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WFDC1 expression identifies memory CD4 T-lymphocytes rendered vulnerable to cell-cell HIV-1 transfer by promoting intercellular adhesive junctions.

Alvarez RA, Thorborn G, Reading JL, Reddy SK, Vyakarnam A - Retrovirology (2011)

Bottom Line: Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer.Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, King's College London, UK.

ABSTRACT

Background: Elucidating mechanisms that promote HIV-1 transfer between CD4+ T-lymphocytes and their subsequent loss is of importance to HIV-1 pathogenesis. We recently reported that whey acidic protein, ps20, promotes cell-free HIV-1 spread through ICAM-1 modulation. Since ICAM-1 is pivotal in cell conjugation and intercellular HIV-1 transfer, this study examines ps20 effects on HIV-1 spread between T lymphocytes.

Results: We demonstrate intrinsic ps20 variability in primary CD4+ T-lymphocyte clonal populations and a significant positive correlation between endogenous ps20 levels and virus transfer involving fusion resulting in a spreading infection that could be reversed by the addition of reverse transcriptase inhibitors. Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer. Conversely, virus transfer was promoted by ectopic ps20 expression or by exogenous addition of recombinant ps20. A higher frequency of virological synapse formation was evident in cocultures of HIV-1 infected donor T-cells with ps20high v ps20low/intermediate targets. Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.

Conclusions: Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

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siRNA-mediated knockdown of ps20 inhibits conjugate and multiple conjugate formation more effectively than siRNA-mediated knockdown of ICAM-1. (A) ps20 and ICAM-1 mRNA levels were measured in a selection of ps20high and ps20inter/low cells at 4 different time points. Data show a two-tailed non-parametric Spearman's r correlation of all data points. CD4 T-cell Jwt ps20inter were treated with either; a non-silencing (NS), ICAM-1 or WFDC1/ps20-silencing siRNA pool for 6 days. (B) After siRNA treatment the expression of ICAM-1, ps20 and GAPDH mRNA was analyzed by qRT-PCR and relative expression to β-actin was measured. Normalized relative expression was calculated in reference to siNS control. Data represent the mean of three replicate assays. (C) Surface expression of ICAM-1 in siRNA treated cells is shown as assessed by standard immunofluorescence. Normalized MFI was calculated in reference to siNS control. Data represent the mean of three replicate assays (D) 8 × 104 NS, ICAM-1, or ps20 siRNA-treated WT Jurkat cells were dye-labelled and co-cultured with donor 40% 2044-infected donor cells at a T:D ratio of 1:0.2. Mean percentage of Gag+ target cells after 4-hour co-culture is shown. Normalized % of Gag+ target cells was calculated in reference to siNS control. Data represent the mean of three replicate assays. (E) 5 × 105 siRNA treated cells were dye-labelled and co-cultured with 5 × 105 60% 2044-infected donor cells. Co-cultures were incubated for 1 hour on Poly-L-lysine coated glass cover slips, then fixed and stained with a FITC anti-Gag (Green) Ab. Conjugates and MCs were assessed as before in at least 500 random target cells per population across triplicate experiments. (F) The panel depicts the mean conjugate interface diameter (μM) between siRNA treated Jurkat cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across triplicate experiments. Asterisk denotes statistically significant data as calculated using a paired t-test (*P ≤0.05) in relation to NS siRNA control.
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Figure 6: siRNA-mediated knockdown of ps20 inhibits conjugate and multiple conjugate formation more effectively than siRNA-mediated knockdown of ICAM-1. (A) ps20 and ICAM-1 mRNA levels were measured in a selection of ps20high and ps20inter/low cells at 4 different time points. Data show a two-tailed non-parametric Spearman's r correlation of all data points. CD4 T-cell Jwt ps20inter were treated with either; a non-silencing (NS), ICAM-1 or WFDC1/ps20-silencing siRNA pool for 6 days. (B) After siRNA treatment the expression of ICAM-1, ps20 and GAPDH mRNA was analyzed by qRT-PCR and relative expression to β-actin was measured. Normalized relative expression was calculated in reference to siNS control. Data represent the mean of three replicate assays. (C) Surface expression of ICAM-1 in siRNA treated cells is shown as assessed by standard immunofluorescence. Normalized MFI was calculated in reference to siNS control. Data represent the mean of three replicate assays (D) 8 × 104 NS, ICAM-1, or ps20 siRNA-treated WT Jurkat cells were dye-labelled and co-cultured with donor 40% 2044-infected donor cells at a T:D ratio of 1:0.2. Mean percentage of Gag+ target cells after 4-hour co-culture is shown. Normalized % of Gag+ target cells was calculated in reference to siNS control. Data represent the mean of three replicate assays. (E) 5 × 105 siRNA treated cells were dye-labelled and co-cultured with 5 × 105 60% 2044-infected donor cells. Co-cultures were incubated for 1 hour on Poly-L-lysine coated glass cover slips, then fixed and stained with a FITC anti-Gag (Green) Ab. Conjugates and MCs were assessed as before in at least 500 random target cells per population across triplicate experiments. (F) The panel depicts the mean conjugate interface diameter (μM) between siRNA treated Jurkat cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across triplicate experiments. Asterisk denotes statistically significant data as calculated using a paired t-test (*P ≤0.05) in relation to NS siRNA control.

