David J. Schnepple

Human Immunodeficiency Virus Type 1 Protease Cleaves Procaspase 8 In Vivo

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection causes apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 and CD8 T cells. It remains unknown what signals cause infected cells to die. We demonstrate that HIV-1 protease specifically cleaves procaspase 8 to create a novel fragment termed casp8p41, which independently induces apoptosis. casp8p41 is specific to HIV-1 protease-induced death but not other caspase 8-dependent death stimuli. In HIV-1-infected patients, casp8p41 is detected only in CD4+ T cells, predominantly in the CD27+ memory subset, its presence increases with increasing viral load, and it colocalizes with both infected and apoptotic cells. These data indicate that casp8p41 independently induces apoptosis and is a specific product of HIV-1 protease which may contribute to death of HIV-1-infected cells.

Differential Effects of Interleukin-7 and Interleukin-15 on NK Cell Anti-Human Immunodeficiency Virus Activity

ABSTRACT The ability of interleukin-7 (IL-7) and IL-15 to expand and/or augment effector cell functions may be of therapeutic benefit to human immunodeficiency virus (HIV)-infected patients. The functional effects of these cytokines on innate HIV-specific immunity and their impact on cells harboring HIV are unknown. We demonstrate that both IL-7 and IL-15 augment natural killer (NK) function by using cells (CD3− CD16+ CD56+) from both HIV-positive and -negative donors. Whereas IL-7 enhances NK function through upregulation of Fas ligand, the effect of IL-15 is mediated through upregulation of tumor necrosis factor-related apoptosis-inducing ligand. The difference in these effector mechanisms is reflected by the ability of IL-15-treated but not IL-7-treated NK cells to reduce the burden of replication-competent HIV in autologous peripheral blood mononuclear cells (PBMC) (infectious units per million for control NK cells, 6.79; for IL-7-treated NK cells, 236.17; for IL-15-treated cells, 1.01; P = 0.01 versus control). In addition, the treatment of PBMC with IL-15-treated but not IL-7-treated NK cells causes undetectable HIV p24 (five of five cases), HIV RNA (five of five cases), or HIV DNA (three of five cases). These results support the concept of adjuvant immunotherapy of HIV infection with either IL-7 or IL-15 but suggest that the NK-mediated antiviral effect of IL-15 may be superior.

Glycoprotein 120 Binding to CXCR4 Causes p38-Dependent Primary T Cell Death That Is Facilitated by, but Does Not Require Cell-Associated CD4

HIV-1 infection causes the depletion of host CD4 T cells through direct and indirect (bystander) mechanisms. Although HIV Env has been implicated in apoptosis of uninfected CD4 T cells via gp120 binding to either CD4 and/or the chemokine receptor 4 (CXCR4), conflicting data exist concerning the molecular mechanisms involved. Using primary human CD4 T cells, we demonstrate that gp120 binding to CD4 T cells activates proapoptotic p38, but does not activate antiapoptotic Akt. Because ligation of the CD4 receptor alone or the CXCR4 receptor alone causes p38 activation and apoptosis, we used the soluble inhibitors, soluble CD4 (sCD4) or AMD3100, to delineate the role of CD4 and CXCR4 receptors, respectively, in gp120-induced p38 activation and death. sCD4 alone augments gp120-induced death, suggesting that CXCR4 signaling is principally responsible. Supporting that model, AMD3100 reduces death caused by gp120 or by gp120/sCD4. Finally, prevention of gp120-CXCR4 interaction with 12G5 Abs blocks p38 activation and apoptosis, whereas inhibition of CD4-gp120 interaction with Leu-3a has no effect. Consequently, we conclude that gp120 interaction with CXCR4 is required for gp120 apoptotic effects in primary human T cells.

Acquired T-cell sensitivity to TRAIL mediated killing during HIV infection is regulated by CXCR4-gp120 interactions.

Background:Sensitivity towards apoptosis induced by ligation of the tumor necrosis factor family of death receptors is controlled in part by death receptor expression. Whereas cellular activation enhances Fas receptor expression and induces Fas sensitivity, such cellular activation neither alters TRAIL receptor expression nor induces TRAIL sensitivity. Cells infected by HIV acquire sensitivity to TRAIL induced death, although the mechanisms by which this is achieved are undefined. Objective:To define the mechanism by which cells from HIV infected patients acquire sensitivity to TRAIL mediated killing. Design:In vitro assessment of TRAIL receptor expression and TRAIL sensitivity. Methods:Treatment of Jurkat T cells, peripheral blood lymphocytes from HIV negative donors, or human osteogenic seroma (HOS) cells expressing CD4, CXCR4 or CCR5 with T tropic gp120, M tropic gp120, or agonistic antibodies against CD4, CXCR4 or CCR5. TRAIL receptors were measured by flow cytometry or reverse transcription-PCR and TRAIL sensitivity was assessed by incubation with recombinant TRAIL followed by Annexin V fluorescein isothiocyanate/Propidium Iodide (PI) staining. Results:Treatment of uninfected Jurkat T cells, as well as primary T cells with gp120 results in the upregulation of TRAIL death receptor expression and acquired sensitivity to TRAIL mediated cell death. The increase in TRAIL death receptor expression and acquisition of TRAIL sensitivity requires the chemokine coreceptor CXCR4 but not CCR5 or the CD4 receptor. Conclusions:These results indicate that chemokine receptor interactions regulate TRAIL receptor expression and provide an explanation for the acquired T cell sensitivity to TRAIL mediated killing death during HIV infection.

