Métallogénie et processus minéralisateurs du Stock de Boyvinet, Desmaraisville, Abitibi, Québec

Ce projet, financé à 100% par SOQUEM Inc., avait pour but de déterminer les facteurs de contrôles sur la formation d’une minéralisation aurifère au sein d’un pluton (Stock de Boyvinet) et de développer un modèle métallogénique pour le secteur du Lac Shortt, à Desmaraisville en Abitibi. Dans un premier temps, une lecture exhaustive de la littérature a permis de préciser les caractéristiques propres à certains modèles métallogéniques basées sur des relations de terrain et d’analyses fines en laboratoire. Ces caractéristiques ont été testées et ont permis de développer un modèle métallogénique pour générer des cibles d’exploration tant à l’échelle locale que régionale. Le Stock de Boyvinet, dans la région du Lac Shortt (SOQUEM Inc.- MDN), renferme une minéralisation aurifère à faible teneur associée à des veinules de quartz et des zones d’altération hydrothermale. Ce stock a longtemps été considéré comme une syénite. Toutefois, une étude minéralogique de l’intrusion a donné lieu à une réinterprétation de la nature de l’intrusion, car composée de feldspaths (albite; ±40%), d’amphiboles (hornblende; ±10%) sous forme de phénocristaux avec en phases interstitielles des feldspaths potassiques à texture perthitique, du quartz, des carbonates (±10%) des minéraux opaques ainsi que de la titanite (±5%). Certaines phases mineures comme l’épidote, le quartz, la chlorite, les carbonates, la pyrite, ilménite, hématite et magnétite (±1-5%) sont également observées. Cette minéralogie est définie comme un résultat de l’altération hydrothermale d’un pluton à priori de composition monzodioritique/monzonitique. Les caractéristiques géochimiques indiquent une affinité calco-alcaline avec une anomalie négative en Nb et Ta, et un fractionnement LaN/YbN = 22. Les roches environnantes sont chimiquement comparables, permettant d’interpréter une source d’origine similaire. Les altérations hydrothermales s’expriment par des assemblages aux proportions variables d’albite, hématite, carbonates, silice, chlorite, pyrite et magnétite. Les zones altérées (n=25) sont caractérisées par des gains forts en Si, Na, K et Ca ainsi que des pertes en FeO. Les enrichissements en or sont en lien avec les zones d’altération, mais les assemblages minéralogiques sont variables, incluant: albite-hématite-pyrite et carbonates-silice-chlorite-pyrite. Les pyrites (n=53) ont été analysées par LA-ICP-MS au LabMater (UQAC). Des zonalités sont identifiées entre les coeurs poreux et les bordures recristallisées. Les assemblages métalliques Bi-Te-Cu-Ag-Sb sont typiquement d’origine magmatique et associés aux coeurs alors que les bordures à As-Ni-Co-Se sont caractéristiques des fluides métamorphiques. Des valeurs aurifères moyennes de 3,81 ppm, jusqu’à un maximum de 95,5 ppm dans les coeurs, par rapport à 0,32 ppm (maximum analysé de 1,32 ppm) dans les bordures permettent de proposer une relation directe entre les pyrites formées à partir des fluides magmatiques et la minéralisation aurifère. Des grains d’or libre se retrouvent également dans des fractures tardives de la pyrite. Les inclusions fluides ont été analysées (n=24) par spectrométrie de masse à sonde-solide au LAMEQ (UQAC). Les fluides hydrothermaux ont été étudiés en fonction de deux groupes: minéralisés (n=14) et stériles (n=10). Cette subdivision été définie selon les altérations hydrothermales et la présence de pyrite en proportion importante (≥2%). Malgré cette distinction, les fluides sont de compositions similaires, composés majoritairement de H2O (50 - 100%; moy. 84,5%) et CO2 (<50%; moy. 12,6%) avec, pour certains, des proportions plus élevées de H2 (<2,1%; moy. 0,44%), de N2 (<5,7%; moy. 2,1%) et de C2H6 (<3,1%; moy. 0,43%). Cette signature est typique des fluides d’origine métamorphique et constituent les derniers fluides enregistrés par les veines. Deux processus minéralisateurs superposés sont proposés : magmatique et métamorphique. Une fénitisation primaire de l’encaissant, induite par des fluides magmatiques alcalins régionaux, est responsable d’un enrichissement aurifère et de l’altération en albite-hématite-pyrite précoce. La présence de plusieurs intrusions alcalines tardives dans la région et de la carbonatite du Lac Shortt, supporte cette interprétation. Une minéralisation orogénique est superposée sur la fénitisation, avec localement le développement de veinules de quartz-carbonates-pyrite et la surimposition des bordures de pyrites primaires

