Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage

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Abstract

Background and purpose

Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells.

Experimental approach

Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca ²⁺) by FLUOR-4, and vascular reactivity with a myography.

Key results

SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca ²⁺ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities.

Conclusion and applications

SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

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Most cited references 63

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Clinical and immunologic features in severe and moderate Coronavirus Disease 2019

Guang Chen, Di Wu, Wei. Guo … (2020)

Journal of Clinical Investigation

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Extrapulmonary manifestations of COVID-19

Aakriti Gupta, Mahesh Madhavan, Kartik Sehgal … (2020)

Although COVID-19 is most well known for causing substantial respiratory pathology, it can also result in several extrapulmonary manifestations. These conditions include thrombotic complications, myocardial dysfunction and arrhythmia, acute coronary syndromes, acute kidney injury, gastrointestinal symptoms, hepatocellular injury, hyperglycemia and ketosis, neurologic illnesses, ocular symptoms, and dermatologic complications. Given that ACE2, the entry receptor for the causative coronavirus SARS-CoV-2, is expressed in multiple extrapulmonary tissues, direct viral tissue damage is a plausible mechanism of injury. In addition, endothelial damage and thromboinflammation, dysregulation of immune responses, and maladaptation of ACE2-related pathways might all contribute to these extrapulmonary manifestations of COVID-19. Here we review the extrapulmonary organ-specific pathophysiology, presentations and management considerations for patients with COVID-19 to aid clinicians and scientists in recognizing and monitoring the spectrum of manifestations, and in developing research priorities and therapeutic strategies for all organ systems involved.

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Multiorgan and Renal Tropism of SARS-CoV-2

Victor G. Puelles, Marc Lütgehetmann, Maja T Lindenmeyer … (2020)

To the Editor: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) preferentially infects cells in the respiratory tract, 1,2 but its direct affinity for organs other than the lungs remains poorly defined. Here, we present data from an autopsy series of 27 patients (see the clinical data in Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org) that show that SARS-CoV-2 can be detected in multiple organs, including the lungs, pharynx, heart, liver, brain, and kidneys. We first quantified the SARS-CoV-2 viral load in autopsy tissue samples obtained from 22 patients who had died from Covid-19. Seventeen patients (77%) had more than two coexisting conditions (Figure 1A), and a greater number of coexisting conditions was associated with SARS-CoV-2 tropism for the kidneys (Table S2), even in patients without a history of chronic kidney disease (Table S3). The highest levels of SARS-CoV-2 copies per cell were detected in the respiratory tract, and lower levels were detected the kidneys, liver, heart, brain, and blood (Figure 1B). These findings indicate a broad organotropism of SARS-CoV-2. Since the kidneys are among the most common targets of SARS-CoV-2, we performed in silico analysis of publicly available data sets of single-cell RNA sequencing. This analysis revealed that RNA for angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), and cathepsin L (CTSL) — RNA of genes that are considered to facilitate SARS-CoV-2 infection 3 — is enriched in multiple kidney-cell types from fetal development through adulthood (Fig. S1). This enrichment may facilitate SARS-CoV-2–associated kidney injury, as previously suggested. 4 We also quantified the SARS-CoV-2 viral load in precisely defined kidney compartments obtained with the use of tissue microdissection from 6 patients who underwent autopsy (1 patient who was included in the previously mentioned 22 patients as an internal negative control, plus 5 additional patients). Three of these 6 patients had a detectable SARS-CoV-2 viral load in all kidney compartments examined, with preferential targeting of glomerular cells (Fig. S2). We also detected viral RNA and protein with high spatial resolution using in situ hybridization and indirect immunofluorescence with confocal microscopy (Figure 1C). Data on additional controls are provided in Figures S3 and S4. On the basis of these findings, renal tropism is a potential explanation of commonly reported new clinical signs of kidney injury in patients with Covid-19, 5 even in patients with SARS-CoV-2 infection who are not critically ill. Our results indicate that SARS-CoV-2 has an organotropism beyond the respiratory tract, including the kidneys, liver, heart, and brain, and we speculate that organotropism influences the course of Covid-19 disease and, possibly, aggravates preexisting conditions.

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Author and article information

Journal

Journal ID (nlm-ta): Vascul Pharmacol

Journal ID (iso-abbrev): Vascul Pharmacol

Title: Vascular Pharmacology

Publisher: Published by Elsevier Inc.

ISSN (Print): 1537-1891

ISSN (Electronic): 1879-3649

Publication date PMC-release: 25 November 2021

Publication date (Electronic): 25 November 2021

Electronic Location Identifier: 106946

Affiliations

[a ]Department of Pharmacology, Ribeirao Preto Medical School, University of São Paulo – USP, Brazil

[b ]Department of Biochemistry and Immunology, Ribeirao Preto Medical School, University of São Paulo – USP, Brazil

[c ]Virology Research Center, Ribeirao Preto Medical School, University of São Paulo – USP, Brazil

[d ]Department of Pharmacology, Institute of Biomedical Science, University of São Paulo – USP, Brazil

[e ]Department of Clinical Medicine, Division of Internal Medicine, Ribeirao Preto Medical School, University of São Paulo – USP, Brazil

[f ]Department of Cell and Molecular Biology, Ribeirao Preto Medical School, University of São Paulo – USP, Brazil

[g ]Department of Surgery and Anatomy, Ribeirao Preto Medical School, University of São Paulo – USP, Brazil

[h ]Institute of Biological and Health Sciences, Federal University of Mato Grosso – UFMT, Brazil

[i ]Department of Biological Chemistry, The Johns Hopkins University School of Medicine, USA

[j ]Institute of Health Sciences, Federal University of Mato Grosso – UFMT, Brazil

Author notes

[* ]Corresponding author at: Department of Pharmacology, Ribeirao Preto Medical School, University of São Paulo, Av. Bandeirantes, 3900, Bairro Monte Alegre, Ribeirão Preto, SP, Brazil.

[1]

These authors equally contributed to the manuscript.

Article

Publisher Item ID: S1537-1891(21)00118-X Publisher ID: 106946

DOI: 10.1016/j.vph.2021.106946

PMC ID: 8612754

PubMed ID: 34838735

SO-VID: aafcba2d-2a16-480d-a8c9-a0cb0118d5eb

License:

Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage

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Abstract

Background and purpose

Experimental approach

Key results

Conclusion and applications

Graphical abstract

Related collections

Novel Coronavirus Disease COVID-19

Most cited references 63

Clinical and immunologic features in severe and moderate Coronavirus Disease 2019

Extrapulmonary manifestations of COVID-19

Multiorgan and Renal Tropism of SARS-CoV-2

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