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Abstract Background Key to the advancement of the field of bioelectronic medicine is the identification of novel pathways of neural regulation of immune function. Sensory neurons (termed nociceptors) recognize harmful stimuli and initiate a protective response by eliciting pain and defensive behavior. Nociceptors also interact with immune cells to regulate host defense and inflammatory responses. However, it is still unclear whether nociceptors participate in regulating primary IgG antibody responses to novel antigens. Methods To understand the role of transient receptor potential vanilloid 1 (TRPV1)-expressing neurons in IgG responses, we generated TRPV1-Cre/Rosa-ChannelRhodopsin2 mice for precise optogenetic activation of TRPV1 + neurons and TRPV1-Cre/Lox-diphtheria toxin A mice for targeted ablation of TRPV1-expressing neurons. Antigen-specific antibody responses were longitudinally monitored for 28 days. Results Here we show that TRPV1 expressing neurons are required to develop an antigen-specific immune response. We demonstrate that selective optogenetic stimulation of TRPV1+ nociceptors during immunization significantly enhances primary IgG antibody responses to novel antigens. Further, mice rendered deficient in TRPV1- expressing nociceptors fail to develop primary IgG antibody responses to keyhole limpet hemocyanin or haptenated antigen. Conclusion This functional and genetic evidence indicates a critical role for nociceptor TRPV1 in antigen-specific primary antibody responses to novel antigens. These results also support consideration of potential therapeutic manipulation of nociceptor pathways using bioelectronic devices to enhance immune responses to foreign antigens.

The pathogenic mechanisms of bacterial infections and resultant sepsis are partly attributed to dysregulated inflammatory responses sustained by some late-acting mediators including the procathepsin-L (pCTS-L). It was entirely unknown whether any compounds of the U.S. Drug Collection could suppress pCTS-L-induced inflammation, and pharmacologically be exploited into possible therapies. Here, we demonstrated that a macrophage cell-based screening of a U.S. Drug Collection of 1360 compounds resulted in the identification of progesterone (PRO) as an inhibitor of pCTS-L-mediated production of several chemokines [e.g., Epithelial Neutrophil-Activating Peptide (ENA-78), Monocyte Chemoattractant Protein-1 (MCP-1) or MCP-3] and cytokines [e.g., Interleukin-10 (IL-10) or Tumor Necrosis Factor (TNF)] in primary human peripheral blood mononuclear cells (PBMCs). In vivo, these PRO-entrapping 2,6-dimethal-β-cyclodextrin (DM-β-CD) nanoparticles (containing 1.35 mg/kg PRO and 14.65 mg/kg DM-β-CD) significantly increased animal survival in both male (from 30% to 70%, n = 20, P = 0.041) and female (from 50% to 80%, n = 30, P = 0.026) mice even when they were initially administered at 24 h post the onset of sepsis. This protective effect was associated with a reduction of sepsis-triggered accumulation of three surrogate biomarkers [e.g., Granulocyte Colony Stimulating Factor (G-CSF) by 40%; Macrophage Inflammatory Protein-2 (MIP-2) by 45%; and Soluble Tumor Necrosis Factor Receptor I (sTNFRI) by 80%]. Surface Plasmon Resonance (SPR) analysis revealed a strong interaction between PRO and pCTS-L (KD = 78.2 ± 33.7 nM), which was paralleled with a positive correlation between serum PRO concentration and serum pCTS-L level (ρ = 0.56, P = 0.0009) or disease severity (Sequential Organ Failure Assessment, SOFA; ρ = 0.64, P = 0.0001) score in septic patients. Our observations support a promising opportunity to explore DM-β-CD nanoparticles entrapping lipophilic drugs as possible therapies for clinical sepsis.

Abstract Background Acute pancreatitis is a common and serious inflammatory condition currently lacking disease modifying therapy. The cholinergic anti-inflammatory pathway (CAP) is a potent protective anti-inflammatory response activated by vagus nerve-dependent α7 nicotinic acetylcholine receptor (α7nAChR) signaling using splenic CD4+ T cells as an intermediate. Activating the CAP ameliorates experimental acute pancreatitis. Galantamine is an acetylcholinesterase inhibitor (AChEI) which amplifies the CAP via modulation of central muscarinic ACh receptors (mAChRs). However, as mAChRs also activate pancreatitis, it is currently unknown whether galantamine would be beneficial in acute pancreatitis. Methods The effect of galantamine (1–6 mg/kg-body weight) on caerulein-induced acute pancreatitis was evaluated in mice. Two hours following 6 hourly doses of caerulein (50 µg/kg-body weight), organ and serum analyses were performed with accompanying pancreatic histology. Experiments utilizing vagotomy, gene knock out (KO) technology and the use of nAChR antagonists were also performed. Results Galantamine attenuated pancreatic histologic injury which was mirrored by a reduction in serum amylase and pancreatic inflammatory cytokines and an increase the anti-inflammatory cytokine IL-10 in the serum. These beneficial effects were not altered by bilateral subdiaphragmatic vagotomy, KO of either choline acetyltransferase+ T cells or α7nAChR, or administration of the nAChR ganglionic blocker mecamylamine or the more selective α7nAChR antagonist methyllycaconitine. Conclusion Galantamine improves acute pancreatitis via a mechanism which does not involve previously established physiological and molecular components of the CAP. As galantamine is an approved drug in widespread clinical use with an excellent safety record, our findings are of interest for further evaluating the potential benefits of this drug in patients with acute pancreatitis.

Abstract Background Vagus nerve stimulation is an investigational anti-inflammatory therapy targeting the nervous system to modulate immune activity. This study evaluated the efficacy and safety of transcutaneous auricular VNS (ta-VNS) in patients with pediatric-onset Crohn’s disease (CD) or ulcerative colitis (UC). Methods Participants were 10–21 years of age with mild/moderate CD or UC and fecal calprotectin (FC) > 200 ug/g within 4 weeks of study entry. Subjects were randomized to receive either ta-VNS targeting the cymba conchae of the external left ear, or sham stimulation, of 5 min duration once daily for a 2-week period, followed by a cross over to the alternative stimulation for an additional 2 weeks. At week 4, all subjects received ta-VNS of 5 min duration twice daily until week 16. Primary study endpoints were clinical remission, and a ≥ 50% reduction in FC level from baseline to week 16. Heart rate variability measurements and patient-reported outcome questionnaires were completed during interval and week 16 assessments. Results Twenty-two subjects were enrolled and analyzed (10 CD, 12 UC). Six of 10 with CD had a wPCDAI > 12.5 and 6/12 with UC had a PUCAI > 10 at baseline, correlating to mild to moderate symptom activity. Among the 12 subjects with active symptomatic disease indices at baseline, clinical remission was achieved in 3/6 (50%) with CD and 2/6 (33%) with UC at week 16. Despite all subjects having FC levels ≥ 200 within 4 weeks of enrollment, five subjects (4 UC, 1 CD) had FC levels

Abstract The emerging field of bioelectronic medicine (BEM) is poised to make a significant impact on the treatment of several neurological and inflammatory disorders. With several BEM therapies being recently approved for clinical use and others in late-phase clinical trials, the 2022 BEM summit was a timely scientific meeting convening a wide range of experts to discuss the latest developments in the field. The BEM Summit was held over two days in New York with more than thirty-five invited speakers and panelists comprised of researchers and experts from both academia and industry. The goal of the meeting was to bring international leaders together to discuss advances and cultivate collaborations in this emerging field that incorporates aspects of neuroscience, physiology, molecular medicine, engineering, and technology. This Meeting Report recaps the latest findings discussed at the Meeting and summarizes the main developments in this rapidly advancing interdisciplinary field. Our hope is that this Meeting Report will encourage researchers from academia and industry to push the field forward and generate new multidisciplinary collaborations that will form the basis of new discoveries that we can discuss at the next BEM Summit.

Abstract 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.

