The report of eradication of HIV from an adult patient [1,2] and long-term suppression of HIV without antiretroviral drugs in a child born with HIV infection [3,4] raises hope that we may eventually develop ways to cure HIV infection. However, for such strategies to be successful, it is critical to develop a better understanding of the tissue reservoirs, particularly the brain. In contrast to reservoirs such as spleen, lymph nodes, and gut, there are no resident lymphoid cells in the brain. HIV infection is restricted to brain macrophages/microglia and astrocytes [5–7], which are long-lived cells [8–10] in which the virus may reside almost indefinitely. Astrocytes can also be infected in vivo by simian and feline immunodeficiency viruses, leading to encephalitis [11–13]. Astrocytes are the most abundant cell types in the brain and outnumber neurons in the ratio of 10 : 1. Infection of a small percentage of astrocytes could result in a sizable reservoir. After the virus establishes latency in astrocytes, exposure to cytokines can result in viral replication without any cytopathic effects [14,15]. The virus emerging from the infected astrocytes can be transmitted to lymphocytes . In an inflammatory environment, astrocytes may proliferate potentially, leading to clonal expansion of HIV in the brain similar to lymphocyte reservoirs . Hence, these cells represent an ideal reservoir for HIV.
The mechanism of HIV infection of astrocytes is, however, poorly understood. Although there is strong evidence showing that astrocytes are infected with HIV in vivo [18–22], in-vitro studies show that infection with cell-free HIV is extremely inefficient in primary astrocytes [15,23–25]. Thus, there might be other mechanisms in vivo by which HIV infects astrocytes. Astrocytes are an integral part of the blood–brain barrier (BBB) and are most commonly infected in the perivascular regions  where astrocytes have the potential to be exposed to the HIV-infected lymphocytes. Here, we report that infection of astrocytes occurred efficiently by cell-to-cell contact with HIV-infected lymphocytes and demonstrate mechanisms by which this interaction promotes HIV transmission.
Primary cells and cell lines
All studies were approved by the Institutional Review Board at the Johns Hopkins University and the Office of Human Subjects Research at the National Institutes of Health (NIH). All brain tissues and blood samples were obtained without identifiers. Astrocytes were cultured from human fetal brain specimens of 10–14 weeks’ gestation of three different individuals. Individual variability was not determined. Cultures derived from human fetal brain and neural progenitor cells contained more than 99% astrocytes as determined by immunostaining for glial fibrillary acidic protein (GFAP) and glutamate transporter.
HIV-1 viruses and infection
X4-using full-length HIV-1 infectious clone pNL4–3 was obtained from the NIH AIDS Reagent Program. HIV-1NL4–3-based reporter virus construct, pNLENG1, was made by inserting an enhanced green fluorescent protein (EGFP) gene linked with internal ribosome entry site between the genes env and nef of pNL4–3 . R5-using HIV-1SF162-based reporter virus, pSF162R3, was constructed in a similar manner . All viral genes including nef are intact in these infectious reporter viruses.
Correlative electron microscopy and three-dimensional electron microscopy
Astrocytes co-cultured with NLENG1-infected Jurkat-tat (JKT) cells were fixed after 3 days and processed for transmission electron microscopy (TEM) at the Johns Hopkins University Microscope Facility. One of the samples described above was processed for three-dimensional electron microscopy (3D-EM) by Renovo Neural Inc. (Cleveland, Ohio, USA).
Infection blocking assay
Antibodies to CD4, CXCR4, Dendritic Cell-specific Intercellular adhesion molecule-3-Grabbing Non-Integrin (DC-SIGN), α4β7 integrin, and antagonists to CD4 and CXCR4 were used to block cell-to-cell transmission of HIV.
Data were analyzed by analysis of variance (ANOVA) with unequal variance or Student's t test. Dunnett's method was used for post-hoc test. Shapiro–Wilk test was applied to test normality of the residuals. SAS version 9.2 (SAS Institute Inc., Cary, North Carolina, USA) was used for the above analysis, and P less than 0.05 was used as significance level.
