Angiotensin II induces reactive oxygen species, DNA damage, and T-cell apoptosis in severe COVID-19

Lucy Kundura, Sandrine Gimenez, Renaud Cezar, Sonia André, Mehwish Younas, Yea-Lih Lin, Pierre Portalès, Claire Lozano, Charlotte Boulle, Jacques Reynes, Thierry Vincent, Clément Mettling, Philippe Pasero, Laurent Muller, Jean-Yves Lefrant, Claire Roger, Pierre-Géraud Claret, Sandra Duvnjak, Paul Loubet, Albert Sotto, Tu-Anh Tran, Jérôme Estaquier, Pierre Corbeau, Lucy Kundura, Sandrine Gimenez, Renaud Cezar, Sonia André, Mehwish Younas, Yea-Lih Lin, Pierre Portalès, Claire Lozano, Charlotte Boulle, Jacques Reynes, Thierry Vincent, Clément Mettling, Philippe Pasero, Laurent Muller, Jean-Yves Lefrant, Claire Roger, Pierre-Géraud Claret, Sandra Duvnjak, Paul Loubet, Albert Sotto, Tu-Anh Tran, Jérôme Estaquier, Pierre Corbeau

Abstract

Background: Lymphopenia is predictive of survival in patients with coronavirus disease 2019 (COVID-19).

Objective: The aim of this study was to understand the cause of the lymphocyte count drop in severe forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Methods: Monocytic production of reactive oxygen species (ROSs) and T-cell apoptosis were measured by flow cytometry, DNA damage in PBMCs was measured by immunofluorescence, and angiotensin II (AngII) was measured by ELISA in patients infected with SARS-CoV-2 at admission to an intensive care unit (ICU) (n = 29) or not admitted to an ICU (n = 29) and in age- and sex-matched healthy controls.

Results: We showed that the monocytes of certain patients with COVID-19 spontaneously released ROSs able to induce DNA damage and apoptosis in neighboring cells. Of note, high ROS production was predictive of death in ICU patients. Accordingly, in most patients, we observed the presence of DNA damage in up to 50% of their PBMCs and T-cell apoptosis. Moreover, the intensity of this DNA damage was linked to lymphopenia. SARS-CoV-2 is known to induce the internalization of its receptor, angiotensin-converting enzyme 2, which is a protease capable of catabolizing AngII. Accordingly, in certain patients with COVID-19 we observed high plasma levels of AngII. When looking for the stimulus responsible for their monocytic ROS production, we revealed that AngII triggers ROS production by monocytes via angiotensin receptor I. ROSs released by AngII-activated monocytes induced DNA damage and apoptosis in neighboring lymphocytes.

Conclusion: We conclude that T-cell apoptosis provoked via DNA damage due to the release of monocytic ROSs could play a major role in COVID-19 pathogenesis.

Keywords: ACE2; DNA oxidation; SARS-CoV-2; angiotensin II receptor; antioxidant; lymphopenia; oxidative stress; programmed cell death.

Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Figures

Fig 1
Fig 1
The monocytes from certain patients with COVID-19 spontaneously produce ROSs. A, Fluorescence in monocytes from an HD that have been preincubated (DPI + LPS [---]) or not (LPS [___]) with the NADPH oxidase inhibitor DPI, exposed to DCFH-DA, and stimulated with LPS. As a negative control, we analyzed fluorescence in the same monocytes preincubated (DPI [...]) or not (None [gray dots]) with DPI and exposed to DCFH-DA. B, Fluorescence in monocytes from an HD (...), a non-ICU patient (non-ICU [___]), and an ICU patient (ICU [---]) exposed to DCFH-DA. C, Mean fluorescence intensity of ROS-producing monocytes from HDs, non-ICU patients (non-ICU), and ICU patients (ICU) exposed to DCFH-DA. One-way ANOVA test (P < .001). D, Mean fluorescence intensity of ROS-producing monocytes from ICU patients who did or did not survive. E, Identification of the classical, intermediate, and alternative monocyte subpopulations by flow cytometry. F, Fluorescence in CD14highCD16low (---), CD14+CD16+ (___), and CD14lowCD16high (...) monocytes from an ICU patient exposed to DCFH-DA. G, Percentages of CD14–CD16+ monocytes circulating in HDs, ICU patients, and non-ICU patients. One-way ANOVA test P = .032. H, Correlation between the proportions of intermediate and ROS-producing monocytes in ICU patients and non-ICU patients.
Fig 2
Fig 2
Monocytes from a patient with COVID-19 may induce DNA damage via ROSs. A, Detection of γ-H2AX foci by immunofluorescence in BJ cells cocultured with PBMCs from an HD or a patient with COVID-19. B, Quantification of the γ-H2AX foci induced in BJ fibroblasts by PBMCs from patients with COVID-19. Welch ANOVA test P < .001. C, γ-H2AX foci induced in BJ cells by PBMCs from a patients with COVID-19 are prevented by N-acetylcysteine or DPI. Kruskal-Wallis test P < .001. D, Monocytes isolated from a patient with COVID-19 induce DNA damage. The ability to induce γ-H2AX foci in the BJ fibroblasts of PBMCs from a patient with COVID-19, of the same PBMCs depleted of monocytes, and of monocytes isolated from these PBMCs was tested. Kruskal-Wallis test P < .001. E, Intensity of phosphatidylserine expression at the surface of HD PBMCs cocultured with PBMCs able to induce DNA damage treated (non-ICU + NAC) or not (non-ICU) with N-acetylcysteine, or with PBMCs unable to induce DNA damage treated (ICU + NAC) or not (ICU) with N-acetylcysteine. One-way ANOVA test P = .002. F, Intensity of phosphatidylserine expression at the surface of HD PBMCs cocultured with COVID-19 monocytes able (patient 3) or not able (patient 4) to induce DNA damage and treated with DPI (+DPI) or not treated.
Fig 3
Fig 3
DNA damage in PBMCs from patients with COVID-19. A, PBMCs from a patient with COVID-19 whose monocytes induce DNA damage in bystander BJ cells spontaneously present with γ-H2AX foci. PBMCs from an HD treated with camptothecin or not treated were used as positive and negative controls, respectively. B, Percentages of PBMCs harboring γ-H2AX foci in HDs, non-ICU patients (non-ICU), and ICU patients (ICU). Kruskal-Wallis test P = .002. C, PBMCs from a patient with COVID-19 whose monocytes induce DNA damage in bystander BJ cells spontaneously present with 53BP1 foci. PBMCs from an HD treated with camptothecin or not treated were used as positive and negative controls, respectively. D, Annexin V expression on peripheral blood CD4+ T cells and CD8+ T cells of HDs, non-ICU patients (non-ICU), and ICU patients (ICU). One-way ANOVA test P < .001 for CD4+ T cells and P < .001 for CD8+ T cells. E, Correlation between the intensity of DNA damage in PBMC and lymphocyte counts. The intensity of DNA damage in PBMCs is expressed as the ratio of the percentage of patient PBMCs presenting γ-H2AX foci to the percentage of HD PBMCs presenting γ-H2AX foci.
Fig 4
Fig 4
AngII induces ROS monocytic production and DNA damage. Fluorescence in monocytes from an HD, preincubated or not (...) with LPS (---) or AngII (___) (A), AngII (___) or DPI and AngII (---) (B), AngII (___), or losartan and AngII (---), and exposed to DCFH-DA (C). D, Plasma levels of AngII in patients and controls. Kruskal-Wallis test P = .001. E, Correlation between plasma levels of AngII and monocytic ROS production in patients and controls. F-H, AngII-activated monocytes induce DNA damage in neighboring cells. Ability of HD monocytes stimulated (monocytes/AngII) or not (monocytes) by AngII to cause γ-H2AX foci in bystander BJ cells (F and G) and HD PBMCs (H). The effect of the preincubation of monocytes with DPI (monocytes/DPI + AngII) (F) or AT1 antagonist (monocytes/anti-AT1 + AngII (G) is shown. F, Welch ANOVA P < .001; G and H, Kruskal-Wallis test P < .001. I, Correlation between plasma levels of AngII and the ability of patient PBMCs to induce DNA damage, expressed as the ratio of the percentage of BJ cells presenting γ-H2AX foci in the presence of patient PBMCs to the percentage of BJ cells presenting γ-H2AX foci in the presence of HD PBMCs. J, Correlation between monocytic ROS production and the percentage of T lymphocytes expressing Fas in patients.
Fig E1
Fig E1
Cascade of events leading to lymphopenia in severe forms of COVID-19. Boxed molecules are blocking the step indicated in the figure.
Fig E2
Fig E2
Kinetics of the events leading to lymphopenia in severe forms of COVID-19. Non-ICU patients are at day 7: their AngII plasma level and monocytic ROS production are strongly increased, DNA damage in their PBMCs and their T-cell apoptosis are moderately elevated, and their lymphocyte count is slightly decreased. ICU patients are at day 12: their AngII plasma level and monocytic ROS production are normal, DNA damage in their PBMCs and their T-cell apoptosis are strongly elevated, and their lymphocyte count is drastically decreased.

