Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19 (2021)
Figure 2

Figure 2: SARS-CoV-2 Spike protein primes NLRP3 inflammasome and induces pyroptosis.

A Chemical inhibition of IL-1β (pg/ml) secretion after pre-incubation of macrophages from COVID-19 patients (red) for 2h with DMSO (n = 21), MMC950 (n = 21) or VX-765 (n = 8), followed by 4h incubation with S-protein and additional 2h with nigericin. For statistical analysis, one-way ANOVA with tukey post hoc test was used.

B Western blot analysis of total cell lysates of macrophages from a SARS-CoV-2 naïve individual (SC-naïve) (top) and COVID-19 infected individual (bottom). Macrophages were stimulated with LPS or S-protein (4h) followed by 2h of nigericin treatment. Control cells were left unstimulated. Antibodies against NLRP3 (110kDa) and β-actin (loading control) were used for detection. Representative example of three individual experiments.

C Representative confocal microscope pictures of macrophages from SC-naïve (top) and COVID-19 patients (bottom). Nuclei are stained with DAPI (blue) and ASC speck are labeled by immunofluorescence staining (green). Macrophages were stimulated with LPS (left) and S-Protein (right) for 4h followed by stimulation with nigericin in both cases. Pictures were taken with a 60x objective using the same microscope settings.

D Quantification of ASC speck from SC-naïve (n=3, blue) and COVID-19 patients (n=8, red) derived macrophages. Percentage of ASC specks was calculated per DAPI stained nuclei. For statistical analysis, two-way ANOVA with tukey post hoc test was used.

E IL-1β gene expression of macrophages from COVID-19 patients (n = 5; red bars) or SC-naïve individuals (n = 3 (S-protein) n = 4 (control); blue bars) stimulated with/without S-Protein were determined by qRT-PCR. Data are normalized to β-actin. Statistical significance was analyzed using the Kolmogorov-Smirnov test.

F Western blot analysis of total cell lysates of macrophages from SARS-CoV-2 naïve individuals (SC-naïve) (top) and COVID-19 infected individuals (bottom). Macrophages were stimulated with LPS or S-protein (1h). Control cells were left unstimulated. Antibodies against pro-IL-1β (31kDa) and β-actin (loading control) were used for detection. Representative example of three experiments performed with individual patients and SARS-CoV-2 naïve controls.

G Western blot analysis comparing macrophage precipitated supernatants from a SC-naïve (top) and a COVID-19 positive patient (bottom). Macrophages were treated with DMSO or MMC950 (2h) and further stimulated with LPS or S-protein (4h) followed by 2h of nigericin treatment. Antibodies binding cleaved IL-1β (17kDa) were used for detection. Representative example of three individual experiments.

H Western blot analysis of total cell lysates of macrophages from a SARS-CoV-2 naïve individuals (SC-naïve) (top) and COVID-19 infected individual (bottom). Macrophages were stimulated with LPS or S-protein (4h) followed by 2h of nigericin treatment. Control cells were left unstimulated. Antibodies targeting cleaved GSDMD (110kDa) and β-actin (loading control) were used for detection. Representative example of three individual experiments.

Data information: Graphs show mean ± SEM. *p<0.05; **p<0.01; ****p<0.0001;.