Figure 1.
Nef induces downregulation of CD36 expression in human PBMC-derived monocytes.
Purified monocytes evaluated for CD14 expression (data not shown) were cultured in presence 50 ng/mL of rNef/myr for three days. (A) Representative dot plots and histogram of monocytes analyzed by flow cytometry. The dot plot on the center shows the forward and side scatter of monocytes. On the left is shown the viability of cells by using SYTOX Blue dead cell stain. CD36 expression, shown in the histogram, was analyzed by using FITC-conjugated anti-CD36 antibody and the fluorescence intensity in Nef-treated (solid grey histogram) was compared to untreated (solid line) cells. Matched isotype (dotted line) was used as control of non-specific fluorescence signals. (A, below the histogram) The column bar graph represents the MFI of cells stained with matched isotype control (Ctr iso), cells stained with FITC-conjugated anti-CD36 antibody (Ctr CD36), and Nef-treated cells stained with FITC-conjugated anti-CD36 antibody (Nef CD36). The results (mean ± standard deviation) are representative of five independent experiments (*p<0.05). In B and C representative histograms of M-CSF and GM-CSF differentiated MDMs evaluated for CD14, CD4 and CD36 expression are shown. Cells were isolated by using CD14 magnetic beads (Miltenyi Biotec) and cultured in presence of human M-CSF (10 ng/mL) or GM-CSF (50 ng/mL) for 5 days before adding rNef/myr protein. CD14, CD4 and CD36 expression was analyzed by using appropriate fluorochrome-conjugated antibodies and the fluorescence intensities in Nef-treated (solid grey histogram) were compared to untreated (solid line) cells. Matched isotype (dotted line) was used as control of non-specific fluorescence signals. (B and C, below the histograms) The column bar graphs show the MFI of untreated cells (Ctr) and Nef-treated cells (Nef) stained with APC- and FITC-conjugated anti-CD4 and CD36 antibodies, respectively. SYTOX Blue has been used to exclude dead cells from the analyses. The results (mean ± standard deviation) are representative of four (MDM M-CSF) and six (MDM GM-CSF) independent experiments (*p<0.05, (**p<0.01).
Figure 2.
PBMCs cultivated under HEMA cell culture conditions produce three main populations.
PBMCs were cultivated in HEMA condition (see Materials and Methods) for three and six days. (A) Representative dot plots PBMCs analyzed at six days in HEMA condition by flow cytometry. The dot plot on the left shows the viability of cells by using SYTOX Blue dead cell stain. The dot plot on the right shows the three main populations identified by characteristic forward and side scatter: a lymphocyte gate (Lym), erythroblast gate (Ery) and MDMs. (B) PBMCs have been cultivated in HEMA culture condition in presence or absence of EPO. The relative percentages of the three populations (Lym, Ery, MDMs) at three (3d) and six (6d) days in HEMA (+/− EPO) culture are presented in the histogram. The results (mean ± standard deviation) are representative of six (HEMA condition) and twelve (HEMA w/o EPO condition) independent experiments (**p<0.01, ***p<0.005).
Figure 3.
CD36 expression on MDM cells cultivated in HEMA condition treated with recombinant Nef.
PBMCs were cultivated in HEMA condition for three days and for additional three days in presence of 50/mL rNef/myr (A) Representative histograms of the three populations (Lym, Ery, MDMs) analyzed for CD36 and CD4 expression by flow cytometry at six days expansion. The respective populations were identified as described in figure 2A and the fluorescence intensities in Nef-treated (solid grey histogram) compared to untreated (solid line) cells are shown. Matched isotype (dotted line) was used as control of non-specific fluorescence signals. In the column bar graphs on the right of the histograms are presented the MFI of cells stained with matched isotype control (iso), untreated cells (Ctr) and Nef-treated cells (Nef) stained with FITC- or APC-conjugated anti-CD36 and CD4 antibodies. The results are representative of ten (CD36) and four (CD4) independent experiments. MDMs were analyzed by flow cytometry for the expression of several specific markers, i.e. CD14, CD11c, CD86, CD68 and CD206. Representative histograms are shown in (B) and the fluorescence intensities of respective antibodies in Nef-treated (solid grey histogram) were compared to untreated (solid line) cells. The results are representative of five independent experiments. (C) Representative histograms of PBMC-derived MDMs analyzed by flow cytometry for the expression of TLR2 and TLR4. The MFI of Nef-treated (+ Nef) compared to untreated (untreated) cells was reported in the respective histograms. The results are representative of five independent experiments (*p<0.05). Matched isotype (dotted line or Ctr iso) was used as control of non-specific fluorescence signals. SYTOX Blue was used to exclude dead cells in all the experiments presented in this figure.
