleveland neural engineering workshop 2017: strategic evaluation of neural engineering.

The Cleveland Neural Engineering Workshop (NEW) was established as a biennial meeting in 2011, with subsequent meetings taking place in 2013, 2015, and most recently, June 2017. This fourth biennial NEW was hosted by the Cleveland Advanced Platform for Technology National Veterans Affairs Center, the Functional Electrical Stimulation National Veterans Affairs Center, the Biomedical Engineering Department at Case Western Reserve University in Cleveland, Ohio, and Northwell Health's Feinstein Institute for Medical Research of New York. The workshop connects leaders and stakeholders in the neural engineering community who are devoted to developing and deploying technological solutions to those with neurological disorders. The meeting in 2017 continued strategic conversations initiated at the third Cleveland NEW conference in 2015. The goal of the 2017 workshop was to was to determine specific actions by which the neural engineering community might advance the goals outlined in 2015, assess progress towards that plan, adjust as necessary, and establish continued strategic direction. This meeting report summarizes the outcomes.

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.

New melanocortin-like peptide of E. coli can suppress inflammation via the mammalian melanocortin-1 receptor (MC1R): possible endocrine-like function for microbes of the gut.

E. coli releases a 33 amino acid peptide melanocortin-like peptide of E. coli (MECO-1) that is identical to the C-terminus of the E. coli elongation factor-G (EF-G) and has interesting similarities to two prominent mammalian melanocortin hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH). Note that MECO-1 lacks HFRW, the common pharmacophore of the known mammalian melanocortin peptides. MECO-1 and the two hormones were equally effective in severely blunting release of cytokines (HMGB1 and TNF) from macrophage-like cells in response to (i) endotoxin (lipopolysaccharide) or (ii) pro-inflammatory cytokine HMGB-1. The in vitro anti-inflammatoty effects of MECO-1 and of alpha-MSH were abrogated by (i) antibody against melanocortin-1 receptor (MC1R) and by (ii) agouti, an endogenous inverse agonist of MC1R. In vivo MECO-1 was even more potent than alpha-MSH in rescuing mice from death due to (i) lethal doses of LPS endotoxin or (ii) cecal ligation and puncture, models of sterile and infectious sepsis, respectively.

HMGB1 signals through toll-like receptor (TLR) 4 and TLR2.

In response to bacterial endotoxin (e.g., LPS) or endogenous proinflammatory cytokines (e.g., TNF and IL-1beta), innate immune cells release HMGB1, a late cytokine mediator of lethal endotoxemia and sepsis. The delayed kinetics of HMGB1 release makes it an attractive therapeutic target with a wider window of opportunity for the treatment of lethal systemic inflammation. However, the receptor(s) responsible for HMGB1-mediated production of proinflammatory cytokines has not been well characterized. Here we demonstrate that in human whole blood, neutralizing antibodies against Toll-like receptor 4 (TLR4, but not TLR2 or receptor for advanced glycation end product) dose-dependently attenuate HMGB1-induced IL-8 release. Similarly, in primary human macrophages, HMGB1-induced TNF release is dose-dependently inhibited by anti-TLR4 antibodies. In primary macrophages from knockout mice, HMGB1 activates significantly less TNF release in cells obtained from MyD88 and TLR4 knockout mice as compared with cells from TLR2 knockout and wild-type controls. However, in human embryonic kidney 293 cells transfected with TLR2 or TLR4, HMGB1 effectively induces IL-8 release only from TLR2 overexpressing cells. Consistently, anti-TLR2 antibodies dose-dependently attenuate HMGB1-induced IL-8 release in human embryonic kidney/TLR2-expressing cells and markedly reduce HMGB1 cell surface binding on murine macrophage-like RAW 264.7 cells. Taken together, our data suggest that there is a differential usage of TLR2 and TLR4 in HMGB1 signaling in primary cells and in established cell lines, adding complexity to studies of HMGB1 signaling which was not previously expected.

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 cytokine activity of HMGB1.

