Following one wash of the beads with wash buffer, the beads were resuspended in 100 L of buffer and analyzed by flow cytometry. ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein ENOX1 reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their capabilities to induce cytokine production. The LPS inhibitor, polymyxin B, clogged this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest extreme caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that relationships of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal cells could promote pathogenic inflammatory cytokine production. 0.05, 0.01, 0.005 and 0.001, respectively. 2.2. Inhibition of SARS CoV-2 Spike Fusion Protein-Induced Cytokine Response by Budesonide and MAPK Inhibitors Early treatment with inhaled budesonide has been reported to dampen swelling in COVID-19 individuals [35]. Consistent with these findings, co-culture of PBMC with budesonide inhibited S1-Fc-induced production of IL-6, IL-8 and TNF (Number 3 and Table S1). As SARS CoV-2 spike protein has been reported to activate the MAPK and NF-B signaling pathways [36], and the NFAT signaling pathway is definitely involved in cytokine induction in many cells [37,38], we next investigated whether S1-Fc-induced cytokine reactions are clogged by inhibitors focusing on these signaling pathways. Treatment with NFAT and NF-B inhibitors did not decrease S1-Fc-induced manifestation of proinflammatory cytokines (Number S1). In contrast, JNK1/2 inhibition reduced the induction of IL-6, IL-8 and TNF; p38 inhibition reduced the induction of IL-6 and Senicapoc (ICA-17043) TNF; and Erk1/2 inhibition reduced TNF induction (Number 3 and Table S1). Open in a separate windows Number 3 Inhibition of S1-Fc-induced cytokine reactions by budesonide and MAPK inhibitors. Rested PBMC were cultured with or without 2.0 g/mL of S1-Fc from Vendor #2 in the presence or absence of budesonide, U0126 (MEK1/2 inhibitor, Erk1/2i), SP600125 (JNK1/2 inhibitor, JNK1/2i) or SB203580 (p38 inhibitor, p38i) for 24 h. The levels of IL-6, TNF and IL-8 were measured as explained in Number 2. Data demonstrated are imply cytokine induction indices (collapse) SE derived from 8 healthy donors, which were determined using cytokine concentrations measured from the CBA assay (IL-6 and TNF) or the ELISA (IL-8) and the method: cytokine concentration of treated PBMC/cytokine concentration of untreated PBMC. Statistical analyses were performed using a two-tailed, College students 0.05 and 0.01, respectively. 2.3. SARS CoV-2 Spike Fusion Protein-Induced Cytokine Response Is definitely Indie of Its Binding to ACE2 During the investigation of the capability of SARS CoV-2 spike protein to induce a cytokine response, we noticed that SARS CoV-2 S1-Fc protein reagent from Merchant #2, but not Merchant #1 (lot 24529-2003), and RBD-Fc from Merchant #1, but not Merchant #2, induced cytokine reactions. In addition, batch variability in PBMC cytokine induction capacity was observed between S1-Fc and RBD-Fc protein lots purchased from Merchant #1 (Number S2). We reasoned that these inconsistencies could Senicapoc (ICA-17043) be due to product quality differences influencing spike protein Senicapoc (ICA-17043) binding to ACE2. To test this hypothesis, we developed an ELISA to measure the binding affinities of spike fusion protein constructs with ACE2. With this assay, ACE2 was coated on a plate, exposed to spike protein-containing test reagents, followed by incubation with HRP-labelled anti-human IgG Fc antibody and substrate development (Number S3). Absorbance ideals at 450 nm correlated linearly with the concentrations of S1-Fc protein (Number S3). By using this ELISA, we observed that SARS CoV-2 S1-Fc and RBD-Fc from both Merchant #1 and Merchant #2, as well as SARS CoV-1 S1-Fc, showed dose-dependent binding to ACE2, which did not correlate with their capabilities to induce cytokine production (Number 4A). Moreover, blockade of S1-Fc binding to ACE2 by soluble ACE2 and an anti-S1 neutralizing antibody did not block S1-induced production of IL-6, IL-8 or TNF, despite obstructing binding of the fusion proteins to ACE2 (Number 4B,C and Number S4). Of notice, low levels of IL-8 production were observed in PBMC cultured with soluble ACE2 (Number 4C) or anti-S1 antibody only (Number S4). Taken collectively, our results suggest that the induction of key pro-inflammatory cytokines in human being PBMC by particular commercial S1-Fc-fusion proteins occurs self-employed of ACE2.