Jérôme Estaquier

Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.

Simian Immunodeficiency Virus Infects Follicular Helper CD4 T Cells in Lymphoid Tissues during Pathogenic Infection of Pigtail Macaques

ABSTRACT T follicular helper (Tfh) cells are a specialized subset of memory CD4+ T cells that are found exclusively within the germinal centers of secondary lymphoid tissues and are important for adaptive antibody responses and B cell memory. Tfh cells do not express CCR5, the primary entry coreceptor for both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), and therefore, we hypothesized that these cells would avoid infection. We studied lymph nodes and spleens from pigtail macaques infected with pathogenic strain SIVmac239 or SIVmac251, to investigate the susceptibility of Tfh cells to SIV infection. Pigtail macaque PD-1high CD127low memory CD4+ T cells have a phenotype comparable to that of human Tfh cells, expressing high levels of CXCR5, interleukin-21 (IL-21), Bcl-6, and inducible T cell costimulator (ICOS). As judged by either proviral DNA or cell-associated viral RNA measurements, macaque Tfh cells were infected with SIV at levels comparable to those in other CD4+ memory T cells. Infection of macaque Tfh cells was evident within weeks of inoculation, yet we confirmed that Tfh cells do not express CCR5 or either of the well-known alternative SIV coreceptors, CXCR6 and GPR15. Mutations in the SIV envelope gp120 region occurred in chronically infected macaques but were uniform across each T cell subset investigated, indicating that the viruses used the same coreceptors to enter different cell subsets. Early infection of Tfh cells represents an unexpected focus of viral infection. Infection of Tfh cells does not interrupt antibody production but may be a factor that limits the quality of antibody responses and has implications for assessing the size of the viral reservoir.

High Levels of Viral Replication Contrast with Only Transient Changes in CD4+ and CD8+ Cell Numbers during the Early Phase of Experimental Infection with Simian Immunodeficiency Virus SIVmnd-1 in Mandrillus sphinx

ABSTRACT Early events during human immunodeficiency virus infections are considered to reflect the capacity of the host to control infection. We have studied early virus and host parameters during the early phase of simian immunodeficiency virus SIVmnd-1 nonpathogenic infection in its natural host, Mandrillus sphinx. Four mandrills were experimentally infected with a primary SIVmnd-1 strain derived from a naturally infected mandrill. Two noninfected control animals were monitored in parallel. Blood and lymph nodes were collected at three time points before infection, twice a week during the first month, and at days 60, 180, and 360 postinfection (p.i.). Anti-SIVmnd-1 antibodies were detected starting from days 28 to 32 p.i. Neither elevated temperature nor increased lymph node size were observed. The viral load in plasma peaked between days 7 to 10 p.i. (2 × 106 to 2 × 108 RNA equivalents/ml). Viremia then decreased 10- to 1,000-fold, reaching the viral set point between days 30 to 60 p.i. The levels during the chronic phase of infection were similar to that in the naturally infected donor mandrill (2 × 105 RNA equivalents/ml). The CD4+ cell numbers and percentages in blood and lymph nodes decreased slightly (<10%) during primary infection, and CD8+ cell numbers increased transiently. All values returned to preinfection infection levels by day 30 p.i. CD8+ cell numbers or percentages, in peripheral blood and lymph nodes, did not increase during the 1 year of follow-up. In conclusion, SIVmnd-1 has the capacity for rapid and extensive replication in mandrills. Despite high levels of viremia, CD4+ and CD8+ cell numbers remained stable in the post-acute phase of infection, raising questions regarding the susceptibility of mandrill T cells to activation and/or cell death in response to SIVmnd-1 infection in vivo.