Focused ultrasound stimulation (FUS) activates mechanosensitive ion channels and is emerging as a method of noninvasive neuromodulation. In preclinical studies, FUS of the spleen (sFUS) activates an anti-inflammatory neural pathway which suppresses acute and chronic inflammation. However, the relevance of sFUS for regulating inflammatory responses in humans is unknown. Here, we used a modified diagnostic ultrasound imaging system to target the spleen of healthy human subjects with 3 min of continuously swept or stationary focused pulsed ultrasound, delivered at three different energy levels within allowable safety exposure limits. Potential anti-inflammatory effects of sFUS were assessed by measuring sFUS-elicited changes in endotoxin-induced tumor necrosis factor (TNF) production in whole blood samples from insonified subjects. We found that stimulation with either continuously swept or focused pulsed ultrasound has an anti-inflammatory effect: sFUS lowers TNF production for >2 h, with TNF returning to baseline by 24 h following sFUS. This response is independent of anatomical target (i.e., spleen hilum or parenchyma) or ultrasound energy level. No clinical, biochemical, or hematological parameters are adversely impacted. This is the first demonstration that sFUS suppresses the normal inflammatory response in humans, with potential implications for noninvasive bioelectronic therapy of inflammatory disorders.

Abstract Background There is emerging evidence that the nervous system regulates immune and metabolic alterations mediating Metabolic syndrome (MetS) pathogenesis via the vagus nerve. This study evaluated the effects of transcutaneous auricular vagus nerve stimulation (TAVNS) on key cardiovascular and inflammatory components of MetS. Methods We conducted an open label, randomized (2:1), two-arm, parallel-group controlled trial in MetS patients. Subjects in the treatment group (n = 20) received 30 min of TAVNS with a NEMOS® device placed on the cymba conchae of the left ear, once weekly. Patients in the control group (n = 10) received no stimulation. Hemodynamic, heart rate variability (HRV), biochemical parameters, and monocytes, progenitor endothelial cells, circulating endothelial cells, and endothelial micro particles were evaluated at randomization, after the first TAVNS treatment, and again after 8 weeks of follow-up. Results An improvement in sympathovagal balance (HRV analysis) was observed after the first TAVNS session. Only patients treated with TAVNS for 8 weeks had a significant decrease in office BP and HR, a further improvement in sympathovagal balance, with a shift of circulating monocytes towards an anti-inflammatory phenotype and endothelial cells to a reparative vascular profile. Conclusion These results are of interest for further study of TAVNS as treatment of MetS.

Vagal fibers travel inside fascicles and form branches to innervate organs and regulate organ functions. Existing vagus nerve stimulation (VNS) therapies activate vagal fibers non-selectively, often resulting in reduced efficacy and side effects from non-targeted organs. The transverse and longitudinal arrangement of fibers inside the vagal trunk with respect to the functions they mediate and organs they innervate is unknown, however it is crucial for selective VNS. Using micro-computed tomography imaging, we tracked fascicular trajectories and found that, in swine, sensory and motor fascicles are spatially separated cephalad, close to the nodose ganglion, and merge caudad, towards the lower cervical and upper thoracic region; larynx-, heart- and lung-specific fascicles are separated caudad and progressively merge cephalad. Using quantified immunohistochemistry at single fiber level, we identified and characterized all vagal fibers and found that fibers of different morphological types are differentially distributed in fascicles: myelinated afferents and efferents occupy separate fascicles, myelinated and unmyelinated efferents also occupy separate fascicles, and small unmyelinated afferents are widely distributed within most fascicles. We developed a multi-contact cuff electrode to accommodate the fascicular structure of the vagal trunk and used it to deliver fascicle-selective cervical VNS in anesthetized and awake swine. Compound action potentials from distinct fiber types, and physiological responses from different organs, including laryngeal muscle, cough, breathing, and heart rate responses are elicited in a radially asymmetric manner, with consistent angular separations that agree with the documented fascicular organization. These results indicate that fibers in the trunk of the vagus nerve are anatomically organized according to functions they mediate and organs they innervate and can be asymmetrically activated by fascicular cervical VNS.

Abstract Background Inflammation, the physiological response to infection and injury, is coordinated by the immune and nervous systems. Interleukin-1β (IL-1β) and other cytokines produced during inflammatory responses activate sensory neurons (nociceptors) to mediate the onset of pain, sickness behavior, and metabolic responses. Although nociceptors expressing Transient Receptor Potential Ankyrin-1 (TRPA1) can initiate inflammation, comparatively little is known about the role of TRPA1 nociceptors in the physiological responses to specific cytokines. Methods To monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into peritoneal cavity of mice. Using transgenic and tissue specific knockouts and chemogenetic techniques, we recorded temperature responses to the potent pro-inflammatory cytokine IL-1β. Using calcium imaging, whole cell patch clamping and whole nerve recordings, we investigated the role of TRPA1 during IL-1β-mediated neuronal activation. Mouse models of acute endotoxemia and sepsis were used to elucidate how specific activation, with optogenetics and chemogenetics, or ablation of TRPA1 neurons can affect the outcomes of inflammatory insults. All statistical tests were performed with GraphPad Prism 9 software and for all analyses, P ≤ 0.05 was considered statistically significant. Results Here, we describe a previously unrecognized mechanism by which IL-1β activates afferent vagus nerve fibers to trigger hypothermia, a response which is abolished by selective silencing of neuronal TRPA1. Afferent vagus nerve TRPA1 signaling also inhibits endotoxin-stimulated cytokine storm and significantly reduces the lethality of bacterial sepsis. Conclusion Thus, IL-1β activates TRPA1 vagus nerve signaling in the afferent arm of a reflex anti-inflammatory response which inhibits cytokine release, induces hypothermia, and reduces the mortality of infection. This discovery establishes that TRPA1, an ion channel known previously as a pro-inflammatory detector of cold, pain, itch, and a wide variety of noxious molecules, also plays a specific anti-inflammatory role via activating reflex anti-inflammatory activity.

The rapid protease degradation of peptides is currently limiting their therapeutic utility. Here, the authors report functionalised thiocarbazate scaffolds as precursors of aza-amino acids that can be integrated in peptide sequences, extending their bioavailability, and demonstrate this on FSSE/P5779 and bradykinin.

Abstract Background Brain metabolic alterations and neuroinflammation have been reported in several peripheral inflammatory conditions and present significant potential for targeting with new diagnostic approaches and treatments. However, non-invasive evaluation of these alterations remains a challenge. Methods Here, we studied the utility of a micro positron emission tomography (microPET) dual tracer ([11C]PBR28 – for microglial activation and [18F]FDG for energy metabolism) approach to assess brain dysfunction, including neuroinflammation in murine endotoxemia. MicroPET imaging data were subjected to advanced conjunction and individual analyses, followed by post-hoc analysis. Results There were significant increases in [11C]PBR28 and [18F]FDG uptake in the hippocampus of C57BL/6 J mice 6 h following LPS (2 mg/kg) intraperitoneal (i.p.) administration compared with saline administration. These results confirmed previous postmortem observations. In addition, patterns of significant simultaneous activation were demonstrated in the hippocampus, the thalamus, and the hypothalamus in parallel with other tracer-specific and region-specific alterations. These changes were observed in the presence of robust systemic inflammatory responses manifested by significantly increased serum cytokine levels. Conclusions Together, these findings demonstrate the applicability of [11C]PBR28 - [18F]FDG dual tracer microPET imaging for assessing neuroinflammation and brain metabolic alterations in conditions “classically” characterized by peripheral inflammatory and metabolic pathogenesis.

