Autonomic Regulation of Inflammation in Conscious Animals.

Bioelectronic medicine is a novel field in modern medicine based on the specific neuronal stimulation to control organ function, cardiovascular, and immune homeostasis. However, most studies addressing neuromodulation of the immune system have been conducted on anesthetized animals, which can affect the nervous system and neuromodulation. Here, we review recent studies involving conscious experimental rodents (rats and mice) to better understand the functional organization of neural control of immune homeostasis. We highlight typical experimental models of cardiovascular regulation, such as electrical activation of the aortic depressor nerve or the carotid sinus nerve, bilateral carotid occlusion, the Bezold-Jarisch reflex, and intravenous administration of the bacterial endotoxin lipopolysaccharide. These models have been used to investigate the relationship between neuromodulation of the cardiovascular and immune systems in conscious rodents (rats and mice). These studies provide critical information about the neuromodulation of the immune system, particularly the role of the autonomic nervous system, i.e., the sympathetic and parasympathetic branches acting both centrally (hypothalamus, nucleus ambiguus, nucleus tractus solitarius, caudal ventrolateral medulla, and rostral ventrolateral medulla), and peripherally (particularly spleen and adrenal medulla). Overall, the studies in conscious experimental models have certainly highlighted to the reader how the methodological approaches used to investigate cardiovascular reflexes in conscious rodents (rats and mice) can also be valuable for investigating the neural mechanisms involved in inflammatory responses. The reviewed studies have clinical implications for future therapeutic approaches of bioelectronic modulation of the nervous system to control organ function and physiological homeostasis in conscious physiology.

Pyridostigmine reduces mortality of patients with severe SARS-CoV-2 infection: A phase 2/3 randomized controlled trial.

BACKGROUND: Respiratory failure in severe coronavirus disease 2019 (COVID-19) is associated with a severe inflammatory response. Acetylcholine (ACh) reduces systemic inflammation in experimental bacterial and viral infections. Pyridostigmine increases the half-life of endogenous ACh, potentially reducing systemic inflammation. We aimed to determine if pyridostigmine decreases a composite outcome of invasive mechanical ventilation (IMV) and death in adult patients with severe COVID-19. METHODS: We performed a double-blinded, placebo-controlled, phase 2/3 randomized controlled trial of oral pyridostigmine (60 mg/day) or placebo as add-on therapy in adult patients admitted due to confirmed severe COVID-19 not requiring IMV at enrollment. The primary outcome was a composite of IMV or death by day 28. Secondary outcomes included reduction of inflammatory markers and circulating cytokines, and 90-day mortality. Adverse events (AEs) related to study treatment were documented and described. RESULTS: We recruited 188 participants (94 per group); 112 (59.6%) were men; the median (IQR) age was 52 (44-64) years. The study was terminated early due to a significant reduction in the primary outcome in the treatment arm and increased difficulty with recruitment. The primary outcome occurred in 22 (23.4%) participants in the placebo group vs. 11 (11.7%) in the pyridostigmine group (hazard ratio, 0.47, 95% confidence interval 0.24-0.9; P = 0.03). This effect was driven by a reduction in mortality (19 vs. 8 deaths, respectively). CONCLUSION: Our data indicate that adding pyridostigmine to standard care reduces mortality among patients hospitalized for severe COVID-19.

Behavioural Fever Promotes an Inflammatory Reflex Circuit in Ectotherms.

BACKGROUND: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. METHODS: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. RESULTS: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfß in target tissues. CONCLUSION: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.

A parallel-group, multicenter randomized, double-blinded, placebo-controlled, phase 2/3, clinical trial to test the efficacy of pyridostigmine bromide at low doses to reduce mortality or invasive mechanical ventilation in adults with severe SARS-CoV-2 infection: the Pyridostigmine In Severe COvid-19 (PISCO) trial protocol.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the causative agent of coronavirus disease 2019 (COVID-19), may lead to severe systemic inflammatory response, pulmonary damage, and even acute respiratory distress syndrome (ARDS). This in turn may result in respiratory failure and in death. Experimentally, acetylcholine (ACh) modulates the acute inflammatory response, a neuro-immune mechanism known as the inflammatory reflex. Recent clinical evidence suggest that electrical and chemical stimulation of the inflammatory reflex may reduce the burden of inflammation in chronic inflammatory diseases. Pyridostigmine (PDG), an ACh-esterase inhibitor (i-ACh-e), increases the half-life of endogenous ACh, therefore mimicking the inflammatory reflex. This clinical trial is aimed at evaluating if add-on of PDG leads to a decrease of invasive mechanical ventilation and death among patients with severe COVID-19. METHODS: A parallel-group, multicenter, randomized, double-blinded, placebo-controlled, phase 2/3 clinical trial to test the efficacy of pyridostigmine bromide 60 mg/day P.O. to reduce the need for invasive mechanical ventilation and mortality in hospitalized patients with severe COVID-19. DISCUSSION: This study will provide preliminary evidence of whether or not -by decreasing systemic inflammation- add-on PDG can improve clinical outcomes in patients with severe COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov NCT04343963 (registered on April 14, 2020).

Neuroautonomic activity evidences parturition as a complex and integrated neuro-immune-endocrine process.

