Fig 1: Neddylation levels tune the immune response in primary peripheral blood mononuclear cells (PBMCs) from healthy donors and COVID-19 patients.a PBMCs in vitro experimental design using mitogen phytohemagglutinin (PHA) (5 µg/ml) as an immune system activation, and MLN4924 (0.5 µM) as a neddylation inhibition during 120 h. 4 groups of study were designed using the combination of the present treatments. b Protein levels of NEDD8-Cullins conjugated, ponceau and quantification form PBMCs from healthy donor, activated with PHA and activated with PHA and treated with MLN4924. c Global Neddylation levels in culture media form PBMCs from healthy donor, activated with PHA and activated with PHA and treated with MLN4924. d PBMCs pictures 120 h after treatments from 4 treatment groups. e Assessment of proliferation of PBMCs from 4 groups of treatments with Carboxyfluorescein succinimidyl ester (CFSE) by flow-cytometry. f Protein levels of NEDD8-Cullins conjugated, ponceau staining and quantification in PBMCs from healthy donors and COVID-19 patients. g Global Neddylation levels in culture media in PBMCs from healthy donors or COVID-19 patients calculated by activation ratio as a basal state versus activation with PHA, and inhibition ratio calculated by activation with PHA versus Activated and treated with MLN4924. Scale bar corresponds to 100 µm. Data are shown as average ± SEM and One-way ANOVA test and T-test respectively, was used to compare groups. *p < 0.05, **p < 0.01 and ***p < 0.001 are shown. Symbols description: - without and + with.
Fig 2: Serum global neddylation levels were increased in COVID-19 patients and positively correlated with IgM levels.a Serum global neddylation levels assessed by ELISA in a cohort of healthy subjects (n = 14), COVID-19 patients (n = 38), convalescents of COVID-19 disease (n = 22), patients with another non-treated immune disease such as systemic lupus erythematosus (SLE) and hepatitis C virus (HCV) (n = 37). b Serum global neddylation levels were evaluated between two groups of COVID-19 severity, non-severe COVID-19 (n = 22) and severe COVID-19 (n = 16). c Ratio between global neddylation levels from the final time of discharge and initial time of hospitalization in the second cohort of COVID-19 patients divided by the severity of the disease progression, moderate (n = 22) and severe (n = 26). d Correlation graphics between serum NEDD8 levels and IgM/IgM levels against SARS-CoV-2 Spike 1 (S1), receptor-binding domain (RBD) and nucleoprotein (N) evaluated from the first cohort of patients represented as R2 and p-value. Data are shown as average ± SEM and One-way ANOVA test was used to compare groups. *p < 0.05, **p < 0.01 and ***p < 0.001 are shown.
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