Background: Vagal reflexes regulate homeostasis in visceral organs and systems through afferent and efferent neurons and nerve fibers. Small, unmyelinated, C-type afferents comprise over 80% of fibers in the vagus and form the sensory arc of autonomic reflexes of the gut, lungs, heart and vessels and the immune system. Selective bioelectronic activation of C-afferents could be used to mechanistically study and treat diseases of peripheral organs in which vagal reflexes are involved, but it has not been achieved. Methods: We stimulated the vagus in rats and mice using trains of kHz-frequency stimuli. Stimulation effects were assessed using neuronal c-Fos expression, physiological and nerve fiber responses, optogenetic and computational methods. Results: Intermittent kHz stimulation for 30 min activates specific motor and, preferentially, sensory vagus neurons in the brainstem. At sufficiently high frequencies (>5 kHz) and at intensities within a specific range (7–10 times activation threshold, T, in rats; 15-25 × T in mice), C-afferents are activated, whereas larger, A- and B-fibers, are blocked. This was determined by measuring fiber-specific acute physiological responses to kHz stimulus trains, and by assessing fiber excitability around kHz stimulus trains through compound action potentials evoked by probing pulses. Aspects of selective activation of C-afferents are explained in computational models of nerve fibers by how fiber size and myelin shape the response of sodium channels to kHz-frequency stimuli. Conclusion: kHz stimulation is a neuromodulation strategy to robustly and selectively activate vagal C-afferents implicated in physiological homeostasis and disease, over larger vagal fibers.

Abstract Background Neuroinflammation is an important driver of acute and chronic pain states. Therefore, targeting molecular mediators of neuroinflammation may present an opportunity for developing novel pain therapies. In preclinical models of neuroinflammatory pain, calcitonin gene-related peptide (CGRP), substance P and high mobility group box 1 protein (HMGB1) are molecules synthesized and released by sensory neurons which activate inflammation and pain. High-frequency electrical nerve stimulation (HFES) has achieved clinical success as an analgesic modality, but the underlying mechanism is unknown. Here, we reasoned that HFES inhibits neuroinflammatory mediator release by sensory neurons to reduce pain. Methods Utilizing in vitro and in vivo assays, we assessed the modulating effects of HFES on neuroinflammatory mediator release by activated sensory neurons. Dorsal root ganglia (DRG) neurons harvested from wildtype or transgenic mice expressing channelrhodopsin-2 (ChR2) were cultured on micro-electrode arrays, and effect of HFES on optogenetic- or capsaicin-induced neuroinflammatory mediator release was determined. Additionally, the effects of HFES on local neuroinflammatory mediator release and hyperalgesia was assessed in vivo using optogenetic paw stimulation and the neuropathic pain model of chronic constriction injury (CCI) of the sciatic nerve. Results Light- or capsaicin-evoked neuroinflammatory mediator release from cultured transgenic DRG sensory neurons was significantly reduced by concurrent HFES (10 kHz). In agreement with these findings, elevated levels of neuroinflammatory mediators were detected in the affected paw following optogenetic stimulation or CCI and were significantly attenuated using HFES (20.6 kHz for 10 min) delivered once daily for 3 days. Conclusion These studies reveal a previously unidentified mechanism for the pain-modulating effect of HFES in the setting of acute and chronic nerve injury. The results support the mechanistic insight that HFES may reset sensory neurons into a less pro-inflammatory state via inhibiting the release of neuroinflammatory mediators resulting in reduced inflammation and pain.

Abstract Background Vagus nerve stimulation (VNS) has shown therapeutic potential in a variety of different diseases with many ongoing clinical trials. The role of VNS in reducing ischemic injury in the brain requires further evaluation. Cardiac arrest (CA) causes global ischemia and leads to the injury of vital organs, especially the brain. In this study, we investigated the protective effects of customized threshold-adjusted VNS (tVNS) in a rat model of CA and resuscitation. Methods Sprague-Dawley rats underwent 12 min asphyxia-CA followed by resuscitation. Rats were assigned to either post-resuscitation tVNS for 2 h or no-tVNS (control). tVNS was applied by electrode placement in the left cervical vagus nerve. To optimize a threshold, we used animal’s heart rate and determined a 15–20% drop from baseline levels as the effective and physiological threshold for each animal. The primary endpoint was 72 h survival; secondary endpoints included neurological functional recovery, reduction in brain cellular injury (histopathology), cardiac and renal injury parameters (troponin I and creatinine levels, respectively). Results In comparison to the control group, tVNS significantly improved 72 h survival and brain functional recovery after 12 minutes of CA. The tVNS group demonstrated significantly reduced numbers of damaged neurons in the CA1 hippocampal region of the brain as compared to the control group. Similarly, the tVNS group showed decreased trend in plasma troponin I and creatinine levels as compared to the control group. Conclusions Our findings suggest that using tVNS for 2 h after 12 minutes of CA attenuates ischemia neuronal cell death, heart and kidney damage, and improves 72 h survival with improved neurological recovery.

IntroductionInflammation is an inherently self-amplifying process, resulting in progressive tissue damage when unresolved. A brake on this positive feedback system is provided by the nervous system which has evolved to detect inflammatory signals and respond by activating anti-inflammatory processes, including the cholinergic anti-inflammatory pathway mediated by the vagus nerve. Acute pancreatitis, a common and serious condition without effective therapy, develops when acinar cell injury activates intrapancreatic inflammation. Prior study has shown that electrical stimulation of the carotid sheath, which contains the vagus nerve, boosts the endogenous anti-inflammatory response and ameliorates acute pancreatitis, but it remains unknown whether these anti-inflammatory signals originate in the brain.MethodsHere, we used optogenetics to selectively activate efferent vagus nerve fibers originating in the brainstem dorsal motor nucleus of the vagus (DMN) and evaluated the effects on caerulein-induced pancreatitis.ResultsStimulation of the cholinergic neurons in the DMN significantly attenuates the severity of pancreatitis as indicated by reduced serum amylase, pancreatic cytokines, tissue damage, and edema. Either vagotomy or silencing cholinergic nicotinic receptor signaling by pre-administration of the antagonist mecamylamine abolishes the beneficial effects.DiscussionThese results provide the first evidence that efferent vagus cholinergic neurons residing in the brainstem DMN can inhibit pancreatic inflammation and implicate the cholinergic anti-inflammatory pathway as a potential therapeutic target for acute pancreatitis.

Abstract Background Severe COVID-19 is characterized by pro-inflammatory cytokine release syndrome (cytokine storm) which causes high morbidity and mortality. Recent observational and clinical studies suggest famotidine, a histamine 2 receptor (H2R) antagonist widely used to treat gastroesophageal reflux disease, attenuates the clinical course of COVID-19. Because evidence is lacking for a direct antiviral activity of famotidine, a proposed mechanism of action is blocking the effects of histamine released by mast cells. Here we hypothesized that famotidine activates the inflammatory reflex, a brain-integrated vagus nerve mechanism which inhibits inflammation via alpha 7 nicotinic acetylcholine receptor (α7nAChR) signal transduction, to prevent cytokine storm. Methods The potential anti-inflammatory effects of famotidine and other H2R antagonists were assessed in mice exposed to lipopolysaccharide (LPS)-induced cytokine storm. As the inflammatory reflex is integrated and can be stimulated in the brain, and H2R antagonists penetrate the blood brain barrier poorly, famotidine was administered by intracerebroventricular (ICV) or intraperitoneal (IP) routes. Results Famotidine administered IP significantly reduced serum and splenic LPS-stimulated tumor necrosis factor (TNF) and IL-6 concentrations, significantly improving survival. The effects of ICV famotidine were significantly more potent as compared to the peripheral route. Mice lacking mast cells by genetic deletion also responded to famotidine, indicating the anti-inflammatory effects are not mast cell-dependent. Either bilateral sub-diaphragmatic vagotomy or genetic knock-out of α7nAChR abolished the anti-inflammatory effects of famotidine, indicating the inflammatory reflex as famotidine’s mechanism of action. While the structurally similar H2R antagonist tiotidine displayed equivalent anti-inflammatory activity, the H2R antagonists cimetidine or ranitidine were ineffective even at very high dosages. Conclusions These observations reveal a previously unidentified vagus nerve-dependent anti-inflammatory effect of famotidine in the setting of cytokine storm which is not replicated by high dosages of other H2R antagonists in clinical use. Because famotidine is more potent when administered intrathecally, these findings are also consistent with a primarily central nervous system mechanism of action.

