Circulating histones play a central role in COVID-19-associated coagulopathy and mortality

COVID-19 has highlighted the lethal consequences of immunothrombosis; i.e., the cross-talk between coagulation, inflammation and the innate immune system. Patients with immunothrombosis have significant immune cell death,¹ which can release pro-coagulant² and cytotoxic³ histones. Histones are small, positivelycharged proteins that are typically found within the cell nucleus and which bind to negatively-charged DNA. We hypothesize that circulating histones play a central role in critically-ill COVID-19 patients. This translational study demonstrates that admission histone levels are significantly elevated with increasing severity of COVID-19 infection (Mild, median=2.6 μg/mL [IQR=0.7-7.6], Moderate, 10.5 μg/mL [3.5-27.2], Critical, 20.0 μg/mL [6.2-33.0], Non-survivors, 29.6 μg/mL [11.2-60.0]; P<0.001). Circulating histones associated with severe coagulopathy, inflammation and organ injury markers, including cardiac troponin. Extracellular histone levels on admission are associated with poor outcomes and independently predict 28-day mortality of hospitalized COVID-19 patients. This is the first report to indicate that circulating histones, released following immune cell death, may play a central pathological role in severe SARS-CoV-2 infection.

COVID-19 was the cause of more than two million deaths worldwide by February 2021,⁴ resulting from respiratory and multi-organ failure,⁵ with evidence of pulmonary thrombosis at post-mortem.⁶ These patients have extensive immune cell death,¹ a strong acute-phase inflammatory response and coagulopathy, as well as cardiac injury.^1,5 Cell death can release histones, and extracellular histones are cytotoxic, pro-inflammatory⁷ and pro-coagulant,² leading to pulmonary thrombosis.⁸ Extracellular histones also trigger interleukin-6 (IL-6) release to induce an acute phase response, including elevation of C-reactive protein (CRP), which, in turn, reduces histone toxicity.⁹ High levels of circulating histones initiate an alternative coagulation pathway during sepsis,² mediate multiple organ injury³ and correlate with adverse clinical outcomes, including death.¹⁰ We therefore hypothesized that high levels of histones are present in severe SARS-CoV-2 infection, and act as major mediators of coagulopathy and mortality in COVID-19 disease.

In this study, adult COVID-19 patients (n=113) were recruited at the Royal Liverpool University Hospital from 30^th March 2020 to 16^th May 2020. Patients were selected using the ISARIC WHO Clinical Characterisation Protocol for Severe Emerging Infections in the UK. Inclusion criteria were: (1) swab positive or high likelihood of infection or (2) ≥1 of the following symptoms: fever ≥38°C, new cough, dyspnea or tachypnea and admitted to a healthcare facility.¹¹ Patients were categorized into four groups: 1) Mild (minor respiratory symptoms to exclude shortness of breath OR incidental finding, where the patient required admission to hospital for reasons other than COVID-19 (such as for frailty) and was otherwise asymptomatic of COVID-19); 2) Moderate (dyspnea, i.e., patient symptomatic with shortness of breath OR hypoxia, defined by oxygen saturations on pulse oximeter of ≤93% or requiring supplementary oxygen to maintain oxygen saturations ≥96%); 3) Critical disease (respiratory failure requiring the administration of continuous positive airway pressure (CPAP) to maintain oxygen saturations ≥96% OR invasive ventilation in a critical care setting); 4) Non-survivors (patients who died within 28 days of hospital admission).

Circulating histones were quantified in patient plasma on admission (as described previously)^8,12 and associations with severity of infection, coagulation, inflammatory and organ injury markers were analyzed. Severity of infection was determined by the patient’s most severe clinical state throughout hospital admission, according to the previously described definitions. Cytokines were measured using a Luminex-based bead array, as per manufacturer’s instructions (Thermo-Fisher Scientific). Outcome measures included ventilator-support days, length of hospital stay, and 28-day mortality. Ethical approval was provided by the South Central - Oxford C Research Ethics Committee in England (Ref 13/SC/0149), the Scotland A Research Ethics Committee (Ref 20/SS/0028), and the WHO Ethics Review Committee (RPC571 and RPC572, 25 April 2013). Local approval was granted by the North West - Haydock Research Ethics Committee (REC reference 20/NW/0332).