La protéine accessoire Vpu du VIH-1 inhibe l'activité antivirale des pDCs à travers un processus ILT7-dépendant

Viral protein U (Vpu) is an accessory protein of HIV‐1 that efficiently targets BST2/Tetherin, a cellular restriction factor that acts as molecular anchor impeding the release of various enveloped viruses from the cell surface. The recently discovered natural receptor of BST2 is ILT7, a molecule exclusively expressed at the surface of the professional type 1 interferon (IFN‐1) producing cells, plasmacytoid dendritic cells (pDCs). The interaction between BST2 and ILT7 has been reported to efficiently induce a repression of IFN­‐1 secretion by pDCs. Here, we investigated the impact of Vpu mediated antagonism of BST2, in regards to this newly described immune function of BST2. Using a system of CD4+ T cell lines infected with wild type or Vpu‐deficient HIV-­1 cultured with peripheral blood mononuclear cells or purified pDCs, we report that the presence of Vpu efficiently reduces IFN-­1 production from sensing pDCs. Furthermore, we observed that this Vpu effect is dependent on the availability of BST2 molecules at the surface of the infected cells, since the Vpu's immunoregulation is abrogated when blocking any potential BST2 trans interaction with anti­‐BST2 antibodies. Similarly, depleting ILT7 from pDCs by means of small interfering RNA treatment equally negates the downregulation of pDC IFN-­1 secretion by Vpu. Finally, the use of recombinant soluble ILT7 competes with pDC‐bound ILT7 for the free BST2 and similarly results in high IFN-­1 production, causing an identical phenotype. Overall, our results demonstrate that Vpu heightens ILT7 activation and subsequent repression of IFN‐1 production by pDCs in response to HIV­‐1 infected CD4+ T cells by promoting it's trans interaction with infected T cell bound BST2, through a yet uncharacterized mechanism. By allowing efficient particle release and restraining pDCs antiviral functions, Vpu exerts a double role on BST2 that seems crucial for the replication and dissemination of HIV‐1.La protéine accessoire U (Vpu) du VIH‐1 cible efficacement BST2/Tetherin, facteur de restriction restreignant la relâche de divers virus enveloppés à même la surface cellulaire. Le récepteur naturel de BST2 récemment découvert est ILT7, une protéine exclusivement exprimée à la surface des cellules produisant l'essentiel de l'interféron de type 1 (IFN-­1) lors d'infections virales, les cellules dendritiques plasmacytoïdes (pDCs). L'interaction entre BST2 et ILT7 réprime la production d'IFN‐1 des pDCs. Considérant le potentiel immunorégulateur de BST2 récemment décrit, nous avons entrepris d'évaluer cet aspect de l'antagonisme de Vpu sur BST2. À l'aide d'un système de co­‐culture entre une lignée de cellules T CD4+ infectée avec un virus exprimant ou n'exprimant pas Vpu et les cellules mononucléées du sang périphérique ou de pDCs purifiés, nous avons observé que Vpu est responsable d'une atténuation majeure de la sécrétion d'interféron de type 1 (IFN-­1) produite en réponse aux cellules infectées. La présence de molécules de BST2 de surface libres est essentielle à ce processus, puisque le bloc de toute interaction en trans de BST2 par des anticorps polyclonaux α­‐BST2 abroge l'effet de Vpu. Similairement, Vpu ne peut exercer cet effet lorsque ILT7 est déplété dans les pDCs à l'aide de petits ARN interférents. Enfin, l'introduction de protéines recombinantes solubles d'ILT7 dans le système de co-culture semble prévenir l'effet inhibiteur de Vpu, suggérant que Vpu exploite l'interaction de BST2 avec ILT7 pour moduler la sécrétion d'IFN-­1 des pDCs. En conclusion, nos résultats démontrent que Vpu exerce un contrôle sophistiqué de la production d'IFN‐1 par les pDCs en réponse aux lymphocytes T CD4+ infectés par le VIH-­1. Il semble ainsi que l'action de Vpu favorise, par un mécanisme encore méconnu, l'activation d'ILT7 à travers BST2. En effet, cette fonction de Vpu semble tout aussi dépendante de BST2 que de l'ILT7. En favorisant la relâche virale et en menant à l'inhibition de la réponse antivirale des pDCs, la régulation ciblée de BST2 par Vpu est non seulement cruciale à la dissémination du virus, mais aussi à sa réplication

Vpu Exploits the Cross-Talk between BST2 and the ILT7 Receptor to Suppress Anti-HIV-1 Responses by Plasmacytoid Dendritic Cells.

International audiencePlasmacytoid dendritic cells (pDCs) constitute a major source of type-I interferon (IFN-I) production during acute HIV infection. Their activation results primarily from TLR7-mediated sensing of HIV-infected cells. However, the interactions between HIV-infected T cells and pDCs that modulate this sensing process remain poorly understood. BST2/Tetherin is a restriction factor that inhibits HIV release by cross-linking virions onto infected cell surface. BST2 was also shown to engage the ILT7 pDC-specific inhibitory receptor and repress TLR7/9-mediated IFN-I production by activated pDCs. Here, we show that Vpu, the HIV-1 antagonist of BST2, suppresses TLR7-mediated IFN-I production by pDC through a mechanism that relies on the interaction of BST2 on HIV-producing cells with ILT7. Even though Vpu downregulates surface BST2 as a mean to counteract the restriction on HIV-1 release, we also find that the viral protein re-locates remaining BST2 molecules outside viral assembly sites where they are free to bind and activate ILT7 upon cell-to-cell contact. This study shows that through a targeted regulation of surface BST2, Vpu promotes HIV-1 release and limits pDC antiviral responses upon sensing of infected cells. This mechanism of innate immune evasion is likely to be important for an efficient early viral dissemination during acute infection

Effect of a BST2 GPI anchor mutant on Vpu-mediated control of IFN-I production by pDCs.

<p><b>(A-B)</b> HEK293T cells were transfected with either an empty plasmid or plasmids expressing BST2 or BST2-dGPI 48 h prior to co-culture with PBMCs. <b>(A)</b> Surface expression of BST2 was evaluated 48 h post transfection by flow cytometry in controls cells (shaded grey histogram), as well as in cell expressing BST2 (solid black histogram), or BST2-dGPI (dashed grey histogram). Mean fluorescence intensity (MFI) values are indicated for each sample <b>(B)</b> After 6 h of co-culture, samples were untreated or treated with Imiquimod (TLR7 agonist) and levels of bioactive IFN-I in supernatants were measured 18 h later. The amount of IFN-I released by PBMCs in contact with HEK293T cells transfected with the empty plasmid in presence of the TLR 7 agonist was set at 100% (n = 4). As a control, transfected HEK293T cells were treated with TLR7 agonist without PBMCs. <b>(C)</b> Percentage of IFN-I released after co-culture of infected SupT1-Empty with PBMCs normalized to the value obtained with dU HIV-infected SuptT1 cells (100%) (n = 4). <b>(D)</b> A representative example of absolute levels of IFN-I produced after co-culture of mock or infected-SupT1,-SupT1-BST2 or-SupT1-BST2-dGPI cells with PBMCs is shown. <b>(E)</b> Relative percentages of IFN-I produced after co-culture of infected-SupT1-BST2 or SupT1-BST2-dGPI cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected SupT1-BST2 cells was set at 100% (n = 4). Two-tailed paired <i>t</i>-test was used in B and C (* p<0.05, ns not significant (p>0.05)). Repeated measures ANOVA with Bonferroni’s multiple comparison test was used in E (*** p<0.001, ns not significant (p>0.05)). Error bars represent SD.</p

Vpu-mediated control of IFN-I production by pDCs requires the presence of BST2 on infected donor cells.

<p><b>(A-C)</b> Control (MT4-shNT) or BST2-depleted (MT4-shBST2) MT4 cells were mock-infected, or infected with GFP-marked NL4.3 WT or dU viruses for 48 h. <b>(A)</b> Surface expression of BST2 on GFP-positive MT4 cells infected with WT (dashed grey histogram) or dU (solid black histogram) was evaluated by flow cytometry. Mean fluorescence intensity (MFI) values are indicated for each sample (staining using pre-immune rabbit serum, PI, shaded grey histograms). <b>(B-C)</b> The indicated MT4 donor cells were co-cultured with PBMCs. After 24 h, levels of bioactive IFN-I were measured in supernatants. A representative example of absolute levels <b>(B)</b> or relative percentages <b>(C)</b> of IFN-I produced after co-culture of the indicated infected MT4 cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected MT4-shNT cells was set at 100% (n = 12). <b>(D-E)</b> MT4-shNT (BST2 +) or MT4-shBST2 (BST2 -) cells were infected with GFP-marked NL4.3 WT or T/F CH077 viruses for 48 h. Similar number of p24+ infected cells were then co-cultured with PBMCs. After 24 h, levels of bioactive IFN-I were measured in supernatants. A representative example of absolute levels <b>(D)</b> or relative percentages <b>(E)</b> of IFN-I produced after co-culture of the indicated infected MT4 cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with infected MT4-shBST2 cells was set at 100% (n = 6). Repeated measures ANOVA with Bonferroni’s multiple comparison test was used. (*** p<0.001, ns not significant (p>0.05)). Error bars represent SD.</p

BST2 at the surface of infected cells is required for Vpu-mediated control of IFN-I production.