Abstract Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker l-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension.

Background: Myelodysplastic syndrome (MDS) is associated with persistent immune activation. High mobility group box-1 (HMGB1) is a ubiquitous, functionally diverse, non-histone intranuclear protein. During acute and chronic inflammatory states, HMGB1 is actively released by inflammatory cells, further amplifying the inflammatory response. A role in MDS and other hypoplastic bone marrow (BM) disorders is incompletely understood. Objectives: The objective of the study is to evaluate whether circulating HMGB1 is elevated in patients with MDS and other BM failure syndromes [namely, aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH)]. Design: This is a observational, cross-sectional, single-center, exploratory study. Methods: We evaluated circulating concentrations of HMGB1, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in patients with MDS and age-matched hematologically healthy controls as well as patients with AA and PNH. Results: We included 66 patients with MDS and 65 age-matched controls as well as 44 patients with other BM failures (AA = 27, PNH = 17). Circulating levels of HMGB1 were higher in patients with MDS [median, 4.9 ng/ml; interquartile range (IQR): 2.3–8.1] than in AA (median, 2.6 ng/ml; IQR: 1.7–3.7), PNH (median, 1.7 ng/ml; IQR: 0.9–2.5), and age-matched healthy individuals (median, 1.9 ng/ml; IQR: 0.9–2.5) ( p = 0.0001). We observed higher concentrations of HMGB1 in the very low/low-risk MDS patients than in the intermediate/high/very high-risk ones ( p = 0.046). Finally, in comparison with patients with AA, those with hypocellular MDS (h-MDS) had significantly higher levels of circulating HMGB1 ( n = 14; median concentration, 5.6 ng/ml, IQR: 2.8–7.3; p = 0.006). We determined a circulating HMGB1 value of 4.095 ng/ml as a diagnostic cutoff differentiator between h-MDS and AA. Conclusion: These observations indicate that circulating HMGB1 is increased in patients with MDS. HMGB1 (but not IL-1β or TNF-α) differentiated between MDS and other BM failures, suggesting that HMGB1 may be mechanistically involved in MDS and a druggable target to decrease inflammation in MDS.

Abstract Background High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is passively released during cell death and secreted by activated cells of many lineages. HMGB1 contains three conserved redox-sensitive cysteine residues: cysteines in position 23 and 45 (C23 and C45) can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Methods Primary human macrophages or murine macrophage-like RAW 264.7 cells were activated in cell cultures by redox-modified or point-mutated (C45A) recombinant HMGB1 preparations or by lipopolysaccharide (E. coli.0111: B4). Cellular phosphorylated NF-κB p65 subunit and subsequent TNF-α release were quantified by commercial enzyme-linked immunosorbent assays. Results Cell cultures with primary human macrophages and RAW 264.7 cells demonstrated that fully reduced HMGB1 with all three cysteines expressing thiol side chains failed to generate phosphorylated NF-КB p65 subunit or TNF-α. Mild oxidation forming a C23-C45 disulfide bond, while leaving C106 with a thiol group, was required for HMGB1 to induce phosphorylated NF-КB p65 subunit and TNF-α production. The importance of a C23–C45 disulfide bond was confirmed by mutation of C45 to C45A HMGB1, which abolished the ability for cytokine induction. Further oxidation of the disulfide isoform also inactivated HMGB1. Conclusions These results reveal critical post-translational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during inflammation.

Abstract A SARS-like coronavirus 2 (SARS-CoV-2) has caused a pandemic Coronavirus Disease 2019 (COVID-19) that killed more than 3.3 million people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its spike protein to bind a host receptor, the angiotensin-converting enzyme 2 (ACE2), to gain entry. Currently, several mRNA or adenoviral vaccines encoding for the spike protein of SARS-CoV-2 are being used to boost antibodies capable of inhibiting spike-ACE2 interaction and viral entry. However, recent evidence has also suggested an anti-inflammatory effect of spike-reactive antibodies, suggesting that some SARS-CoV-2 spike-based vaccines may elicit protective antibodies capable of inhibiting GM-CSF production and COVID-19 progression.

Abstract There is a broad and growing interest in Bioelectronic Medicine, a dynamic field that continues to generate new approaches in disease treatment. The fourth bioelectronic medicine summit “Technology targeting molecular mechanisms” took place on September 23 and 24, 2020. This virtual meeting was hosted by the Feinstein Institutes for Medical Research, Northwell Health. The summit called international attention to Bioelectronic Medicine as a platform for new developments in science, technology, and healthcare. The meeting was an arena for exchanging new ideas and seeding potential collaborations involving teams in academia and industry. The summit provided a forum for leaders in the field to discuss current progress, challenges, and future developments in Bioelectronic Medicine. The main topics discussed at the summit are outlined here.

Abstract Obesity, a growing health concern, is associated with an increased risk of morbidity and mortality. Chronic low-grade inflammation is implicated in obesity-driven metabolic complications. Peripheral focused ultrasound stimulation (pFUS) is an emerging non-invasive technology that modulates inflammation. Here, we reasoned that focused ultrasound stimulation of the liver may alleviate obesity-related inflammation and other comorbidities. After 8 weeks on a high-fat high-carbohydrate “Western” diet, C57BL/6J mice were subjected to either sham stimulation or focused ultrasound stimulation at the porta hepatis. Daily liver-focused ultrasound stimulation for 8 weeks significantly decreased body weight, circulating lipids and mitigated dysregulation of adipokines. In addition, liver-focused ultrasound stimulation significantly reduced hepatic cytokine levels and leukocyte infiltration. Our findings demonstrate the efficacy of hepatic focused ultrasound for alleviating obesity and obesity-associated complications in mice. These findings suggest a previously unrecognized potential of hepatic focused ultrasound as a possible novel noninvasive approach in the context of obesity.

The pathogenesis of microbial infections and sepsis is partly attributable to dysregulated innate immune responses propagated by late-acting proinflammatory mediators such as procathepsin L (pCTS-L). It was previously not known whether any natural product could inhibit pCTS-L-mediated inflammation or could be strategically developed into a potential sepsis therapy. Here, we report that systemic screening of a NatProduct Collection of 800 natural products led to the identification of a lipophilic sterol, lanosterol (LAN), as a selective inhibitor of pCTS-L-induced production of cytokines [e.g., Tumor Necrosis Factor (TNF) and Interleukin-6 (IL-6)] and chemokines [e.g., Monocyte Chemoattractant Protein-1 (MCP-1) and Epithelial Neutrophil-Activating Peptide (ENA-78)] in innate immune cells. To improve its bioavailability, we generated LAN-carrying liposome nanoparticles and found that these LAN-containing liposomes (LAN-L) similarly inhibited pCTS-L-induced production of several chemokines [e.g., MCP-1, Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES) and Macrophage Inflammatory Protein-2 (MIP-2)] in human blood mononuclear cells (PBMCs). In vivo, these LAN-carrying liposomes effectively rescued mice from lethal sepsis even when the first dose was given at 24 h post the onset of this disease. This protection was associated with a significant attenuation of sepsis-induced tissue injury and systemic accumulation of serval surrogate biomarkers [e.g., IL-6, Keratinocyte-derived Chemokine (KC), and Soluble Tumor Necrosis Factor Receptor I (sTNFRI)]. These findings support an exciting possibility to develop liposome nanoparticles carrying anti-inflammatory sterols as potential therapies for human sepsis and other inflammatory diseases.