Figure 4.
CD36 expression on MDM cells cultivated in HEMA condition in presence of the LPS inhibitor polymyxin B.
PBMCs were cultivated in HEMA condition for three days followed by additional three days in presence of 50/mL rNef/myr, 100 ng/mL LPS or 10 μg/ml polymyxin B. In some cultures, polymyxin B was added 15 min before the Nef and LPS treatment. (A) Representative histogram of MDMs analyzed by flow cytometry for the expression of CD36. The histogram on the right shows the respective treatment with LPS, polymyxin B and polymyxin B+LPS. The fluorescence intensities were compared to untreated cells (Ctr). In (B) similar analysis by replacing LPS with rNef/myr is shown. Both in (A) and (B) matched isotypes (Isotype) were used as control of non-specific fluorescence signals. SYTOX Blue was used to exclude dead cells. The results are representative of three independent experiments.
Figure 5.
Comparison of myristoylated and non-myristoylated Nef activity on CD36 expressed by MDM and erythroblast cells cultivated in HEMA condition.
PBMCs were cultivated in HEMA condition for three days followed by additional three or five days in presence of 50/mL rNef/myr and rNef. Representative histogram of MDM (A) and Ery (B) cells analyzed by flow cytometry for the expression of CD36 after three days of Nef treatment while in (C) and (D) MDM and Ery cells analyzed after five days of Nef treatment are shown. The fluorescence intensities were compared to untreated cells (Ctr). In all experiments matched isotypes (Isotype) were used as control of non-specific fluorescence signals. SYTOX Blue was used to exclude dead cells. The results are representative of three independent experiments.
Figure 6.
CD36 and CD4 expression in MDM GM-CSF cells infected in vitro with Nef(+)-HIV-1.
MDMs differentiated in presence of GM-CSF for 5 days were infected with VSV-G pseudotyped HIV-1-expressing (Nef (+)-HIV-1) or not expressing the nef gene (ΔNef-HIV-1). In the left histogram the infection efficiency is shown; it is evaluated by estimating the levels of intracytoplasmic HIV-1 Gag-related products (HIV-1 CAp24) by flow cytometry analysis at 48 h postinfection. CD4 and CD36 expression was analyzed by using APC- and FICT-conjugated antibodies (center and right histogram, respectively) and the fluorescence intensities in Nef(+)-HIV-1-infected (solid grey histogram) was compared to ΔNef-HIV-1-infected (solid line) cells. Matched isotype (dotted line) was used as control of non-specific fluorescence signals. The column bar graphs, below the respective histograms, represent: the MFI of untreated cells (Ctr), and Nef(+)-HIV-1- and ΔNef-HIV-1-infected cells stained with APC-conjugated anti-CD4 and FITC-conjugated CD36 antibodies. SYTOX Blue has been used to exclude dead cells from the analyses. The results (mean ± standard deviation) are representative of four (MDM M-CSF) and six (MDM GM-CSF) independent experiments (**p<0.01).
Figure 7.
CD36 RNA transcriptional levels in Nef treated cells.
PBMCs were cultivated in HEMA condition w/o EPO for three days followed by additional three days in presence of 50 ng/mL rNef/myr. (A) Representative dot plots and histogram of PBMCs analyzed by flow cytometry. The viability of cells, evaluated by SYTOX Blue dead cell stain (R1), is shown on the left dot plot. The dot plot on the center shows the forward and side scatter of expanded PBMCs (R1-gated cells). The R2 (Lym) and R3 (MDM) populations were identified as described in figure 2A. The smaller dot plots show the Lym (R2-sorted) and MDM (R3-sorted) cells separated by cell sorting and the purity was >99% and >95%, respectively. The histogram on the right shows the CD36 expression, analyzed on MDMs by using FITC-conjugated anti-CD36 antibody, and the fluorescence intensity in Nef-treated (solid grey histogram) was compared to untreated (solid line) cells. Matched isotype (dotted line) was used as control of non-specific fluorescence signals. (B) The histogram reports the CD36 relative mRNA levels expression of CD36 assessed in expanded PBMCs (Total cells), MDMs (R3-sorted cells) and Lymphocytes (R2-sorted cells) cultivated in presence or absence of Nef. RT-PCR results are normalized to the GADPH housekeeping gene. The results (mean ± standard deviation) are representative of three independent experiments (*p<0.05, **p<0.01); b.d., below of detection.
