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. 2020 Nov 24;218(3):e20201707. doi: 10.1084/jem.20201707

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© 2020 Rodrigues et al.

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Figure 1. — Infection of primary human monocytes with SARS-CoV-2 triggers NLRP3 inflammasome activation and cell death. Human CD14⁺ monocytes were primed or not with Pam3Cys (300 ng/ml) for 4 h and infected with SARS-CoV-2 at an MOI of 5 (or as indicated) for 24 h. Mock was used as a negative infection control and nigericin as a positive control for NLRP3 activation. The NLRP3 inhibitor MCC950 (10 µM) was added 1 h after viral infection and maintained (S.CoV-2 + 950). When indicated, the SARS-CoV-2 was inactivated by UV irradiation (U.V. Inat.). (A) Caspase-1 activity was measured in the tissue culture supernatants using the Caspase-Glo 1 assay. (B) IL-1β production in the tissue culture supernatants. (C) LDH release in the tissue culture supernatants. Triton X-100 (9%) was used to induce cell death and estimate 100% death. (D) Percentage of cells containing NLRP3 puncta was estimated by microscopy scoring. (E) A representative image of a monocyte containing NLRP3 puncta (green, indicated by arrows) is shown. Inset (red) shows a higher magnification of a cell containing NLRP3 puncta. DAPI stains cell nuclei (blue). Scale bar, 10 µm. (F) Viral loads in the cell culture supernatants were estimated by RT-PCR in monocytes infected for 8 and 24 h at the indicated MOI. #, P < 0.05 compared with mock treated cells; *, P < 0.05 comparing the indicated groups, as determined by Student’s t test. A, Pam3cys: Mock × S.CoV-2, P = 0.032; U.V. Inat. × S.Cov-2, P = 0.018; S.CoV-2 × S.Cov-2 + 950, P = 0.014; Mock × Nigericin, P = 0.0005. B, Pam3cys: Mock × S.CoV-2, P = 0.0022; U.V. Inat. × S.Cov-2, P = 0.0014; S.CoV-2 × S.Cov-2 + 950, P = 0.0004; Mock × Nigericin, P = 0.0194. C, unprimed: Mock × S.CoV-2, P = 0.0049; U.V. Inat. × S.Cov-2, P = 0.0104. C, Pam3cys: Mock × S.CoV-2, P = 0.0027; Mock × Nigericin, P = 0.0007. D, unprimed: Mock × S.CoV-2, P = 0.0013; U.V. Inat. × S.Cov-2, P = 0.0013; S.CoV-2 × S.Cov-2 + 950, P = 0.0075. D, Pam3cys: Mock × S.CoV-2, P = 0.0075; U.V. Inat. × S.Cov-2, P = 0.0474; S.CoV-2 × S.Cov-2 + 950, P = 0.0075; Mock × Nigericin, P = 0.0194. Box shows the average of triplicates ± SD of the values. Each technical triplicate is indicated as a square in the figure. Shown is one representative experiment out of three (A–F) experiments performed with similar results. p.i., postirradiation; RLU, relative light unit; S.CoV-2, SARS-CoV-2.