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This letter refers to the recent demonstration that HIV-1 infected macrophages form specialized conduits that connect to B-cells (1). The conduit selectively transports the HIV-1 nef protein, providing nef with numerous means to interfere with cellular processes. Currently, no consideration of the connection between the conduit and the development of AIDS-related lymphoma (ARL) has been offered. ARL is one of the primary causes of death in the HIV-infected population and is related to B-cell proliferation and activation. In this letter we discuss several studies that link HIV-infected macrophages and specific forms of the nef protein to the development of ARL. The conduits discovered by Xu et al.  may lead to a better understanding of how HIV infection results in lymphomagenesis.
A recent study by Xu et al. presented a series of exciting live-cell confocal images of HIV-1 infected macrophages reaching out to make contact with B-cells . The group defined a major purpose for these macrophage protrusions: the creation of a conduit that selectively transfers HIV-1 nef protein into B-cells. These conduits then exclusively carry nef from macrophages to B-cells in order to evade protective virus-specific immunoglobin responses (IgG2 and IgA) at the mucosal sites of entry. The transfer of nef to B-cells also decreases CD40-dependent activation, interferes with germinal center factors such as Bcl-6, AID, IRF4 and increases differentiation factors and receptors Blimp-1 and CD138 [1–3]. Swingler et al. also described an intricate relationship between nef, macrophages and B-cells, indicating that the HIV-1 nef protein carries a pathogenic determinant that governs B-cell defects in HIV-1 infection .
The publication and follow-up commentaries to Xu et al.’s work, however, failed to address the important relationship of macrophage-mediated B-cell activation to the development of AIDS-related lymphoma (ARL) [1–3]. Lymphoma is the second most common cause of death among HIV-infected individuals (60 times more likely to occur in the HIV-infected population than in the uninfected population, highly metastatic, and always fatal). Although HIV-1 does not productively infect B-cells, they are influenced by the presence of HIV-infected macrophages to overproduce stimulatory cytokines that may result in B-cell activation via cytidine deaminase-associated DNA modification errors and oncogenic translocations; mechanisms associated to ARL . HIV-infected individuals are frequently co-infected with the Epstein Barr virus (EBV), a known contributor to tumorogenesis through B-cell immortalization and proliferation. However we recently reported that 23% of ARL tumor biopsies were HIV+/EBV−, suggesting that a novel class of ARL tumors exists that is linked to HIV infection alone .
Monocyte-derived macrophages are the primary differentiated cell in the mononuclear phagocyte system. They are derived from bone marrow, distributed throughout the body and display great structural and functional heterogeneity . Macrophages are the first immune cells to recognize pathogens and signal other immune cells, primarily T-cells, to mount an immune response. They contribute to many disease processes and usually increase in number during wound healing, inflammation, or malignancy. Consequently, macrophages have great potential to modulate the immune response, which can be either positive or can mediate tissue destruction. In a recent review, Herbein and Varin  described three different macrophage activation states: M1 is the IFN-γ classically activated macrophage that displays a pro-inflammatory response, M2 is the activation of macrophages by IL-4 and IL-13 that display an anti-inflammatory response and dM represent macrophages deactivated by IL-10, which leads to immune suppression. Polarization of macrophages into classically activated M1 and alternatively activated M2 macrophages is critical in mediating an effective immune response against invading pathogens. However, during HIV infection, the virus uses these pathways to facilitate viral dissemination and pathogenesis . M1 macrophages that are recruited to sites of infection typically have a short half-life and may cause tissue damage. M2 macrophages produce immunosuppressive cytokines that may promote tumor growth and progression [8–10]. dM macrophages strongly express CD206, CD36 and TGF-β and do not produce inflammatory cytokines . dM macrophages are typically observed only at very late stages of HIV disease .