The Kruskall-Wallis test was used to compare continuous variables, presented as median (interquartile range; IQR); the Fisher Exact/χ² test for comparison of categorical variables, presented as counts (percentage). Circulating histone levels were measured by Western Blot, using purified histone as the standard, and analyzed either as continuous variables or categorized based on a previously-determined threshold for cytotoxicity (30 μg/mL).^3,7 The Mann-Whitney U test was used to compare categorical histone levels to continuous clinical variables. Correlation analysis was performed using Spearman’s rank. A Receiver Operating Characteristic (ROC) curve analysis and multivariate regression (adjusted for age, gender, ethnicity and co-morbidities) assessed admission histone levels in predicting 28-day mortality. Kaplan-Meier survival curve analysis was performed to analyze the probability of mortality over time. Statistical tests were performed on SPSS (IBM, version 25). A 2-tailed P value of <0.05 was considered significant.

The study involved 113 COVID-19 patients (Table 1): median age 65.0 years (IQR=51.0-78.0 years), 65 patients were male (57.5%), 96 of white ethnicity (85.0%). Disease severity was associated with coagulation activation (Table 1), characterized by elevated D-dimer (P=0.017) and prolonged prothrombin time (P=0.005), and a pro-inflammatory phenotype characterized by elevated CRP (P<0.001) and IL-6 (P=0.002) on hospital admission, as well as with hypoxia and cardiac injury (Table 1). The median hospital stay was 10 days (IQR, 3-20 days) and 25 patients (22.1%) died within 28 days.

Circulating histone levels on admission were significantly elevated in COVID-19 patients compared to normal controls and were associated with increasing severity of infection (Figure 1A and B; Healthy controls, median= 2.9 μg/mL [IQR=1.5-3.3]; Mild, 2.6 μg/mL [0.7-7.6]; Moderate, 10.5 μg/mL [3.5-27.2]; Critical, 20.0 μg/mL [6.2-33.0]; Non-survivors, 29.6 μg/mL [11.2-60.0]; P<0.001). Circulating histone levels strongly correlated with D-dimer levels (R=0.606), indicating the potential involvement of extracellular histones in COVID-19 coagulopathy. Positive association with organ injury markers, including bilirubin (R=0.531), creatinine (R=0.501) and cardiac troponin (R=0.486), indicates the possible role of histone-induced cytotoxicity in multiple organ injury. Strong associations with fibrinogen (R=0.632), CRP (R=0.735) and IL-6 (R=0.677) confirmed histone-initiated acute phase response.⁹ Negative correlation with lymphocyte count (R=-0.446) suggests that lymphocyte and other immune cell death might be a major source of circulating histones in COVID-19 infection.

Adopting a 30 μg/mL cytotoxic histone threshold,^3,7 patients over the threshold (n=29) had significantly higher D-dimer (2267.0 ng/mL [1227.0-5235.0] vs. 1128.0 ng/ml [589.0-1844.3], P=.001), fibrinogen (6.6 g/L [4.6-7.6] vs. 4.8 g/L [3.9-5.7], P=0.012), IL-6 (226.2 pg/mL [90.6-518.9] vs. 71.8 pg/mL [35.2-111.4], P<0.001) and CRP levels (186 mg/L [108.5-247.5] vs. 48.0 mg/L [10.0-107.5], P<0.001) than those patients below the threshold (Table 2). These patients also had significantly reduced SpO2 compared to those with circulating histones <30 μg/mL (oxygen saturations 92.0% [85.8-94.0] vs. 95.0% [93.5-97.0], P=0.001), required critical care admission (P<0.001), with a longer duration of mechanical ventilation (R=0.635) and longer hospital stay (R=0.654).