Sepsis is a potentially life-threatening systemic inflammatory syndrome characterized by dysregulated host immunological responses to infection. Uncontrolled immune cell activation and exponential elevation in circulating cytokines can lead to sepsis, septic shock, multiple organ dysfunction syndrome, and death. Sepsis is associated with high re-hospitalization and recovery may be incomplete, with long term sequelae including post-sepsis syndrome. Consequently, sepsis continues to be a leading cause of morbidity and mortality across the world. In our recent review of human chorionic gonadotropin (hCG), we noted that its major properties including promotion of fertility, parturition, and lactation were described over a century ago. By contrast, the anti-inflammatory properties of this hormone have been recognized only more recently. Vasopressin, a hormone best known for its anti-diuretic effect, also has anti-inflammatory actions. Surprisingly, vasopressin’s close cousin, oxytocin, has broader and more potent anti-inflammatory effects than vasopressin and a larger number of pre-clinical studies supporting its potential role in limiting sepsis-associated organ damage. This review explores possible links between oxytocin and related octapeptide hormones and sepsis-related modulation of pro-inflammatory and anti-inflammatory activities.

Abstract Background Peripheral nerve reflexes enable organ systems to maintain long-term physiological homeostasis while responding to rapidly changing environmental conditions. Electrical nerve stimulation is commonly used to activate these reflexes and modulate organ function, giving rise to an emerging class of therapeutics called bioelectronic medicines. Dogma maintains that immune cell migration to and from organs is mediated by inflammatory signals (i.e. cytokines or pathogen associated signaling molecules). However, nerve reflexes that regulate immune function have only recently been elucidated, and stimulation of these reflexes for therapeutic effect has not been fully investigated. Methods We utilized both electrical and ultrasound-based nerve stimulation to activate nerve pathways projecting to specific lymph nodes. Tissue and cell analysis of the stimulated lymph node, distal lymph nodes and immune organs is then utilized to measure the stimulation-induced changes in neurotransmitter/neuropeptide concentrations and immune cellularity in each of these sites. Results and conclusions In this report, we demonstrate that activation of nerves and stimulated release of neurotransmitters within a local lymph node results in transient retention of immune cells (e.g. lymphocytes and neutrophils) at that location. Furthermore, such stimulation results in transient changes in neurotransmitter concentrations at distal organs of the immune system, spleen and liver, and mobilization of immune cells into the circulation. This report will enable future studies in which stimulation of these long-range nerve connections between lymphatic and immune organs can be applied for clinical purpose, including therapeutic modulation of cellularity during vaccination, active allergic response, or active auto-immune disease.

Abstract Background Oxygen therapy, using supraphysiological concentrations of oxygen (hyperoxia), is routinely administered to patients who require respiratory support including mechanical ventilation (MV). However, prolonged exposure to hyperoxia results in acute lung injury (ALI) and accumulation of high mobility group box 1 (HMGB1) in the airways. We previously showed that airway HMGB1 mediates hyperoxia-induced lung injury in a mouse model of ALI. Cholinergic signaling through the α7 nicotinic acetylcholine receptor (α7nAChR) attenuates several inflammatory conditions. The aim of this study was to determine whether 3–(2,4 dimethoxy-benzylidene)-anabaseine dihydrochloride, GTS-21, an α7nAChR partial agonist, inhibits hyperoxia-induced HMGB1 accumulation in the airways and circulation, and consequently attenuates inflammatory lung injury. Methods Mice were exposed to hyperoxia (≥99% O2) for 3 days and treated concurrently with GTS-21 (0.04, 0.4 and 4 mg/kg, i.p.) or the control vehicle, saline. Results The systemic administration of GTS-21 (4 mg/kg) significantly decreased levels of HMGB1 in the airways and the serum. Moreover, GTS-21 (4 mg/kg) significantly reduced hyperoxia-induced acute inflammatory lung injury, as indicated by the decreased total protein content in the airways, reduced infiltration of inflammatory monocytes/macrophages and neutrophils into the lung tissue and airways, and improved lung injury histopathology. Conclusions Our results indicate that GTS-21 can attenuate hyperoxia-induced ALI by inhibiting extracellular HMGB1-mediated inflammatory responses. This suggests that the α7nAChR represents a potential pharmacological target for the treatment regimen of oxidative inflammatory lung injury in patients receiving oxygen therapy.

Abstract Background The 2019 novel coronavirus disease (COVID-19) causes for unresolved reasons acute respiratory distress syndrome in vulnerable individuals. There is a need to identify key pathogenic molecules in COVID-19-associated inflammation attainable to target with existing therapeutic compounds. The endogenous damage-associated molecular pattern (DAMP) molecule HMGB1 initiates inflammation via two separate pathways. Disulfide-HMGB1 triggers TLR4 receptors generating pro-inflammatory cytokine release. Extracellular HMGB1, released from dying cells or secreted by activated innate immunity cells, forms complexes with extracellular DNA, RNA and other DAMP or pathogen-associated molecular (DAMP) molecules released after lytic cell death. These complexes are endocytosed via RAGE, constitutively expressed at high levels in the lungs only, and transported to the endolysosomal system, which is disrupted by HMGB1 at high concentrations. Danger molecules thus get access to cytosolic proinflammatory receptors instigating inflammasome activation. It is conceivable that extracellular SARS-CoV-2 RNA may reach the cellular cytosol via HMGB1-assisted transfer combined with lysosome leakage. Extracellular HMGB1 generally exists in vivo bound to other molecules, including PAMPs and DAMPs. It is plausible that these complexes are specifically removed in the lungs revealed by a 40% reduction of HMGB1 plasma levels in arterial versus venous blood. Abundant pulmonary RAGE expression enables endocytosis of danger molecules to be destroyed in the lysosomes at physiological HMGB1 levels, but causing detrimental inflammasome activation at high levels. Stress induces apoptosis in pulmonary endothelial cells from females but necrosis in cells from males. Conclusion Based on these observations we propose extracellular HMGB1 to be considered as a therapeutic target for COVID-19.

Abstract Background Electrical stimulation of peripheral nerves is a widely used technique to treat a variety of conditions including chronic pain, motor impairment, headaches, and epilepsy. Nerve stimulation to achieve efficacious symptomatic relief depends on the proper selection of electrical stimulation parameters to recruit the appropriate fibers within a nerve. Recently, electrical stimulation of the vagus nerve has shown promise for controlling inflammation and clinical trials have demonstrated efficacy for the treatment of inflammatory disorders. This application of vagus nerve stimulation activates the inflammatory reflex, reducing levels of inflammatory cytokines during inflammation. Methods Here, we wanted to test whether altering the parameters of electrical vagus nerve stimulation would change circulating cytokine levels of normal healthy animals in the absence of increased inflammation. To examine this, we systematically tested a set of electrical stimulation parameters and measured serum cytokine levels in healthy mice. Results Surprisingly, we found that specific combinations of pulse width, pulse amplitude, and frequency produced significant increases of the pro-inflammatory cytokine tumor necrosis factor (TNF), while other parameters selectively lowered serum TNF levels, as compared to sham-stimulated mice. In addition, serum levels of the anti-inflammatory cytokine interleukin-10 (IL-10) were significantly increased by select parameters of electrical stimulation but remained unchanged with others. Conclusions These results indicate that electrical stimulation parameter selection is critically important for the modulation of cytokines via the cervical vagus nerve and that specific cytokines can be increased by electrical stimulation in the absence of inflammation. As the next generation of bioelectronic therapies and devices are developed to capitalize on the neural regulation of inflammation, the selection of nerve stimulation parameters will be a critically important variable for achieving cytokine-specific changes.

Abstract The recent opioid crisis is one of the rising challenges in the history of modern health care. New and effective treatment modalities with less adverse effects to alleviate and manage this modern epidemic are critically needed. The FDA has recently approved two non-invasive electrical nerve stimulators for the adjunct treatment of symptoms of acute opioid withdrawal. These devices, placed behind the ear, stimulate certain cranial nerves with auricular projections. This neural stimulation reportedly generates a prompt effect in terms of alleviation of withdrawal symptoms resulting from acute discontinuation of opioid use. Current experimental evidence indicates that this type of non-invasive neural stimulation has excellent potential to supplement medication assisted treatment in opioid detoxification with lower side effects and increased adherence to treatment. Here, we review current findings supporting the use of non-invasive neural stimulation in detoxification from opioid use. We briefly outline the neurophysiology underlying this approach of auricular electrical neural stimulation and its role in enhancing medication assisted treatment in treating symptoms of opioid withdrawal. Considering the growing deleterious impact of addictive disorders on our society, further studies on this emerging treatment modality are warranted.

