Markers of NETosis and DAMPs are altered in critically ill COVID-19 patients

Background Coronavirus disease 19 (COVID-19) is known to present with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels of extracellular histone H3 (H3), neutrophil elastase (NE) and cfDNA in relation to other plasma parameters, including the immune modulators GAS6 and AXL, ICU scoring systems and mortality in patients with severe COVID-19. Methods We measured plasma H3, NE, cfDNA, GAS6 and AXL concentration in plasma of 83 COVID-19-positive and 11 COVID-19-negative patients at admission to the Intensive Care Unit (ICU) at the Uppsala University hospital, a tertiary hospital in Sweden and a total of 333 samples obtained from these patients during the ICU-stay. We determined their correlation with disease severity, organ failure, mortality and other blood parameters. Results H3, NE, cfDNA, GAS6 and AXL were increased in plasma of COVID-19 patients compared to controls. cfDNA and GAS6 decreased in time in in patients surviving to 30 days post ICU admission. Plasma H3 was a common feature of COVID-19 patients, detected in 40% of the patients at ICU admission. Although these measures were not predictive of the final outcome of the disease, they correlated well with parameters of tissue damage (H3 and cfDNA) and neutrophil counts (NE). A subset of samples displayed H3 processing, possibly due to proteolysis. Conclusions Elevated H3 and cfDNA levels in COVID-19 patients illustrate the severity of the cellular damage observed in critically ill COVID-19 patients. The increase in NE indicates the important role of neutrophil response and the process of NETosis in the disease. GAS6 appears as part of an early activated mechanism of response in Covid-19.The study was supported through grants from the dedSciLifeLab/KAW national COVID-19 research program project grant (MH), by Scilifelab, the Knut and Alice Wallenberg Foundation and in part by the Swedish Research Council (RF, grant no 2014-02569 and 2014-07606), and the Netherlands Thrombosis Foundation (GN).N

Presence and evolution of NET markers and DAMPS in critically ill COVID-19 patients

Resumen del trabajo presentado en el 4th European Congress on Thrombosis and Haemostasis, celebrado en Gante (Bélgica), los días 14 y 15 de octubre de 2021Background: The coronavirus disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection presents with a wide range of disease symptoms. In the more severe patients, COVID-19 is associated with respiratory failure, neutrophil extracellular trap (NET) formation, and multiple organ failure (MOF). Aims: We investigated the presence and evolution of several damage associated molecular patterns (DAMPs) neutrophil markers and immune modulators in a group of 100 COVID-19-positive ICU patients. Methods: Citrated plasma was collected from adult patients with confirmed COVID-19 by PCR detection of SARS-CoV-2 E and N-genes in nasopharyngeal swabs admitted to the intensive care unit (ICU) at Uppsala University hospital, Sweden. Written informed consent was obtained from the patients, or next of kin if the patient was unable to give consent. The Declaration of Helsinki and its subsequent revisions were followed. Plasma concentration of cell free DNA (cfDNA), extracellular histone H3 (H3), neutrophil elastase (NE), myeloperoxidase (MPO) and the cfDNA-MPO complex, and the immune modulators GAS6, and sAXL were measured in all COVID-19-positive and in COVID-19-negative patients and healthy controls. We determined marker levels upon admission, of their evolution, and correlation with disease severity, organ failure, thromboembolic events, mortality, and other blood parameters. Results: The level of cfDNA, H3, NE, MPO, cfDNA-MPO complex, GAS6, and sAXL were all significantly increased in plasma of COVID-19 patients compared to controls. Importantly, a diminution of cfDNA and GAS6 levels over time was observed in patients surviving 30 days after ICU admission. Histone H3 levels were detected in 40% of the COVID-19 patient plasma at ICU admission and the presence of histone H3 during ICU stay was associated with an increased risk of thromboembolic events and secondary infection. Though NET markers were not predictive of 30-day mortality, they correlated with several parameters of tissue damage and neutrophil counts. Summary/Conclusion: The increased presence of cfDNA, H3 and NE, MPO, and MPO-DNA illustrates the severity of cellular damage and indicates activation of NETosis in severe COVID-19 ICU patients. The evolution of cfDNA and Gas6 is able to predict disease prognosis of severely ill COVID-19 patients, where GAS6 appears to be part of an early activated mechanism in response to COVID-19. These data support treatment aimed at the reduction of NET formation in severe COVID-19 patients