Circulating histone levels were significantly higher in non-survivors than those who survived (29.6 μg/mL 11.2-60.0] vs. 8.6 μg/ml [3.1-24.8], P=0.002), and, accordingly, patients with histones >30 μg/mL were more likely to die (13/29 [44.8%] vs. 12/84 [14.3%], P=0.001). Patients who died were significantly older than those who survived (Table 2, 76 years [66-86] vs. 59 years [46-72] P<0.001). Compared to survivors, non-survivors had evidence of consumptive coagulopathy with lower platelet counts (P=0.003), prolonged prothrombin time (P=0.028), elevated D-dimer (P=0.017) and reduced antithrombin levels (P=0.048). Furthermore, in non-survivors, lymphocyte counts (P=0.001), and oxygen saturations (P=0.005) were significantly reduced, and IL-6 (P=0.021), CRP (P=0.013), troponin (P<0.001), bilirubin (P=0.041) and creatinine (P=0.024) were elevated when compared to survivors (Table 2).

Table 1.Demographics, peripheral blood measurements and outcomes for disease severity groups in COVID-19 infection.

Univariate analysis using continuous circulating histones demonstrated that rising histone levels were associated with mortality (odds ratio =1.031 (95% CI=1.013-1.049, P=0.001). Using categorical data where patients were stratified based on a ≥30 μg/mL threshold,^3,7 similar results were obtained (Figure 1C, OR=4.875 (95% CI=1.879-12.649, P=0.001), demonstrating that patients with high circulating histone levels on admission had a higher risk of mortality. Subsequent multivariate analysis demonstrated that histones were independently associated with mortality after adjustment for age, gender, ethnicity and co-morbidity when histone levels were treated as either continuous (odds ratio=1.032; 95% CI=1.013-1.051, P=0.001) or categorical variables (odds ratio=5.404; 95% CI=1.852-15.770, P=0.002). ROC curve analysis shows an area under the curve [AUC] of 0.708 (95% CI=0.589-0.827, P=0.002). A Kaplan-Meier survival curve demonstrated a significant increase in the probability of mortality during the 28-day period in patients with histones ≥30 μg/mL (Figure 1D, P<0.001).

Figure 1.High levels of circulating histones on hospital admission are associated with disease severity and mortality in COVID-19. Typical Western Blots (A) and quantification (B) of histone levels in healthy controls (n=12), mild (n=30), moderate (n=38), critical disease (n=20) and non-survivors (n=25) with COVID-19 infection. Circulating histone levels were higher with increasing disease severity (P<0.001). Histone levels were higher in non-survivors compared to the moderate (P=0.023), mild groups (P<0.001) and to normal healthy controls (P<0.001). Histone levels were higher in the critical group compared to mild groups (P<0.001) and normal healthy controls (P<0.001). Histone levels were higher in the moderate group compared to the mild group (P=0.007) and normal healthy controls (P=0.002). (C) Multivariate analysis of crude and adjusted odds ratios (with patients adjusted for age, gender, Black and Ethnic Minorities (BAME) and comorbidities including smoking, hypertension, asthma/COPD, diabetes, ischemic heart disease and chronic kidney disease). Circulating histone levels ≥30 μg/mL were independently associated with 28-day mortality. (D) Kaplan-Meier survival curve for the probability of mortality during the 28-day period. Patients were stratified based on circulating histones levels on admission (<30 μg/mL vs. ≥ 30 μg/mL). (E) Diagram to propose that circulating histones play a central pathological role in the development of severe COVID-19.

Table 2.Demographics, peripheral blood measurements and outcomes of COVID-19 patients.

