key: cord-0955861-qsge9mg8
authors: Lipskaia, Larissa; Maisonnasse, Pauline; Fouillade, Charles; Sencio, Valentin; Pascal, Quentin; Flaman, Jean-Michel; Born, Emmanuelle; Londono-Vallejo, Arturo; Le Grand, Roger; Bernard, David; Trottein, François; Adnot, Serge
title: Evidence That SARS-CoV-2 Induces Lung Cell Senescence: Potential Impact on COVID-19 Lung Disease
date: 2021-09-29
journal: Am. j. respir. cell mol. biol
DOI: 10.1165/rcmb.2021-0205le
sha: 153fc777f89104aaa588ce34ea195680aa8e2daa
doc_id: 955861
cord_uid: qsge9mg8

nan

Older age is a major risk factor for severe coronavirus disease (COVID-19) (1) . Understanding the biological mechanisms linking age to the pathogenesis of COVID-19 is essential for developing preventive and therapeutic strategies. We hypothesized that cell senescence, a basic aging process that plays a pivotal role in health deterioration and diseases, particularly those targeting the lung (2) , is involved in the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced lung disease, including the development of long-lasting lung alterations. Senescent cells exhibit a stable proliferation arrest and acquire a specific senescence-associated secretory phenotype characterized by the release of inflammatory cytokines, immune modulators, proteases, profibrotic factors, and various effectors that can alter tissue organization and function (3) . Senescence is triggered by a myriad of stressors that promote a DNA damage response leading to p53-dependent upregulation of the CDK inhibitor p21 and/or expression of p16, which is used as a reliable marker of senescent cells. Cell senescence is pivotal in age-associated lung diseases, notably lung emphysema, fibrosis, and chronic obstructive pulmonary disease (2, (4) (5) (6) . In recent studies, SARS-CoV-2 Spike protein-1 was shown to exacerbate the senescence-associated secretory phenotype of human senescent cells, thereby contributing to the exuberant inflammatory response seen in severe COVID-19. Targeting senescent cells using senolytic drugs reduced mortality in old mice infected with a mouse b-coronavirus (7) . To further evaluate potential links between SARS-CoV-2 infection and cell senescence, we analyzed publicly available single-cell RNA sequencing data sets obtained using BAL fluid (BALF) cells from patients with moderate or severe-to-critical COVID-19 (8) . We also monitored lung cell senescence in SARS-CoV-2-infected macaques, which constitute a relevant model for studying human COVID-19 (9) .

First, we extracted data from publicly available, BALF cell, single-cell RNA sequencing data sets from patients with moderate or severe-to-critical COVID-19 versus healthy control subjects to analyze senescence-related genes (8) . In BALFs collected 10-16 days after symptom onset, mRNA of the senescence marker CDKN2A encoding p16 was mainly detected in epithelial cells, macrophages, and T cells, with higher levels in epithelial cells from patients with severe-to-critical disease compared with control subjects ( Figure 1A) . Expression of the senescence markers CDKN2A, CDKN1A (encoding p21), uPAR (urokinase plasminogen activator surface receptor), CXCL8, IGFBP3, and GDF15 was significantly increased in epithelial ciliated and club cells from patients with severe COVID-19 compared with those with moderate disease and with healthy control subjects, suggesting that lung cell senescence induction coincided with virus detection ( Figure 1B ). Of note, patients with severe COVID-19 were older than those with moderate disease, whereas age was comparable between patients with moderate disease and healthy control subjects ( Figure 1 ). In single-cell data sets from another study (10) , which compared same-age patients with mild versus critical disease (see Fig.  E1 in the data supplement), variations were similar, although CDKN1A and CDKN2A were less affected than in the first data set. To further assess the extent of SARS-CoV-2-induced lung cell senescence and the fate of senescent lung cells over time, we investigated macaques at 4 and 30 dpi, or in other words, at the viral load peak and at the first negative airway sample qRT-PCR, respectively (9) . Immunohistochemical studies of lung sections at 4 dpi revealed SARS-CoV-2 antigen-stained cells, including lung endothelial cells (ECs) and parenchymal cells, as well as numerous p16-and p21-immunofluorescence-stained cells predominating at sites of alveolar damage ( Figure 2A ). Cells positive for p16 were also positive for SARS-CoV-2 Spike protein-1 at 4 dpi, indicating that senescent lung cells were infected with the virus. SARS-CoV-2 antigen-stained cells were rarer at 30 dpi, whereas massive accumulation of p16-and p21-positive cells throughout the lung indicated persistence of senescent lung cells after virus clearance ( Figure 2A ). Cells stained for p16 were also stained for the DNA damage markers g-H2AX protein and p53-binding protein 1 at both 4 and 30 dpi ( Figure 2A) .

Interestingly, the lungs at 30 dpi no longer exhibited the consolidated parenchymal areas seen at 4 dpi but showed extensive lung parenchyma remodeling, with thickening of the alveolar and pulmonary vessel walls and abundant extracellular matrix deposits as assessed by collagen staining ( Figure 2B and Figure E2 ). These advanced lesions were accompanied by massive accumulation of p16and p21-positive cells, most of which were alveolar type II cells and ECs, as shown by double immunofluorescence staining for p16 and mucin 1 and for von Willebrand factor, respectively ( Figure 2B ). Of note, most ECs stained for p16 in many lung vessels, notably those occluded by thrombi and showing intraluminal von Willebrand factor and fibrin staining. Collectively, our data constitute the first evidence of temporal and topographic relations between senescent cell accumulation and pulmonary lesions induced by SARS-CoV-2.

Cell senescence is usually viewed as a response to chronic stressors that severely impedes healthy aging and promotes agerelated noncommunicable diseases (11) . Here, BALF cells from patients with severe COVID-19 expressed high levels of senescent cell markers. This original observation was confirmed in a macaque COVID-19 model: early massive senescent lung cell accumulation occurred in areas of severe COVID-19-related lung damage. Moreover, senescent cells persisted in the lungs over time, and many of them appeared concomitantly with the development of long-term lung alterations, including remodeling of the alveolar septa and pulmonary vessels. Given the deleterious effect of cell senescence on tissue repair and inflammation, these results suggest that senescent cell accumulation may contribute to the early lung alterations caused by SARS-CoV-2 infection and, potentially, to the postviral lung pathology seen in a substantial proportion of patients (12) . Most ECs in thrombosed vessels were senescent, suggesting a causal relationship between EC senescence and vascular thrombosis. Thus, counteracting the cell senescence process or eliminating senescent lung cells might lessen lung damage severity. This may be of therapeutic importance as strategies are now proposed to control senescence in various lung diseases, as well as in acute respiratory distress syndrome due to other causes (13, 14) . A recent study in mice showed that lung inflammation caused by a mouse b-coronavirus was markedly reduced by senolytic treatment, which also decreased mortality in old mice (7) . These findings also support senescence as a major mechanism in the pathogenesis of COVID-19 and of other viral infections (15) and suggest that senescent lung cell persistence after virus clearance may contribute to postviral lung disease, namely emphysema or fibrosis. 

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