Twenty-four hours post transfection, cells and supernatants had been collected for further analysis. TUNEL assay Apoptosis in SARS-CoV-2-infected Calu-3 cells was detected by a TUNEL Assay kit purchased from Thermo Fisher (“type”:”entrez-nucleotide”,”attrs”:”text”:”C10617″,”term_id”:”1535688″,”term_text”:”C10617″C10617) according to the manufacturers protocols. common of immune pathogenesis in the lung. The SARS-CoV-2 contamination brought on a dual mode of cell death pathways and caspase-8-dependent inflammatory responses may lead to the lung damage in the COVID-19 patients. These discoveries might aid the development of therapeutic strategies to treat COVID-19. and em Aspergillus fumigatus /em .9 Whether caspase-8 activation can also mediate virus-induced inflammatory responses has not been reported before. Necroptosis is an immunogenic cell death pathway that AZ5104 can eliminate virus-infected cells and mobilize both innate and adaptive immune responses to restrict computer virus replication.10 The benefits of necroptosis to the host, however, may sometimes be outweighed by the potentially deleterious hyper-inflammatory consequences of activating this death pathway in pulmonary and other tissues.11 For example, the influenza A computer virus (IAV) infection-induced necroptosis in airway epithelial cells is associated with lung damage and hyper-inflammatory responses. Comparable with IAV, SARS-CoV-2 can cause severe lung damage and disease-associated hyper-inflammatory AZ5104 responses.12 Whether SARS-CoV-2 contamination also induces necroptosis pathway to trigger cell death and inflammatory responses are currently unknown. Here we statement that SARS-CoV-2 contamination of lung epithelial cells induces caspase-8 activation that triggers cell apoptosis and processing of inflammatory cytokines including IL-1 into the bioactive form. IL-1 was then secreted through the SARS-CoV-2-induced necroptosis pathway resulting in inflammatory responses. The SARS-CoV-2 contamination induced a dual mode of cell death pathways, apoptosis, and necroptosis, and inflammatory responses were also observed in the infected HFH4-hACE2 transgenic mouse model and in the postmortem lung sections of fatal COVID-19 Mmp2 patients. Thus, the cell death and inflammatory responses are intimately linked during SARS-CoV-2 contamination. The inflammatory responses from the infected epithelial cells may further induce infiltration of inflammatory cells inducing strong immune pathogenesis as revealed in the postmortem lung sections of fatal COVID-19 patients. These discoveries have shed light on the underlying mechanisms of COVID-19 pathogenesis and will offer useful insights for the rational development of effective therapeutic strategies to treat COVID-19. Results SARS-CoV-2 contamination induces inflammatory responses in lung epithelial cells To investigate the mechanisms of SARS-CoV-2-induced inflammatory responses, Calu-3 cells, a lung epithelial cell model, were infected and the cell lysates were collected at different time points post contamination. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed a time-dependent upregulation of inflammatory cytokines and chemokines including IL-7, IL-8, tumor necrosis factor- (TNF-), CXCL10, and CCL5 in the virus-infected cells (Fig. ?(Fig.1a),1a), of which upregulation were also reported in the COVID-19 patients.13 Furthermore, SARS-CoV-2 infection of Calu-3 cells induced IL-1 processing and secretion in an multiplicity of infection (MOI)-dependent manner (Fig. ?(Fig.1b).1b). The expression level of pro-IL-1 was also upregulated in the infected cells (Fig. ?(Fig.1c).1c). To analyze whether SARS-CoV-2-induced inflammatory responses depend on viral replication, SARS-CoV-2 was inactivated with ultraviolet (UV) treatment. UV inactivation abolished computer virus replication (Fig. ?(Fig.1d),1d), virus-induced upregulation of TNF- and IL-1 (Fig. 1e, f), and also prevented pro-IL-1 processing and secretion (Fig. ?(Fig.1g).1g). On the other hand, treatment with MLN120B, an inhibitor of nuclear factor-B (NFB), inhibited virus-induced upregulation of pro-IL-1 and TNF- without affecting viral replication (Fig. 1hCj). Taken together, these results suggested that SARS-CoV-2-induced AZ5104 inflammatory responses depend on viral replication and activation of the NFB pathway. Open in a separate windows Fig. 1 SARS-CoV-2 contamination induces inflammatory responses through activation of the AZ5104 NFB pathway. a Calu-3 cells were infected with SARS-CoV-2 (MOI?=?0.1) with indicated time; intracellular mRNA levels of IL-7, IL-8, TNF-, CCL5, and CXCL10 were measured with quantitative RT-PCR (qRT-PCR). b, c Calu-3 cells were treated with LPS (5?g/ml) or infected with SARS-CoV-2 with indicated MOIs for 48?h. Mature IL-1 (P17) levels in the supernatants and intracellular levels of pro-IL-1 were determined by western blotting (b). Intracellular mRNA levels of IL-1 were.