Detailed protocols are described in the Supplemental Information (http://links.lww.com/QAD/A657).
Efficient infection of astrocytes occurs in co-cultures with HIV-infected lymphocytes
To investigate HIV transmission from lymphocytes to astrocytes, primary human astrocytes were co-cultivated with HIVNL4–3-based reporter virus (NLENG1)-infected JKT cells in the absence of any other treatment (Fig. 1a). Infection of astrocytes appeared 3 days after co-culture. EGFP was distinctly expressed in the entire cytoplasm, nucleus and in all the processes of the infected astrocyte (Fig. 1a–c). Although astrocyte infection was only approximately 1%, the actual rate was higher because some of the cells did not express EGFP, but express HIV-1 p24 (∼5%). However, the infection was consistently observed in the co-cultures compared with the absence of visible infection using cell-free HIV alone. Similar rates of infection were seen in both human fetal astrocytes and progenitor-derived astrocytes (PDAs); hence, subsequent experiments were performed using human fetal astrocytes.
The infection of astrocytes by cell-to-cell contact was further verified with other NLENG1-infected lymphocytic cell lines, such as MT4 (Fig. 1b), and peripheral blood mononuclear cells (PBMCs) (Fig. 1c). And this was also true for other X4 or X4R5-using viruses (Fig. S1, http://links.lww.com/QAD/A657). This cell-to-cell transmission was not observed in the co-cultures with R5-using HIVSF162R3-infected PBMCs (data not shown). Therefore, cell-to-cell contact with HIV-infected lymphocytes represents an efficient mechanism for HIV infection of astrocytes.
Lymphocytes transmit HIV to astrocytes through a dynamic process of interactions between two types of cells
To study the interactions between lymphocytes and astrocytes in real time, we visualized the co-cultures by time-lapse video microscopy. We observed that lymphocytes frequently made contact with astrocytes, but both cell types were in motion (Movies S1a, http://links.lww.com/QAD/A651 and S1b, http://links.lww.com/QAD/A652). Multiple lymphocytes attached to a single astrocyte and conversely a single lymphocyte made contact with more than one astrocyte. The process of attachment and detachment of lymphocytes to astrocytes was frequently noted in the co-cultures with both uninfected and NLENG1-infected JKT cells (Movies S1a, http://links.lww.com/QAD/A651 and S1b, http://links.lww.com/QAD/A652). However, some lymphocytes remained attached to astrocytes for a long period (Movie S1b, http://links.lww.com/QAD/A652), or eventually immobilized on astrocytes, resulting in invagination of astrocytes and subsequent HIV infection of astrocytes (Movie S1b, http://links.lww.com/QAD/A652, Fig. 2a and S2a, http://links.lww.com/QAD/A657). The area of contact between astrocytes and HIV-infected lymphocytes manifested diverse morphological features. In some aggregates, the infected lymphocytes produced processes that spread over the astrocyte surface and formed a large synapse with an interdigitated interface (Fig. 2b and S2b, http://links.lww.com/QAD/A657). In others, long processes extended from the infected lymphocytes and made contacts with astrocytes (Fig. 2c). Some intermediary features of contact were seen between these two extremes (Fig. 2d). Thus, HIV transmission from lymphocytes to astrocytes requires active interactions between the two cell types through a dynamic process, leading to immobilization of the cells by formation of tight areas of contact.
Ultrastructural features of HIV transmission from lymphocytes to astrocytes
We further characterized ultrastructural features of the contacts between astrocytes and HIV-infected lymphocytes by correlative electron microscopy/3D-EM. Serial block-face scanning electron microscopy (SBF-SEM) generated large sets of electron microscopy images from the co-culture sample, from which 3D images were reconstructed.
