Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Expressing, Purification, Staining, Fluorescence

Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Infection, Cell Culture, Western Blot, Expressing, Control, Staining

Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Activation Assay, Inhibition, Cell Culture, Western Blot, Expressing, Control, Infection, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Infection, Cell Culture, Staining, Fluorescence, Expressing, Western Blot, Control

Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Infection, Cell Culture, Staining, Expressing, Fluorescence

Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Infection, Cell Culture, Expressing, Western Blot, Control, Staining

Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Cell Culture, Western Blot, Expressing, Staining, Fluorescence, Control, Infection

Journal: Autophagy

Article Title: EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

doi: 10.1080/15548627.2018.1536530

Figure Lengend Snippet: 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.

Article Snippet: Sixteen h after transfection, monocytes were infected at an MOI of 10 genome equivalents/cell, seeded into 12-well plates at a density of 5 × 106 cells/well for 1 h at 37°C and then cultured for an additional 72 h, in complete medium supplemented with CSF2 (50 ng/mL; Miltenyi Biotec, 130–093-865) and IL4 (20 ng/mL; Miltenyi Biotec, 130–095-373).

Techniques: Cell Culture, Expressing, Western Blot, Control, Infection, Over Expression, Fluorescence

Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 4 The effect of monosodium urate (MSU) crystals on Caspase 1 activity and IL-1β secretion is time-of-day dependent. (A) Schematic of the experimental design to assess the time-of-day differences in NLRP3 inflammasome activity. Following 18 h serum starvation, cells were media changed to serum-replete media and either treated immediately with MSU crystals (500 μg/mL) or rested for 12 h before media-changing again with/without MSU crystal addition. Caspase 1 activity was measured following 12 h of MSU crystal exposure and normalized for differences in cell number as determined by CyQuant assay with reference to a standard curve. (B) The magnitude of Caspase 1 activity induced by MSU crystal exposure in cells at the 0–12 h compared with 12–24 h timepoints. (C) The magnitude of increase in secreted IL-1β levels induced by MSU crystal exposure in THP-1 macrophages at the 0–12 h compared with 12–24 h timepoints. Data shown are mean ± SEM. Data were analyzed by t-test with p < .05 considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Activity Assay, CyQUANT Assay

Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 6 BMAL1 represses expression of pro-IL-1β and CASP1 but not NLRP3 in THP-1 macrophages BMAL1 was knocked down or over-expressed in THP-1 macrophages using adenoviral-mediated gene delivery. Following gene transduction, cells were serum starved for 18 h then media changed to serum-replete media and either treated immediately with MSU crystals (500 μg/mL) (0–12 h timepoint) or rested for 12 h before media-changing again with/without MSU crystal addition (12–24 h timepoint). NLRP3 expression measured by RT-qPCR following BMAL1 knockdown at (A) the 0–12 h timepoint and (B) 12–24 h timepoint. NLRP3 expression measured by RT-qPCR following BMAL1 overexpression at (C) the 0–12 h timepoint and (D) 12–24 h timepoint. Pro-IL-1β expression measured by RT-qPCR following BMAL1 knockdown at (E) the 0–12 h timepoint and (F) 12–24 h timepoint. Pro-IL-1β expression measured by RT-qPCR following BMAL1 overexpression at (G) the 0–12 h timepoint and (H) 12–24 h timepoint. CASP1 expression measured by RT-qPCR following BMAL1 knockdown at (I) the 0–12 h timepoint and (J) 12–24 h timepoint. CASP1 expression measured by RT-qPCR following BMAL1 overexpression at (K) the 0–12 h timepoint and (L) 12–24 h timepoint. Data shown are mean ± SEM for 3 experimental replicates. Data were analyzed by one-way ANOVA (post-hoc Tukey) with p < .05 considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Knockdown, Over Expression

Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 7 REV-ERBα represses NLRP3 expression and inflammasome activity in THP-1 macrophages. Following 18 h serum starvation, cells were re-fed with serum-replete media and either treated immediately with/without MSU crystals (500 μg/mL) and heme (30 μM)), SR8278 (1 μM), heme+SR8278 or vehicle (0.1M NaOH + 0.1% DMSO) for 12 h (0–12 h timepoint) or rested for 12 h and then media-changed to fresh serum-replete media with/without MSU crystals and heme/SR8278/vehicle (12–24 h timepoint). (A) Caspase-1 activity normalized to cell number as measured at the 0–12 h timepoint and (B) 12–24 h timepoint. (C) Levels of secreted IL-1β measured by ELISA at the 0–12 h and (D) 12–24 h timepoint. To ensure all cells were exposed to the same vehicles, SR8278 vehicle (0.1%) DMSO) was added to the heme-only treatment and heme vehicle (0.1M NaOH) added to the SR8278-only treatment. Data shown are mean ± SEM for 3 experimental replicates. All data were analyzed by one-way ANOVA (post-hoc Tukey) p < .05 was considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Cytokine response to vitamin E supplementation is dependent on pre-supplementation cytokine levels

doi:

Figure Lengend Snippet: Description of SNPs, primers and probes

Article Snippet: IL-1β was detected using mouse anti-human IL-1β MAb and biotinylated anti-human IL-1β Ab (R&D Systems, Minneapolis, MN).

Techniques:

Journal:

Article Title: Cytokine response to vitamin E supplementation is dependent on pre-supplementation cytokine levels

doi:

Figure Lengend Snippet: Genotype frequencies

Article Snippet: IL-1β was detected using mouse anti-human IL-1β MAb and biotinylated anti-human IL-1β Ab (R&D Systems, Minneapolis, MN).

Techniques:

Journal:

Article Title: Cytokine response to vitamin E supplementation is dependent on pre-supplementation cytokine levels

doi:

Figure Lengend Snippet: The effect of vitamin E supplementation on cytokine production depends on baseline cytokine production. Cytokine production was measured from whole blood at the beginning and end of a one year vitamin E supplementation in the elderly. TNF-α, IL-1β, and IL-6 was measured from whole blood elicited for 24 hours with lipopolysacchride (LPS; 1.0 µg/mL). IFN-γ was measured from whole blood elicited for 48 hours with phytohemagluttinin (PHA; 20 µg/mL) or concalavinA (ConA; 40 µg/mL). Cytokines production was corrected for the number of monocytes and lymphocytes in the blood (pg/ 106 lymphocyte and monocyte). P values (P) for the interaction are unadjusted.

Article Snippet: IL-1β was detected using mouse anti-human IL-1β MAb and biotinylated anti-human IL-1β Ab (R&D Systems, Minneapolis, MN).

Techniques: