Inflammasome activation in neutrophils of patients with severe COVID-19

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Aymonnier, K., Ng, J., Fredenburgh, L. E., Zambrano-Vera, K., Muenzer, P., Gutch, S., Fukui, S., Desjardins, M., Subramaniam, M., Baron, R. M., Raby, B. A., Perrella, M. A., Lederer, J. A., & Wagner, D. D. Inflammasome activation in neutrophils of patients with severe COVID-19. Blood Advances, 6(7), (2022): 2001–2013, https://doi.org/10.1182/bloodadvances.2021005949.Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-CoV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation (ie, apoptosis-associated speck-like protein containing a CARD [ASC] speck assembly) and timing relative to NETosis in stimulated neutrophils by real-time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that ∼2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in lipopolysaccharide -stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.P.M. received an Individual Marie Skłodowska-Curie Actions fellowship by the European Commission (796365 - COAGULANT). This work was supported by the National Institutes of Health (NIH)/Research Program Award grant R35 HL135765 (D.W.), by the NIH/National Heart, Lung, and Blood Institute grant T32 HL007633-35 (J.N.), and by the NIH/National Institute of Allergy and Infectious Diseases grant U01AI138318 (J.L and M.P); by the Massachusetts Consortium on Pathogen Readiness (MassCPR) Evergrande COVID‐19 Response Fund Award to B.R.; and by a generous gift to D.W. from the Steven Berzin family

Circulating N-formylmethionine and metabolic shift in critical illness : a multicohort metabolomics study

Funding Information: KN is supported by Foundation for the National Institutes of Health (NIH)/National Center for Advancing Translational Sciences grant KL2-TR-002385, R01 HL123915. AJR is supported by NIH grant R01 HL152083. LEF is supported by NIH grant R01 HL114839. RMB is supported by NIH grants R01 HL142093 and R01 GM115605. KBC is supported by NIH grant R01 GM115774. The VITdAL-ICU trial was supported by the European Society for Clinical Nutrition and Metabolism (ESPEN), a research grant including provision of study medication from Fresenius Kabi (Germany), and the Austrian National Bank (Jubiläumsfonds, Project Nr. 14143). Landspitali University Hospital Science Fund: A2021-03 Publisher Copyright: © 2022, The Author(s). © 2022. The Author(s).BACKGROUND: Cell stress promotes degradation of mitochondria which release danger-associated molecular patterns that are catabolized to N-formylmethionine. We hypothesized that in critically ill adults, the response to N-formylmethionine is associated with increases in metabolomic shift-related metabolites and increases in 28-day mortality. METHODS: We performed metabolomics analyses on plasma from the 428-subject Correction of Vitamin D Deficiency in Critically Ill Patients trial (VITdAL-ICU) cohort and the 90-subject Brigham and Women's Hospital Registry of Critical Illness (RoCI) cohort. In the VITdAL-ICU cohort, we analyzed 983 metabolites at Intensive Care Unit (ICU) admission, day 3, and 7. In the RoCI cohort, we analyzed 411 metabolites at ICU admission. The association between N-formylmethionine and mortality was determined by adjusted logistic regression. The relationship between individual metabolites and N-formylmethionine abundance was assessed with false discovery rate correction via linear regression, linear mixed-effects, and Gaussian graphical models. RESULTS: Patients with the top quartile of N-formylmethionine abundance at ICU admission had a significantly higher adjusted odds of 28-day mortality in the VITdAL-ICU (OR, 2.4; 95%CI 1.5-4.0; P = 0.001) and RoCI cohorts (OR, 5.1; 95%CI 1.4-18.7; P = 0.015). Adjusted linear regression shows that with increases in N-formylmethionine abundance at ICU admission, 55 metabolites have significant differences common to both the VITdAL-ICU and RoCI cohorts. With increased N-formylmethionine abundance, both cohorts had elevations in individual short-chain acylcarnitine, branched chain amino acid, kynurenine pathway, and pentose phosphate pathway metabolites. CONCLUSIONS: The results indicate that circulating N-formylmethionine promotes a metabolic shift with heightened mortality that involves incomplete mitochondrial fatty acid oxidation, increased branched chain amino acid metabolism, and activation of the pentose phosphate pathway.Peer reviewe

Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation

Background: Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients. Methods and Findings: Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women's Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6–15.8, p = 1×10−7) and ME ARDS (OR 8.4, 95% CI 2.9–24.2, p = 9×10−5) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10−4) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers. Conclusions: Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients. Please see later in the article for the Editors' Summar

Plasma mitochondrial DNA and metabolomic alterations in severe critical illness

Abstract Background Cell-free plasma mitochondrial DNA (mtDNA) levels are associated with endothelial dysfunction and differential outcomes in critical illness. A substantial alteration in metabolic homeostasis is commonly observed in severe critical illness. We hypothesized that metabolic profiles significantly differ between critically ill patients relative to their level of plasma mtDNA. Methods We performed a metabolomic study with biorepository plasma samples collected from 73 adults with systemic inflammatory response syndrome or sepsis at a single academic medical center. Patients were treated in a 20-bed medical ICU between 2008 and 2010. To identify key metabolites and metabolic pathways related to plasma NADH dehydrogenase 1 (ND1) mtDNA levels in critical illness, we first generated metabolomic data using gas and liquid chromatography-mass spectroscopy. We performed fold change analysis and volcano plot visualization based on false discovery rate-adjusted p values to evaluate the distribution of individual metabolite concentrations relative to ND1 mtDNA levels. We followed this by performing orthogonal partial least squares discriminant analysis to identify individual metabolites that discriminated ND1 mtDNA groups. We then interrogated the entire metabolomic profile using pathway overrepresentation analysis to identify groups of metabolite pathways that were different relative to ND1 mtDNA levels. Results Metabolomic profiles significantly differed in critically ill patients with ND1 mtDNA levels ≥ 3200 copies/μl plasma relative to those with an ND1 mtDNA level < 3200 copies/μl plasma. Several analytical strategies showed that patients with ND1 mtDNA levels ≥ 3200 copies/μl plasma had significant decreases in glycerophosphocholines and increases in short-chain acylcarnitines. Conclusions Differential metabolic profiles during critical illness are associated with cell-free plasma ND1 mtDNA levels that are indicative of cell damage. Elevated plasma ND1 mtDNA levels are associated with decreases in glycerophosphocholines and increases in short-chain acylcarnitines that reflect phospholipid metabolism dysregulation and decreased mitochondrial function, respectively