<p>MT4 cells were mock-infected or infected with GFP-marked NL4.3 WT or dU viruses and pre-incubated with anti-BST2 rabbit polyclonal (Rb BST2 Ab) or pre-immune (Rb PI) Abs or left untreated (No Ab). <b>(A)</b> Mock cells were subsequently stained for surface BST2 using mAb 26F8 and analyzed by flow cytometry. As a positive control, cells were directly stained with mAb 26F8. <b>(B-C)</b> The indicated MT4 cells were co-cultured with PBMCs. After 24 h, levels of IFN-I released in supernatants were measured. A representative example of absolute levels <b>(B)</b> or relative percentages <b>(C)</b> of IFN-I produced after co-culture of PBMCs with infected MT4 cells pre-treated with the indicated Abs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of Rb PI was set at 100% (n = 4). Two-tailed paired <i>t</i>-test was used (** p<0.01, * p<0.05, ns not significant (p>0.05)). Error bars represent SD.</p

BST2 binds and effectively activates ILT7.

<p><b>(A-E)</b> BST2 binds ILT7. <b>(A)</b> Purified GST-BST2 and bacILT7 were analyzed by SDS-PAGE and visualized by Coomassie brilliant blue staining. <b>(B)</b> Recombinant GST-BST2 pre-coated on the surface of Biacore sensor chips, was mixed with the indicated concentrations of bacILT7. The kinetic response data after subtracting the value from a reference cell coated with GST alone are shown. Kinetic constants (K_D = 2.33 μM, k_on = 1.25×10³ M^-1s^-1, k_off = 3.08×10⁻³ s^-1) were derived by fitting the data (dotted lines) to a 1:1 Langmuir model (black lines) using local R_max parameters (chi² = 14). <b>(C)</b> Control (293T) or ILT7-expressing HEK 293T cells (293T-ILT7) were incubated with control supernatant (CTRL) or with BST2-Fc-containing supernatant (BST2-Fc) prior to crosslinking with DTSSP. Cells were then stained for surface BST2-Fc and analyzed by flow cytometry. <b>(D-E)</b> ILT7+ or ILT7- NFAT-GFP reporter cells were incubated with control supernatant (CTRL) or with BST2-Fc-containing supernatant (BST2-Fc). Proximity ligation assay (PLA) was performed using mouse ILT7 mAb and rabbit polyclonal anti-BST2 Abs. A fluorochrome-labeled probe (red) was used to reveal locations of close proximity, and nuclei were highlighted with DAPI staining (blue). <b>(D)</b> Images were acquired by confocal microscopy using a 63Å~ objective. Images shown are representative of multiple fields. A magnification of the section marked in yellow is shown beside the panel. White bar = 10 μm. <b>(E)</b> The percentage of cells with PLA red staining (% positive cells) was calculated from at least 50 cells per condition. <b>(F-K)</b> BST2 effectively activates ILT7. <b>(F)</b> ILT7+ NFAT-GFP reporter cells were cultured in the presence or absence of plate-bound BST2-Fc for 24 h and analyzed for GFP expression using flow cytometry. <b>(G-H)</b> ILT7+ NFAT-GFP reporter cells were cultured in the presence of plate-bound or soluble anti-ILT7_alexa647 Abs (grey shaded or solid black histograms, respectively) or soluble isotype_alexa647 Ab as negative control (dotted lines). Twenty-four hours later, cells were harvested and samples in contact with the isotype Ab were stained for surface ILT7 only, using the above mentioned Abs for 30 min at 4°C (isotype_alexa647: dotted black histogram and aILT7_alexa647: dotted red histogram). All sample were analyzed by flow cytometry to detect <b>(G)</b> the percentage of GFP positive cells and <b>(H)</b> anti-ILT7 Abs (surface or total: surface + internalized). <b>(I-K)</b> ILT7+ NFAT-GFP reporter cells were co-cultured for 24 h with <b>(I)</b> control Hela or BST2-depleted Hela (Hela shBST2) cells or <b>(J-K)</b> HEK293T cells expressing either <b>(J)</b> increasing amounts of BST2 or <b>(K)</b> a fixed amount of BST2 and analyzed by flow cytometry (n = 2). <b>(K)</b> Prior to co-cultures, HEK293T-BST2 cells were incubated with rabbit anti-BST2 Abs (aBST2) or ILT7+ NFAT-GFP reporter cells were incubated with anti-ILT7 Abs (aILT7) for 1h or cells were left untreated as control (noAb). Relative percentage of ILT7 activation was plotted as % of GFP+ cells in each condition relative to the no Ab condition, which was set at 100%. Error bars represent SD.</p

Innate sensing of WT or Vpu-defective HIV-infected T cells requires Env-dependent viral fusion and is largely dependent on TLR7.