Systemic Human Neutrophil Lipocalin Associates with Severe Acute Kidney Injury in SARS-CoV-2 Pneumonia

Neutrophils have been suggested mediators of organ dysfunction in COVID-19. The current study investigated if systemic neutrophil activity, estimated by human neutrophil lipocalin (HNL) concentration in peripheral blood, is associated with acute kidney injury (AKI) development. A total of 103 adult patients admitted to intensive care, with PCR-confirmed SARS-CoV-2 infection, were prospectively included (Clinical Trials ID: NCT04316884). HNL was analyzed in plasma (P-HNL Dimer) and in whole blood (B-HNL). The latter after ex vivo activation with N-formyl-methionine-leucine-phenylalanine. All patients developed respiratory dysfunction and 62 (60%) were treated with invasive ventilation. Sixty-seven patients (65%) developed AKI, 18 (17%) progressed to AKI stage 3, and 14 (14%) were treated with continuous renal replacement therapy (CRRT). P-HNL Dimer was higher in patients with invasive ventilation, vasopressors, AKI, AKI stage 3, dialysis, and 30-day mortality (p < 0.001-0.046). B-HNL performed similarly with the exception of mild AKI and mortality (p < 0.001-0.004). The cohort was dichotomized by ROC estimated cutoff concentrations of 13.2 mu g/L and 190 mu g/L for P-HNL Dimer and B-HNL respectively. Increased cumulative risks for AKI, AKI stage 3, and death were observed if above the P-HNL cutoff and for AKI stage 3 if above the B-HNL cutoff. The relative risk of developing AKI stage 3 was nine and 39 times greater if above the cutoffs in plasma and whole blood, respectively, for CRRT eight times greater for both. In conclusion, systemically elevated neutrophil lipocalin, interpreted as increased neutrophil activity, was shown to be associated with an increased risk of severe AKI, renal replacement therapy, and mortality in COVID-19 patients with respiratory failure

Plasma endostatin correlates with hypoxia and mortality in COVID-19-associated acute respiratory failure

Background: The contribution of endothelial injury in the pathogenesis of COVID-19-associated acute respiratory distress syndrome (ARDS) and resulting respiratory failure remains unclear. Plasma endostatin, an endogenous inhibitor of angiogenesis and endothelial dysfunction is upregulated during hypoxia, inflammation and progress of pulmonary disease. Aim: To investigate if plasma endostatin is associated to hypoxia, inflammation and 30-day mortality in patients with severe COVID-19 infection. Method: Samples for blood analysis and plasma endostatin quantification were collected from adult patients with ongoing COVID-19 (n = 109) on admission to intensive care unit (day 1). Demographic characteristics and 30-day mortality data were extracted from medical records. The ability of endostatin to predict mortality was analyzed using receiving operating characteristics and Kaplan-Meier analysis with a cutoff at 46.2 ng/ml was used to analyze the association to survival. Results: Plasma endostatin levels correlated with; PaO2/FiO2 (r = -0.3, p < 0.001), arterial oxygen tension (r = -0.2, p = 0.01), lactate (r = 0.2, p = 0.04), C-reactive protein (r = 0.2, p = 0.04), ferritin (r = 0.2, p = 0.09), D-dimer (r = 0.2, p = 0.08) and IL-6 (r = 0.4, p < 0.001). Nonsurvivors at 30 days had higher plasma endostatin levels than survivors (72 ± 26 vs 56 ± 16 ng/ml, p = 0.01). Receiving operating characteristic curve (area under the curve 0.7) showed that plasma endostatin >46.2 ng/ml predicts mortality with a sensitivity of 92% and specificity of 71%. In patients with plasma endostatin >46.2 ng/ml probability of survival was lower (p = 0.02) in comparison to those with endostatin <46.2 ng/ml. Conclusion: Our results suggest that plasma endostatin is an early biomarker for disease severity in COVID-19

Immuno-Modulatory Effects of Dexamethasone in Severe COVID-19—A Swedish Cohort Study

