Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly, cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also cleave and inactivate the pyroptosis executioner Gasdermin (GSDM)-D. Consequently, Caspase-3 and GSDM-E promote alternative cell pyroptosis, a process exacerbated in cells exhibiting imparied type I interferon production. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to Interferon alterations identify GSDM-E/Caspase-3 as biological markers of disease severity. Overall, our findings identify NLRP1 as a key sensor of SARS-CoV-2 infection in lung epithelia.Funding Information: This project has been funded on lab own funds from unrelated grants from the Fondation pour la Recherche Médicale (FRM) and ERC StG (INFLAME) to EM, from ERC StG (ANTIViR) to CG, by the French Ministry of Health with the participation of the Groupement Interrégional de Recherche Clinique et d'Innovation Sud-Ouest Outre-Mer (PHRCI 2020 IMMUNOMARK-COV) to G-M.B. The ASB3 structure is supported by LABEX, Investissement d'Avenir and foundation Bettencourt grants to ON. MP and RP were respectively funded by a CIFRE PhD fellowship and a research grant from Invivogen. SB is supported by a PhD fellowship from Mali ministry of education and from the FRM (FDT 12794). SALC is supported by a Vaincre La Mucoviscidose (VLM) PhD fellowship.Declaration of Interests: Authors declare no conflict of interest. Ethics Approval Statement: All donors had given written informed consent and the study was approved by the ethical review board "Comité de Protection des Personnes Est-III" (ID-RCB 2020-A01292-37).
HIV-1 Tat protein induces the production of CXCL8 chemokine in a TLR4/MD2 and PKC dependent manner. The objective of this study was to understand whether these two pathways were distinct or constituted a single common pathway, and to determine the nature of the PKC isoforms involved and their interrelation with the activation of NF-κB and CXCL8 gene product expression. Here, we show that Tat-induced CXCL8 production is essentially dependent on the activation of PKC delta isoform, as shown a) by the capacity of PKC delta dominant negative (DN), and Rottlerin, a selective PKC delta pharmacological inhibitor, to inhibit Tat-induced CXCL8 production and b) by the ability of the constitutively active (CAT) isoform of PKC delta to induce CXCL8 production in a HEK cell line in the absence of Tat stimulation. The finding that comparable amounts of CXCL8 were produced following stimulation with either Tat protein, PKC-delta CAT transfection, or both, argue for the implication of one common pathway where PKC delta is activated downstream of TLR4 recruitment and leads to the activation of NF-κB. Altogether, our results underline the crucial role of PKC delta isoform in activating gene expression of CXCL8, a cytokine largely implicated in the physiopathology of HIV-1 infection.
Abstract In 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.
Human Immunodeficiency Virus (HIV) is the causative agent of AIDS (Acquired Immune Deficiency Syndrome).Currently, the infection by HIV is considered as one of the major problem of public health and social stability.The scale of HIV/AIDS epidemic has exceeded all expectations since its identification 30 years ago.Currently, according to the report on HIV/AIDS by UNAIDS and World Health Organization (WHO) in 2009, we estimated approximately 33 million people are currently living with HIV-1 and about 25 millions have already died (Piot, Feachem et al. 2004).HIV infection leads to a decline in the number of T helper CD4 cells leading to a progressive dysfunction of the immune system.This weakening of the immune system results in the development of opportunistic infections leading to death of the patient.Despite prevention policies and anti-retroviral therapies, AIDS still remains one of the most serious humanitarian crises that we have never known. HIV taxonomy, structure and genome organizationIdentified in 1983, HIV belongs to lentivirus genus of Retroviridaea (Barre-Sinoussi, Chermann et al. 1983).Lentiviruses are host-specific viruses which cause slowly progressive diseases in their hosts and are characterized by selective T CD4 depletion (Fauci 1988) associated with a severe immunological and neurological disorders.These disorders are amplified by the fact that the virus infects the key cells of the immune system.As consequences, immunological disorders in HIV infected patients are observed since the asymptomatic stage.Two major subtypes of HIV have been identified: HIV-1 and HIV-2 (Clavel, Guetard et al. 1986).The first, HIV-1, is world wide, virulent and responsible of HIV infection globally.However, the second, HIV-2, is less pathogenic and is largely confined to West Africa.These two types of virus share a 40% homology in their sequences.HIV-1 strain is subdivided into three genetically groups: M (Major), N (New or non M non O) and O (Outlier) (Clavel, Guetard et al. 1986).The M strain is the most spread worldwide, it represents more than 95% of the global virus isolates.The variability within the sequence of env gene of HIV-1, allowed it to be subdivided into 10 clades A to L (McCutchan 2000).At least 15 additional circulating recombinant forms (CRF) has been identified in HIV-1 patients living in different geographic regions of the world (Peeters and Sharp 2000).However, the presence of these CRF seems to be more present in Africa, Asia and South of America.Despite the high sequence homology between HIV-1 and HIV-2 and the www.intechopen.com
In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) βII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10.In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC-βII, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it may be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein.
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.