We took phase and fluorescent combined photomicrographs before the samples were processed for EM/3D-EM (Fig. 3a). A 3D-EM image was reconstructed using the described techniques, by which the relationship between astrocytes and T lymphocytes was observed (Fig. 3b and c, Movie S2a, http://links.lww.com/QAD/A654). From the sets of images, astrocytes (A1 and A2) and lymphocytes (T1–T3) were analyzed by reconstructing high-resolution 3D images to elucidate the mechanisms of HIV transmission from lymphocytes to astrocytes. T1 was an HIV-infected, and JKT syncytium confirmed in high-resolution electron microscopy images. This cell was partially wrapped by A1, and both T1 and A1 produced processes that stretched out and interdigitated (Fig. 3b and d; Movies S2a, http://links.lww.com/QAD/A654 and S2b, http://links.lww.com/QAD/A653). A long process from A1 was seen extending and formed a claw-like structure to grab T2, and many viral particles were seen adherent to these processes, suggesting that the processes might aid in the spread of the virus from the lymphocyte to the astrocyte (Fig. 3a–c, e; Movie S2a, http://links.lww.com/QAD/A654 and S2c, http://links.lww.com/QAD/A655). T3 was making contact with A2 and A1, and multiple filopodium-like processes from A02 wrapped around T3, whereas a single thick process from T3 protruded into A2 (Fig. 3a–c, f; and Movies S2a, http://links.lww.com/QAD/A654 and S2d, http://links.lww.com/QAD/A656).
In the areas where the cell membranes of A2 and T3 seemed to be in a tight opposition (Fig. 3g), serial SBF-SEM images showed that there were multiple small protrusions extending from either of these cells and reaching out to the other cell (Fig. 3h, i–iv). This produced appearance of a Velcro-like structure (Fig. 3h, ii) with tight contacts of cell membranes (Fig. 3h, ii–iv) interspersed with cleft-like spaces (Fig. 3h, iii and iv). Each individual unit gave the appearance of a virological synapse. Within these synapses, fully mature HIV particles as well as immature viruses that had not been released yet from the lymphocyte membrane were visualized (Fig. 3h, i–iv). Importantly, the viruses were seen budding from the lymphocyte directly onto the astrocyte membrane and making direct contact with the astrocyte membrane (Fig. 3h, iii and iv). The accompanying diagram is a composite of the SBF-SEM images and illustrates this unique phenomenon (Fig. 3i).
A similar mode of HIV transmission from lymphocytes to astrocytes was observed in the context of filopodial extensions by correlative, transmission electron microscopy. The processes from HIV-infected lymphocytes were noted making contacts with the astrocyte and forming filopodial connections (Fig. 4a and b). The filopodia indented the astrocyte membrane (Fig. 4b) or established contact with an astrocytic process (Fig. 4c and d). Viral particles were concentrated and lined up along the filopodia (Fig. 4b and e). At the sites of contacts between the lymphocyte filopodia and the astrocyte membrane, crescent-shaped hyperdensities protruded from the lymphocyte membranes, suggesting budding of the viruses (Fig. 4d and e). These immature or incomplete viral particles made direct contact with the astrocyte membrane. A diagram representing a composite of these photomicrographs illustrates the mode of HIV cell-to-cell transmission (Fig. 4f).