Dexamethasone (Dex) has been shown to decrease mortality in severe coronavirus disease 2019 (COVID-19), but the mechanism is not fully elucidated. We aimed to investigate the physiological and immunological effects associated with Dex administration in patients admitted to intensive care with severe COVID-19. A total of 216 adult COVID-19 patients were included—102 (47%) received Dex, 6 mg/day for 10 days, and 114 (53%) did not. Standard laboratory parameters, plasma expression of cytokines, endothelial markers, immunoglobulin (Ig) IgA, IgM, and IgG against SARS-CoV-2 were analyzed post-admission to intensive care. Patients treated with Dex had higher blood glucose but lower blood lactate, plasma cortisol, IgA, IgM, IgG, D-dimer, cytokines, syndecan-1, and E-selectin and received less organ support than those who did not receive Dex (Without-Dex). There was an association between Dex treatment and IL-17A, macrophage inflammatory protein 1 alpha, syndecan-1 as well as E-selectin in predicting 30-day mortality. Among a subgroup of patients who received Dex early, within 14 days of COVID-19 debut, the adjusted mortality risk was 0.4 (95% CI 0.2–0.8), i.e., 40% compared with Without-Dex. Dex administration in a cohort of critically ill COVID-19 patients resulted in altered immunological and physiologic responses, some of which were associated with mortality

Increased levels of plasma cytokines and correlations to organ failure and 30-day mortality in critically ill Covid-19 patients

BACKGROUND: The infection caused by SARS CoV-2 has been postulated to induce a cytokine storm syndrome that results in organ failure and even death in a considerable number of patients. However, the inflammatory response in Corona virus disease-19 (Covid-19) and its potential to cause collateral organ damage has not been fully elucidated to date. This study aims to characterize the acute cytokine response in a cohort of critically ill Covid-19 patients. METHOD: 24 adults with PCR-confirmed Covid-19 were included at time of admission to intensive care a median of eleven days after initial symptoms. Eleven adult patients admitted for elective abdominal surgery with preoperative plasma samples served as controls. All patients were included after informed consent was obtained. 27 cytokines were quantified in plasma. The expression of inflammatory mediators was then related to routine inflammatory markers, SAPS3, SOFA score, organ failure and 30-day mortality. RESULTS: A general increase in cytokine expression was observed in all Covid-19 patients. A strong correlation between respiratory failure and IL-1ra, IL-4, IL-6, IL-8 and IP-10 expression was observed. Acute kidney injury development correlated well with increased levels of IL-1ra, IL-6, IL-8, IL-17a, IP-10 and MCP-1. Generally, the cohort demonstrated weaker correlations between cytokine expression and 30-day mortality out of which IL-8 showed the strongest signal in terms of mortality. CONCLUSION: The present study found that respiratory failure, acute kidney injury and 30-day mortality in critically ill Covid-19 patients are associated with moderate increases of a broad range of inflammatory mediators at time of admission

Urinary cytokines correlate with acute kidney injury in critically ill COVID-19 patients

BACKGROUND: Acute kidney injury is common in COVID-19 patients admitted to the ICU. Urinary biomarkers are a non-invasive way of assaying renal damage, and so far, urinary cytokines are not fully investigated. The current study aimed to assess urinary cytokine levels in COVID-19 patients. METHODS: Urine was collected from COVID-19 patients (n = 29) in intensive care and compared to a preoperative group of patients (n = 9) with no critical illness. 92 urinary cytokines were analyzed in multiplex using the Olink Target 96 inflammation panel and compared to clinical characteristics, and urinary markers of kidney injury. RESULTS: There were strong correlations between proinflammatory cytokines and between urinary cytokines and urinary kidney injury markers in 29 COVID-19 patients. Several cytokines were correlated to kidney injury, 31 cytokines to AKI stage and 19 cytokines correlated to maximal creatinine. CONCLUSIONS: Urinary inflammatory cytokines from a wide range of immune cell lineages were significantly upregulated during COVID-19 and the upregulation correlated with acute kidney injury as well as urinary markers of kidney tissue damage

Renal mitochondrial dysfunction in ovine experimental sepsis-associated acute kidney injury