Parturition in mammals demands a precise coordination of several neuro-immune-endocrine interactions including: a sterile inflammatory response that involves secretion of inflammation mediators like cytokines/chemokines; changes in the secretion of hormones such as progestogen, estrogens, cortisol, and oxytocin; as well as adjustments of the neuroautonomic function. Specifically, the so-called cholinergic anti-inflammatory pathway seems to play a key role in the homeostasis of the neuro-immune-endocrine axis by adjusting the vagus nerve activity during parturition. Here, we provide insights into the importance of the vagus during parturition from an autonomic, endocrine, and immune interplay perspective, and describe the potential role of heart rate variability analysis to explore these interactions noninvasively, economically, and accessibly.

Sign and magnitude scaling properties of heart rate fluctuations following vagus nerve stimulation in a patient with drug-resistant epilepsy.

Vagus nerve stimulation (VNS) therapy has been recently incorporated in Latin America as a treatment for drug-resistant epilepsy. In particular, it is known that linear analysis and fractal parameters of heart rate variability (HRV) are able to indirectly measure cardiac autonomic activity. This case report presents a 17-year-old female with drug-resistant epilepsy implanted with a VNS device. In order to explore cardiac autonomic changes due to VNS, linear and fractal HRV indices were calculated in the presence and absence of neurostimulation. Novel fractal scaling exponents from HRV analysis were obtained from this patient and from a healthy control subject. Our results indicate that fractal indices of HRV, such as short-term scaling parameters from magnitude and sign analyses seem to be sensitive to the presence or absence of VNS, being confirmed by linear classical methods. This study shows that VNS therapy increases the complexity of cardiac fluctuations in a patient with drug-resistant epilepsy, reflecting an augmented HRV non-linearity and a diminished anticorrelated pattern in heart rate fluctuations. A potential clinical use of these parameters includes the early identification of bradycardia, sudden unexpected death (SUDEP) risk and preoperative VNS approaches. Thus, the scaling and magnitude properties of HRV have potential importance as a non-invasive and easy method for adequate diagnostic/prognostic implications in epilepsy treatment.

Stimulation of nAchRα7 Receptor Inhibits TNF Synthesis and Secretion in Response to LPS Treatment of Mast Cells by Targeting ERK1/2 and TACE Activation.

The cholinergic anti-inflammatory pathway is recognized as one of the main mechanisms of neuromodulation of the immune system. Activation of the α7 nicotinic acetylcholine receptor (nAchRα7) suppresses cytokine synthesis in distinct immune cells but the molecular mechanisms behind this effect remain to be fully described. Mast cells (MCs) are essential players of allergic reactions and innate immunity responses related to chronic inflammation. Activation of TLR4 receptor in MCs leads to the rapid secretion of pre-synthesized TNF from intracellular pools and to the activation of NFκB, necessary for de novo synthesis of TNF and other cytokines. Here we report that the nAchRα7 receptor specific agonist GTS-21 inhibits TLR4-induced secretion of preformed TNF from MCs in vivo and in vitro. Utilizing bone marrow-derived mast cells (BMMCs) it was found that GTS-21 also diminished secretion of de novo synthesized TNF, TNF mRNA accumulation and IKK-dependent p65-NFκB phosphorylation in response to LPS. nAchRα7 triggering prevented TLR4-induced ERK1/2 phosphorylation, which resulted an essential step for TNF secretion due to the phosphorylation of the metallopeptidase responsible for TNF maturation (TACE). Main inhibitory actions of GTS-21 were prevented by AG490, an inhibitor of JAK-2 kinase. Our results show for the first time, that besides the prevention of NFκB-dependent transcription, inhibitory actions of nAchRα7 triggering include the blockade of pathways leading to exocytosis of granule-stored cytokines in MCs.

Increase in cholinergic modulation with pyridostigmine induces anti-inflammatory cell recruitment soon after acute myocardial infarction in rats.

We tested the hypothesis that an increase in the anti-inflammatory cholinergic pathway, when induced by pyridostigmine (PY), may modulate subtypes of lymphocytes (CD4+, CD8+, FOXP3+) and macrophages (M1/M2) soon after myocardial infarction (MI) in rats. Wistar rats, randomly allocated to receive PY (40 mg·kg(-1)·day(-1)) in drinking water or to stay without treatment, were followed for 4 days and then were subjected to ligation of the left coronary artery. The groups-denominated as the pyridostigmine-treated infarcted (IP) and infarcted control (I) groups-were submitted to euthanasia 3 days after MI; the heart was removed for immunohistochemistry, and the peripheral blood and spleen were collected for flow cytometry analysis. Noninfarcted and untreated rats were used as controls (C Group). Echocardiographic measurements were registered on the second day after MI, and heart rate variability was measured on the third day after MI. The infarcted groups had similar MI areas, degrees of systolic dysfunction, blood pressures, and heart rates. Compared with the I Group, the IP Group showed a significant higher parasympathetic modulation and a lower sympathetic modulation, which were associated with a small, but significant, increase in diastolic function. The IP Group showed a significant increase in M2 macrophages and FOXP3(+)cells in the infarcted and peri-infarcted areas, a significantly higher frequency of circulating Treg cells (CD4(+)CD25(+)FOXP3(+)), and a less extreme decrease in conventional T cells (CD25(+)FOXP3(-)) compared with the I Group. Therefore, increasing cholinergic modulation with PY induces greater anti-inflammatory cell recruitment soon after MY in rats.