Mentions: We assessed the potency of ps20- vs. ICAM-1- mediated virus transfer and determined the relative importance of each in T-T conjugate formation, using an si-RNA targeted knock-down strategy in the Jurkat system. Treatment of Jwt-ps20inter cells with siRNA against ICAM-1 led to a significant 50% reduction in the levels of ICAM-1 mRNA, with no significant reduction of either ps20 or GAPDH expression (Figure 6A). However, siRNA against ps20 led to a significant 60% reduction in ps20 mRNA, and a concomitant 40% reduction in ICAM-1 mRNA, with no reduction in GAPDH. This confirms our previous observations that blocking ps20 can inhibit ICAM-1 expression [23]. Surface ICAM-1 protein expression was reduced by 50% and 45% with siRNA against ICAM-1 or ps20, respectively (Figure 6B). Ps20 vs. ICAM-1 knockdown resulted in a 36% vs. 30% reduction in the levels of virus transfer respectively (Figure 6C). In addition, ICAM-1 versus ps20 siRNA inhibited single conjugates by 50% vs. 61% respectively (Figure 6D). ICAM-1 siRNA inhibited multiple conjugates by 50% versus a marked 92% by ps20 siRNA (Figure 6D). Lastly, the size of the conjugate interface was not affected by ICAM-1 knockdown, whereas ps20 knockdown had a small but consistent effect; a significant 1.2-fold reduction in mean conjugate diameter from 3.601 (± 0.1871) μm in NS siRNA treated control to 2.933 (± 0.2179) μm in ps20 siRNA treated cells was noted (Figure 6E).


WFDC1 expression identifies memory CD4 T-lymphocytes rendered vulnerable to cell-cell HIV-1 transfer by promoting intercellular adhesive junctions.

Alvarez RA, Thorborn G, Reading JL, Reddy SK, Vyakarnam A - Retrovirology (2011)