Role of HIV receptors and adhesion molecules in cell-to-cell transmission of HIV from lymphocytes to astrocytes
To further elucidate the mechanism for cell-to-cell transmission of HIV from lymphocytes to astrocytes, a series of blocking assays were performed using specific antibodies or pharmacological antagonists. We found that anti-CXCR4 antibody almost completely blocked this transmission (Fig. 5a). However, the continuous presence of anti-CXCR4 antibody was necessary, since removing the antibody resulted in an increased number of infected astrocytes (Fig. 5b). Similarly, a CXCR4 antagonist, ADM3100, significantly inhibited cell-to-cell infection of astrocytes. However, there was no effect of anti-CXCR4 antibody on the infection with cell-free HIV in astrocytes (Fig. S3, http://links.lww.com/QAD/A657). Anti-CD4 antibody and HIV fusion inhibitor, T-20, partially abolished cell-to-cell infection in astrocytes (Fig. 5a). In contrast, antibodies to DC-sign and α4β7 integrin had no effect on this cell-to-cell infection (Fig. 5a). Anti-intercellular adhesion molecule-1 (ICAM-1) and anti-lymphocyte function associated antigen-1 (LFA-1) antibodies partially inhibited this cell-to-cell infection (Fig. 5a). Taken together, CXCR4 is considered as a primary receptor for HIV entry into astrocytes in the context of cell-to-cell infection.
Furthermore, the focused ion beam scanning electron microscopy/3D study showed that the contact zones between astrocytes and HIV-infected T cells had a smooth configuration in the co-cultures treated with anti-CXCR4 antibody for 1 day. However, membrane extensions from astrocytes lined with HIV were observed in the co-cultures free of antibody or treated with anti-CD4 antibody (Fig. 5c), indicating that anti-CXCR4 antibody might inhibit the formation of filopodia from astrocytes. Thus, CXCR4 possibly plays a dual role in mediating HIV transmission to astrocytes from the infected lymphocytes.
To determine if the CXCR4-mediated transmission of HIV from lymphocytes to astrocytes may be due to an aberrant mutation in the envelope leading to a conformational change, we sequenced the env gene of NLENG1. No mutations were found in the gp120 region compared to the parental NL4–3.
On the basis of these observations, a novel mechanism is proposed for cell-to-cell transmission of HIV from lymphocytes to astrocytes, by which the budding or immature virus with ‘open’ CXCR4-binding sites on the envelope might bind directly to CXCR4 on the astrocyte membrane and trigger the process of fusion during HIV maturation (Fig. 5d). However, the mature HIV particle cannot gain entry into an astrocyte without expressing the CD4+ receptor.
Infection of astrocytes may be associated with migration of HIV-infected lymphocytes into the brain
HIV infection of astrocytes occurs primarily in the perivascular regions of the brain. For this infection to occur, the infected lymphocytes need to either cross the BBB or engage them across the endothelial tight junctions. Hence, we used an in-vitro BBB model to determine if HIV-infected T lymphocytes can cross the BBB and monitored their response to specific chemokines. A significant amount of NLENG1-infected JKT cells migrated across the BBB in the presence of stromal cell-derived factor-1α (150 ng/ml) (P < 0.05 for 1 day, P < 0.001 for 2 days). In comparison, no significant migration was noted with regulated on activation, normal T-cell expressed and secreted (RANTES) at the same concentration (Fig. S4a, http://links.lww.com/QAD/A657). When the transwell inserts were examined by confocal microscopy, cell-to-cell contacts were frequently observed between NLENG1-infected JKT cells and astrocytes in the presence of SDF1α (Fig. S4b, http://links.lww.com/QAD/A657), but less frequently with RANTES (Fig. S4c, http://links.lww.com/QAD/A657). Since chemokine SDF-1α is a ligand of CXCR4, this further suggests that CXCR4 plays a central role in cell-to-cell interaction and HIV transmission from lymphocytes to astrocytes.
