La protéine Tat du VIH-1 active la voie TLR4 : effets sur l'expression des facteurs immunosuppressifs IL-10, PD-L1 et IDO

Au cours de l'infection par le virus de l'immunodéficience humaine de type 1 (VIH-1) on observe une augmentation progressive de l'expression des molécules inhibitrices du système immunitaire (IL-10, PD-L1 et IDO) principalement par les cellules présentatrices d'antigènes (CPA) telles que les cellules dendritiques (DCs). Ces molécules agissent négativement sur la réponse immunitaire anti-VIH et sont associées à la persistance du virus et à l'évolution de la maladie vers le stade SIDA. Une des protéines virales impliquée dans ce mécanisme est la protéine Tat. En plus de son rôle direct dans la transactivation du génome viral, Tat est sécrétée par les cellules infectées et se retrouve à des concentrations de l'ordre du nanomolaire dans le sérum des patients. Dans la circulation, Tat interagit avec les cellules immunitaires et module l'expression de nombreux facteurs incluant des facteurs immunosuppressifs. Les travaux précédents du laboratoire, ont montré que Tat agit au niveau membranaire, par son domaine N-terminal 1-45, afin de stimuler l'expression des cytokines pro-inflammatoires TNF-a, et immunosuppressives IL-10 par les monocytes et les macrophages humains. Plus récemment, la recherche du récepteur membranaire recruté par Tat a permis de mettre en évidence l'implication du TLR4/MD2 dans l'interaction avec Tat. Objectifs : Mon projet de thèse qui s'inscrit dans la continuité des travaux du laboratoire a pour objectif 1) de caractériser davantage le rôle du TLR4 comme récepteurs de Tat en utilisant une approche basée sur des modèles murins invalidés (KO) pour le TLR4 ou ses partenaires moléculaires (CD14, MD2, MyD88, TRIF) 2) d'étudier l'effet de la protéine Tat et de son interaction avec le TLR4 sur la modulation de la fonction des DCs incluant : la production de cytokines, la maturation, l'expression des facteurs immunosuppressifs et la capacité à activer/inhiber les lymphocytes T. Résultats : L'utilisation du modèle de souris KO nous a permis de confirmer le rôle central du TLR4/MD2 comme récepteur de la protéine Tat ainsi que de mieux comprendre le mécanisme moléculaire mis en jeu par Tat pour induire la production de l'IL-10 et du TNF-a impliquant le TLR4 mais également les cofacteurs CD14 et MD2 ainsi que l'activation des deux voies de signalisation MyD88 et TRIF dépendantes. L'étude des effets de l'interaction Tat-TLR4 sur la modulation de la réponse immunitaire des DCs nous a permis de montrer que i) Tat induit la production des cytokines pro-inflammatoires IL-6, IL-12 et des IFN-a et IFN-? en plus de l'IL-10 et du TNF-a ; ii) Tat induit la maturation des DCs en augmentant l'expression de CD83, CD80, CD86 ; iii) Tat stimule l'expression des facteurs immunosuppressifs IL-10, PD-L1 et IDO associées à une inhibition de la prolifération des LT ; iv) l'ensemble de ces effets sont abolis ou inhibés en bloquant l'interaction Tat-TLR4. Conclusions : Nos résultats suggèrent que le VIH-1, par l'intermédiaire de sa protéine Tat, détourne le TLR4 à la surface des CPA pour induire la production de facteurs pro-inflammatoires et stimuler l'expression des molécules immunosuppressives directement associées à la perte de fonction du système immunitaire. Il n'est donc pas exclu que Tat, à cause de son expression précoce, contribue à instaurer un état immunosuppressif très tôt après l'infection et serait aussi impliquée dans l'affaiblissement du système immunitaire et la persistance virale.Human immunodeficiency virus type 1 (HIV-1) infection is associated with a gradual increase in the expression of inhibitory molecules including IL-10, PD-L1 and IDO essentially by antigen presenting cells (APCs) such as dendritic cells (DCs). These molecules act negatively on the anti-HIV immune response and are associated with viral persistence and disease progression towards AIDS. One of the viral protein involved in this mechanism is Tat. In HIV-1 infected patients, Tat is secreted in the sera at nanomolar levels. In addition to its role in viral cycle, Tat interacts with immune cells to modulate the expression of many host genes including immunosuppressive factors. Previous work from our laboratory have shown that Tat protein, by acting at the membrane level, via its 1-45 N-terminal domain, stimulates the expression of pro-inflammatory cytokines TNF-a, and immunosuppressive IL-10 production in human monocytes and macrophages. More recently, we have shown the involvement of TLR4/MD2 in the interaction with Tat. Aim of the study: The objective of my thesis was to 1) Further characterize the implication of TLR4/MD2 as potential receptor of Tat protein. To this end mouse models invalidated (KO) for TLR4 or its molecular partners (CD14, MD2, MyD88, TRIF) were used 2) Study the effect of Tat protein and its interaction with TLR4/MD2 on the modulation of DCs functions including cytokine production, maturation, and the expression of immunosuppressive factors and their effects on the stimulation or inhibition of T cells activation. Results: The use of knockout mouse models has allowed us to confirm the central role of TLR4/MD2 as receptor for Tat protein and to better understand the signalling pathways recruited by Tat to stimulate the production of IL-10 and TNF-a. Our results showed the importance of CD14 and MD2 in addition to TLR4 and demonstrated that Tat activates both TRIF and MyD88-dependent pathways. In parallel, the study of the effects of Tat-TLR4 interaction on the modulation of the immune response of DCs allowed us to show that i) Tat induces the production of pro-inflammatory cytokines IL-6, IL-12 and IFN-a and IFN-? in addition to IL-10 and TNF-a ii) Tat induces the maturation of DCs by increasing the expression of CD83, CD80, CD86 and iii) Tat stimulates the expression of immunosuppressive factors IL-10, PD-L1 and IDO in association with the inhibition of T cells proliferation. iv) All these effects were abolished or inhibited following the blockade of Tat-TLR4 interaction. Conclusions: Our results suggest that HIV-1 by its Tat protein hijacks TLR4 pathway in APCs to induce the production of pro-inflammatory and immunosuppressive factors which are associated with the impairment of immune functions. It is therefore possible that, because of its early expression, Tat may help to establish an immunosuppressive state early after infection and thus contributed in the weakening of the immune system and viral persistence