Effects of Antiretroviral Drugs on Human Immunodeficiency Virus Type 1-Induced CD4+ T-Cell Death

ABSTRACT Apoptosis of peripheral blood T cells plays an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. In this study, we found that HIV type 1 (HIV-1) primes CD4+ T cells from healthy donors for apoptosis, which occurs after CD95 ligation or CD3-T-cell receptor (TCR) stimulation. CD95-mediated death did not depend on CD4 T-cell infection, since it occurred in the presence of the reverse transcriptase inhibitor didanosine (ddI). In contrast, apoptosis induced by productive infection (CD3-TCR stimulation) is prevented by both CD95 decoy receptor and ddI. Our data suggest that HIV-1 triggers at least two distinct death pathways: a CD95-dependent pathway that does not require viral replication and a viral replication-mediated cell death independent of the CD95 pathway. Further experiments indicated that saquinavir, a protease inhibitor, at a 0.2 μM concentration, decreased HIV-mediated CD95 expression and thus cell death, which is independent of its role in inhibiting viral replication. However, treatment of peripheral blood mononuclear cells from healthy donors with a higher concentration (10 μM) of an HIV protease inhibitor, saquinavir or indinavir, induced both a loss in mitochondrial membrane potential (ΔΨm) and cell death. Thus, protease inhibitors have the potential for both beneficial and detrimental effects on CD4+ T cells independent of their antiretroviral effects.

Lyssavirus Matrix Protein Induces Apoptosis by a TRAIL-Dependent Mechanism Involving Caspase-8 Activation

ABSTRACT Lyssaviruses, which are members of the Rhabdoviridae family, induce apoptosis, which plays an important role in the neuropathogenesis of rabies. However, the mechanisms by which these viruses mediate neuronal apoptosis have not been elucidated. Here we demonstrate that the early induction of apoptosis in a model of lyssavirus-infected neuroblastoma cells involves a TRAIL-dependent pathway requiring the activation of caspase-8 but not of caspase-9 or caspase-10. The activation of caspase-8 results in the activation of caspase-3 and caspase-6, as shown by an increase in the cleavage of the specific caspase substrate in lyssavirus-infected cells. However, neither caspase-1 nor caspase-2 activity was detected during the early phase of infection. Lyssavirus-mediated cell death involves an interaction between TRAIL receptors and TRAIL, as demonstrated by experiments using neutralizing antibodies and soluble decoy TRAIL-R1/R2 receptors. We also demonstrated that the decapsidation and replication of lyssavirus are essential for inducing apoptosis, as supported by UV inactivation, cycloheximide treatment, and the use of bafilomycin A1 to inhibit endosomal acidification. Transfection of cells with the matrix protein induced apoptosis using pathways similar to those described in the context of viral infection. Furthermore, our data suggest that the matrix protein of lyssaviruses plays a major role in the early induction of TRAIL-mediated apoptosis by the release of a soluble, active form of TRAIL. In our model, Fas ligand (CD95L) appears to play a limited role in lyssavirus-mediated neuroblastoma cell death. Similarly, tumor necrosis factor alpha does not appear to play an important role.

The relevance of apoptosis to AIDS pathogenesis

Several recent experimental findings support the hypothesis that apoptosis induced by human immune deficiency virus (HIV) is important in the pathogenesis of acquired immune deficiency syndrome (AIDS). Thus, one potential therapeutic strategy against AIDS may be to block the HIV-mediated apoptosis signal transduction pathway. Induction of apoptosis by HIV infection may prove a useful paradigm for the pathogenesis of a wide range of diseases that involve cell depletion and tissue atrophy.

Nonpathogenesis of Simian Immunodeficiency Virus Infection Is Associated with Reduced Inflammation and Recruitment of Plasmacytoid Dendritic Cells to Lymph Nodes, Not to Lack of an Interferon Type I Response, during the Acute Phase

ABSTRACT Divergent Toll-like receptor 7 (TLR7) and TLR9 signaling has been proposed to distinguish pathogenic from nonpathogenic simian immunodeficiency virus infection in primate models. We demonstrate here that increased expression of type I interferon in pathogenic rhesus macaques compared to nonpathogenic African green monkeys was associated with the recruitment of plasmacytoid dendritic cells in the lymph nodes and the presence of an inflammatory environment early after infection, instead of a difference in the TLR7/9 response.

