Fig. 1.

SARS-CoV-2 infection activates p38 MAPK signaling and induces a pro-inflammatory cytokine response. A) Comparisons of a priori selected blood cytokine levels in COVID-19 patients with moderate/severe (IMC, n = 7) or critical (ICU, n = 16) disease and healthy controls (n = 11). Levels of IL6, CXCL8, CXCL10 and TNF were measured by multiplex proximity extension assay. Statistical significance was determined using non-parametric Mann-Whitney-U-test. **p < 0.01, ***p < 0.001, ****p < 0.0001. AU: arbitrary units, IMC: intermediate care wards, ICU: intensive care units. B) Replication kinetic in Calu-3 cells infected with SARS-CoV-2 strain hCoV-19/Germany/FI1103201/2020 at 0.01 MOI. Virus titers are expressed as plaque forming units (PFU/ml), n = 3. C) Phosphorylation of p38, MSK1 and NF-κB p65 during SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.01 for 48 h or the highly pathogenic influenza A virus KAN-1 with MOI 0.001 for 48 h. Viral infections were verified by immuno-detection of the SARS-CoV-2 Nucleoprotein (N) and the IAV polymerase basic 1 (PB1) protein. Signals for total and phosphorylated p38, MSK1 and NF-κB p65 were detected by Western blot using (phospho-) specific antibodies. Quantification of p38, MSK1 and NF-κB p65 phosphorylation is provided below the blot. D) Phosphorylation and signal transduction of p38 at early time points during SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 at MOI 2 for 2 h. Signals for total and phosphorylated p38 and MSK1 were detected by Western blot using (phospho-) specific antibodies. Quantification of p38 and MSK1 phosphorylation is shown below the blot. E) Phosphorylation of p38 and MSK1 following infection with Delta S pseudotyped VSV. Vero E6 cells were infected with VSV WT or VSV expressing the spike protein of the Delta variant with MOI 1.5 for 60 min or 75 min, respectively. As a positive control, cells were exposed to 1 kJ/m² ultraviolet light 30 min prior to lysis. Tubulin was used as a loading control. Quantification of p38, MSK1 and TRIM28 phosphorylation is shown below the blot. F) and G) Cytokine response over time during SARS-CoV-2 infection. Calu-3 cells were infected with SARS-CoV-2 with MOI 0.01 for the indicated time points and mRNA expression of pro-inflammatory cytokines, IFNB1 and ISGs OAS1 and MX1 was determined by qRT-PCR. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test. Data are presented as n-fold gene induction over non-infected cells; bars indicate means ± SEM; n = 4.