The Phospholipid Scramblases 1 and 4 Are Cellular Receptors for the Secretory Leukocyte Protease Inhibitor and Interact with CD4 at the Plasma Membrane

Secretory leukocyte protease inhibitor (SLPI) is secreted by epithelial cells in all the mucosal fluids such as saliva, cervical mucus, as well in the seminal liquid. At the physiological concentrations found in saliva, SLPI has a specific antiviral activity against HIV-1 that is related to the perturbation of the virus entry process at a stage posterior to the interaction of the viral surface glycoprotein with the CD4 receptor. Here, we confirm that recombinant SLPI is able to inhibit HIV-1 infection of primary T lymphocytes, and show that SLPI can also inhibit the transfer of HIV-1 virions from primary monocyte-derived dendritic cells to autologous T lymphocytes. At the molecular level, we show that SLPI is a ligand for the phospholipid scramblase 1 (PLSCR1) and PLSCR4, membrane proteins that are involved in the regulation of the movements of phospholipids between the inner and outer leaflets of the plasma membrane. Interestingly, we reveal that PLSCR1 and PLSCR4 also interact directly with the CD4 receptor at the cell surface of T lymphocytes. We find that the same region of the cytoplasmic domain of PLSCR1 is involved in the binding to CD4 and SLPI. Since SLPI was able to disrupt the association between PLSCR1 and CD4, our data suggest that SLPI inhibits HIV-1 infection by modulating the interaction of the CD4 receptor with PLSCRs. These interactions may constitute new targets for antiviral intervention

Spotlight on the NLRP3 inflammasome pathway

International audienceInflammation is triggered by a repertoire of receptors detecting infections and damages. Some of these receptors directly bind microbial ligands, while others recognize endogenous molecules exposed under stress conditions, including infections. Most of these receptors can be engaged by a relatively limited number of stimuli. Differently, NLRP3 acts as a broad sensor of cell homeostasis rupture and can be activated downstream of a plethora of stimuli. NLRP3 then assembles a multiprotein platform resulting in caspase-1 activation, which controls, by direct cleavage, the maturation of cytosolic pro-cytokines including pro-interleukin-1β. In addition, caspase-1 processes cytosolic gasdermin-D and unleashes its pore-forming N-terminal domain, leading to the release of mature cytosolic cytokines and alarmins, as well as pyroptotic cell lysis. Accumulating evidences of the aggravating role of NLRP3-mediated inflammation in various highly prevalent human conditions, including diabetes, neurodegenerative and cardiovascular diseases, raises a huge clinical interest. Nevertheless, the molecular mechanism governing NLRP3 activation remains insufficiently understood. In line with the detrimental consequences of NLRP3 activation illustrated by the aforementioned pathologies, this process is tightly regulated. In this review, we address the current understanding of the control of NLRP3 activity which can be divided into two coordinated processes referred to as priming and activation. In particular, we detail the emerging role of NLRP3 post-translational modifications critical in inflammasome assembly regulation

NLRP3, un inflammasome sous contrôle

International audienceThe innate immunity constitutes an efficient barrier by rapidly detecting pathogens and tissue damages through pattern recognition receptors including NLRP3. Moreover, inappropriate NLRP3 activation causes deleterious inflammation and contributes to various conditions including atherosclerosis, diabetes, gout and Alzheimer's diseases. NLRP3 assembles a multimeric inflammasome complex serving as an activation platform for caspase-1 that controls processing and release of cytosolic inflammatory factors and cytokines including IL-1β Inflammasome assembly is tightly controlled and requires coordinated NLRP3 priming, through cytokine or other pattern recognition receptors, followed by activation by cellular stress. Here, we describe recent advances in the understanding of the signalling pathways supporting the priming and activation of NLRP3, with a special focus on the key role of post-translational modifications of NLRP3, including phosphorylation and ubiquitination, in inflammasome regulation.La réponse immunitaire innée protège l’organisme par la détection rapide des agents pathogènes et des lésions via des récepteurs spécialisés, dont NLRP3. Celui-ci assemble un inflammasome, un complexe cytosolique de signalisation qui active la caspase-1, contrôle la libération de cytokines et de facteurs inflammatoires produits dans le cytosol comme les interleukines 1α/β Les pathologies inflammatoires associées à NLRP3, dont la goutte, révèlent la nécessité d’un contrôle étroit de son activité. Cette revue présente les avancées sur la signalisation du priming (ou amorçage) du complexe puis de son activation avec une attention particulière sur le rôle des modifications post-traductionnelles de NLRP3

A critical role of eEF-2K in mediating autophagy in response to multiple cellular stresses

International audienceThe phosphorylation of the subunit alpha of eukaryotic translation initiation factor 2 (eIF2alpha), a critical regulatory event in controlling protein translation, has recently been found to mediate the induction of autophagy. However, the mediators of autophagy downstream of eIF2alpha remain unknown. Here, we provide evidence that eIF2alpha phosphorylation is required for phosphorylation of eukaryotic elongation factor 2 (eEF-2) during nutrient starvation. In addition, we show that eukaryotic elongation factor 2 kinase (eEF-2K) is also required for autophagy signaling during ER stress, suggesting that phosphorylation of eEF-2 may serve as an integrator of various cell stresses for autophagy signaling. On the other hand, although the activation of eEF-2K in response to starvation requires the phosphorylation of eIF2alpha, additional pathways relying partly on Ca(2+) flux may control eEF-2K activity during ER stress, as eIF2alpha phosphorylation is dispensable for both eEF-2 phosphorylation and autophagy in this context