siRNA-mediated knockdown of ps20 inhibits conjugate and multiple conjugate formation more effectively than siRNA-mediated knockdown of ICAM-1. (A) ps20 and ICAM-1 mRNA levels were measured in a selection of ps20high and ps20inter/low cells at 4 different time points. Data show a two-tailed non-parametric Spearman's r correlation of all data points. CD4 T-cell Jwt ps20inter were treated with either; a non-silencing (NS), ICAM-1 or WFDC1/ps20-silencing siRNA pool for 6 days. (B) After siRNA treatment the expression of ICAM-1, ps20 and GAPDH mRNA was analyzed by qRT-PCR and relative expression to β-actin was measured. Normalized relative expression was calculated in reference to siNS control. Data represent the mean of three replicate assays. (C) Surface expression of ICAM-1 in siRNA treated cells is shown as assessed by standard immunofluorescence. Normalized MFI was calculated in reference to siNS control. Data represent the mean of three replicate assays (D) 8 × 104 NS, ICAM-1, or ps20 siRNA-treated WT Jurkat cells were dye-labelled and co-cultured with donor 40% 2044-infected donor cells at a T:D ratio of 1:0.2. Mean percentage of Gag+ target cells after 4-hour co-culture is shown. Normalized % of Gag+ target cells was calculated in reference to siNS control. Data represent the mean of three replicate assays. (E) 5 × 105 siRNA treated cells were dye-labelled and co-cultured with 5 × 105 60% 2044-infected donor cells. Co-cultures were incubated for 1 hour on Poly-L-lysine coated glass cover slips, then fixed and stained with a FITC anti-Gag (Green) Ab. Conjugates and MCs were assessed as before in at least 500 random target cells per population across triplicate experiments. (F) The panel depicts the mean conjugate interface diameter (μM) between siRNA treated Jurkat cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across triplicate experiments. Asterisk denotes statistically significant data as calculated using a paired t-test (*P ≤0.05) in relation to NS siRNA control.
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Figure 6: siRNA-mediated knockdown of ps20 inhibits conjugate and multiple conjugate formation more effectively than siRNA-mediated knockdown of ICAM-1. (A) ps20 and ICAM-1 mRNA levels were measured in a selection of ps20high and ps20inter/low cells at 4 different time points. Data show a two-tailed non-parametric Spearman's r correlation of all data points. CD4 T-cell Jwt ps20inter were treated with either; a non-silencing (NS), ICAM-1 or WFDC1/ps20-silencing siRNA pool for 6 days. (B) After siRNA treatment the expression of ICAM-1, ps20 and GAPDH mRNA was analyzed by qRT-PCR and relative expression to β-actin was measured. Normalized relative expression was calculated in reference to siNS control. Data represent the mean of three replicate assays. (C) Surface expression of ICAM-1 in siRNA treated cells is shown as assessed by standard immunofluorescence. Normalized MFI was calculated in reference to siNS control. Data represent the mean of three replicate assays (D) 8 × 104 NS, ICAM-1, or ps20 siRNA-treated WT Jurkat cells were dye-labelled and co-cultured with donor 40% 2044-infected donor cells at a T:D ratio of 1:0.2. Mean percentage of Gag+ target cells after 4-hour co-culture is shown. Normalized % of Gag+ target cells was calculated in reference to siNS control. Data represent the mean of three replicate assays. (E) 5 × 105 siRNA treated cells were dye-labelled and co-cultured with 5 × 105 60% 2044-infected donor cells. Co-cultures were incubated for 1 hour on Poly-L-lysine coated glass cover slips, then fixed and stained with a FITC anti-Gag (Green) Ab. Conjugates and MCs were assessed as before in at least 500 random target cells per population across triplicate experiments. (F) The panel depicts the mean conjugate interface diameter (μM) between siRNA treated Jurkat cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across triplicate experiments. Asterisk denotes statistically significant data as calculated using a paired t-test (*P ≤0.05) in relation to NS siRNA control.
Mentions: We assessed the potency of ps20- vs. ICAM-1- mediated virus transfer and determined the relative importance of each in T-T conjugate formation, using an si-RNA targeted knock-down strategy in the Jurkat system. Treatment of Jwt-ps20inter cells with siRNA against ICAM-1 led to a significant 50% reduction in the levels of ICAM-1 mRNA, with no significant reduction of either ps20 or GAPDH expression (Figure 6A). However, siRNA against ps20 led to a significant 60% reduction in ps20 mRNA, and a concomitant 40% reduction in ICAM-1 mRNA, with no reduction in GAPDH. This confirms our previous observations that blocking ps20 can inhibit ICAM-1 expression [23]. Surface ICAM-1 protein expression was reduced by 50% and 45% with siRNA against ICAM-1 or ps20, respectively (Figure 6B). Ps20 vs. ICAM-1 knockdown resulted in a 36% vs. 30% reduction in the levels of virus transfer respectively (Figure 6C). In addition, ICAM-1 versus ps20 siRNA inhibited single conjugates by 50% vs. 61% respectively (Figure 6D). ICAM-1 siRNA inhibited multiple conjugates by 50% versus a marked 92% by ps20 siRNA (Figure 6D). Lastly, the size of the conjugate interface was not affected by ICAM-1 knockdown, whereas ps20 knockdown had a small but consistent effect; a significant 1.2-fold reduction in mean conjugate diameter from 3.601 (± 0.1871) μm in NS siRNA treated control to 2.933 (± 0.2179) μm in ps20 siRNA treated cells was noted (Figure 6E).

Bottom Line: Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer.Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, King's College London, UK.

ABSTRACT

Background: Elucidating mechanisms that promote HIV-1 transfer between CD4+ T-lymphocytes and their subsequent loss is of importance to HIV-1 pathogenesis. We recently reported that whey acidic protein, ps20, promotes cell-free HIV-1 spread through ICAM-1 modulation. Since ICAM-1 is pivotal in cell conjugation and intercellular HIV-1 transfer, this study examines ps20 effects on HIV-1 spread between T lymphocytes.

Results: We demonstrate intrinsic ps20 variability in primary CD4+ T-lymphocyte clonal populations and a significant positive correlation between endogenous ps20 levels and virus transfer involving fusion resulting in a spreading infection that could be reversed by the addition of reverse transcriptase inhibitors. Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer. Conversely, virus transfer was promoted by ectopic ps20 expression or by exogenous addition of recombinant ps20. A higher frequency of virological synapse formation was evident in cocultures of HIV-1 infected donor T-cells with ps20high v ps20low/intermediate targets. Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.

Conclusions: Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

Show MeSH
Related in: MedlinePlus