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

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 TLR...

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Published in:	Vascular pharmacology Vol. 142; p. 106946
Main Authors:	Costa, Tiago J., Potje, Simone R., Fraga-Silva, Thais F.C., da Silva-Neto, Júlio A., Barros, Paula R., Rodrigues, Daniel, Machado, Mirele R., Martins, Ronaldo B., Santos-Eichler, Rosangela A., Benatti, Maira N., de Sá, Keyla S.G., Almado, Carlos Eduardo L., Castro, Ítalo A., Pontelli, Marjorie C., Serra, Leonardo La, Carneiro, Fernando S., Becari, Christiane, Louzada-Junior, Paulo, Oliveira, Rene D.R., Zamboni, Dario S., Arruda, Eurico, Auxiliadora-Martins, Maria, Giachini, Fernanda R.C., Bonato, Vânia L.D., Zachara, Natasha E., Bomfim, Gisele F., Tostes, Rita C.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01.02.2022 Elsevier Science Ltd Published by Elsevier Inc
Subjects:	ACE2 Angiotensin-converting enzyme 2 Animals Arteries Calcium (intracellular) Calcium ions Cardiovascular Complications Coronaviruses COVID-19 Cytokines DNA probes DNA, Mitochondrial - genetics DNA, Mitochondrial - metabolism Endothelial cells Endothelial Cells - metabolism Endothelial dysfunction Endothelin 1 Enzyme-linked immunosorbent assay Humans Immunofluorescence Infections Inflammation Interleukin 6 Membrane potential Metabolic pathways Mice Mitochondria Mitochondria - metabolism Mitochondrial DNA NF-κB protein Nitric oxide Nitric-oxide synthase Polymerase chain reaction Proteins SARS-CoV-2 Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Signaling Superoxide anions TLR9 protein Toll like receptor 9 Toll-Like Receptor 9 - genetics Toll-Like Receptor 9 - metabolism Toll-like receptors Umbilical vein Viral diseases Endothelial dysfunction SARS-CoV-2 Mitochondria Toll like receptor 9
ISSN:	1537-1891, 1879-3649, 1879-3649
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Summary:	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. 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 (Ca2+) by FLUOR-4, and vascular reactivity with a myography. 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 Ca2+ 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. 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. [Display omitted] •Serum of COVID-19 patients present high levels of mtDNA•SARS-CoV-2 infects endothelial cells, which express ACE2 and TMPRSS2 proteins•SARS-CoV-2 promotes mitochondrial dysfunction, mtDNA release, TLR9 activation, and cytokines release•mtDNA induces vascular dysfunction in wild-type, but not TLR9 KO mice•TLR9 antagonism reduces IL-6 production in endothelial cells infected by SARS-CoV-2
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 These authors equally contributed to the manuscript.
ISSN:	1537-1891 1879-3649 1879-3649
DOI:	10.1016/j.vph.2021.106946