References

    1. Zhou F., Yu T., Du R., Fan G., Liu Y., Liu Z., et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020;395:1054–1062.
    1. Zhang X., Tan Y., Ling Y., Lu G., Liu F., Yi Z., et al. Viral and host factors related to the clinical outcome of COVID-19. Nature. 2020;583:437–440.
    1. Fan B.E., Chong V.C.L., Chan S.S.W., Lim G.H., Lim K.G.E., Tan G.B., et al. Hematologic parameters in patients with COVID-19 infection. Am J Hematol. 2020;95:E131–E134.
    1. Reshi M.L. RNA viruses: ROS-mediated cell death. Int J Cell Biol. 2014;2014
    1. Casola A., Burger N., Liu T., Jamaluddin M., Brasier A.R., Garofalo R.P. Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus role in viral-induced interferon regulatory factor activation. J Biol Chem. 2001;276:19715–19722.
    1. Hosakote Y.M., Jantzi P.D., Esham D.L., Spratt H., Kurosky A., Casola A., et al. Viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis. Am J Resp Crit Care Med. 2011;183:1550–1560.
    1. Li Q., Wang L., Dong C., Che Y., Jiang L., Liu L., et al. The interaction of the SARS coronavirus non-structural protein 10 with the cellular oxido-reductase system causes an extensive cytopathic effect. J Clin Virol. 2005;34:133–139.
    1. Vijay R., Hua X., Meyerholz D.K., Miki Y., Yamamoto K., Gelb M., et al. Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome-CoV infection. J Exp Med. 2015;212:1851–1868.
    1. Codo A.C., Davanzo G.G., Monteiro L.B., de Souza G.F., Muraro S.P., Virgilio-da-Silva J.V., et al. Elevated glucose levels favor SARS-CoV-2 infection and monocyte response through a HIF-1alpha/glycolysis-dependent axis. Cell Metab. 2020;32:437–446.e5.
    1. Violi F., Oliva A., Cangemi R., Ceccarelli G., Pignatelli P., Carnevale R., et al. Nox2 activation in Covid-19. Redox Biol. 2020;36
    1. Moghaddam A., Heller R.A., Sun Q., Seelig J., Cherkezov A., Seibert L., et al. Selenium deficiency is associated with mortality risk from COVID-19. Nutrients. 2020;12:2098.
    1. Thomas T., Stefanoni D., Reisz J.A., Nemkov T., Bertolone L., Francis R.O., et al. COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status. JCI Insight. 2020;5
    1. Darzynkiewicz Z., Zhao H., Halicka H.D., Rybak P., Dobrucki J., Wlodkowic D. DNA damage signaling assessed in individual cells in relation to the cell cycle phase and induction of apoptosis. Crit Rev Clin Lab Sci. 2012;49:199–217.
    1. Valdiglesias V., Giunta S., Fenech M., Neri M., Bonassi S. GammaH2AX as a marker of DNA double strand breaks and genomic instability in human population studies. Mutation Res. 2013;753:24–40.
    1. Panier S., Boulton S.J. Double-strand break repair: 53BP1 comes into focus. Nat Rev Mol Cell Biol. 2014;15:7–18.
    1. Xavier L.L., Ribas Neves P.F., Paz L.V., Neves L.T., Bagatini P.B., Saraiva Macedo Timmers L.F., et al. Does angiotensin II peak in response to SARS-CoV-2? Front Immunol. 2021;11
    1. Chen Y., Zhang A.-H., Huang S.-M., Ding G.-X., Zhang W.-Z., Bao H.-V., et al. NADPH oxidase-derived reactive oxygen species involved in angiotensin II-induced monocyte chemoattractant protein-1 expression in mesangial cells. Zhonghua Bing Li Xue Za Zhi. 2009;38:456–461.
    1. Amati F., Vancheri C., Latini A., Colona V.L., Grelli S., D'Apice M.R., et al. Expression profiles of the SARS-CoV-2 host invasion genes in nasopharyngeal and oropharyngeal swabs of COVID-19 patients. Heliyon. 2020;6
    1. Ziegler C.G.K., Allon S.J., Nyquist S., Mbano I.M., Miao V.N., Tzouanas C.N., et al. SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Cell. 2020;181:1016–1035.
    1. Wing P.A.C., Keeley T.P., Zhuang X., Lee J.Y., Prange-Barczynska M., Tsukuda S., et al. Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells Cell. Rep. 2021;35
    1. Andre S., Picard M., Cezar R., Roux-Dalvai F., Alleaume-Butaux A., Soundaramourty C., et al. T cell apoptosis characterizes severe Covid-19 disease. Cell Death Differ. 2022:1–14. doi: 10.1038/s41418-022-00936-x.
    1. Tsuruya K., Tokumoto M., Ninomiya T., Hirakawa M., Masutani K., Taniguchi M., et al. Antioxidant ameliorates cisplatin-induced renal tubular cell death through inhibition of death receptor-mediated pathways. Am J Physiol Renal Physiol. 2003;285:F208–F218.