HIV-1 nef is a multi-factorial protein that is required to maintain high viral loads . It is the first HIV protein to accumulate following infection of macrophages, at which time it is estimated to represent three-quarters of the viral load . Additionally, nef promotes the survival of infected cells  and migration [16, 17]. In the cell, nef can bind MHC class II receptors, allowing it to interfere with a variety of cellular processes such as triggering rapid CD4 endocytosis, targeting the trans-golgi network (TGN) and activating phosphatidylinositol 3-kinases (P13K). The MHC class II molecules may also bind nef at the plasma membrane resulting in lysosomal degradation [18, 19].
Nef consists of two primary domains: 1) a myristoylation domain that allows it to bind to lipid membranes and 2) a transmembrane domain including a conserved core that is cleaved from the protein after attachment and inserted into cellular membranes. The two domains have been crystallized independently (PDB# 1EFN, 1QA4, 2NEF). In one of these structures (1EFN), nef is shown binding the SH3 domain of a Fyn tyrosine kinase, thus demonstrating one of the important interactions of the protein . Bentham et al. stated that the myristoylation of nef alone is insufficient for lipid binding and suggests that it is an interaction of basic residues at the N-terminus, including electrostatic interactions that allow nef to migrate and bind to target membranes . Giese et al. agreed and elaborated further that the association of nef to lipid membranes is governed by a complex pattern of amino acid motifs that contribute differentially to individual nef activities . The association of nef with cellular membranes is believed to be essential for virtually all of its effects in infected cells ; however, a significant amount of nef is cytosolic, unbound to any membrane [21, 23–26]. Fig. (1) summarizes some of the known interactions of nef within a M2 activated macrophage.
The population dynamics of HIV-1 within ARL tumors suggest that different tumor sites within the same individual contain closely related HIV genetic populations. The HIV population within ARL tumors expands at a 100× faster rate than at non-tumor locales . As Salemi et al. stated, “The different population dynamics between the viruses found in tumors versus the non-tumor associated viruses suggest that there is a significant relationship between HIV evolution and lymphoma pathogenesis.” Furthermore, at the recent 2010 International AIDS Conference in Austria, we described the finding of specific signatures in nef genomes found in ARL-tissues when we compared ARL viruses to viruses derived from brain tissues of patients with dementia . Dementia is an inflammatory disease where macrophages are classically M1 activated. The ARL signatures mapped to regions of the nef structure involved with SH3 binding, the internal core, and the flexible loop domain. Interestingly, this latter amino acid signature exists in a nef domain analyzed in  and was considered by Xu et al. to be an important motif for nanotube formation. In another study , we used a neural network (details of the approach are described in ) to determine if positional hydrophobicity in the nef protein could separate sequences derived from lymphoma tissues from sequences derived from non-lymphoma within ARL patients. The result was a 97% correct identification of HIV sequences derived from ARL tumors. When taken together, these findings support a link between the HIV-1 nef protein and ARL development; however, it remained unclear exactly how nef alone could promote B-cell activation.
The determination that nef is responsible for nanotube formation and transfer of nef proteins from macrophages to B-cells further defines the growing list of ways that nef manipulates host-cell machinery during HIV progression. Since nef proteins can remain liberated, this may explain why it can easily move through the macrophage conduits described by Xu et al. . As the number of known cellular interactions associated with nef increases, it is likely that both the myristolic and cytosolic forms of nef will be found to contribute to the biological activity of the protein and will be linked to HIV disease pathogenesis. Due to the notoriously high mutation rate of HIV, further studies should address how specific nef variants, especially those associated with disease pathogenesis, might impact nanotube formation or if an abundance of such structures are found in tumor tissues. Additionally, if nef can move through nanotubes into B-cells, can HIV small RNAs move through as well? A recent study that used a deep sequencing approach identified over 100 non-coding HIV small RNAs, including two previously published HIV microRNAs . One of these microRNAs, HIV1-miR-N367, is found within the nef gene. It will interesting to further understand the impact of the newly discovered macrophage nanotube connection to B-cells and the associated mechanisms that might allow this structure to contribute to ARL pathogenesis.
The project was funded by NIH grants U01 CA066529 and U19 MH081835.