BackgroundHepatic ischemia and reperfusion (I/R) injury is commonly associated with surgical liver resection or transplantation, and represents a major cause of liver damage and graft failure. Currently, there are no effective therapies to prevent hepatic I/R injury other than ischemic preconditioning and some preventative strategies. Previously, we have revealed the anti-inflammatory activity of a sweat gland-derived peptide, dermcidin (DCD), in macrophage/monocyte cultures. Here, we sought to explore its therapeutic potential and protective mechanisms in a murine model of hepatic I/R.MethodsMale C57BL/6 mice were subjected to hepatic ischemia by clamping the hepatic artery and portal vein for 60 min, which was then removed to initiate reperfusion. At the beginning of reperfusion, 0.2 ml saline control or solution of DCD (0.5 mg/kg BW) or DCD-C34S analog (0.25 or 0.5 mg/kg BW) containing a Cys (C)→Ser (S) substitution at residue 34 was injected via the internal jugular vein. For survival experiments, mice were subjected to additional resection to remove non-ischemic liver lobes, and animal survival was monitored for 10 days. For mechanistic studies, blood and tissue samples were collected at 24 h after the onset of reperfusion, and subjected to measurements of various markers of inflammation and tissue injury by real-time RT-PCR, immunoassays, and histological analysis.ResultsRecombinant DCD or DCD-C34S analog conferred a significant protection against lethal hepatic I/R when given intravenously at the beginning of reperfusion. This protection was associated with a significant reduction in hepatic injury, neutrophilic CXC chemokine (Mip-2) expression, neutrophil infiltration, and associated inflammation. Furthermore, the administration of DCD also resulted in a significant attenuation of remote lung inflammatory injury. Mechanistically, DCD interacted with epidermal growth factor receptor (EGFR), a key regulator of liver inflammation, and significantly inhibited hepatic I/R-induced phosphorylation of EGFR as well as a downstream signaling molecule, protein kinase B (AKT). The suppression of EGFR expression by transducing Egfr-specific shRNA plasmid into macrophages abrogated the DCD-mediated inhibition of nitric oxide (NO) production induced by a damage-associated molecular pattern (DAMP), cold-inducible RNA-binding protein, CIRP.ConclusionsThe present study suggests that human DCD and its analog may be developed as novel therapeutics to attenuate hepatic I/R-induced inflammatory injury possibly by impairing EGFR signaling.

Abstract Background Animal models of diet‐induced obesity (DIO) are commonly used in medical research for mimicking human diseases. There is no universal animal model, and careful evaluation of variety of factors needs to be considered when designing new experiments. Here, we investigated the effect of 9 weeks high‐fat diet (HFD) intervention, providing 60% energy from fat, on parameters of inflammation and insulin resistance in male C57BL/6J mice. Methods Six weeks old mice were initiated on regular diet (RD) or HFD providing 60 kcal energy from fat for 9 weeks. Fasting blood glucose levels were measured by glucometer, and fasting plasma levels of insulin and proinflammatory cytokines by Luminex assay. Insulin sensitivity was evaluated by using QUICKI and HOMA2 indexes. Results HFD mice showed ~ 40% higher body weight and ~ 20% larger abdominal circumference, due to an increase in the white adipose tissue mass. Liver examination revealed increased size and higher hepatic lipid accumulation in livers from HFD mice compared to their RD counterparts. Animals from the HFD group were characterized with significantly higher presence of crown‐like structures (CLS) in WAT and higher plasma levels of proinflammatory cytokines (TNF‐α, IL‐6, leptin, MCP‐1, PAI‐1, and resistin). HFD‐fed mice also demonstrated impaired insulin sensitivity (lower QUICKI, higher HOMA‐insulin resistance (HOMA‐IR), and lower HOMA‐percent sensitivity (HOMA‐%S)) index values. Conclusion Male C57BL/6J mice on 9 weeks HFD providing 60 kcal energy from fat display impaired insulin sensitivity and chronic inflammation, thus making this DIO mouse model appropriate for studies of early stages of obesity‐related pathology.

Abstract Background Glucose is a crucial energy source. In humans, it is the primary sugar for high energy demanding cells in brain, muscle and peripheral neurons. Deviations of blood glucose levels from normal levels for an extended period of time is dangerous or even fatal, so regulation of blood glucose levels is a biological imperative. The vagus nerve, comprised of sensory and motor fibres, provides a major anatomical substrate for regulating metabolism. While prior studies have implicated the vagus nerve in the neurometabolic interface, its specific role in either the afferent or efferent arc of this reflex remains elusive. Methods Here we use recently developed methods to isolate and decode specific neural signals acquired from the surface of the vagus nerve in BALB/c wild type mice to identify those that respond robustly to hypoglycemia. We also attempted to decode neural signals related to hyperglycemia. In addition to wild type mice, we analyzed the responses to acute hypo- and hyperglycemia in transient receptor potential cation channel subfamily V member 1 (TRPV1) cell depleted mice. The decoding algorithm uses neural signals as input and reconstructs blood glucose levels. Results Our algorithm was able to reconstruct the blood glucose levels with high accuracy (median error 18.6 mg/dl). Hyperglycemia did not induce robust vagus nerve responses, and deletion of TRPV1 nociceptors attenuated the hypoglycemia-dependent vagus nerve signals. Conclusion These results provide insight to the sensory vagal signaling that encodes hypoglycemic states and suggest a method to measure blood glucose levels by decoding nerve signals. Trial registration Not applicable.

Abstract Background Rheumatoid arthritis (RA) is a chronic and debilitating inflammatory disease characterized by extensive joint tissue inflammation. Implantable bioelectronic devices targeting the inflammatory reflex reduce TNF production and inflammation in preclinical models of inflammatory disease, and in patients with RA and Crohn’s disease. Here, we assessed the effect of applying a vibrotactile device to the cymba concha of the external ear on inflammatory responses in healthy subjects, as well as its effect on disease activity in RA patients. Methods Six healthy subjects received vibrotactile treatment at the cymba concha, and TNF production was analyzed at different time points post-stimulation. In a separate study, nineteen healthy subjects were enrolled in a randomized cross-over study, and effects of vibrotactile treatment at either the cymba concha or gastrocnemius on cytokine levels were assessed. In addition, the clinical efficacy of vibrotactile treatment on disease activity in RA was assessed in nine patients with RA in a prospective interventional study. Results Vibrotactile treatment at the cymba concha reduced TNF levels, and the suppressive effect persisted up to 24 h. In the cross-over study with 19 healthy subjects, vibrotactile treatment at the cymba concha but not at the gastrocnemius significantly reduced TNF, IL-1β, and IL-6 levels compared to pre-treatment baseline (TNF p

Abstract Background Extracellular high mobility group box 1 protein (HMGB1) serves a central role in inflammation as a transporter protein, which binds other immune-activating molecules that are endocytosed via the receptor for advanced glycation end-products (RAGE). These pro-inflammatory complexes are targeted to the endolysosomal compartment, where HMGB1 permeabilizes the lysosomes. This enables HMGB1-partner molecules to avoid degradation, to leak into the cytosol, and to reach cognate immune-activating sensors. Lipopolysaccharide (LPS) requires this pathway to generate pyroptosis by accessing its key cytosolic receptors, murine caspase 11, or the human caspases 4 and 5. This lytic, pro-inflammatory cell death plays a fundamental pathogenic role in gram-negative sepsis. The aim of the study was to identify molecules inhibiting HMGB1 or HMGB1/LPS cellular internalization. Methods Endocytosis was studied in cultured macrophages using Alexa Fluor-labeled HMGB1 or complexes of HMGB1 and Alexa Fluor-labeled LPS in the presence of an anti-HMGB1 monoclonal antibody (mAb), recombinant HMGB1 box A protein, acetylcholine, the nicotinic acetylcholine receptor subtype alpha 7 (α7 nAChR) agonist GTS-21, or a dynamin-specific inhibitor of endocytosis. Images were obtained by fluorescence microscopy and quantified by the ImageJ processing program (NIH). Data were analyzed using student’s t test or one-way ANOVA followed by the least significant difference or Tukey’s tests. Results Anti-HMGB1 mAb, recombinant HMGB1 antagonist box A protein, acetylcholine, GTS-21, and the dynamin-specific inhibitor of endocytosis inhibited internalization of HMGB1 or HMGB1-LPS complexes in cultured macrophages. These agents prevented macrophage activation in response to HMGB1 and/or HMGB1-LPS complexes. Conclusion These results demonstrate that therapies based on HMGB1 antagonists and the cholinergic anti-inflammatory pathway share a previously unrecognized molecular mechanism of substantial clinical relevance.

