Tunneling nanotubes (TNT) are induced by HIV-infection of macrophages: a potential mechanism for intercellular HIV trafficking

Abstract

Cell to cell communication is essential for the organization/coordination of multicellular systems and cellular development. Cellular communication is mediated by soluble factors, including growth factors, neurotransmitters, cytokines/chemokines, gap junctions, and the recently described tunneling nanotubes (TNT). TNT are long cytoplasmatic bridges that enable long range directed communication between cells. The proposed function for TNT is the cell-to-cell transfer of large cellular structures such as vesicles and organelles. We demonstrate that HIV-infection of human macrophages results in an increased number of TNT, and show HIV particles within these structures. We propose that HIV "highjacks" TNT communication to spread HIV through an intercellular route between communicated cells, contributing to the pathogenesis of AIDS.

Fig. 1

HIV-infection of macrophages results in HIV-p24 production as detected by ELISA and immunofluorescence. Cultures of human macrophages were exposed to HIV_ADA (20 ng/ml p24) for 24 h and then washed extensively to eliminate unbound virus. Medium was collected every day to assay p24 production by ELISA. (A) HIV-infected macrophages produce significant amounts of p24, after 1-3 days that decrease after 4-8 days post-infection (red line, ●) (n = 8 independent experiments, mean ± SD). No p24 was detected in uninfected cultures (black line, ■). (B) HIV-infected macrophages were stained at different time points with antibodies for HIV-p24 (green staining, see inset), actin (Texas Red-phalloidin, red staining, see inset) and with DAPI (blue staining). A representative picture of 2 days post-infection is shown to illustrate intracellular vesicular staining as well as HIV-p24 staining inside long processes (B). No background or nonspecific staining was detected using isotype-matched irrelevant antibodies (C). Bar = 25 μm.

Fig. 2

Uninfected and HIV-infected macrophages have three types of processes, filopodia, short and long TNT. Based on the length of the processes formed by macrophages, we categorized them into three groups, filopodia, short and long TNT. The criteria for these groups were based on the length of the processes as well as on whether the process connected one or more cells, TNT. (A-F) Macrophages stained for HIV-p24 (green staining), actin (Texas Red-phalloidin, red staining) and DAPI (blue staining). Panel (A) shows filopodia in uninfected macrophages and (D), is the enlargement of the boxed area denoted in (A). Panel (B) shows a representative picture 3 days post-HIV-infection of short TNT, and E, is the enlargement of the boxed area denoted in (E). Panel (C) shows a representative picture 3 days post-HIV-infection of long TNT, and (F), is the enlargement of the boxed area denoted in (C). Bars = 25 μm for (A and B) and 50 μm for (C).

Fig. 3

HIV-infection induces the transient formation of short and long range TNT. The percentage of cells with processes was quantified using confocal microscopy to detect staining for HIV-p24 (green staining), actin (Texas Red-phalloidin, red staining) and DAPI (blue staining). Three dimensional reconstruction, using the imaging program NIS, was performed to visualize all TNTs and filopodia in these cultures. (A) In uninfected (■, black line) and HIV-infected (, red line) macrophage cultures, ∼15-25% of the cells have filopodia. (B) HIV-infection of macrophages induces the formation of additional short TNT (, red line, *p < 0.001, n = 4) as compared to uninfected cultures of macrophages (■, black line). (C) HIV-infection of macrophages induces the formation of an increased number of long TNT (, red line, *p < 0.003, n = 4) as compared to uninfected cultures (■, black line). Statistical significance in (B and C) graphs, between uninfected and HIV-infected macrophages, was found at days 2-6 and 2-4, respectively, and returned to control levels (uninfected cells) after 5 and 6 days post-infection. (D) An example of filopodia at time 0 in uninfected cells stained for actin and DAPI. (E) An example of short range TNT, 3 days post-infection with HIV stained for HIV-p24 (green staining), actin (Texas Red-phalloidin, red staining) and DAPI (blue staining). (F) An example of a decrease in short and long TNT, 7 days post-infection. Arrow in (D) denotes the filopodia structures at time 0. Bar = 25 μm.

Fig. 4

HIV-infection, although alter the numbers of TNT, did not alter the length of the processes. Using the process classification described in Fig. 2, we determined whether HIV-infection alters the length of the processes induced. Length of the processes (μm) in uninfected cells (white bars) and HIV-infected cells (black bars) at days 2 and 3 post-infection. No changes in the length of the processes were detected at any other time points (data not shown). No significant changes in the length of the processes were found between uninfected and HIV-infected macrophages, suggesting that the length of the processes is not altered (day 3, p < 0.7, n = 5) by HIV-infection.

Fig. 5

Distinct HIV vesicles localization in TNT processes. Staining of macrophages infected with HIV for HIV-p24 (green staining), actin (Texas Red-phalloidin, red staining) and DAPI (blue staining) and subsequent 3D reconstruction demonstrated that one cell can have both filopodia and TNT in different optical planes. It also demonstrated HIV particles between connected cells. (A and B) These pictures correspond to the same field showing that the same cell can have both TNT (top optical section) and filopodia (bottom optical section) (see arrows). Panel (C) represents a picture of short TNT in HIV-infected macrophages at day 3 post-infection stained for HIV-p24 (green staining), actin (Texas Red-phalloidin, red staining) and DAPI (blue staining). The white box in (C), enlarged in (D-F), corresponds to short range TNT, stained for actin (Texas Red-phalloidin, red staining, (D)), HIV-p24 (green staining, E), and DAPI (blue staining, no shown). Panel (F) corresponds to the merge of (D and E), showing HIV-p24 positive vesicles in short TNT (see arrows). The white box in (G), as enlarged in (H), corresponds to long range TNT, stained for actin (Texas Red-phalloidin, red staining), HIV-p24 (green staining), and DAPI (blue staining). Arrows denote HIV-p24 positive vesicles being transported across long range TNT.