Figure 8.
Recombinant Nef reduces oxLDL uptake in MDMs.
PBMCs were cultivated in HEMA condition w/o EPO for three days followed by additional three days in presence of 50 ng/mL rNef/myr. Cells were then incubated with 25 μg/mL of DiI-nLDL or DiI-oxLDL in delipidized serum for different time (15, 30 and 60 min). (A) Representative histograms of fluorescent lipids uptake by Nef-treated (solid grey histogram) compared to untreated (solid line) cells evaluated by flow cytometry. Cells w/o DiI-conjugated lipids (dotted line) were used as control for autofluorescent signal. (B) The MFI of Nef-treated (Nef) compared to untreated (Ctr) cells at 60 min from the DiI-conjugated lipids addition were reported in the histogram. SYTOX Blue was used to exclude dead cells. The results (mean ± standard deviation) are representative of three independent experiments (*p<0.05).
Figure 9.
Recombinant Nef reduces phagocytosis in MDMs.
PBMCs were cultivated in HEMA condition w/o EPO for three days followed by additional three days in presence of 50 ng/mL rNef/myr. Cells were then incubated with FITC-labeled beads or GFP-S. typhimurium for 30 min. (A) Representative dot plots of fluorescent beads and S. typhimurium uptake by Nef-treated (+Nef) compared to untreated (Ctr) cells evaluated by flow cytometry. Cells not incubated with beads or S. typhimurium were used as control for auto-fluorescent signal. The gates indicate the respective percent of phagocytosis. (B) The phagocytosis capability of Nef-treated (Nef) expressed as percent of control (Crt) is reported in the histogram. Where required by experimental procedures, control cells were pre-incubated with blocking anti-CD36 antibody for 20 min before the phagocytosis assay (Crt+anti-CD36). SYTOX Blue was used to exclude dead cells. The results (mean ± standard deviation) are representative of four independent experiments (*p<0.05).
Figure 10.
Nef induces TNF-α release and anti-human TNF-α antibody neutralizes rhTNF-α-induced CD36 downregulation.
TNF-α release was measured into supernatants collected from MDM cultures in presence or absence of Nef protein, (A) The column bar graph shows the amount of TNF-α released in the medium by untreated cells (Ctr) or Nef-treated cells (Nef) derived from PBMCs cultivated in HEMA condition w/o EPO for three days and in presence of rNef/myr for additional three days. (B) The column bar graph shows the amount of TNF-α released in the medium by MDMs differentiated in presence of M-CSF (10 ng/mL) for 5 days and treated with rNef/myr or infected with VSV-G pseudotyped HIV-1-expressing or not expressing the nef gene. (Ctr) untreated cells, (Nef(+)-HIV-1) infected MDMs with VSV-G pseudotyped HIV-1-expressing Nef, (ΔNef-HIV-1) infected MDMs with VSV-G pseudotyped HIV-1-not expressing Nef, (Nef) rNef/myr-treated cells. The levels of the cytokine are expressed as picograms/mL. The results (mean ± standard deviation) are representative of three independent experiments (*p<0.05). (C) Cells isolated by using CD14 magnetic beads (Miltenyi Biotec) were cultured in presence of human M-CSF (10 ng/mL) for 5 days followed by additional three days in presence of different concentrations of rhTNF-α (10, 3, 1, 0.3 ng/mL). In the column bar graph are presented the MFI of untreated cells (Ctr CD36) and TNF-α-treated cells at different cytokine concentrations (TNFα 10, 3, 1, 0.3 ng/mL) stained with FITC-conjugated anti-CD36. Matched isotype (Ctr isotype) was used as control of non-specific fluorescence signals and SYTOX Blue was used to exclude dead cells. The results (mean ± standard deviation) are representative of three independent experiments (*p<0.05). (D) Measurement of cytotoxic activity of serial diluted concentrations of rhTNF-α on WEHI-164 cells by using a MTT assay. In the line graph the absorbance of multiwells containing cells pre-incubated in presence of actinomycin D (♦, 1 μg/mL) with addition of rhTNF-α alone (▪) or together with anti-human TNF-α antibody (▴) is reported. The data shown are representative of two independent experiments carried out in triplicate. (E) M-CSF-derived MDMs were treated for three days with different concentrations of rhTNF-α alone (10, 3, 1, 0.3 ng/mL) or together with anti-human TNF-α antibody (1 μg/mL). The column bar graph represent the MFI of untreated cells (Ctr CD36), TNF-α-treated cells at different cytokine concentrations (TNFα 10, 3, 1, 0.3 ng/mL) or cells incubate with both rhTNF-α and 1 μg/mL of anti-human TNF-α antibody (TNFα 10, 3, 1 ng/mL+anti TNFα) stained with FITC-conjugated anti-CD36. Matched isotype (Ctr isotype) was used as control of non-specific fluorescence signals and SYTOX Blue was used to exclude dead cells. The results (mean ± standard deviation) are representative of three independent experiments (*p<0.05).