Regulation of immunity during visceral Leishmania infection

Unicellular eukaryotes of the genus Leishmania are collectively responsible for a heterogeneous group of diseases known as leishmaniasis. The visceral form of leishmaniasis, caused by L. donovani or L. infantum, is a devastating condition, claiming 20,000 to 40,000 lives annually, with particular incidence in some of the poorest regions of the world. Immunity to Leishmania depends on the development of protective type I immune responses capable of activating infected phagocytes to kill intracellular amastigotes. However, despite the induction of protective responses, disease progresses due to a multitude of factors that impede an optimal response. These include the action of suppressive cytokines, exhaustion of specific T cells, loss of lymphoid tissue architecture and a defective humoral response. We will review how these responses are orchestrated during the course of infection, including both early and chronic stages, focusing on the spleen and the liver, which are the main target organs of visceral Leishmania in the host. A comprehensive understanding of the immune events that occur during visceral Leishmania infection is crucial for the implementation of immunotherapeutic approaches that complement the current anti-Leishmania chemotherapy and the development of effective vaccines to prevent disease.

Poliovirus induces Bax-dependent cell death mediated by c-Jun NH2-terminal kinase.

ABSTRACT Poliovirus (PV) is the causal agent of paralytic poliomyelitis, a disease that involves the destruction of motor neurons associated with PV replication. In PV-infected mice, motor neurons die through an apoptotic process. However, mechanisms by which PV induces cell death in neuronal cells remain unclear. Here, we demonstrate that PV infection of neuronal IMR5 cells induces cytochrome c release from mitochondria and loss of mitochondrial transmembrane potential, both of which are evidence of mitochondrial outer membrane permeabilization. PV infection also activates Bax, a proapoptotic member of the Bcl-2 family; this activation involves its conformational change and its redistribution from the cytosol to mitochondria. Neutralization of Bax by vMIA protein expression prevents cytochrome c release, consistent with a contribution of PV-induced Bax activation to mitochondrial outer membrane permeabilization. Interestingly, we also found that c-Jun NH2-terminal kinase (JNK) is activated soon after PV infection and that the PV-cell receptor interaction alone is sufficient to induce JNK activation. Moreover, the pharmacological inhibition of JNK by SP600125 inhibits Bax activation and cytochrome c release. This is, to our knowledge, the first demonstration of JNK-mediated Bax-dependent apoptosis in PV-infected cells. Our findings contribute to our understanding of poliomyelitis pathogenesis at the cellular level.

CD8+ T Cell Dynamics during Primary Simian Immunodeficiency Virus Infection in Macaques: Relationship of Effector Cell Differentiation with the Extent of Viral Replication

Immunological and virological events that occur during the earliest stages of HIV-1 infection are now considered to have a major impact on subsequent disease progression. We observed changes in the frequencies of CD8bright T cells expressing different chemokine receptors in the peripheral blood and lymph nodes of rhesus macaques during the acute phase of the pathogenic SIVmac251 infection; the frequency of CD8bright T cells expressing CXCR4 decreased, while the frequency of those expressing CCR5 increased. These reciprocal changes in chemokine receptor expression were associated with changes in the proportion of cycling (Ki67+) CD8bright T cells, and with the pattern of CD8bright T cell differentiation as defined by expression of CCR7 and CD45RA. In contrast, during the primary phase of the attenuated SIVmac251Δnef infection, no major change was observed. Whereas during the acute phase of the infection with pathogenic SIV (2 wk postinfection) no correlate of disease protection was identified, once the viral load set points were established (2 mo postinfection), we found that the levels of cycling and of CCR5- and CXCR4-positive CD8bright T cells were correlated with the extent of viral replication and therefore with SIV-infection outcome. Our data reveal that, during primary SIV infection, despite intense CD8 T cell activation and an increase in CCR5 expression, which are considered as essential for optimal effector function of CD8+ T cells, these changes are associated with a poor prognosis for disease progression to AIDS.