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...

In semen, proteolytic peptide fragments from prostatic acid phosphatase can form amyloid fibrils termed SEVI (semen-derived enhancer of viral infection). These fibrils greatly enhance human immunodeficiency virus (HIV) infectivity by increasing the attachment of virions to target cells. Therefore, SEVI may have a significant impact on whether HIV is successfully transmitted during sexual contact. Here, we demonstrate that surfen, a small molecule heparan sulfate proteoglycan antagonist, inhibits both SEVI-and semen-mediated enhancement of HIV type 1 infection. Surfen interferes with the binding of SEVI to both target cells and HIV type 1 virions but does not deaggregate SEVI fibrils. Because SEVI can increase HIV infectivity by several orders of magnitude, supplementing current HIV microbicide candidates with SEVI inhibitors, such as surfen, might greatly increase their potency. HIV2 is primarily a sexually transmitted disease. Worldwide, estimates suggest that the majority of all HIV infections are acquired through sexual contact. This includes sexual transmission from male to female, from male to male, and from female to male. In all these routes of infection, semen is either the vehicle carrying HIV (in the case of male-to-female and male-to-male transmission) or is often present during the infection process (in the case of female-to-male transmission).Recently, semen has been reported to enhance HIV infection (1). Fractionation of semen from healthy donors led to the identification of a factor that can enhance HIV infection up to 10 5 -fold in cell culture when viral inocula are limiting. This factor, termed SEVI (semen-derived enhancer of viral infection), corresponds to amyloid fibrils composed of internal 34 -40 amino acid proteolytic fragments from prostatic acid phosphatase (PAP), a protein present at a concentration of ϳ1-2 mg/ml in semen (1, 2). The predominant peptide fragment, PAP-(248 -286), has eight basic residues, rendering it very cationic (isoelectric point ϭ 10.21). The positively charged SEVI fibrils bind to both target cells and HIV virions and augment infection by increasing physical contact between these two components (1, 3), much in the same way that synthetic cationic polymers promote retrovirus attachment to target cells (4).Although we demonstrated that the cationic nature of SEVI is important for its pro-attachment effects (3), the surface components of target cells that interact with SEVI remained unknown. We previously observed that anionic polymers, such as heparin sulfate, interfere with the binding of SEVI to target cells (3). This led us to hypothesize that the fibrils may bind target cells by interacting with cell-surface heparan sulfate proteoglycans (HSPG), naturally occurring anionic carbohydrate polymers that are closely related in structure to heparin sulfate.We therefore sought to examine whether antagonists of HSPG might inhibit the viral enhancing activity of SEVI. Bis-2-methyl-4-amino-quinolyl-6-carbamide (surfen), a recently identified small molecule antagonist of H...

Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.

Summary Progression to AIDS is driven by CD4 T-cell depletion, mostly involving pyroptosis elicited by abortive HIV infection of CD4 T cells in lymphoid tissues. Inefficient reverse transcription in these cells leads to cytoplasmic accumulation of viral DNAs that are detected by the DNA sensor IFI16, resulting in inflammasome assembly, caspase-1 activation, and pyroptosis. Unexpectedly, we found that peripheral blood-derived CD4 T cells naturally resist pyroptosis. This resistance is partly due to their deeper resting state, resulting in fewer HIV-1 reverse transcripts and lower IFI16 expression. However, when co-cultured with lymphoid-derived cells, blood-derived CD4 T cells become sensitized to pyroptosis, likely recapitulating interactions occurring within lymphoid tissues. Sensitization correlates with higher levels of activated NF-κB, IFI16 expression, and reverse transcription. Blood-derived lymphocytes re-purified from co-cultures lose sensitivity to pyroptosis. These differences highlight how the lymphoid tissue microenvironment encountered by trafficking CD4 T lymphocytes dynamically shapes their biological response to HIV.