Autophagy Limits Listeria monocytogenes Intracellular Growth in the Early Phase of Primary Infection

International audienceAutophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a DeltaactA mutant strain, impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria colocalize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a DeltaplcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy

Roles of Caspases in Necrotic Cell Death

International audienceCaspases were originally identified as important mediators of inflammatory response and apoptosis. Recent discoveries, however, have unveiled their roles in mediating and suppressing two regulated forms of necrotic cell death, termed pyroptosis and necroptosis, respectively. These recent advances have significantly expanded our understanding of the roles of caspases in regulating development, adult homeostasis, and host defense response

The double sides of NLRP3 inflammasome activation in sepsis

International audienceAbstract Sepsis is defined as a life-threatening organ dysfunction induced by a dysregulated host immune response to infection. Immune response induced by sepsis is complex and dynamic. It is schematically described as an early dysregulated systemic inflammatory response leading to organ failures and early deaths, followed by the development of persistent immune alterations affecting both the innate and adaptive immune responses associated with increased risk of secondary infections, viral reactivations, and late mortality. In this review, we will focus on the role of NACHT, leucin-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome in the pathophysiology of sepsis. NLRP3 inflammasome is a multiproteic intracellular complex activated by infectious pathogens through a two-step process resulting in the release of the pro-inflammatory cytokines IL-1β and IL-18 and the formation of membrane pores by gasdermin D, inducing a pro-inflammatory form of cell death called pyroptosis. The role of NLRP3 inflammasome in the pathophysiology of sepsis can be ambivalent. Indeed, although it might protect against sepsis when moderately activated after initial infection, excessive NLRP3 inflammasome activation can induce dysregulated inflammation leading to multiple organ failure and death during the acute phase of the disease. Moreover, this activation might become exhausted and contribute to post-septic immunosuppression, driving impaired functions of innate and adaptive immune cells. Targeting the NLRP3 inflammasome could thus be an attractive option in sepsis either through IL-1β and IL-18 antagonists or through inhibition of NLRP3 inflammasome pathway downstream components. Available treatments and results of first clinical trials will be discussed

Deubiquitination of NLRP3 by BRCC3 Critically Regulates Inflammasome Activity

International audienceNLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various proinflammatory stimuli associated with tissue damage or malfunction. Upon activation, NLRP3 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. Although NLRP3 expression is induced by the NF-κB pathway, the posttranscriptional molecular mechanism controlling the activation of NLRP3 remains elusive. Using both pharmacological and molecular approaches, we show that the activation of NLRP3 inflammasome is regulated by a deubiquitination mechanism. We further identify the deubiquitinating enzyme, BRCC3, as a critical regulator of NLRP3 activity by promoting its deubiquitination and characterizing NLRP3 as a substrate for the cytosolic BRCC3-containing BRISC complex. Our results elucidate a regulatory mechanism involving BRCC3-dependent NLRP3 regulation and highlight NLRP3 ubiquitination as a potential therapeutic target for inflammatory diseases

Deubiquitination of NLRP3 by BRCC3 Critically Regulates Inflammasome Activity

International audienceNLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various proinflammatory stimuli associated with tissue damage or malfunction. Upon activation, NLRP3 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. Although NLRP3 expression is induced by the NF-κB pathway, the posttranscriptional molecular mechanism controlling the activation of NLRP3 remains elusive. Using both pharmacological and molecular approaches, we show that the activation of NLRP3 inflammasome is regulated by a deubiquitination mechanism. We further identify the deubiquitinating enzyme, BRCC3, as a critical regulator of NLRP3 activity by promoting its deubiquitination and characterizing NLRP3 as a substrate for the cytosolic BRCC3-containing BRISC complex. Our results elucidate a regulatory mechanism involving BRCC3-dependent NLRP3 regulation and highlight NLRP3 ubiquitination as a potential therapeutic target for inflammatory diseases

Role of Protein Misfolding in DFNA9 Hearing Loss*

Mutations in the COCH (coagulation factor C homology) gene have been attributed to DFNA9 (deafness, autosomal-dominant 9), an autosomal-dominant non-syndromic hearing loss disorder. However, the mechanisms responsible for DFNA9 hearing loss remain unknown. Here, we demonstrate that mutant cochlin, the protein product of the COCH gene, forms a stable dimer that is sensitive to reducing agent. In contrast, wild-type (WT) cochlin may form only dimers transiently. Interestingly, the presence of mutant cochlin can stabilize WT cochlin in dimer conformation, providing a possible mechanism for the dominant nature of DFNA9 mutations. Furthermore, the expression of mutant cochlin eventually induces WT cochlin to form stable oligomers that are resistant to reducing agent. Finally, we show that mutant cochlin is cytotoxic in vitro and in vivo. Our study suggests a possible molecular mechanism underlying DFNA9 hearing loss and provides an in vitro model that may be used to explore protein-misfolding diseases in general