<p>MT4 cells were mock-infected (m) or infected with GFP-encoding NL4.3 variants (WT or dU) as indicated. <b>(A-B)</b> Cells were left un-treated (no Tx) or were treated with T-20 prior to co-culture with PBMCs. To assess the effect of inhibiting reverse transcription, PBMCs were treated with 3TC prior to co-culture with infected MT4 cells. As a positive control, CpG was added to inhibitor-treated or untreated mock-infected cells. A representative example of absolute levels <b>(A)</b> or relative percentages <b>(B)</b> of IFN-I detected after co-culture of WT or dU HIV-infected MT4 cells with PBMCs in the presence or absence of inhibitors are shown. Results are expressed relative to values obtained in the no-Tx samples (n = 8). <b>(C-F)</b> PBMCs were pre-treated with either TLR9 or TLR7/8/9 antagonists (antag.) or their respective controls (antag. Ctrl) prior to TLR agonist treatment <b>(C-D)</b> or to co-culture with the indicated infected cells <b>(E-F)</b>. A representative example of absolute levels of IFN-I detected after treatment with either TLR9 agonist (CpG-A) <b>(C)</b> or TLR7 agonist (R848) <b>(D)</b> is shown. A representative example of absolute levels <b>(E)</b> or relative percentages <b>(F)</b> of IFN-I produced in the indicated co-cultures in the presence of TLR antagonists or controls are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in the presence of the TLR7/8/9 antagonist control was set at 100% (n = 3). Two-tailed paired <i>t</i>-test was used. (*** p<0.001, * p<0.05, ns not significant (p>0.05)). Error bars represent SD.</p

Vpu-mediated control of IFN-I production by pDCs involves engagement and activation of ILT7 by BST2.

<p><b>(A-C)</b> Vpu-mediated BST2 antagonism enhances activation of ILT7. ILT7+ NFAT-GFP reporter cells were co-cultured with HEK293T (mock) or BST2-expressing HEK293T cells mock-transfected or transfected with the indicated pNL4.3 constructs (WT or dU). <b>(A)</b> Representative example of ILT7 activation as determined by the percentage of NFAT-GFP positive cells measured by flow cytometry. <b>(B)</b> Percentage of ILT7 activation after co-culture with the indicated HIV/BST2 expressing HEK 293T cells relative to WT HIV-producing cells (100%) (n = 4). <b>(C)</b> Percentage of BST2 surface expression in HEK293T cells after co-transfection of BST2 with the indicated HIV provirus relative to dU HIV-producing cells (100%) (n = 4). <b>(D-F)</b> Effect of ILT7 depletion on IFN-I production by pDCs. <b>(D)</b> Non-pDC fraction (BDCA-2-) and siRNA-treated enriched pDCs (CD14-/BDCA-2+) were stained using anti-ILT7 Abs as indicated. A representative example of absolute levels <b>(E)</b> or relative percentages <b>(F)</b> of IFN-I produced after co-culture of control pDCs (pDC-siCTRL) or ILT7-depleted pDCs (pDC-siILT7) with the indicated infected MT4 cells are shown. The amount of IFN-I released by pDC siCTRL in contact with dU HIV-infected cells was set at 100% (n = 4). <b>(G-I)</b> Effect of recombinant soluble ILT7 on IFN-I production by pDCs. <b>(G)</b> Expression of a HA-tagged soluble ILT7 (soILT7-HA) in HEK 293T cells. Cells and supernatants (sup) were analyzed by Western blot (WB) using anti-HA Abs. Purity of secreted soILT7-HA was confirmed by Coomassie staining (sup Coom). A representative example of <b>(H)</b> absolute levels or <b>(I)</b> relative percentages of IFN-I production after co-culture of PBMCs with the indicated infected MT4 cells pre-treated with control (CTRL) or soILT7-HA-containing supernatants are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of CTRL supernatant was set at 100% (n = 6). Two-tailed paired <i>t</i>-test was used in B-C while repeated measures ANOVA with Bonferroni’s multiple comparison test was used in F and I (*** p<0.001, ** p<0.01, ns not significant (p>0.05)). Error bars represent SD.</p