Stimulation of peripheral nerve activity may be used to treat metabolic and inflammatory disorders, but current approaches need implanted devices. Here, the authors present a non-invasive approach, and show that ultrasound-mediated stimulation can be targeted to specific sub-organ locations in preclinical models and alter the response of metabolic and inflammatory neural pathways.

Acute respiratory distress syndrome (ARDS) is a critical illness complication that is associated with high mortality. ARDS is documented in severe cases of COVID-19. No effective pharmacological treatments for ARDS are currently available. Dysfunctional immune responses and pulmonary and systemic inflammation are characteristic features of ARDS pathogenesis. Recent advances in our understanding of the regulation of inflammation point to an important role of the vagus-nerve-mediated inflammatory reflex and neural cholinergic signaling. We examined whether pharmacological cholinergic activation using a clinically approved (for myasthenia gravis) cholinergic drug, the acetylcholinesterase inhibitor pyridostigmine alters pulmonary and systemic inflammation in mice with lipopolysaccharide (LPS)-induced ARDS. Male C57Bl/6 mice received one intratracheal instillation of LPS or were sham manipulated (control). Both groups were treated with either vehicle or pyridostigmine (1.5 mg/kg twice daily, 3 mg/day) administered by oral gavage starting at 1 h post-LPS and euthanized 24 h after LPS administration. Other groups were either sham manipulated or received LPS for 3 days and were treated with vehicle or pyridostigmine and euthanized at 72 h. Pyridostigmine treatment reduced the increased total number of cells and neutrophils in the bronchoalveolar lavage fluid (BALF) in mice with ARDS at 24 and 72 h. Pyridostigmine also reduced the number of macrophages and lymphocytes at 72 h. In addition, pyridostigmine suppressed the levels of TNF, IL-1β, IL-6, and IFN-γ in BALF and plasma at 24 and 72 h. However, this cholinergic agent did not significantly altered BALF and plasma levels of the anti-inflammatory cytokine IL-10. Neither LPS nor pyridostigmine affected BALF IFN-γ and IL-10 levels at 24 h post-LPS. In conclusion, treatments with the cholinergic agent pyridostigmine ameliorate pulmonary and systemic inflammatory responses in mice with endotoxin-induced ARDS. Considering that pyridostigmine is a clinically approved drug, these findings are of substantial interest for implementing pyridostigmine in therapeutic strategies for ARDS.

Background: The metabolic syndrome (MetS) is an obesity-associated disorder of pandemic proportions and limited treatment options. Oxidative stress, low-grade inflammation and altered neural autonomic regulation, are important components and drivers of pathogenesis. Galantamine, an acetylcholinesterase inhibitor and a cholinergic drug that is clinically-approved (for Alzheimer's disease) has been implicated in neural cholinergic regulation of inflammation in several conditions characterized with immune and metabolic derangements. Here we examined the effects of galantamine on oxidative stress in parallel with inflammatory and cardio-metabolic parameters in subjects with MetS.Trial Design and Methods: The effects of galantamine treatment, 8 mg daily for 4 weeks or placebo, followed by 16 mg daily for 8 weeks or placebo were studied in randomly assigned subjects with MetS (n = 22 per group) of both genders. Oxidative stress, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase activities, lipid and protein peroxidation, and nitrite levels were analyzed before and at the end of the treatment. In addition, plasma cytokine and adipokine levels, insulin resistance (HOMA-IR) and other relevant cardio-metabolic indices were analyzed. Autonomic regulation was also examined by heart rate variability (HRV) before treatment, and at every 4 weeks of treatment.Results: Galantamine treatment significantly increased antioxidant enzyme activities, including SOD [+1.65 USOD/mg protein, [95% CI 0.39–2.92], P = 0.004] and CAT [+0.93 nmol/mg, [95% CI 0.34–1.51], P = 0.01], decreased lipid peroxidation [thiobarbituric acid reactive substances [log scale 0.72 pmol/mg, [95% CI 0.46–1.07], P = 0.05], and systemic nitrite levels [log scale 0.83 μmol/mg protein, [95% CI 0.57–1.20], P = 0.04] compared with placebo. In addition, galantamine significantly alleviated the inflammatory state and insulin resistance, and decreased the low frequency/high frequency ratio of HRV, following 8 and 12 weeks of drug treatment.Conclusion: Low-dose galantamine alleviates oxidative stress, alongside beneficial anti-inflammatory, and metabolic effects, and modulates neural autonomic regulation in subjects with MetS. These findings are of considerable interest for further studies with the cholinergic drug galantamine to ameliorate MetS.

Abstract Background High Mobility Group Box 1 (HMGB1) was first identified as a nonhistone chromatin-binding protein that functions as a pro-inflammatory cytokine and a Damage-Associated Molecular Pattern molecule when released from necrotic cells or activated leukocytes. HMGB1 consists of two structurally similar HMG boxes that comprise the pro-inflammatory (B-box) and the anti-inflammatory (A-box) domains. Paradoxically, the A-box also contains the epitope for the well-characterized anti-HMGB1 monoclonal antibody “2G7”, which also potently inhibits HMGB1-mediated inflammation in a wide variety of in vivo models. The molecular mechanisms through which the A-box domain inhibits the inflammatory activity of HMGB1 and 2G7 exerts anti-inflammatory activity after binding the A-box domain have been a mystery. Recently, we demonstrated that: 1) the TLR4/MD-2 receptor is required for HMGB1-mediated cytokine production and 2) the HMGB1–TLR4/MD-2 interaction is controlled by the redox state of HMGB1 isoforms. Methods We investigated the interactions of HMGB1 isoforms (redox state) or HMGB1 fragments (A- and B-box) with TLR4/MD-2 complex using Surface Plasmon Resonance (SPR) studies. Results Our results demonstrate that: 1) intact HMGB1 binds to TLR4 via the A-box domain with high affinity but an appreciable dissociation rate; 2) intact HMGB1 binds to MD-2 via the B-box domain with low affinity but a very slow dissociation rate; and 3) HMGB1 A-box domain alone binds to TLR4 more stably than the intact protein and thereby antagonizes HMGB1 by blocking HMGB1 from interacting with the TLR4/MD-2 complex. Conclusions These findings not only suggest a model whereby HMGB1 interacts with TLR4/MD-2 in a two-stage process but also explain how the A-box domain and 2G7 inhibit HMGB1.