Flying in the Face of Resistance: Antiviral-independent Benefit of HIV Protease Inhibitors on T-cell Survival

Human immunodeficiency virus (HIV) infection results in excessive apoptosis of infected and uninfected cells, mediated by host and viral factors present in plasma. As HIV protease inhibitors (PIs) have intrinsic antiapoptotic properties, we questioned whether HIV PIs could block HIV‐induced CD4+ T‐cell death independent of their effects on HIV replication. We demonstrate that HIV PIs block the death of CD4+ T cells induced by HIV glycoprotein 120 (gp120), Vpr, and Tat, as well as host signals Fas ligand, tumor necrosis factor, and tumor necrosis factor‐related apoptosis‐inducing ligand. Using gp120/CXCR4 as a model, we show that the HIV PIs specifically block mitochondrial apoptosis signaling. Furthermore, HIV PIs inhibit CD4+ T‐cell death induced by viruses with high‐level resistance to PIs (P<0.01) and apoptosis induced by serum of HIV patients with known resistance to HIV PIs (P=0.01). Together, these results show that HIV PIs block CD4+ T‐cell death and have a beneficial effect on CD4+ T‐cell survival despite PI resistance.

Nelfinavir/ritonavir reduces acinar injury but not inflammation during mouse caerulein pancreatitis

There is no clinical treatment that reduces acinar injury during pancreatitis. Human immunodeficiency virus (HIV) protease inhibitors (PI), including nelfinavir (NFV) and ritonavir (RTV), may reduce the rate of pancreatitis in HIV-infected patients. Since permeability transition pore (PTPC)-mediated mitochondrial dysfunction occurs during pancreatitis, and we have shown that PI prevents PTPC opening, we studied its effects in a model of pancreatitis. The effect of NFV plus RTV (NFV/RTV) or vehicle on caerulein-induced pancreatitis in mice was compared by measuring changes in mitochondrial membrane potential in vitro and cytochrome c leakage in vivo. Histological and inflammatory makers were also compared. NFV/RTV improved DiOC6 retention in acini exposed to caerulein in vitro. In vivo NFV prevented cytosolic leakage of cytochrome c and reduced pancreatic acinar injury, active caspase-3 staining, TUNEL-positive acinar cells, and serum amylase (P < 0.05). Conversely, trypsin activity, serum cytokine levels, and pancreatic and lung inflammation were unaffected. NFV/RTV reduces pancreatic injury and acinar cell death in experimental mouse caerulein-induced pancreatitis but does not impact inflammation.

Isolation of a TRAIL Antagonist from the Serum of HIV-infected Patients

Background: The TRAIL:TRAIL receptor system has been implicated in the pathogenesis of a variety of malignant and infectious disorders, including HIV infection. Results: We show that HIV causes production of a novel TRAIL splice variant, that we call TRAIL-short, which binds TRAIL R2, antagonizes TRAIL signaling, and is present in HIV patient samples. Conclusion: Introduction of TRAIL-short causes resistance to TRAIL, whereas knockdown restores sensitivity. Significance: The identification of TRAIL-short impacts our understanding of TRAIL sensitivity and has implications for the pathogenesis of both infectious and malignant pathogenesis. Virus-host interactions are characterized by the selection of adaptive mechanisms by which to evade pathogenic and defense mechanisms, respectively. In primary T cells infected with HIV, HIV infection up-regulates TNF-related apoptosis inducing ligand (TRAIL) and death-inducing TRAIL receptors, but blockade of TRAIL:TRAIL receptor interaction does not alter HIV-induced cell death. Instead, HIV infection results in a novel splice variant that we call TRAIL-short (TRAIL-s), which antagonizes TRAIL-R2. In HIV patients, plasma TRAIL-s concentration increases with increasing viral load and renders cells resistant to TRAIL-induced death. Knockdown of TRAIL-s abrogates this resistance. We propose that TRAIL-s is a novel adaptive mechanism of apoptosis resistance acquired by HIV-infected cells to avoid their elimination by TRAIL-dependent effector mechanism.