Sheep develop sepsis-associated acute kidney injury (SA-AKI) during experimental sepsis despite normal to increased renal oxygen delivery. A disturbed relation between oxygen consumption (V_ O2) and renal Na thorn transport has been demonstrated in sheep and in clinical studies of AKI, which could be explained by mitochondrial dysfunction. We investigated the function of isolated renal mitochondria compared with renal oxygen handling in an ovine hyperdynamic model of SA-AKI. Anesthetized sheep were randomized to either an infusion of live Escherichia coli with resuscitative measures (sepsis group; n = 13 animals) or served as controls (n = 8 animals) for 28 h. Renal V_ O2 and Na thorn transport were repeatedly measured. Live cortical mitochondria were isolated at baseline and at the end of the experiment and assessed in vitro with high-resolution respirometry. Sepsis markedly reduced creatinine clearance, and the relation between Na thorn transport and renal V_ O2 was decreased in septic sheep compared with control sheep. Cortical mitochondrial function was altered in septic sheep with a reduced respiratory control ratio (6.0 & PLUSMN; 1.5 vs. 8.2 & PLUSMN; 1.6, P = 0.006) and increased complex II-to-complex I ratio during state 3 (1.6 & PLUSMN; 0.2 vs. 1.3 & PLUSMN; 0.1, P = 0.0014) mainly due to decreased complex I-dependent state 3 respiration (P = 0.016). However, no differences in renal mitochondrial efficiency or mitochondrial uncoupling were found. In conclusion, renal mitochondrial dysfunction composed of a reduction of the respiratory control ratio and an increased complex II/complex I relation in state 3 was demonstrated in an ovine model of SA-AKI. However, the disturbed relation between renal V_ O2 and renal Na thorn transport could not be explained by a change in renal cortical mitochondrial efficiency or uncoupling. NEW & NOTEWORTHY We studied the function of renal cortical mitochondria in relation to oxygen consumption in an ovine model of sepsis with acute kidney injury. We demonstrated changes in the electron transport chain induced by sepsis consisting of a reduced respiratory control ratio mainly by a reduced complex I-mediated respiration. Neither an increase in mitochondrial uncoupling nor a reduction in mitochondrial efficiency was demonstrated and cannot explain why oxygen consumption was unaffected despite reduced tubular transport

Systemic Human Neutrophil Lipocalin Associates with Severe Acute Kidney Injury in SARS-CoV-2 Pneumonia

Neutrophils have been suggested mediators of organ dysfunction in COVID-19. The current study investigated if systemic neutrophil activity, estimated by human neutrophil lipocalin (HNL) concentration in peripheral blood, is associated with acute kidney injury (AKI) development. A total of 103 adult patients admitted to intensive care, with PCR-confirmed SARS-CoV-2 infection, were prospectively included (Clinical Trials ID: NCT04316884). HNL was analyzed in plasma (P-HNL Dimer) and in whole blood (B-HNL). The latter after ex vivo activation with N-formyl-methionine-leucine-phenylalanine. All patients developed respiratory dysfunction and 62 (60%) were treated with invasive ventilation. Sixty-seven patients (65%) developed AKI, 18 (17%) progressed to AKI stage 3, and 14 (14%) were treated with continuous renal replacement therapy (CRRT). P-HNL Dimer was higher in patients with invasive ventilation, vasopressors, AKI, AKI stage 3, dialysis, and 30-day mortality (p < 0.001–0.046). B-HNL performed similarly with the exception of mild AKI and mortality (p < 0.001–0.004). The cohort was dichotomized by ROC estimated cutoff concentrations of 13.2 µg/L and 190 µg/L for P-HNL Dimer and B-HNL respectively. Increased cumulative risks for AKI, AKI stage 3, and death were observed if above the P-HNL cutoff and for AKI stage 3 if above the B-HNL cutoff. The relative risk of developing AKI stage 3 was nine and 39 times greater if above the cutoffs in plasma and whole blood, respectively, for CRRT eight times greater for both. In conclusion, systemically elevated neutrophil lipocalin, interpreted as increased neutrophil activity, was shown to be associated with an increased risk of severe AKI, renal replacement therapy, and mortality in COVID-19 patients with respiratory failure

Evolution of NETosis markers and DAMPs have prognostic value in critically ill COVID-19 patients

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days