Phosphatidylcholine Liposomes Down-Modulate CD4 Expression Reducing HIV Entry in Human Type-1 Macrophages

Abstract

A strategy adopted to combat human immunodeficiency virus type-1 (HIV-1) infection is based on interfering with virus entry into target cells. In this study, we found that phosphatidylcholine (PC) liposomes reduced the expression of the CD4 receptor in human primary type-1 macrophages but not in CD4⁺ T cells. The down-regulation was specific to CD4, as any effect was not observed in CCR5 membrane expression. Moreover, the reduction of membrane CD4 expression required the Ca²⁺-independent protein kinase C (PKC), which in turn mediated serine phosphorylation in the intracytoplasmic tail of the CD4 receptor. Serine phosphorylation of CD4 was also associated with its internalization and degradation in acidic compartments. Finally, the observed CD4 downregulation induced by PC liposomes in human primary macrophages reduced the entry of both single-cycle replication and replication competent R5 tropic HIV-1. Altogether, these results show that PC liposomes reduce HIV entry in human macrophages and may impact HIV pathogenesis by lowering the viral reservoir.

Keywords: HIV entry; host-directed therapy; liposome; macrophage; phosphatidylcholine.

Figure 1

Downregulation of CD4 expression on type-1 macrophages. (A, B) Lymphocytes (10⁶ cells/0.5 ml) were stimulated or not with liposome formulations at the ratio 1:1 (liposome:cell) for 18 h. Cells were collected, stained with anti-CD3-FITC, anti-CD4-PerCP-Vio700, and anti-CCR5-PE and analyzed by flow cytometry. Figure shows the percentage of CD4 positive cells (A, n = 4) and CCR5 positive cells (B, n = 3) of CD3 positive cells. p-value was obtained by one-sided Wilcoxon matched-pairs signed rank test (p = ns, not significant). (C, D) Type-1 macrophages (5 × 10⁵ cells/ml) were stimulated or not with liposome formulations at the ratio 1:1 (liposome:cell) for 18 h. Cells were collected, stained with anti-CD4-PerCP-Vio700 and anti-CCR5-PE and analyzed by flow cytometry. Figure shows the percentage of CD4 (C, n = 5) and CCR5 (D, n = 5) positive cells. p-value was obtained by one-sided Wilcoxon matched-pairs signed rank test (p = ns, not significant). (E, F) Macrophages (5 × 10⁵ cells/ml) were stimulated or not with PC liposomes at the ratio 1:1 (liposome:cell) for 1, 3, 6, and 18 h and then collected. Cells were stained with anti-CD4-PerCP-Vio700, fixed, permeabilized, stained with anti-CD4-APC and finally analyzed by flow cytometry. Data show the percentage of cells expressing surface CD4 (E) and intracellular CD4 (F) and are representative of five experiments performed on cells from different healthy donors.

Figure 2

Downregulation of CD4 expression involves acidic compartments by serine phosphorylation. (A, B) Macrophages (5 × 10⁵ cells/ml) were stimulated or not with PC liposomes at the ratio 1:1 (liposome:cell) for 5, 10, 20, 30 min, 1, and 18 h. Cells were collected (2 × 10⁶), stored at −80°C and then analyzed by western blotting, following lysis. Figure shows a representative Western blotting (A) and the densitometric analysis of the levels phosphorylated CD4 on serine 433 (p-CD4 Ser433) normalized to CD4 basal signal (B) shown as mean ± Standard Deviation (SD) of values obtained from four different healthy donors. GADPH was used as loading control. *p <0.05 and **p <0.01 by Kruskal–Wallis Uncorrected Dunn’s test, in comparison with non-stimulated control. (C) Macrophages (5 × 10⁵ cells/ml) were pre-treated or not with 10 nM RO 31-8220 and 100 nM Gö 6976 for 30 min and then stimulated or not with PC liposomes at the ratio 1:1 (liposome:cell) for 18 h. Cells were collected, stained with anti-CD4-PerCP-Vio700 and analyzed by flow cytometry. Data are shown as mean ± SD of fold variation of percentage of CD4 positive cells calculated by normalizing the percentage of CD4 positive cells obtained from each healthy donor on their own non-stimulated control (n = 5). *p <0.05 and **p <0.01 by Student’s t-test. (D) Macrophages (5 × 10⁵ cells/ml) were pre-treated or not with 10 nM concanamycin A (ConcA) for 30 min, stimulated or not with PC liposomes at the ratio 1:1 (liposome:cell) for 18 h and then were collected. Cells were stained with anti-CD4-PerCP-Vio700, fixed, permeabilized, stained with anti-CD4-APC and finally analyzed by flow cytometry. Data are shown as mean ± SD of fold variation of percentage of positive cells expressing intracellular CD4 calculated by normalizing the percentage of CD4 positive cells obtained from each healthy donor on their own non-stimulated control (n = 5). ***p <0.0001 by Student’s t-test.

Figure 3

Down-modulation of CD4 expression in PC liposomes pre-treated macrophages reduces HIV-1 entry. (A) Macrophages (5 × 10⁵ cells/ml) were pre-treated or not with PC liposomes for 18 h at the ratio 1:1 (liposome:cell), washed and then infected with HIV-1 Env (Ba-L)- or VSV-G-pseudotyped luciferase-encoding HIV-1. After infection, cells were washed, cultured for 6 days, and infection was quantified by luciferase activity. RLUs were normalized to the value for luciferase obtained in the VSV-G-pseudotyped virus-infected cells. Mean relative luciferase activities ± SD are shown (three independent experiments performed in quadruplicate on cells from three different healthy donors; ***p <0.0001 by Student’s t-test). A nonfunctional envelope (HIV-1-ΔKS) was used as a negative control to determine the background levels of luciferase activity. (B) Type-1 macrophages (3 × 10⁵ cells/ml) were pre-treated or not with PC liposomes for 18 h at the ratio 1:1 (liposome:cell), washed and then infected with replication competent R5 HIV-1 81A strain. After infection, cells were washed, cultured for 4 days, and intracellular HIV-DNA was quantified by ddPCR. Total amount of HIV-DNA was normalized on cell number obtained by the quantification of Albumin. Results are shown as mean HIV-DNA values ± SD of the values obtained from 4 replicates and are representative of experiments performed on cells from two healthy donors (**p = 0.0005 by Student’s t-test).