Figures & data
Figure 1. EBV infects differentiating monocytes and impairs cell survival. Primary monocytes (CD14+) were infected with recombinant EBV particles purified from 293/EBV-WT cells or mock-infected. After 5 days of EBV infection, (a) cells were analysed by using a fluorescence microscopy showing that about 25% of the cells were infected (GFP-positive), Bars: 10 mm. EBV-infected differentiating monocytes and mock control cells were then analysed for EBV protein expression (b) by western blot for the early lytic EA-D expression, where also B95.8 and BJAB were included as positive and negative controls (lane 1 mock monocytes, lane 2 EBV-infected monocytes, lane 3 BJAB and lane 4 B95.8) and (c) by IFA for EBV gp350/220 late lytic antigen. DAPI staining is shown in blue and gp350/220 late lytic antigen in red, Bars: 10 mm; (d) the supernatant of EBV-infected monocytes was able to infect monocytes, as indicated by DNA-PCR analysis. Lane 1 and 2 B95.8 positive controls, lane 3 and 4 monocytes infected with supernatant at day 5 and 3 respectively, lane 5 negative control. (e and f) After 3 and 5 days of culture, cell survival was evaluated by trypan blue exclusion assay in EBV and mock-infected differentiating monocytes. The histograms represent the mean plus S.D. of more than 3 experiments * P value < 0.05 ^ alpha value < 0.05; ANXA5 staining in the gate of Propidium Iodide (PI)-negative cells and PARP cleavage were also evaluated in these cells. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of EA-D:ACTB and cleaved PARP:ACTB of 3 different experiments. * P value < 0.05.
Table 1. Kinetics of EBV-infected and control monocyte differentiation into DCs.
Figure 2. EBV reduces DC formation. (a) FACS profiles of CD14 and CD1A expression in purified monocytes before initiating the differentiating process; (b, c and d) time-dependent FACS analysis of CD14 and CD1A surface expression on differentiating monocytes exposed or unexposed to EBV, after 3, 5 and 7 days of culture in the presence of the differentiation cocktail; in (d) also HLA-DR, CD86 and CD83 expression is indicated; (e) CD14 and CD1A expression in monocytes exposed to UV-inactivated EBV after 7 days of culture. (f) CD14 expression on differentiating monocytes exposed or unexposed to EBV, following 5 days of culture in the presence of CSF2 and IL4, as evaluated by IFA. DAPI staining is shown in blue and CD14 in red; bars: 10 µm; (g) IFA showing CD14 expression in GFP-EBV-positive monocytes. CD14 is red stained. Bars: 10 mm; (h) FACS analysis of FITC-dextran uptake by differentiating monocytes exposed or unexposed to EBV; (i) FACS analysis of CD86 surface expression on differentiating monocytes exposed or unexposed to EBV, stimulated with LPS for 24 h. The mean of fluorescence intensity is reported. Solid grey peaks represent the isotype controls. One representative experiment out of 3 is shown.
Figure 3. Autophagy reduction by EBV interferes with monocyte differentiation. Monocytes infected with EBV were cultured for 3 and 5 days with CSF2 and IL4 and analysed by western blot (a) for LC3-II expression in the presence or absence of bafylomicin A1 (BAF) (added for the last 2 h at 20 nM) and (b) for SQSTM1 expression by western blot and (c) by IFA in EBV- and mock-infected control cells. SQSTM1 staining is shown in red; bars: 10 µm; ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of LC3-II:ACTB, SQSTM1:ACTB, of 3 different experiments. * P value < 0.05. (d) IFA for EBV gp350/220 late lytic antigen in EBV and EBV-UV-infected cells. DAPI staining is shown in blue and gp350/220 late lytic antigen in red, Bars: 10 mm; and (e) SQSTM1 expression by western blot in EBV- and EBV-UV-infected cells. ACTB was used as loading control. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB, of 3 different experiments. * P value < 0.05.
Figure 4. The decrease of RAB7 and ATG5 and the activation of STAT3 correlate with EBV-mediated autophagy inhibition in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV were cultured for 5 days with CSF2 and IL4 and analyzed by western blot for (a) RAB7 and (b) ATG5 and BECN1 expression and (c) pSTAT3 (Tyr705), pSTAT3 (Ser727) and total STAT3 expression. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of RAB7:ACTB, ATG5:ACTB, BECN1:ACTB, p-STAT3 (Tyr705): STAT3, p-STAT3 (Ser727):STAT3 and total STAT3:ACTB of 3 different experiments. * P value < 0.05. (d) IL6 release, by EBV- and mock-infected monocytes as measured by ELISA. The histograms represent the mean plus S.D. of more than 3 experiments * P value < 0.05 ^ alpha value < 0.05.
Figure 5. STAT3 silencing partially prevents EBV-mediated inhibition of DC formation. Monocytes silenced for STAT3 with specific siRNA or scrambled siRNA-treated (SCR) were exposed to EBV and cultured in the presence of the differentiation cocktail for 3 days. (a) Western blot analysis of STAT3 and SQSTM1 expression and (b) FACS profiles of CD14 and CD1A expression in EBV-infected, scrambled siRNA-treated or STAT3-silenced cells. The mean of fluorescence intensity is indicated. Solid grey peaks represent the isotype controls. One representative experiment out of 3 is shown. For western blots ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of STAT3:ACTB and of SQSTM1:ACTB of 3 different experiments. * P value < 0.05.