Multiple studies show HIV infection of astrocytes in vivo in adults and children [18–22,29]. These cells are predominantly infected in the perivascular regions, and the number of infected cells correlates with the presence of HIV dementia and encephalitis . However, in-vitro studies show a very low infectivity of astrocytes with cell-free virus [15,23,24]. Because astrocytes do not express detectable levels of CD4 on their membranes [30,31], cell-free virus enters astrocytes via endocytosis and becomes trapped and degraded in endosomes/lysosomes . This barrier can be overcome by transfection of astrocytes with HIV proviral DNA [32,33] or by infection with pseudotyped viruses [25,34]. HIV can infect astrocytes when the cells are co-cultured with HIV-infected lymphocytes [35,36]; however, the mechanisms of these interactions are poorly understood. Here, we show that co-culturing astrocytes with HIV-infected lymphocytes overcame the barrier of infection with cell-free virus. This cell-to-cell transmission was observed in X4 or X4R5-using viruses. Considering that HIV transmission in lymphocytes by cell-to-cell contact is 3–5 orders of magnitude more efficient than the infection with cell-free virus [37–39], cell-to-cell infection could be an important mechanism for HIV infection of astrocytes in vivo.
HIV-infected lymphocytes could make multiple dynamic contacts with astrocytes. A key ultrastructural observation was the direct budding of incomplete viral particles from the lymphocyte membrane onto the astrocyte membrane, such that these immature viruses were in contact with the astrocyte membrane while still connected to the lymphocyte. Such interactions were mainly seen in virological synapses with two types of distinct structures. The first type of synapse was observed in the Velcro-like structures where clefts were surrounded by tight membrane junctions formed by the two type cells. This synapse is clearly different from other forms of virological synapses described in HIV transmission between lymphocytes or from dendritic cells to lymphocytes; and viral transmission from lymphocytes or dendritic cells to lymphocytes is CD4 and coreceptor-dependent [40–43]. The differences between the lymphocyte–lymphocyte, dendritic cell–lymphocyte, and lymphocyte-astrocyte interactions were recently published . Another type of structure was seen at the contact interfaces between the astrocyte membrane and the filopodial extension tips from the infected lymphocytes, where the viruses were observed budding onto the astrocyte membrane. These observations indicate that cell-to-cell infection might occur before complete formation or detachment of HIV particles from the donor lymphocytes, which establishes a unique mechanism of HIV transmission from lymphocyte to astrocyte.
X4-tropic HIV uses CXCR4 as a co-receptor which is abundantly expressed on the astrocyte membrane . We found that blockade of CXCR4 completely abolished HIV transmission in the co-cultures. Previous studies implicated a number of receptors in HIV infection of astrocytes with cell-free virus, but the role of CXCR4 has not been investigated [30,46–49]. Our results indicate that cell-to-cell contact could result in a change in HIV receptor utilization for viral entry in astrocytes. On the basis of the observation that HIV directly budded onto the astrocyte membrane, we hypothesize that the CXCR4-binding sites on the envelope of immature HIV might be exposed and accessible for its binding to CXCR4 independent of CD4. Studies show that immature viral particles are resistant to fusion with target cells and thus not infectious until undergoing a proteolytic maturation to form a functional core [50–52]. The immature virus contains a highly stable spherical Gag lattice, and the process of maturation may trigger a conformational change in the ectodomain of the envelope complex leading to its conversion into a fusion-competent state [50,53]. Typically, the envelope protein binds to CD4, inducing a conformational change in gp120, by which the exposed epitopes bind to CXCR4 . However, it is possible that gp120 in the immature virus may be in a conformational state that allows it to bind to CXCR4 independent of CD4. In the co-culture, the immature viruses that have not completely detached from the cell membrane would have the opportunity to bind to CXCR4 and then trigger viral entry while completing maturation. This may be a key factor in HIV transmission from lymphocytes to astrocytes. These observations are also consistent with a recent study of SHIV-infected macaques showing the expansion of CD4-independent virus with infection of astrocytes .
