SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes

Abstract

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.

Fig. 1. SARS-CoV-2 induces lytic monocyte cell death.

Human monocytes were infected with SARS-CoV-2 (MOI 0.1) for 24 h. As a positive control, monocytes were stimulated with LPS (500 ng/mL) for 23 h and, after this time, incubated with ATP (2 mM) for 1 h. A Cell viability was assessed through the measurement of LDH levels in the culture supernatant. B–D Monocytes were stained with propidium iodide (PI) and Annexin V. E Monocytes were stained with PI and DAPI, scale bar 20 μm. Images and graph data are representative of six independent experiments. Data are presented as the mean ± SEM *P < 0.05 versus control group (MOCK-infected).

Fig. 2. Lytic cell death in SARS-CoV-2-infected monocytes associates with inflammasome engagement and pyroptosis.

Human monocytes were treated with pharmacological inhibitors to impair the function of the following proteins: NLPR3 (Gliburyde; 100 µM), caspase-1 (AC-YVAD-CMK; 1 µM), or pan-caspase (Z-VAD-FMK; 10 µM) or RIPK (Nec-1; 25 µM). Monocytes were treated since 1 h prior to infection with SARS-CoV-2 (MOI 0.1), for 24 h. As a control, monocytes were stimulated with LPS (500 ng/mL) for 23 h and, after this time, stimulated with ATP (2 mM) for 1 h. A Cell viability was assessed through the measurement of LDH levels in the supernatant of monocytes. B, C Monocytes were lysed and used for determination of pro-caspase-1 and cleaved caspase-1 levels by western blotting. D, E Monocytes were stained with FAM-YVAD-FLICA to determine the caspase-1 activity by flow cytometry. F, G Monocytes were stained with FAM-FLICA to determine caspase-3/7 activity by flow cytometry. H Cell culture supernatants were collected for the measurement of the levels of IL-1β. I, J Monocytes were lysed and cleaved GSDMD levels were determined western blotting. Western blotting images, histogram and graph data are representative of six independent experiments. Data are presented as the mean ± SEM ^#P < 0.05 versus infected and untreated group; ^*P < 0.05 versus control group (MOCK-infected).

Fig. 3. Caspase-1- and IL-1 receptor-dependent amplification pro-inflammatory cytokines secretion in SARS-CoV-2-infected monocytes.

Human monocytes were pharmacologically treated to impair the function of caspase-1 (AC-YVAD-CMK; 1 µM), pan-caspase (ZVAD-FMK; 10 µM), RIPK (Nec-1; 25 µM), IL-1ß receptor (IL-1RA; 1 µM), or NLPR3 (glyburide 100 µM) since 1 h prior to infection at MOI of 0.1 with SARS-CoV-2. Monocytes were stimulated with LPS (500 ng/mL) for 23 h and after this time were stimulated with ATP (2 mM) for 1 h as a positive control. Cell culture supernatants were collected for the measurement of the levels of A IL-6 and B TNF-α. Graphs data are representative of six independent experiments. Data are presented as the mean ± SEM ^#P < 0.05 versus infected and untreated group.

Fig. 4. Atazanavir prevented capase-1-dependent lytic monocyte death.

Human monocytes were infected with SARS-CoV-2 and treated with atazanavir (ATV; 10 µM), ribavirina (10 µM), or Lopinavir (10 µM) for 24 h. A, B Monocytes were stained with FAM-YVAD-FLICA to determine caspase-1 activity. C Monocytes were stained with FAM-FLICA to determine caspase-3/7 activity. D–G Culture supernatants were collected and the levels of IL-1β, IL-6, TNF-α, and IL-8 were determined by ELISA. H Assessment of cell viability through the measurement of LDH levels in the supernatant of monocytes. Histogram and graphs data are representative of six independent experiments. Data are presented as the mean ± SEM ^* P < 0.05 versus control group (MOCK-infected); ^#P < 0.05 versus only infected group.

Fig. 5. Caspase 1 activation, lytic monocyte death, and increased Il-1β lvels associate with severe COVID-19.

Monocytes were isolated from blood samples of critically ill patients with COVID-19 and healthy donors. These cells were stained with FAM-YVAD-FLICA (A, B) or propidium iodide (PI) (C, D). Plasma samples were analyzed for IL-1β levels (E). Western blotting, histogram and graphs data are representative of nine critically ill patients and eight healthy donors. Data are presented as the mean ± SEM ^*P < 0.05 versus healthy donors (HD).