The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood. Apoptosis has been proposed as the key mechanism for CD4 T-cell loss. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of productively infected cells. The remaining >95% of quiescent lymphoid CD4 T-cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death where cytoplasmic contents and pro-inflammatory cytokines including IL-1β, are released. This death pathway thus links the two signature events in HIV infection––CD4 T-cell depletion and chronic inflammation––and creates a vicious pathogenic cycle where dying CD4 T-cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase-1 inhibitors shown to be safe in humans, raising the possibility of a new class of “anti-AIDS” therapeutics targeting the host rather than the virus.

Transmission of HIV-1 via intercellular connections has been estimated as 100-1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virological synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication (for example, by trafficking vesicles or transmitting calcium-mediated signals), but they can also contribute to pathologies (for example, by directing the spread of viruses). Recent data have revealed considerable heterogeneity in their structures, processes of formation and functional properties, in part dependent on the cell types involved. Despite recent progress in this young research field, further research is sorely needed.

IntroductionNatural killer (NK) cells are important effector cells of the innate immune response through their production of cytokines and lysis of transformed or infected cells without prior sensitization. NK cells exert killing by sensing "missing self" and/or by triggering of activating receptors upon interaction with specific ligands. 1 One of the best-characterized activating receptors is NKG2D, expressed on NK, NKT, and T cells, which recognizes stress-inducible class I major histocompatibility complex (MHC)-like proteins. 2 Other activating receptors, natural cytotoxicity receptors (NCRs), are expressed almost exclusively on NK cells. 3 Ligands for activating NK cell receptors are found on many cancer cell lines 4 and cells infected with bacteria 5 or viruses. [6][7][8] Immunoregulatory crosstalk between NK cells and dendritic cells (DCs) has emerged as important in both innate and adaptive immune responses. 9,10 However, the extent of crosstalk between human NK cells and macrophages has been less studied. Macrophages are also important effector cells of the innate immune response, exerting their function by using a range of receptors that recognize pathogen molecules such as bacterial lipopolysaccharide (LPS). 11 LPS is a powerful endotoxin that activates macrophages, although at high doses macrophages become refractory to further stimulation (ie, endotolerant). 12 Here, we set out to examine the potential for immunoregulatory crosstalk between human NK cells and macrophages or macrophages activated with LPS and probe the molecular basis for this.At the intercellular contact between immune cells, proteins are commonly seen to segregate into central and peripheral supramolecular activating clusters (c-and p-SMACs) at the immune synapse (IS). Functions of the NK cell IS could be to provide a framework for establishing checkpoints for cellular activation and/or directing secretion of lytic granules or cytokines in some circumstances. [13][14][15] Here we define, for the first time, 2 distinct NK cell-activating synapses. The macrophage-NK cell IS associated with priming, but not triggering, NK cytolysis and the IS between NK cells and macrophages treated with a high dose of LPS that triggers NK cytolysis. Materials and methods Generation of macrophages and DCsPeripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll-Paque Plus; Amersham Pharmacia Biotech, Piscataway, NJ). Serum was collected, heat inactivated for 30 minutes at 56°C, and filtered. PBMCs were incubated for 2 hours in plastic plates An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on March 31, 2019. by guest www.bloodjournal.org From previously coated overnight with 2% gelatin (Sigma-Aldrich, St Louis, MO). After 2 hours, the flask was was...