Abstract Background The vagus nerve plays an important role in the regulation of organ function, including reflex pathways that regulate immunity and inflammation. Recent studies using genetically modified mice have improved our understanding of molecular mechanisms in the neural control of immunity. However, mapping neural signals transmitted in the vagus nerve in mice has been limited by technical challenges. Here, we have standardized an experimental protocol to record compound action potentials transmitted in the vagus nerve. Methods The vagus nerve was isolated in Balb/c and B6.129S mice, and placed either on a hook or cuff electrode. The electrical signals from the vagus nerve were digitized using either a Neuralynx or Plexon data acquisition system. Changes in the vagus nerve activity in response to anesthesia, feeding and administration of bacterial endotoxin were analyzed. Results We have developed an electrophysiological recording system to record compound action potentials from the cervical vagus nerve in mice. Cuff electrodes significantly reduce background noise and increase the signal to noise ratio as compared to hook electrodes. Baseline vagus nerve activity varies in response to anesthesia depth and food intake. Analysis of vagus neurograms in different mouse strains (Balb/c and C57BL/6) reveal no significant differences in baseline activity. Importantly, vagus neurogramactivity in wild type and TLR4 receptor knock out mice exhibits receptor dependency of endotoxin mediated signals. Conclusions These methods for recording vagus neurogram in mice provide a useful tool to further delineate the role of vagus neural pathways in a standardized murine disease model.

Abstract Background The NLRP3 inflammasome, a cytosolic complex that mediates the maturation of IL-1β and IL-18 as well as the release of high mobility group box 1 (HMGB1), contributes to the lethality of endotoxic shock. Ethyl pyruvate (EP) was previously shown to inhibit HMGB1 release and promote survival during endotoxemia and experimental sepsis. However, the underlying protective mechanism remains elusive. Result EP dose-dependently inhibited the ATP-, nigericin-, alum-, and silica-induced caspase-1 activation and HMGB1 release in mouse macrophages. EP failed to inhibit DNA transfection- or Salmonella Typhimurium-induced caspase-1 activation and HMGB1 release. Mechanistically, EP significantly attenuated mitochondrial damage and cytoplasmic translocation of mitochondrial DNA, a known NLRP3 ligand, without influencing the potassium efflux, the lysosomal rupture or the production of mitochondrial reactive oxygen species (mtROS). Conclusion Ethyl pyruvate acts as a novel NLRP3 inflammasome inhibitor that preserves the integrity of mitochondria during inflammation.

High mobility group box 1 protein (HMGB1), a highly conserved nuclear DNA-binding protein, is a “damage-associated molecular pattern” molecule (DAMP) implicated in both stimulating and inhibiting innate immunity. As reviewed here, HMGB1 is an oxidation-reduction sensitive DAMP bearing three cysteines, and the post-translational modification of these residues establishes its proinflammatory and anti-inflammatory activities by binding to different extracellular cell surface receptors. The redox-sensitive signaling mechanisms of HMGB1 also occupy an important niche in innate immunity because HMGB1 may carry other DAMPs and pathogen-associated molecular pattern molecules (PAMPs). HMGB1 with DAMP/PAMP cofactors bind to the receptor for advanced glycation end products (RAGE) which internalizes the HMGB1 complexes by endocytosis for incorporation in lysosomal compartments. Intra-lysosomal HMGB1 disrupts lysosomal membranes thereby releasing the HMGB1-transported molecules to stimulate cytosolic sensors that mediate inflammation. This HMGB1-DAMP/PAMP cofactor pathway slowed the development of HMGB1-binding antagonists for diagnostic or therapeutic use. However, recent discoveries that HMGB1 released from neurons mediates inflammation via the TLR4 receptor system, and that cancer cells express fully oxidized HMGB1 as an immunosuppressive mechanism, offer new paths to targeting HMGB1 for inflammation, pain, and cancer.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s10020-020-00264-1

Abstract The axons of the sensory, or afferent, vagus nerve transmit action potentials to the central nervous system in response to changes in the body’s metabolic and physiological status. Recent advances in identifying neural circuits that regulate immune responses to infection, inflammation and injury have revealed that vagus nerve signals regulate the release of cytokines and other factors produced by macrophages. Here we record compound action potentials in the cervical vagus nerve of adult mice and reveal the specific activity that occurs following administration of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin 1β (IL-1β). Importantly, the afferent vagus neurograms generated by TNF exposure are abolished in double knockout mice lacking TNF receptors 1 and 2 (TNF-R1/2KO), whereas IL-1 β-specific neurograms are eliminated in knockout mice lacking IL-1 β receptor (IL-1RKO). Conversely, TNF neurograms are preserved in IL-1RKO mice, and IL-1 β neurograms are unchanged in TNF-R1/2KO mice. Analysis of the temporal dynamics and power spectral characteristics of afferent vagus neurograms for TNF and IL-1β reveals cytokine-selective signals. The nodose ganglion contains the cell bodies of the sensory neurons whose axons run through the vagus nerve. The nodose neurons express receptors for TNF and IL-1β, and we show that exposing them to TNF and IL-1β significantly stimulates their calcium uptake. Together these results indicate that afferent vagus signals in response to cytokines provide a basic model of nervous system sensing of immune responses.

Abstract When pathogens and toxins breech the epithelial barrier, antigens are transported by the lymphatic system to lymph nodes. In previously immunized animals, antigens become trapped in the draining lymph nodes, but the underlying mechanism that controls antigen restriction is poorly understood. Here we describe the role of neurons in sensing and restricting antigen flow in lymph nodes. The antigen keyhole-limpet hemocyanin (KLH) injected into the mouse hind paw flows from the popliteal lymph node to the sciatic lymph node, continuing through the upper lymphatics to reach the systemic circulation. Re-exposure to KLH in previously immunized mice leads to decreased flow from the popliteal to the sciatic lymph node as compared with naïve mice. Administering bupivacaine into the lymph node region restores antigen flow in immunized animals. In contrast, neural activation using magnetic stimulation significantly decreases antigen trafficking in naïve animals as compared with sham controls. Ablating NaV1.8 + sensory neurons significantly reduces antigen restriction in immunized mice. Genetic deletion of FcγRI/FcεRI also reverses the antigen restriction. Colocalization of PGP9.5-expressing neurons, FcγRI receptors and labeled antigen occurs at the antigen challenge site. Together, these studies reveal that neuronal circuits modulate antigen trafficking through a pathway that requires NaV1.8 and FcγR.

Neural reflexes regulate inflammation and electrical activation of the vagus nerve reduces inflammation in models of inflammatory disease. These discoveries have generated an increasing interest in targeted neurostimulation as treatment for chronic inflammatory diseases. Data from the first clinical trials that use vagus nerve stimulation (VNS) in treatment of rheumatoid arthritis and Crohn’s disease suggest that there is a therapeutic potential of electrical VNS in diseases characterized by excessive inflammation. Accordingly, there is an interest to further explore the molecular mechanisms and therapeutic potential of electrical VNS in a range of experimental settings and available genetic mouse models of disease. Here, we describe a method for electrical VNS in experimental inflammation in mice.

The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNFα) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer's disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation.

High-mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that is released during tissue damage from immune and non-immune cells — including microglia and neurons. HMGB1 can contribute to progression of numerous chronic inflammatory and autoimmune diseases which is mediated in part by interaction with the receptor for advanced glycation endproducts (RAGE). There is increasing evidence from in vitro studies that HMGB1 may link the two main pathophysiological components of Parkinson's disease (PD), i.e. progressive dopaminergic degeneration and chronic neuroinflammation which underlie the mechanistic basis of PD progression.Analysis of tissue and biofluid samples from PD patients, showed increased HMGB1 levels in human postmortem substantia nigra specimens as well as in the cerebrospinal fluid and serum of PD patients. In a mouse model of PD induced by sub-acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), systemic administration of neutralizing antibodies to HMGB1 partly inhibited the dopaminergic cell death, and reduced the increase of RAGE and tumour necrosis factor-alpha. The small natural molecule glycyrrhizin, a component from liquorice root which can directly bind to HMGB1, both suppressed MPTP-induced HMGB1 and RAGE upregulation while reducing MPTP-induced dopaminergic cell death in a dose dependent manner.These results provide first in vivo evidence that HMGB1 serves as a powerful bridge between progressive dopaminergic neurodegeneration and chronic neuroinflammation in a model of PD, suggesting that HMGB1 is a suitable target for neuroprotective trials in PD.