key: cord-0986862-kbxglb9j authors: Corriden, Ross; Schmidt, Benjamin E.; Olson, Joshua; Okerblom, Jonathan; Silva, Jorge Masso; Nizet, Victor; Meier, Angela title: Dexmedetomidine does not directly inhibit neutrophil extracellular trap production date: 2021-11-19 journal: Br J Anaesth DOI: 10.1016/j.bja.2021.11.015 sha: bc0b6cae8f9c937656cdba375a5368a9169b2eee doc_id: 986862 cord_uid: kbxglb9j nan Editor-Dexmedetomidine is a highly selective α2-adrenergic receptor agonist widely used in clinical anaesthesia as a sedative and analgesic agent 1 . It has also been associated with numerous anti-inflammatory effects in preclinical models, including protection against leukocyte-mediated acute lung injury (ALI) following caecal ligation-puncture 2 , reducing pulmonary oedema in lipopolysaccharide-induced ALI 3 , and attenuating cell injury in experimental severe acute pancreatitis via the cholinergic anti-inflammatory pathway 4 . In a recent case report, clinical improvement upon dexmedetomidine treatment was suggested to have spared a patient with COVID-19 with worsening hypoxaemia from mechanical ventilation 5 . Indeed, there are ongoing clinical trials registered to examine dexmedetomidine in palliative sedation for severe COVID-19 (NCT04350086) and to evaluate its immunomodulatory profile in patients recovering from COVID-19-elated acute respiratory distress syndrome (ARDS) (NCT04413864). Neutrophil Extracellular Trap formation (NETosis) is a specialized cell death process in which release of chromatin components such as DNA and histones provides a framework for trapping and killing invading microbes 6 . However, when dysregulated, NETosis can also aggravate harmful inflammatory responses, including those driving the pathogenesis and thrombosis of severe COVID-19 in lungs and other major organs 7, 8 . In 2020, Jain and collegaues 9 hypothesized that "given the anti-inflammatory effects of dexmedetomidine, it too may inhibit NETosis and be beneficial in COVID-19 patients". They went on to provide a detailed schematic illustration of the many feedforward mechanisms potentiating NETosis during COVID-19 and the molecular pathways through which they predicted dexmedetomidine could act to inhibit NET activation. Our research group has a longstanding interest in the biology and pathobiology of NETs in animal models of infectious diseases such as necrotizing fasciitis 10 and bacterial pneumonia 11 , and recently we studied NET phenotypes in critically ill patients with COVID-19 12 . In parallel, we have examined how NETosis is modulated by common medications including statins 13 , tamoxifen 14 , desferoxamine 15 , and propofol 16 . With this background, we tested the hypothesis that dexmedetomidine inhibits human NETosis. Blood was collected from healthy adults under a protocol approved by the UC San Diego IRB, and neutrophils were isolated using the PolyMorphPrep TM Kit (Fresenius Kabi, location) per manufacturer's instructions. The effective sedative concentration of dexmedetomidine in plasma has been estimated to be 0.2 to 3.2 ng ml -1 (x-xx nM). 17 We stimulated neutrophils to produce NETs by exposure to live methicillin-resistant Staphylococcus aureus (MRSA) or to the classical NET inducer phorbol myristate acetate (PMA) at 25 nM, in the presence or absence of dexmedetomidine at final concentrations of 0.5, 5, 50 and 500 ng ml -1 . For all dexmedetomidine exposures, no inhibition of NET production by MRSA-or PMA-stimulated neutrophils was seen by PICO-green quantification of extracellular DNA release (Fig. 1A) or immunocytochemistry using antibodies against myeloperoxidase (Fig. 1B) . Neutrophil oxidative burst/generation of reactive oxygen species (ROS) can promote NETosis 18 . We found that dexmedetomidine at final concentrations of 0.5, 5.0 and 50 ng ml -1 did not inhibit PMA-induced neutrophil ROS production as measured by a 2',7'dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma-Aldrich, location) fluorescence assay (Fig. 1C) . Examining broader neutrophil functions, we found that similar dexmedetomidine concentrations did not significantly affect neutrophil chemotaxis across a Transwell membrane toward N-formylmethionyl-leucyl-phenylalanine (fMLP) (Fig. 1D) , nor did it influence the efficiency of neutrophil phagocytosis of S. aureus-coated particles (pHrodo™ Red S. aureus Bioparticles; Invitrogen, location; Fig. 1E ). In an ex vivo bactericidal assay, dexmedetomidine (5 ng ml -1 ) impaired neutrophil killing of MRSA, an effect that was reversed by the α2-adrenergic receptor antagonist yohimbine (Fig. 1F) while yohimbine alone did not significantly affect killing (Fig. 1G) . Finally, in a murine intraperitoneal MRSA infection model approved by the UC San Diego IACUC, treatment with 166 μg kg -1 of dexmedetomidine i.p. at time of infection and again 1 h after bacterial challenge was associated with significantly increased recovery of bacterial colony-forming units (CFU) from kidneys 24 h later (Fig. 1H) , though no change was seen in CFUs recovered from the liver. We conclude that dexmedetomidine at therapeutically relevant concentrations and