Antiviral and Anti-Inflammatory Activities of Fluoxetine in a SARS-CoV-2 Infection Mouse Model

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world’s population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis

HIV-1 Tat protein induces the production of IDO in human monocyte derived-dendritic cells through a direct mechanism: effect on T cells proliferation.

During HIV-1 infection, an increase of indoleamine 2,3 dioxygenase (IDO) expression, and dendritic cells (DC) dysfunction were often associated with AIDS disease progression. In this work, we investigated the effect of HIV-1 Tat protein on the expression of IDO, in MoDCs. We show that Tat induces IDO protein expression and activity in a dose dependent manner by acting at the cell membrane. Using Tat-mutants, we show that the N-Terminal domain, Tat 1-45, but not the central region, Tat 30-72, is sufficient to induce the expression of active IDO. Tat protein is also able to induce several cytokines in MoDCs, including IFN-γ, a strong inducer of IDO. In order to understand whether IDO is induced directly by Tat protein or indirectly following IFN-γ production, complementary experiments were performed and showed that: i) at the kinetic level, Tat induced IDO expression before the production of IFN-γ ii) treatment of MoDCs with Tat-conditioned medium was unable to stimulate IDO expression, iii) coculture of MoDCs in a transwell cell system did not allow IDO expression in MoDCs not previously treated by Tat, iv) direct contact between Tat-treated and untreated MoDCs was not sufficient to induce IDO expression in a Tat-independent manner, and v) treatment of MoDCs in the presence of IFN-γ pathway inhibitors, Jak I and Ly294002, inhibited IFN-γ-induced IDO but had no effect on Tat-induced IDO. At the functional level, our data showed that treatment of MoDCs with Tat led to the inhibition of their capacity to stimulate T cell proliferation. This impairement was totally abolished when the stimulation was performed in the presence of 1MT, an inhibitor of IDO activity, arguing for the implication of the kynurenine pathway. By inducing IDO, Tat protein may be considered, as a viral pathogenic factor, in the dysregulation of the DC functions during HIV-1 infection

HIV-1 Tat – TLR4/MD2 interaction drives the expression of IDO-1 in monocytes derived dendritic cells through NF-κB dependent pathway

International audienceIn the present study we showed that HIV-1 Tat protein stimulated the expression of Indoleamine 2,3 dioxygenase (IDO) -1 in human monocytes derived dendritic cells (MoDC) but not IDO-2 by acting directly at the cell membrane level. This induction of IDO-1 is dependent on the secondary structure of Tat protein, since stimulation with a chemically oxidized Tat protein loses its capacity to induce the production of IDO-1. Among the variety of candidate receptors described for Tat, we demonstrated that Tat protein interacted physically with TLR4/MD2 complex. Strikingly, blockade of Tat-TLR4 interaction by anti-TLR4 antibodies (clone HTA125), LPS-RS, a known TLR4 antagonist, or by soluble recombinant TLR4/MD2 complex inhibited strongly or totally the capacity of Tat to induce IDO-1 in MoDC while such treatments had no effect on IFN-γ-induced IDO-1. Furthermore, we showed that the activation of the transcription factor NF-κB by Tat is essential for the production of IDO-1 by human MoDC. Indeed, Tat activated NF-κB pathway in MoDC as demonstrated by the phosphorylation of p65 in Tat-treated MoDC. Further, we demonstrate that the stimulation of IDO-1 by Tat or by IFN-γ was totally or partially inhibited in the presence of NF-κB inhibitor respectively. These results suggest that Tat and IFN-γ act probably by two distinct mechanisms to induce the production of IDO-1. Our results clearly demonstrated that, although TLR4 pathway is necessary for Tat-induced IDO-1 in MoDC, it seems not to be sufficient since stable transfection of a functional TLR4/MD2 pathway in HEK or HeLa cell lines which are endogenously defectives for TLR4, did not restore the capacity of Tat to induce IDO-1 while IFN-γ treatment induces IDO-1 in HeLa cells independently of TLR4 pathway. These results suggest the involvement of additional stimuli in addition to TLR4 pathway which remain to be identified. Altogether our results demonstrated that, in human MoDC, HIV-1 Tat protein induced IDO-1 expression and activity in a NF-κB dependent-manner by recruiting TLR4 pathway