Importantly, CXCR4 can be up-regulated by cytokines interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α [56,57]. These pro-inflamma cytokines are markedly elevated in the brain of patients with HIV-associated neurocognitive disorders [58,59]. This is consistent with the observation that HIV infection of astrocytes is more abundant in the later stages of HIV infection , which is also the stage when X4-using virus appears . In contrast, we did not observe any significant infection of astrocytes co-cultured with R5-using virus-infected PBMCs, likely because astrocytes express very low level of CCR5 compared to abundant CXCR4. Although autopsy studies show that most HIV strains isolated from the brain are R5-using viruses [7,61], some studies have reported X4 or R5X4-using HIV-1 from the brain or cerebrospinal fluid (CSF) of patients with HIV-associated dementia [7,62,63] and some R5X4 viruses preferentially use CXCR4 for entry [7,63]. In one study, both R5 and X4 viruses were found in CSF of selected patients, whereas only R5 virus was present in the blood, suggesting autonomous CSF viral evolution . CXCR4-using simian/human immunodeficiency viruses (SHIVs) have also been shown to cause an encephalitis in rhesus macaques . Antiretroviral therapy induces HIV to switch from co-receptor CCR5 to CXCR4 usage, and this switch may appear later in the central nervous system (CNS) compartment compared to the periphery . In general, a switch from R5 to R5X4 or X4 is associated with acceleration of disease progression .
Studies show that CD4 and adhesion molecules (LFA1, ICAM1) contribute to cell-to-cell transmission of HIV between lymphocytes [39–41]. Since astrocytes do not express CD4 [30,31], it is interesting that a partial blockade with anti-CD4 antibody was noted in the co-cultures. It is possible that a transreceptor mechanism may contribute to viral entry into astrocytes, by which CD4 expressed on neighboring cells primes HIV envelope protein to fuse with a target cell that expresses appropriate co-receptors . Another possibility is that astrocytes may express very low level of CD4 since CD4 mRNA can be detected in astrocytes . ICAM1 expression can be up-regulated upon activation by pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (INF)-γ, and HIV proteins gp120 [69,70]. Adhesion molecules contributed to HIV transmission from lymphocytes to astrocytes because they can promote cell-to-cell contact.
Lymphocytes migrate into the brain  with a frequency that is significantly higher in asymptomatic carriers [71,72]. Lymphocyte infiltration is also observed in SIV/feline immunodeficiency virus (FIV)-infected animal models [73,74]. Because HIV-1-infected and/or immune-activated macrophages produce IL-1β, secretion of SDF-1 from astrocytes is further induced . We found that SDF-1 significantly triggered the migration of HIV-infected lymphocytes through the BBB. This may explain the predominance of HIV-infected astrocytes in the perivascular compartment .
In conclusion, the virus is transmitted from lymphocytes to astrocytes via a mechanism by which the immature virus buds from the lymphocyte membrane and binds to CXCR4 directly and triggers the fusion process in a CD4-independent manner during viral maturation. This could be a major mechanism for HIV entry into other cells with co-receptor expression and extremely low or no CD4 expression [76–78]. CXCR4 also plays a critical role in the migration of lymphocytes across the BBB and in the formation of cellular processes from astrocytes that engage HIV-infected lymphocytes. Hence, CXCR4 may be a therapeutic target to prevent formation of an HIV reservoir in astrocytes.
G.L. conceived the project, conducted experiments, analyzed data, and wrote the manuscript. C.A., L.J., and T.D. conducted experiments and analyzed data. E.O. helped with conceptual design and provided logistical support. A.N. conceived the project, analyzed the data, and wrote the manuscript.
The study was supported by NIMH P30 Pilot Award (G.L.) and the NINDS intramural funds. We thank David Levy at the New York University and Amanda Brown at the Johns Hopkins University (JHU) for providing HIV-1 reporter viruses NLENG1 and SF162R3, respectively; Michael Delannoy at the JHU Microscope Facility, Andrew Roholt at Renovo Neural Inc. and Sriram Subramaniam and Lesley Earl at the NCI forcorrelative EM and 3D-EM; Joseph Steiner and Suneil Hosmane at the JHU for time-lapse imaging; Alan Hoofring at NIH for editing pictures, movies, and cartoons; and Tianxia Wu for statistical analysis.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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