High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein present in the nuclei and cytoplasm of nearly all cell types. We recently discovered that HMGB1 is secreted into the extracellular milieu and acts as a proinflammatory cytokine. Administration of HMGB1 to normal animals causes inflammatory responses, including fever, weight loss and anorexia, acute lung injury, epithelial barrier dysfunction, arthritis, and death. Anti-HMGB1 treatment, with antibodies or specific antagonists, rescues mice from lethal endotoxemia or sepsis and ameliorates the severity of collagen-induced arthritis and endotoxin-induced lung injury. Here, we give an abridged review of the cytokine activity of HMGB1, its secretion and release into the extracellular milieu, the putative signal transduction pathways, including interaction with cell-surface receptors and intracellular signaling, and its role in several inflammatory diseases. Finally, the therapeutic potential of blocking HMGB1 in the treatment of inflammatory diseases is discussed.

HMGB1 as a cytokine and therapeutic target.

HMGB1 is an abundant nuclear and cytoplasmic protein present in mammalian cells. It is traditionally known as a DNA binding protein involved in maintenance of nucleosome structure and regulation of gene transcription. Beyond these intracellular roles, we recently discovered that HMGB1 is released from activated macrophages and functions as a late mediator of lethal endotoxemia. Addition of HMGB1 to macrophage cultures activates cytokine release. When released into the extracellular milieu, HMGB1 causes systemic inflammatory responses including acute lung injury, epithelial barrier dysfunction, and death. Passive immunization with anti-HMGB1 antibodies confers significant protection against lethality induced by LPS administration and sepsis caused by cecal perforation in mice. Truncation of HMGB1 into individual structural domains revealed that the HMGB1 A box, a DNA-binding motif, specifically antagonizes the activity of HMGB1 and rescues mice from lethal sepsis caused by cecal perforation. Thus, strategies that target HMGB1 with specific antibodies or antagonists have potential for treating lethal systemic inflammatory diseases characterized by excessive HMGB1 release.

Neural inhibition of inflammation: the cholinergic anti-inflammatory pathway.

The innate immune system is activated by infection and injury to release pro-inflammatory cytokines, which activate macrophages and neutrophils and modulate specific cellular responses. The magnitude of the cytokine response is critical, because a deficient response may result in secondary infections, while an excessive response may be more injurious than the original insult. We recently described a neural pathway, termed the "cholinergic anti-inflammatory pathway", that reflexively monitors and adjusts the inflammatory response by inhibiting pro-inflammatory cytokine synthesis. Efferent signals in the vagus nerve provide a direct mechanism for neural regulation of the immune response that is rapid, localized, and integrated. Vagus nerve stimulation inhibits the release of TNF, HMGB1, and other cytokines, and protects against endotoxemia and ischemia-reperfusion injury. This newly identified physiological mechanism of maintaining immunological homeostasis suggests that novel therapeutics may effectively modulate inflammatory responses by activating the cholinergic anti-inflammatory pathway.

Recombinant HMGB1 with cytokine-stimulating activity.

We describe methods for the isolation, purification, and characterization of full-length high-mobility group box 1 (HMGB1) and truncated mutants expressed in bacteria and in mammalian Chinese Hamster Ovary (CHO) cells. HMGB1 is an abundant nuclear and cytoplasmic protein, highly conserved across species and widely distributed in eukaryotic cells from yeast to man. As a ubiquitous nuclear DNA binding protein, HMGB1 binds DNA, facilitates gene transcription, and stabilizes nucleosome structure. In addition to these intracellular roles, HMGB1 can be released into the extracellular milieu by activated innate immune cells (i.e., macrophages, monocytes) and functions as a mediator of lethal endotoxemia and sepsis. The proinflammatory cytokine activity of HMGB1 has become an intense area of research and recombinant protein can be a useful tool to probe HMGB1 functions. Due to its dipolar charged properties, HMGB1 isolated by some methods can be contaminated with bacterial products (such as CpG DNA or lipopolysaccharide [LPS]) that may interfere with immunological analyses. Here we report our newly developed methods for the isolation and purification of biologically active HMGB1 from bacteria or mammalian CHO cells that is essentially free of contaminants. This strategy provides an important advance in methodology to facilitate future HMGB1 studies.