Tat treated MoDC inhibits T cell proliferation in an IDO dependent mechanism.

<p>Immature MoDCs were incubated for 48(50 nM), IFN-γ (500 ng/ml), or PBS at 37°C. After washing with PBS, MoDCs were put back into culture with or without IDO inhibitor 1MT (500 µM). After 2 hr, 2.10⁵ MoDCs were cocultured with 4.10⁵ autologous PBL (a CD14 negative fraction) labelled with 2 µM CFSE, and stimulated with anti-CD3, OKT3, Mab (10 ng/ml) or left unstimulated. Direct treatment with kynurenine (500 µM) was used as a control. After 5 days, autologous T cell proliferation was monitored by FACS analysis by following CFSE dilution analysis in the CD3 positive population. Overlay show T cell proliferation performed in the absence (purple) or in the presence (red) of 1MT. Histogram plots are representative of two independent experiments.</p

HIV-1 Tat induces IDO protein expression and activity in MoDCs.

<p>Tat protein specifically induces IDO expression/activity in MoDCs. (A) MoDCs (2.10⁶) were treated with 50 nM of Tat 1–86 protein (Lai strain) for 24 hr. Untreated and IFN-γ-treated (500 ng/ml) cells were used as negative and positive controls respectively. The specificity of Tat was evaluated by treating MODCs with Tat (50 nM) previously incubated with anti-Tat antibodies (3 µg/ml) for 30 min at 37°C. (B) MoDCs (2.10⁶) were treated with increasing amounts (10, 100, and 200 nM) of full length recombinant GST-Tat 1–101 (SFII strain) or with the truncated forms GST-Tat 1–45, and GST-Tat 30–72 (100 nM). GST protein alone (100 nM) and IFN-γ (100 ng/ml) were used as negative and positive controls respectively. Eluent corresponds to the fraction of GST-Tat not retained following incubation of GST-Tat (100 nM) with anti-Tat/anti-GST coupled to protein A sepharose beads (pharmacia biotech). (C) MoDCs (2.10⁶) were treated with LPS (1 µg/ml), IFN-γ (1 µg/ml), GST-Tat 1–101 (50 nM), or kept untreated for 24 hr. For each experiment, the upper panel shows IDO protein expression by immuno-blot, and the loading control (β-actine) in the second line. Lower panels’ shows the tryptophan catabolism activity determined by Ehrlich’s spetrophotometric assay. (D) Intracellular IDO protein expression was assessed by flow cytometry in CD11c positive MoDCs after stimulation for 24 hr with GST (100 nM), GST-Tat (100 nM), LPS (1 µg/ml) IFN-γ (500 ng/ml), or untreated MoDC. The settings were made on the control isotype. Data are representative of three to four independent experiments.</p

HIV-1 Envelope Glycoproteins Induce the Production of TNF-α and IL-10 in Human Monocytes by Activating Calcium Pathway.

Human HIV-1 infection leads inevitably to a chronic hyper-immune-activation. However, the nature of the targeted receptors and the pathways involved remain to be fully elucidated. We demonstrate that X4-tropic gp120 induced the production of TNF-α and IL-10 by monocytes through activation of a cell membrane receptor, distinct from the CD4, CXCR4, and MR receptors. Gp120 failed to stimulate IL-10 and TNF-α production by monocytes in Ca2+ free medium. This failure was total for IL-10 and partial for TNF-α. However, IL-10 and TNF-α production was fully restored following the addition of exogenous calcium. Accordingly, addition of BAPTA-AM and cyclosporine-A, fully and partially inhibited IL-10 and TNF-α respectively. The PKA pathway was crucial for IL-10 production but only partially involved in gp120-induced TNF-α. The PLC pathway was partially and equivalently involved in gp120-induced TNF-α and IL-10. Moreover, the inhibition of PI3K, ERK1/2, p38 MAP-kinases and NF-κB pathways totally abolished the production of both cytokines. In conclusion, this study revealed the crucial calcium signaling pathway triggered by HIV-1 gp120 to control the production of these two cytokines: TNF-α and IL-10. The finding could help in the development of a new therapeutic strategy to alleviate the chronic hyper-immune-activation observed in HIV-1 infected patients