Table 1.
TNF-α released by MDMs.
Figure 11.
Nef-induced TNF-α release does not explain the downregulation of CD36 expression in MDMs.
Cells isolated by using CD14 magnetic beads (Miltenyi Biotec) were cultured in presence of human M-CSF (10 ng/mL) for 5 days and for additional three days in presence of two rNef/myr from different manufactures, the inactivated rNef/myr proteins by boiling or in presence of the rNef/myr proteins together with anti-human TNF-α antibody (1 μg/mL). (A) The dot plot shows the viability of cells by using SYTOX Blue dead cell stain. MDMs are identified by rectangular gate (MDM M-CSF) and analyzed for CD36 expression by using FITC-conjugated CD36 antibody. (B) The relative fluorescence intensities of CD36 in Nef-treated (solid line), Nefa-treated (solid grey histogram) and untreated (dash line) cells are shown in the representative histogram. Matched isotype (dotted line) was used as control of non-specific fluorescence signals (Nef refers to the protein from Dr. M. Federico [22]; Nefa, to the protein from Jena Bioscience). (C) The column bar graph presents the MFI of untreated cells (Ctr CD36), Nef- and Nefa-treated cells (Nef CD36 and Nefa CD36), cells incubated with inactivated Nef proteins (boiled Nef CD36 and boiled Nefa CD36) and cells incubated with the Nef proteins and anti-humanTNF-α (Nef+anti TNFα CD36) stained with FITC-conjugated anti-CD36. The results (mean ± standard deviation) are representative of three independent experiments (*p<0.05). (D) PBMCs were cultivated in HEMA condition w/o EPO for three days and for additional three days in presence of TNF-α (10 ng/mL) or rNef/myr as control of CD36 downregulation. The viability of cells, assessed by SYTOX Blue dead cell stain, is shown in the panel D. MDMs are identified by rectangular gate (MDM) and analyzed for CD36 expression by using FITC-conjugated CD36 antibody. (E) The relative fluorescence intensities of CD36 in Nef-treated (solid line), TNF-α-treated (solid grey histogram) and untreated (dash line) cells are shown in the representative histogram. Matched isotype (dotted line) was used as control of non-specific fluorescence signals. The data shown are representative of three independent experiments with similar results. PBMCs were cultivated in HEMA condition without EPO for three days and for additional three days in presence of both polyclonal rabbit anti-human TNF-α (1 μg/mL) and rNef/myr or in presence of rNef/myr alone as control of CD36 downregulation. MDMs were then analyzed for CD36 expression by using FICT-conjugated CD36 antibody. (F) The histogram reports the relative fluorescence intensities of CD36 in Nef-treated (solid line), Nef- and anti TNF-α-treated (solid grey histogram) or untreated (dotted line) cells. SYTOX Blue was used to exclude dead cells. (G) The column bar graph represents the MFI of untreated cells (Ctr CD36), Nef-treated cells (Nef CD36), Nef- and anti-humanTNF-α-treated cells (Nef+anti TNF-α CD36) stained with FITC-conjugated CD36 antibody. The results (mean ± standard deviation) are representative of three independent experiments (*p<0.05).