Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells.

International audienceChronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration

Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection

Contact with HIV-1 envelope protein elicits release of ATP through pannexin-1 channels on target cells; by activating purinergic receptors and Pyk2 kinase in target cells, this extracellular ATP boosts HIV-1 infectivity

Etude des mécanismes moléculaires d'apoptose au cours de la phase de primo-infection chez le macaque rhésus infecté par la souche pathogène SIVmac251

La primo-infection par le virus d'immunodéficience humaine (VIH) ou un virus d'immunodéficience simienne (SIV) pathogène est caractérisée par un pic de réplication virale à 2 semaines post-infection suivi d'une stabilisation de la charge virale à 2 mois (J60) à un niveau prédictif de la vitesse d'évolution vers le SIDA. Une déplétion lymphocytaire T CD4+ progressive est mesurée au cours de la phase chronique, en partie due à une apoptose exacerbée. Dans le modèle du macaque rhésus infecté par la souche pathogène SIVmac251, nous avons mesuré l'apoptose lymphocytaire, et montré que le taux d'apoptose des lymphocytes T CD4+, mesuré dans les ganglions lymphatiques à J60, était prédictif de l'évolution vers le SIDA et corrélé avec la réplication virale. Cette apoptose est indépendante de l'activation des caspases et de l'AIF et est associée à une augmentation de la forme active de la molécule pro-apoptotique Bak. Les lymphocytes T CD4+ qui meurent sont des cellules mémoires effectrices et leur déplétion entraîne une perte de la réponse envers les antigènes de rappel de SIV et de la tuberculine ainsi qu'une altération de la réponse T CD8+. L'étude des co-récepteurs CXCR4 et CCR5 du virus a montré que des variations de leur expression à la surface des lymphocytes T CD8+ entre JO et J60 étaient liées à l'évolution des animaux.PARIS7-Bibliothèque centrale (751132105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

The Mitochondrial Pathways of Apoptosis

International audienceApoptosis is a process of programmed cell death that serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Studies in nematode, Drosophila and mammals have shown that, although regulation of the cell death machinery is somehow different from one species to another, it is controlled by homologous proteins and involves mitochondria. In mammals, activation of caspases (cysteine pro-teases that are the main executioners of apoptosis) is under the tight control of the Bcl-2 family proteins , named in reference to the fi rst discovered mammalian cell death regulator. These proteins mainly act by regulating the release of caspases activators from mitochondria. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of apoptosis. In this chapter, we present the current view on the mitochondrial pathway of apoptosis with a particular attention to new aspects of the regulation of the Bcl-2 proteins family control of mitochondrial membrane permeabilization: the mechanisms implicated in their mitochondrial targeting and activation during apoptosis, the function(s) of the oncosuppressive protein p53 at the mitochondria and the role of the processes of mitochondrial fusion and fi ssion

Increased neutrophil apoptosis in chronically SIV-infected macaques

Abstract Polymorphonuclear neutrophils (PMN) from chronically HIV-infected individuals have been reported to be more prone to die. However, although non-human primates models have been extensively used for improving our knowledge on T cell immunity, the impact of SIV-infection on PMN, in relationships with disease severity, has never been assessed. In our study, we demonstrate that PMN from Rhesus macaques (RMs) of Chinese origin chronically infected with the virulent strain SIVmac251 display increased susceptibility to undergo apoptosis as compared to PMN from RMs infected with the non-pathogenic SIVΔnef strain. PMN apoptosis was significantly increased in RMs progressing faster to AIDS as compared to non-progressors RMs. Furthermore, the percentage of apoptotic cells correlated with PMN activation state reflected by increased CD11b expression and reactive oxygen species production. Interestingly, whereas inflammatory cytokines IL-8 and IL-1β prevent in vitro PMN death, the levels of those cytokines were low in RMs progressing towards AIDS. Altogether, increased PMN death during SIV infection is a new pathogenic effect associated with AIDS progression, adding to the long list of markers associated with disruption of defense against infection.</p

Increased Immunogenicity of Full-Length Protein Antigens through Sortase-Mediated Coupling on the PapMV Vaccine Platform

Background: Flexuous rod-shape nanoparticles&mdash;made of the coat protein of papaya mosaic virus (PapMV)&mdash;provide a promising vaccine platform for the presentation of viral antigens to immune cells. The PapMV nanoparticles can be combined with viral antigens or covalently linked to them. The coupling to PapMV was shown to improve the immune response triggered against peptide antigens (&lt;39 amino acids) but it remains to be tested if large proteins can be coupled to this platform and if the coupling will lead to an immune response improvement. Methods: Two full-length recombinant viral proteins, the influenza nucleoprotein (NP) and the simian immunodeficiency virus group-specific protein antigen (GAG) were coupled to PapMV nanoparticles using sortase A. Mice were immunized with the nanoparticles coupled to the antigens and the immune response directed to the antigens were analyzed by ELISA and ELISPOT. Results: We showed the feasibility of coupling two different full-length proteins (GAG and NP) to the nanoparticle. We also showed that the coupling to PapMV nanoparticles improved significantly the humoral and the cytotoxic T lymphocyte (CTL) immune response to the antigens. Conclusion: This proof of concept demonstrates the versatility and the efficacy of the PapMV vaccine platform in the design of vaccines against viral diseases

The density of coreceptors at the surface of CD4+ T cells contributes to the extent of human immunodeficiency virus type 1 viral replication-mediated T cell death