Coagulopathy has emerged as a key feature of severe COVID-19 and has been linked to increased mortality.¹³ It has been documented that extracellular histones, released following cell death, are drivers of coagulation by activating platelets,⁷ generating thrombin² and damaging endothelial cells⁸ to induce coagulopathy in critical illness. ³ This is the first report to demonstrate high levels of circulating histones in SARS-CoV-2 infection, with levels strongly associated with coagulopathy. This suggests their involvement in thrombosis in severe cases.¹⁴

High levels of circulating histones reflect the extent of cellular death, such as lymphopenia or NETosis,¹⁵ which may be a major source of circulating histones in COVID-19. Histone release following cell death triggers IL-6 release to induce an acute-phase response.⁸ We found that circulating histone levels significantly correlated with IL-6 and acute-phase protein levels, including fibrinogen and CRP, indicating histone-induced acute phase response in patients with COVID-19.

Extracellular histones disrupt cell membranes through phospholipid binding to induce cytotoxic effects on cells, including endothelial cells⁸ and cardiomyocytes.¹² This study demonstrates circulating histones associated with cardiac injury, which is frequently observed in severe COVID-19 and associated with poor outcomes.⁵ Therefore, the cytotoxic and pro-coagulant properties of circulating histones may be an underlying molecular mechanism contributing to disease severity and poor outcomes (Figure 1E).

In conclusion, this is the first report to quantify high levels of circulating histones in viral infection and demonstrate that extracellular histones play a central role in the development of immunothrombosis and critical illness in COVID-19.

Footnotes

• Received February 5, 2021
• Accepted April 1, 2021

Correspondence

*RJS, STA and JA contributed equally as co-first authors.

^# MGS and C-HT contributed equally as-senior authors.

CHENG-HOCK TOH - toh@liverpool.ac.uk

Disclosures: all authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.

Contributions: RJS, JT, TD, CD and STA collected and interpreted the clinical data and performed data analysis. RJS, STA and SL performed statistical analysis. JA, STA, RJS and MD measured the levels of circulating histones. RJS, STA, TD, GW and CHT wrote, edited and reviewed the manuscript and figures. PJMO, JKB, LT and MS edited and reviewed the manuscript. STA, GW and CHT designed and supervised the work. STA and CHT had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. STA, GW, CHT, PJMO, JKB, and MS obtained funding for this work.

Data-sharing: this work uses data provided by patients and collected by the NHS as part of their care and support #DataSavesLives. The CO-CIN data was collated by ISARIC4C Investigators. ISARIC4C welcomes applications for data and material access through our Independent Data and Material Access Committee (https://isaric4c.net).

Funding

this work was funded by University of Liverpool COVID-19 strategic funding, the British Heart Foundation [PG/16/65/32313], Bayer AG (Germany) and the Royal Liverpool & Broadgreen University Hospitals NHS Trust. It was funded in whole, or in part, by the Wellcome Trust [205228/Z/16/Z]. This research is supported by grants from: the National Institute for Health Research (NIHR) [award CO-CIN-01]; the Medical Research Council [grant MC_PC_19059] and by the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool, in partnership with Public Health England (PHE), in collaboration with the Liverpool School of Tropical Medicine and the University of Oxford [award 200907]; NIHR HPRU in Respiratory Infections at Imperial College London with Public Health England (PHE) [award 200927]; the Wellcome Trust and Department for International Development [215091/Z/18/Z]; the Bill and Melinda Gates Foundation [OPP1209135]; the Liverpool Experimental Cancer Medicine Centre (Grant Reference: C18616/A25153); the NIHR Biomedical Research Centre at Imperial College London [IS-BRC- 1215-20013]; the EU Platform foR European Preparedness Against (Re-) emerging Epidemics (PREPARE) [FP7 project 602525] and NIHR Clinical Research Network for providing infrastructure support for this research. PJMO is supported by an NIHR Senior Investigator Award [award 201385]. The views expressed are those of the authors and not necessarily those of the DHSC, DID, NIHR, MRC, Wellcome Trust or PHE. The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Acknowledgments

This work uses data provided by patients and collected by the NHS as part of their care and support #DataSavesLives. We are extremely grateful to the 2,648 frontline NHS clinical and research staff and volunteer medical students, who collected this data in challenging circumstances; and the generosity of the participants and their families for their individual contributions in these difficult times. We also acknowledge the support of Jeremy J Farrar and Nahoko Shindo.