ABSTRACT The intracellular inflammasome complex have been implicated in the maladaptive tissue damage and inflammation observed in chronic Pseudomonas aeruginosa infection. Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by P. aeruginosa , specifically express the inflammasome sensor NLRP1. Here, together with a companion study, we report that the NLRP1 inflammasome detects Exotoxin A (EXOA), a ribotoxin released by P. aeruginosa Type 2 Secretion System (T2SS) during chronic infection. Mechanistically, EXOA-driven Eukaryotic Elongation Factor 2 (EEF2) ribosylation and covalent inactivation promotes ribotoxic stress and subsequent NLRP1 inflammasome activation, a process shared with other EEF2-inactivating toxins, Diphtheria Toxin and Cholix Toxin. Biochemically, irreversible EEF2 inactivation triggers ribosome stress-associated kinases ZAKα- and P38-dependent NLRP1 phosphorylation and subsequent proteasome-driven functional degradation. Finally, Cystic Fibrosis cells from patients exhibit exacerbated P38 activity and hypersensitivity to EXOA-induced ribotoxic stress-dependent NLRP1 inflammasome activation, a process inhibited by the use of ZAKα inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAKα as a critical sensor of virulence-inactivated EEF2. KEY POINTS P. aeruginosa induces NLRP1-dependent pyroptosis in human corneal and nasal epithelial cells P. aeruginosa Exotoxin A (EXOA) and other EEF2-inactivating bacterial exotoxins activate the human NLRP1 inflammasome EEF2 inactivation promotes ribotoxic stress response and ZAKα kinase-dependent NLRP1 inflammasome activation. Bronchial epithelial cells from Cystic Fibrosis patients show extreme sensitivity to ribotoxic stress-dependent NLRP1 inflammasome activation in response to Exotoxin A P38 and ZAKα inhibition protects Cystic Fibrosis epithelial cell from EXOA-induced pyroptosis
Au cours de l'infection par le virus de l'immunodeficience humaine de type 1 (VIH-1) on observe une augmentation progressive de l'expression des molecules inhibitrices du systeme immunitaire (IL-10, PD-L1 et IDO) principalement par les cellules presentatrices d'antigenes (CPA) telles que les cellules dendritiques (DCs). Ces molecules agissent negativement sur la reponse immunitaire anti-VIH et sont associees a la persistance du virus et a l'evolution de la maladie vers le stade SIDA. Une des proteines virales impliquee dans ce mecanisme est la proteine Tat. En plus de son role direct dans la transactivation du genome viral, Tat est secretee par les cellules infectees et se retrouve a des concentrations de l'ordre du nanomolaire dans le serum des patients. Dans la circulation, Tat interagit avec les cellules immunitaires et module l'expression de nombreux facteurs incluant des facteurs immunosuppressifs. Les travaux precedents du laboratoire, ont montre 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 recemment, la recherche du recepteur membranaire recrute par Tat a permis de mettre en evidence l'implication du TLR4/MD2 dans l'interaction avec Tat. Objectifs : Mon projet de these qui s'inscrit dans la continuite des travaux du laboratoire a pour objectif 1) de caracteriser davantage le role du TLR4 comme recepteurs de Tat en utilisant une approche basee sur des modeles murins invalides (KO) pour le TLR4 ou ses partenaires moleculaires (CD14, MD2, MyD88, TRIF) 2) d'etudier l'effet de la proteine 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 capacite a activer/inhiber les lymphocytes T. Resultats : L'utilisation du modele de souris KO nous a permis de confirmer le role central du TLR4/MD2 comme recepteur de la proteine Tat ainsi que de mieux comprendre le mecanisme moleculaire mis en jeu par Tat pour induire la production de l'IL-10 et du TNF-a impliquant le TLR4 mais egalement les cofacteurs CD14 et MD2 ainsi que l'activation des deux voies de signalisation MyD88 et TRIF dependantes. L'etude des effets de l'interaction Tat-TLR4 sur la modulation de la reponse 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 associees a une inhibition de la proliferation des LT ; iv) l'ensemble de ces effets sont abolis ou inhibes en bloquant l'interaction Tat-TLR4. Conclusions : Nos resultats suggerent que le VIH-1, par l'intermediaire de sa proteine Tat, detourne le TLR4 a la surface des CPA pour induire la production de facteurs pro-inflammatoires et stimuler l'expression des molecules immunosuppressives directement associees a la perte de fonction du systeme immunitaire. Il n'est donc pas exclu que Tat, a cause de son expression precoce, contribue a instaurer un etat immunosuppressif tres tot apres l'infection et serait aussi impliquee dans l'affaiblissement du systeme immunitaire et la persistance virale.
Dendritic cells have the unique ability to pick up dead cells carrying antigens in tissue and migrate to the lymph nodes where they can cross-present cell-associated antigens by MHC class I to CD8+ T cells. There is strong in vivo evidence that the mouse XCR1+ dendritic cells subset acts as a key player in this process. The intracellular processes underlying cross-presentation remain controversial and several pathways have been proposed. Indeed, a wide number of studies have addressed the cellular process of cross-presentation in vitro using a variety of sources of antigen and antigen presenting cells. Here we review the in vivo and in vitro evidence supporting the current mechanistic models and disscuss their physiological relevance to the cross-presentation of cell-associated antigens by dendritic cells subsets
