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Rationale SARS-CoV-2 infection of the respiratory system can progress to a life threatening multi-systemic disease, mediated via an excess of cytokines (“cytokine storm”), but the molecular mechanisms are poorly understood. Objectives To investigate whether SARS-CoV-2 may induce cellular senescence in lung epithelial cells, leading to secretion of inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). Methods Autopsy lung tissue samples from eleven COVID-19 patients and sixty age-matched non-infected controls were analysed by immunohistochemistry for SARS-CoV-2 and markers of cellular senescence (SenTraGor, p16INK4A) and key SASP cytokines (interleukin-1β, interleukin-6). We also investigated whether SARS-CoV-2 infection of an epithelial cell line induces senescence and cytokine secretion. Measurements and Main Results SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in alveolar type-2 (AT2) cells, which also expressed the angiotensin-converting-enzyme 2 (ACE2), a critical entry receptor for this virus. In COVID-19 samples, AT2 cells displayed increased markers of senescence [p16INK4A, SenTraGor staining positivity in 12±1.2% of cells compared to 1.7±0.13% in non-infected controls (p Conclusions We demonstrate that in severe COVID-19 patients, AT2 cells are infected with SARS-CoV-2 and show senescence and expression of proinflammatory cytokines. We also show that SARS-CoV-2 infection of epithelial cells may induce senescence and inflammation, indicating that cellular senescence may be an important molecular mechanism of severe COVID-19.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory system can progress to a multisystemic disease with aberrant inflammatory response. Cellular senescence promotes chronic inflammation, named senescence-associated secretory phenotype (SASP). We investigated whether coronavirus disease 2019 (COVID-19) is associated with cellular senescence and SASP.MethodsAutopsy lung tissue samples from 11 COVID-19 patients and 43 age-matched non-COVID-19 controls with similar comorbidities were analysed by immunohistochemistry for SARS-CoV-2, markers of senescence and key SASP cytokines. Virally induced senescence was functionally recapitulated in vitro, by infecting epithelial Vero-E6 cells and a three-dimensional alveosphere system of alveolar type 2 (AT2) cells with SARS-CoV-2 strains isolated from COVID-19 patients.ResultsSARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in AT2 cells. Infected AT2 cells expressed angiotensin-converting enzyme 2 and exhibited increased senescence (p16INK4A and SenTraGor positivity) and interleukin (IL)-1β and IL-6 expression. In vitro, infection of Vero-E6 cells with SARS-CoV-2 induced senescence (SenTraGor), DNA damage (γ-H2AX) and increased cytokine (IL-1β, IL-6, CXCL8) and apolipoprotein B mRNA-editing (APOBEC) enzyme expression. Next-generation sequencing analysis of progenies obtained from infected/senescent Vero-E6 cells demonstrated APOBEC-mediated SARS-CoV-2 mutations. Dissemination of the SARS-CoV-2-infection and senescence was confirmed in extrapulmonary sites (kidney and liver) of a COVID-19 patient.ConclusionsWe demonstrate that in severe COVID-19, AT2 cells infected by SARS-CoV-2 exhibit senescence and a proinflammatory phenotype. In vitro, SARS-CoV-2 infection induces senescence and inflammation. Importantly, infected senescent cells may act as a source of SARS-CoV-2 mutagenesis mediated by APOBEC enzymes. Therefore, SARS-CoV-2-induced senescence may be an important molecular mechanism of severe COVID-19, disease persistence and mutagenesis.