higher does not directly inhibit production of NETs by human neutrophils in response to commonly used NETosis inducers, nor does it significantly alter neutrophil behaviour in selected other common phenotypic assays including ROS generation, chemotaxis, and phagocytosis. Dexmedetomidine slightly but significantly (a) impaired human neutrophil killing of MRSA in an α2-adrenergic receptor-dependent manner and (b) reduced kidney bacterial burden in a murine systemic infection model, but it is premature to conclude whether these modest phenotypes are related or clinically significant for humans. Of note, dexmedetomidine is mainly hepatically metabolised and can reach liver concentrations much higher than plasma, after which its metabolites are primarily excreted through the kidneys 19 . Our study has several limitations. First, we describe in vitro studies with purified human neutrophils and in vivo studies using mice, both relatively distant from the clinical setting. Second, the stimuli used to trigger NETosis and other neutrophil effector functions, while commonly used in the field, are not of viral origin. Follow-up ex vivo studies using COVID-19 patient blood, along with in vitro studies using activators of viral origin, will be important. Several anaesthetic drugs are known to possess important anti-inflammatory and immunomodulatory properties, including those acting on neutrophils 20 , that can influence their pharmacodynamics and clinical effectiveness. Of immediate impact, there is emerging clinical opinion that the immunomodulatory activities of dexmedetomidine might be harnessed to improve patient outcomes in severe COVID-19 9, 21 . Our studies, with the stated limitations, suggest that the proposed benefits do not include direct inhibition of extracellular trap formation by human neutrophils. RC: conceptualized and conducted experiments, analysed data, edited manuscript BES: conducted experiments, analysed data JOl: conducted experiments JOk: helped to conduct initial concept experiments JMS: conducted imaging experiment VN: conceptualized project, interpreted data, co-wrote manuscript AM * : conceptualized project, conducted experiments, analysed data, co-wrote manuscript Dexmedetomidine: A Review of Its Use for Sedation in the Intensive Care Setting Dose-related effects of dexmedetomidine on sepsis-initiated lung injury in rats Dexmedetomidine alleviates pulmonary edema through the epithelial sodium channel (ENaC) via the PI3K/Akt/Nedd4-2 pathway in LPSinduced acute lung injury Dexmedetomidine attenuates inflammation and pancreatic injury in a rat model of experimental severe acute pancreatitis via cholinergic anti-inflammatory pathway Dexmedetomidine and worsening hypoxemia in the setting of COVID-19: A case report Neutrophil extracellular traps kill bacteria The Emerging Role of Neutrophils in the Pathogenesis of Thrombosis in COVID-19 The Role of Neutrophil NETosis in Organ Injury: Novel Inflammatory Cell Death Mechanisms Dexmedetomidine: another arrow in the quiver to fight COVID-19 in intensive care units DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps Increased peripheral blood neutrophil activation phenotypes and NETosis in critically ill COVID-19 patients: a case series and review of the literature Statins enhance formation of phagocyte extracellular traps Tamoxifen augments the innate immune function of neutrophils through modulation of intracellular ceramide Iron-chelating agent desferrioxamine stimulates formation of neutrophil extracellular traps (NETs) in human blood-derived neutrophils Inhibition of Human Neutrophil Extracellular Trap (NET) Production by Propofol and Lipid Emulsion The effects of increasing plasma concentrations of dexmedetomidine in humans The impact of various reactive oxygen species on the formation of neutrophil extracellular traps Clinical Pharmacokinetics and Pharmacodynamics of Dexmedetomidine Impact of Anesthetics on Human Neutrophil Function Potential therapeutic value of dexmedetomidine in COVID-19 patients admitted to ICU Blue: DAPI (staining nuclei). C: Time course of reactive oxygen species (ROS) production by human neutrophils (measured at indicated time points using H2DCFDA, n =3) in the presence or absence of dexmedetomidine either alone or with PMA. D: Chemotaxis of human neutrophils in response to 100 nM N-formylmethionyl-leucyl phenylalanine (fMLP) in the presence or absence of several concentrations of dexmedetomidine (assessed using Transwell inserts with a 3 µm pore size as described previously, n =5). E: Phagocytosis time course of S. aureus bioparticles in the presence and absence of several concentrations of dexmedetomidine, n =4. F MRSA killing by human neutrophils (MOI 10) in the presence of dexmedetomidine at 5 ng ml -1 and dexmedetomidine and yohimbine as compared to the respective control without cells expressed as % colony-forming units (CFU)/ml, n =8. G: MRSA killing by human neutrophils (MOI 10) in the presence of several concentrations of yohimbine as compared to the respective control without cells expressed as % CFU/ml The authors declare no conflicts of interest.