Fig. 3

CFZ impairs the monocyte-mediated antiviral response without affecting T cell activation. (A) Representative merge of immunofluorescence fields showing p65 staining (green) as indicated and DAPI staining for nucleic acids (blue) in HD and RRMM CD14+  isolated cells treated with CFZ for 30 min, then infected or not with RV (5 MOI) for 2–4 h. TNF-α (50 ng/ml) was used as a positive internal control showing p65 nuclear translocation; (B-C) Imaging-based quantification up to 2 h of fluorescence staining intensity vs. pixel position of 50 representative cells in HD (B) and 4 h for RRMM CD14+  isolated cells (C). Comparisons among groups were performed by one-way ANOVA: ∗∗∗≤0.01, ∗∗∗∗p ≤ 0.0001; (D-E) Histograms showing IFN-α (D) and IFN-β (E) mRNA expression of RV infected (5 MOI) or uninfected HD-PBMCs and treated with different concentrations (1 or 2.5 nM) of Bay-11 for 24 h and normalized to control GAPDH. Data represent the mean ± SD expressed in F.C. compared to the control; (F) Flow cytometry-based killing assay was performed using HD PBMCs co-cultured (8:1) with MM.1S GFP+ for 24 h and treated or not with RV (5 MOI) and Bay-11 (2.5 nM) alone or in combination; the experiment was repeated in n = 3 independent replicates (G) Bar graph showing the killing rate as 7-AAD in % expressed as the mean ± SEM of triplicates ∗∗∗∗p ≤ 0.0001; (H) q-RT-PCR showing RV capsid formation in the same experimental conditions used in F. Data are normalized and expressed as mean ± SD in F.C. compared to the control GAPDH; (I) Western blot assay on THP-1 cells showing RV (σ-NS) protein detection after treatment with Bay-11, BTZ, CFZ 2.5 nM and RV 5 MOI alone or in combination; (J) Luciferase reporter assays in HEK293 cells transfected with plasmids encoding a NF-κB luciferase reporter gene, and treated with RV (5MOI) and CFZ (10 nM and 2.5 nM) up to 72 h; TNF-α (5 ng/ml) was added to all the wells. NF-κB activity was determined by luciferase assay (mean ± SD (n = 3), ∗∗∗∗p ≤ 0.0001, ∗∗∗≤0.01, ∗∗p ≤ 0.01; (K-L) q-RT-PCR showing IFNs type I (IFN-α and IFN-β) induction after RV (5 MOI) and CFZ (2.5 nM) treatments in HD PBMCs at different time points (2-4-12–24 h). Data are normalized to control GAPDH and expressed as mean ± SD in F.C. compared to the control; (M) Heatmap of multiplex cytokine profile performed on supernatant from PBMCs from an HD treated for 4 h with CFZ, RV or both, showing 14 out 22 of the analyzed cytokines, in which the signal was detected; (N-O-P) Mass cytometry t-SNE heatmaps showing CD69 and CD80 expression in RRMM isolated PBMCs with or without RV infection (10 MOI) and CFZ treatment (2.5 nM) for 24 h; Bar graphs showing CD69 and CD80 relative expression in the monocytes (O) and CD69 in the T cells (P) of n = 4 RRMM patients. Data are expressed as mean ± SD in F.C. compared to the control; (Q-R) Three representative fields for each treatment conditions of live-cell imaging of phagocytic activity of CD14+  cells isolated from one patient showing higher phagocytic ability of CFZ-treated macrophages compared to the control macrophages as shown by the significant difference in the mean fluorescence intensity (MFI) (right) (R); (S-T) Representative flow cytometry analysis showing the gating strategy for one out of n = 4 different healthy donors analyzed and dot plot (S) showing increased CD14+  co-localization with MM.1S GFP + cells after overnight incubation with RV (5 MOI) + CFZ (2.5nM) treatments. Data analyzed by one-way ANOVA