Figure 6. EBV infection reduces the production intracellular of ROS that promotes monocyte differentiation and autophagy. (a) FACS analysis of ROS production by differentiating monocytes exposed or unexposed to EBV and cultured with CSF2 and IL4 for 3 and 5 days, measured by DCFDA staining. The mean of fluorescence intensity is indicated. Solid grey peaks represent the isotype controls. One representative experiment out of 3 is shown; (b) FACS analysis for CD14 and CD1A expression of differentiating monocytes cultured for 5 days with CSF2 and IL4 in the presence or absence of the ROS scavenger NAC. The mean of fluorescence intensity is indicated. Solid grey peaks represent the isotype controls. One representative experiment out of 3 is shown; (c) FACS analysis of ROS production by differentiating monocytes in the presence or absence of NAC, measured by DCFDA staining. The mean of fluorescence intensity is indicated. Solid grey peaks represent the isotype controls. One representative experiment out of 3 is shown; (d) western blot analysis of SQSTM1, BECN1, pSTAT3 (Tyr705) and total STAT3 expression of differentiating monocytes cultured with CSF2 and IL4 in the presence or absence of NAC. TUBA1A was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:TUBA1A, BECN1:TUBA1A, p-STAT3 (Tyr705):STAT3 and STAT3: TUBA1A of 3 different experiments. * P value < 0.05.
Figure 7. ROS reduction by metformin prevents monocyte differentiation, while H2O2 rescues it in EBV-infected cells. (a) FACS analysis of ROS production by differentiating monocytes exposed or unexposed to metformin (MET) and cultured with CSF2 and IL4 for 5 days, as measured by DCFDA staining. (b) FACS analysis for CD14 and CD1A expression of differentiating monocytes cultured for 5 days with CSF2 and IL4 in the presence or absence of metformin (MET) (10 mM). (c) FACS analysis for CD14 and CD1A expression of EBV-infected monocytes cultured for 5 days with CSF2 and IL4 in the presence or absence of H2O2 (200 mM). Solid grey peaks represent the isotype controls. The mean of fluorescence intensity is indicated and one representative experiment out of 3 is shown.
Figure 8. SQSTM1 accumulation activates the SQSTM1-KEAP1-NFE2L2 axis that reduces ROS production in EBV-infected monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) KEAP1 and NFE2L2 expression by western blot, (b) NFE2L2 localization by IFA and (c) CAT and GSR expression by western blot. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of KEAP1:ACTB, NFE2L2:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. SQSTM1 staining is shown in red; bars: 10 mm. Differentiating monocytes exposed to EBV were silenced for siSQSTM1 with specific siRNA or scrambled siRNA and cultured for 5 days with CSF2 and IL4 before analysing (d) NFE2L2 localization by IFA and (e) SQSTM1, CAT and GSR expression by western blot. NFE2L2 staining is shown in red; bars: 10 mm. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB, CAT:ACTB, GSR:ACTB of 3 different experiments. * P value < 0.05.
Figure 9. Autophagy manipulation affects DC differentiation. Differentiating monocytes were silenced for ATG5 with specific siRNA or scrambled siRNA-treated, cultured for 5 days with CSF2 and IL4 and analyzed by western blot analysis for (a) ATG5, SQSTM1, GSR, pSTAT3 (Tyr705) and total STAT3 expression (b) by FACS analysis for ROS production by DCFDA staining and (c) CD14 and CD1A expression. The mean of fluorescence intensity is indicated. Solid grey peaks represent the isotype controls. One representative experiment out of 3 is shown; TUBA1A was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of ATG5:TUBA1A, SQSTM1:TUBA1A, p-STAT3 (Tyr705):STAT3, STAT3:TUBA1A, GSR:TUBA1A of 3 different experiments.* P value < 0.05; (d) EBV-infected differentiating monocytes silenced for ATG5 or scrambled siRNA-treated and cultured for 5 days with CSF2 and IL4 were analyzed CD14 and CD1A expression by FACS analysis and (e) for ATG5 and SQSTM1 expression by western blot; EBV-infected monocytes were differentiated for 5 days in the presence or in the absence of Rapamycin (Rapa) (50 nM) and analysed (f) for SQSTM1 expression by western blot and (g) for CD14 and CD1A expression by FACS analysis. The mean of fluorescence intensity is indicated. Solid grey peaks represent the isotype controls. ACTB was used as loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of ATG5:ACTB and SQSTM1:ACTB, of 3 different experiments. * P value < 0.05.
Figure 10. EBV reduces mitochondrial biogenesis in differentiating monocytes. Differentiating monocytes exposed or unexposed to EBV and cultured for 5 days with CSF2 and IL4 were analyzed for (a) CYCS and (b) for NRF1 and TFAM expression by western blot. ACTB was used as a loading control. One representative experiment out of 3 is shown. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of CYCS:ACTB, NRF1:ACTB and TFAM:ACTB of 3 different experiments. * P value < 0.05. (c) The mitochondrial pool was evaluated by using MitoTracker Red dye in control and EBV-infected monocytes with or without TFAM overexpression; bars: 10 mm; (d) CD14 and CD1A expression was evaluated by FACS analysis and (e) SQSTM1 expression in EBV-infected monocytes with or without TFAM overexpression, was analysed by western blot. The mean of fluorescence intensity is indicated. Solid grey peaks represent the isotype controls. The histograms represent the mean plus S.D. of the densitometric analysis of the ratio of SQSTM1:ACTB of 3 different experiments. * P value < 0.05.
Figure 11. Schematic model depicting the molecules involved in the EBV-mediated impairment of monocyte differentiation into DCs.