Chemokine receptors serve as coreceptors for HIV-1 entry into CD4(+) T cells. Several reports have mentioned that density of CCR5 expression modulates in vitro viral replication and in vivo the course of the disease. Our goal was to investigate the impact of coreceptor density at the surface of a CD4(+) cell line on HIV-1 entry, replication, spreading, and programmed cell death. We engineered a CEM cell line that expresses constitutively CD4 and CXCR4 and CCR5 after transfection. This model allows us to compare the effect of the X4 and R5 strains to induce T cell death in the same T cell host. We show here that the extent of T cell death correlates with the rate of virus replication. X4 induces faster T cell death than R5 that depends at least in part on the higher density of CXCR4 compared to CCR5. Furthermore, sorting CEM populations expressing low, intermediate, and high densities of CCR5 molecules but constant amount of CD4, we found that the capacity to induce T cell death depends at least in part on the level of CCR5 when low amount of virus was used to infect the CEM cells. Moreover, viral transcription, assessed by cell-associated HIV-1 RNA/DNA ratio, was increased in CCR5high as compared to CCR5low cells, while inhibition of replication by zidovudine was more effective in CCR5low cells. Our data indicate that the density of chemokine receptors expressed on CD4(+) T cells may be a critical parameters for the cytopathic effect of HIV strains and may have major impact on CD4 T cell depletion during HAART

Highly active antiretroviral treatment against STLV-1 infection combining reverse transcriptase and HDAC inhibitors.

International audienceApproximately 3% of all human T-lymphotropic virus type 1 (HTLV-1)-infected persons will develop a disabling inflammatory disease of the central nervous system known as HTLV-1-associated myelopathy/tropical spastic paraparesis, against which there is currently no efficient treatment. As correlation exists between the proviral load (PVL) and the clinical status of the carrier, it is thought that diminishing the PVL could prevent later occurrence of the disease. We have conducted a study combining valproate, an inhibitor of histone deacetylases, and azidothymidine, an inhibitor of reverse transcriptase, in a series of baboons naturally infected with simian T-lymphotropic virus type 1 (STLV-1), whose PVL was equivalent to that of HTLV-1 asymptomatic carriers. We show that the combination of drugs caused a strong decrease in the PVL and prevented the transient rise in PVL that is seen after treatment with histone deacetylases alone. We then demonstrate that the PVL decline was associated with an increase in the STLV-1-specific cytotoxic T-cell population. We conclude that combined treatment with valproate to induce viral expression and azidothymidine to prevent viral propagation is a safe and effective means to decrease PVL in vivo. Such treatments may be useful to reduce the risk of HAM/TSP in asymptomatic carriers with a high PVL

Effect of silencing (A–E) and overexpressing (F–I) TSPO in HT-29 cells.

<p>(A) Time dependence of TSPO mRNA expression in control cells (open bars), after mock transfection (grey bars) or siRNA-targeting TSPO transfection (dashed bars). (B) HT-29 cell proliferation (controls, black circles; mock-transfected, grey squares; and siRNA-targeting TSPO transfected, grey triangles). Time course of mRNA expression of <i>TSPO</i> (C), <i>IL-8</i> (D), and <i>MnSOD</i> (E) relative to <i>GAPDH</i> after 3–6 hours, 24 hours, or 72 hours of treatment with 10 ng/mL of TNF performed 24 hours after transfection by siRNA-targeting TSPO (controls, open bars; siRNA alone, short dashed bars; TNF-treated, black bars; siRNA and TNF-treated, long dashed bars). (F) HT-29 cell proliferation (controls, black circles; mock-transfected, grey squares; empty vector plasmid, open squares; and TSPO cDNA-containing vector, grey triangles) in the presence of TNF (nontransfected cells or siRNA-targeting TSPO transfected cells). Time dependence of mRNA expression of <i>TSPO</i> (G), <i>IL-8</i> (H), and <i>MnSOD</i> (I) relative to <i>GAPDH</i> after 3–6 hours, 24 hours, or 72 hours of treatment with TNF performed 24 hours after transfection by vector plasmid (controls, open bars; cDNA alone, short dashed bars; TNF-treated, black bars; cDNA and TNF-treated, long dashed bars). The results are expressed as the means ± standard error of the mean.</p

HT-29 cells’ mitochondrial networks and fusion proteins upon TNF treatment.

<p>(A), Representative images of HT-29 cells expressing green fluorescent protein (GFP) targeted to mitochondria in control and treated cells. (B and C) Quantitative analysis of mitochondrial structure using morphological parameters (Aspect Ratio and Form Factor) following Koopman et al. treatment. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152919#pone.0152919.ref023" target="_blank">23</a>] At least 500–600 mitochondria were analyzed per condition and normalized to control conditions. The time course of the Aspect Ratio (B) and Form Factor (C) changes in TNF-treated cells (black bars), 1 μM of PK 11195-treated cells (grey bars), and cells treated simultaneously with 10 ng/mL of TNF and 1 μM of PK 11195 (dashed bars). mRNA expression of mitofusins (<i>Mfn1</i> and <i>Mfn2</i> in panels D and E, respectively) relative to <i>GAPDH</i> as a function of time of treatment with 10 ng/mL of TNF (open square), the simultaneous presence of TNF and PK 11195 (open diamonds), and controls without treatment (closed circle). (F) Western blot analysis of mitofusins (1 and 2) in DDM and Digit-solubilized mitochondrial membranes after 72 hours of treatment with TNF [T], PK 11195 [P], both TNF and PK 11195 [T+P], and controls [C]. (G) Densitometry evaluation of western blot of mitofusins. The results are expressed as the means ± standard error of the mean.</p