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. 2021 Jul 14;22(14):7540. doi: 10.3390/ijms22147540

Figure 4.

Figure 4

SARS CoV-2 spike protein-induced cytokine production is independent of its binding to ACE2. (A) The binding activities of coronaviral spike Fc fusion proteins to ACE2 were measured using an ELISA. The binding activities of 16, 80, 400 and 2000 ng/mL of the indicated proteins were presented as absorbance at 450 nm (O.D. 450). (B) Inhibition of S1-Fc binding to ACE2 by soluble ACE2 or a neutralizing anti-S1 antibody; 400 ng/mL of S1-Fc were preincubated with or without 2 µg/mL of soluble ACE2, 1 µg/mL of neutralizing (nAb) or non-neutralizing (non-nAb) anti-S1 antibody before incubation with plate-bound ACE2. Statistical analyses were performed using a two-tailed, Student’s t-test. *** depicts p < 0.001. (C) Rested PBMC were cultured with or without 2.0 µg/mL of S1-Fc in the presence or absence of 5 µg/mL of soluble ACE2, 2 µg/mL of nAb or non-nAb for 24 h. The levels of IL-6, IL-8 and TNFα in the supernatants of cultured PBMC were assessed using the CBA human inflammatory cytokine kit and flow cytometric analysis.