    1. Denning T.L., Takaishi H., Crowe S.E., Boldogh I., Jevnikar A., Ernst P.B. Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithelial cells. Free Radic Biol Med. 2002;33:1641–1650.
    1. Wang G., Jiang L., Song J., Zhou S.F., Zhang H., Wang K., et al. Mipu1 protects H9c2 myogenic cells from hydrogen peroxide-induced apoptosis through inhibition of the expression of the death receptor Fas. Int J Mol Sci. 2014;15:18206–18220.
    1. Facchinetti F., Furegato S., Terrazzino S., Leon A. H(2)O(2) induces upregulation of Fas and Fas ligand expression in NGF-differentiated PC12 cells: modulation by cAMP. J Neurosci Res. 2002;69:178–188.
    1. Montes-Berrueta D., Ramirez L., Salmen S., Berrueta L. Fas and FasL expression in leukocytes from chronic granulomatous disease patients. Invest Clin. 2012;53:157–167.
    1. De Zio D., Cianfanelli V., Cecconi F. New insights into the link between DNA damage and apoptosis. Antioxid Redox Signal. 2013;19:559–571.
    1. Boef A.G.C., van Wezel E.M., Gard L., Netkova K., Lokate M., van der Voort P.H.J., et al. Viral load dynamics in intubated patients with COVID-19 admitted to the intensive care unit. J Crit Care. 2021;64:219–225.
    1. Abouelkhair M.A. Non-SARS-CoV-2 genome sequences identified in clinical samples from COVID-19 infected patients: Evidence for co-infections. PeerJ. 2020;8
    1. Rodriguez-Nava G., Yanez-Bello M.A., Trelles-Garcia D.P., Chul Won Chung C.W., Goar Egoryan G., Harvey J., et al. A retrospective study of coinfection of SARS-CoV-2 and Streptococcus pneumoniae in 11 hospitalized patients with severe COVID-19 pneumonia at a single center. Med Sci Monit. 2020;26
    1. Segrelles-Calvo G., de S Araújo G.R., Frases S. Systemic mycoses: a potential alert for complications in COVID-19 patients. Future Microbiol. 2020;15:1405–1413.
    1. Meckiff B.J., Ramírez-Suástegui C., Fajardo V., Chee S.J., Kusnadi A., Simon H., et al. Imbalance of regulatory and cytotoxic SARS-CoV-2-rReactive CD4 + T cells in COVID-19. Cell. 2020;183:1–14.
    1. Crouse J., Bedenikovic G., Wiesel M., Ibberson M., Xenarios I., Von Laer D., et al. Type I interferons protect T cells against NK cell attack mediated by the activating receptor NCR1. Immunity. 2014;40:961–973.
    1. Madera S., Rapp M., Firth M.A., Beilke J.N., Lanier L.L., Sun J.C. Type I IFN promotes NK cell expansion during viral infection by protecting NK cells against fratricide. J Exp Med. 2016;213:225–233.
    1. Soy M., Atagündüz P., Atagündüz I., Sucak G.T. Hemophagocytic lymphohistiocytosis: a review inspired by the COVID-19 pandemic. Rheumatol Int. 2021;41:7–18.
    1. Duan Y.-Q., Xia M.-H., Ren L., Zhang Y.-F., Ao Q.-L., Xu S.-P., et al. Deficiency of Tfh cells and germinal center in deceased COVID-19 patients. Curr Med Sci. 2020;40:618–624.
    1. Newell K.L., Clemmer D.C., Cox J.B., Kayode Y.I., Zoccoli-Rodriguez V., Taylor H.E., et al. Switched and unswitched memory B cells detected during SARS-CoV-2 convalescence correlate with limited symptom duration. medRxiv. 2020 :2020.09.04.20187724.
    1. Morgan M.J., Liu Z.-G. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 2011;21:103–115.
    1. Harijith A., Ebenezer D.L., Natarajan V. Reactive oxygen species at the crossroads of inflammasome and inflammation. Front Physiol. 2014;5:352.
    1. Rendeiro A.F., Ravichandran H., Bram Y., Salvatore S., Borczuk A., Elemento O., et al. The spatio-temporal landscape of lung pathology in SARS-CoV-2 infection. medRxiv. 2020 :2020.10.26.20219584.
    1. Verdecchia P., Cavallini C., Spanevello A., Angeli F. The pivotal link between ACE2 deficiency and SARS-CoV-2 infection. Eur J Intern Med. 2020;76:14–20.
    1. Saavedra J.M. Angiotensin receptor blockers are not just for hypertension anymore. Physiology (Bethesda) 2021;36:160–173.
    1. Mohanty R.R., Padhy B.M., Das S., Meher B.R. Therapeutic potential of N-acetyl cysteine (NAC) in preventing cytokine storm in COVID-19: review of current evidence. Eur Rev Med Pharmacol Sci. 2021;25:2802–2807.

Source: PubMed

Sponsorit ja yhteistyökumppanit

Sairaudet

Huumeiden interventiot

3
Tilaa