Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury
Temesgen E. Andargie, Naoko Tsuji, Fayaz Seifuddin, Moon Kyoo Jang, Peter S.T. Yuen, Hyesik Kong, Ilker Tunc, Komudi Singh, Ananth Charya, Kenneth Wilkins, Steven Nathan, Andrea Cox, Mehdi Pirooznia, Robert A. Star, Sean Agbor-Enoh
Temesgen E. Andargie, Naoko Tsuji, Fayaz Seifuddin, Moon Kyoo Jang, Peter S.T. Yuen, Hyesik Kong, Ilker Tunc, Komudi Singh, Ananth Charya, Kenneth Wilkins, Steven Nathan, Andrea Cox, Mehdi Pirooznia, Robert A. Star, Sean Agbor-Enoh
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Clinical Research and Public Health COVID-19 Inflammation

Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury

Abstract

INTRODUCTION The clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODS We conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTS We found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSION cfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19–induced tissue injury.FUNDING Intramural Targeted Anti–COVID-19 grant, NIH.

Authors

Temesgen E. Andargie, Naoko Tsuji, Fayaz Seifuddin, Moon Kyoo Jang, Peter S.T. Yuen, Hyesik Kong, Ilker Tunc, Komudi Singh, Ananth Charya, Kenneth Wilkins, Steven Nathan, Andrea Cox, Mehdi Pirooznia, Robert A. Star, Sean Agbor-Enoh

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Figure 10

Time- and concentration-dependent generation of mitochondrial ROS.

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Time- and concentration-dependent generation of mitochondrial ROS. Cell ...
Cell culture of mouse primary proximal tubular cells (mPPTCs) was treated with serial COVID-19 patient plasma. (A) mtcfDNA and ncfDNA dynamics of plasma from COVID-19 patient A at day 1 and day 8 after admission. Day 8 plasma contained higher ncfDNA and mtcfDNA than day 1. (B) Representative images of mitochondrial superoxide in mPPTCs treated with control (no treatment), 3% healthy volunteer plasma, 1% and 3% COVID-19 patient A’s plasma of day 8 after 3 and 12 hours of incubation: a. Control (no treatment) at 24 hours; b. 24 hours after adding healthy volunteer plasma (3%); c. 3 hours after adding day 8 COVID-19 patient A plasma (1%); d. 3 hours after adding day 8 COVID-19 patient A plasma (3%); e. 12 hours after adding day 8 COVID-19 patient A plasma (1%); f. 12 hours after adding day 8 COVID-19 patient A plasma (3%). Red: MitoSOX Red representing mitochondrial superoxide, blue: Hoechst3342 representing nuclei. Original magnification: ×400. (C and D) Comparison of MitoSOX Red intensity in mPPTCs (n = 18–29 cells from 3–5 fields/group) with (C) 1% and (D) 3% serial COVID-19 patient A plasma and after 0, 3, 12, and 24 hours of incubation. Each point represents total MitoSOX Red background-corrected intensity for 1 tubule cell. The bar in scatter plots is expressed as mean ± SEM. Statistical significance was determined using a Kruskal-Wallis test and a post hoc Mann-Whitney test. P value less than 0.05 considered statistically significant; *P < 0.05: day 1 versus day 8. (E–F) Incubation time (0, 3, 12, and 24 hours) and plasma dose (0.3%, 1%, 3%, and 10%) dependency of MitoSOX Red intensity in mPPTCs (n = 18–29 cells/3–5 fields/group). The 3D bar graph is expressed as mean. Statistical significance was determined using Tukey’s multiple-comparison test after 2-way ANOVA. *P < 0.05 versus control, #P < 0.05 versus 3% healthy plasma, †P < 0.05 versus 0 hours of incubation.