Development of broad‐spectrum human monoclonal antibodies for rabies post‐exposure prophylaxis

Currently available rabies post‐exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad‐spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non‐RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post‐vaccination antibody response

The role of the nuclear receptor Rev-erb-alpha in inflammatory diseases : the circadian regulation of the NLRP3 inflammasome complex in fulminant hepatitis and the repression of the development of atherosclerosis and vascular calcification

L'athérosclérose consiste en l'accumulation de lipoprotéines de faible densité (LDL) dans la paroi vasculaire qui provoque l'infiltration de leucocytes, principalement des monocytes. Ces cellules se différencient alors en macrophages pour éliminer les LDL infiltrées en favorisant le transport inverse du cholestérol vers les lipoprotéines de haute densité.Cependant, lorsque l'efflux de cholestérol devient inefficace, les macrophages s’engorgent delipides, forment des cellules spumeuses qui meurent par apoptose ou nécrose. Ils déversent alorsleur contenu lipidique dans la matrice extracellulaire, formant ainsi le coeur nécrotique. Descristaux de cholestérol peuvent se former dans l’intima et sont phagocytés par les macrophages.Ils provoquent alors des dommages lysosomiaux qui activent le complexe inflammasomeNLRP3. Ce complexe entraîne la maturation de la caspase 1 qui clive les pro-cytokines en IL1Bet IL18 actives. Ces interleukines sont alors libérées et stimulent la sécrétion d'interféron γ(IFNγ) par les lymphocytes T CD4+, provoquant la prolifération et la migration des CellulesMusculaires Lisses (CML) vers le coeur nécrotique pour former la chape fibreuse. En plus desnombreux facteurs de risque des maladies cardiovasculaires comme le diabète ou le tabagisme,la perturbation des rythmes nycthéméraux comme chez les travailleurs postés, augmentel’incidence de ces maladies. Le récepteur nucléaire Rev-erb-α est un composant central del’horloge moléculaire. Il est en outre un régulateur important du métabolisme des lipides et dela réponse inflammatoire. Il représente donc une cible thérapeutique de choix dans le traitementde ces maladies multifactorielles liées à un dérèglement de l’horloge. Mes travaux montrentque l’absence de Rev-erbα dans un modèle murin pro-athérogène LDLr-/- entraînel’augmentation des taux plasmatiques de lipides et accélère le processus d'athérogenèse. Deplus, Rev-erb-α régule l'expression circadienne des composants de la voie NLRP3 et lasécrétion d'IL1B et IL18 dans les macrophages. Au niveau moléculaire, Rev-erb-α réprimedirectement la voie NLRP3, tandis que la déficience en Rev-erbα augmente l'activation de cettevoie. Ce mécanisme régulerait la susceptibilité aux stimuli inflammatoires dans des modèlesd'inflammation aiguë comme la péritonite ou l'hépatite fulminante en fonction du moment dela journée auquel les souris ont été stimulées. L'absence de Rev-erbα induit égalementl’expression de Nlrp3, Il1β et Il18 dans des modèles d'inflammation chronique commel'athérosclérose ainsi que l’expression de NLRP3 dans les macrophages des lésions d'athérome.De plus, IL1B et IL18 stimulent la différenciation des CML en cellules ostéoblastiques quiforment de la calcification intimale. Il est intéressant de noter que l'absence de Rev-erbα dansle fond génétique LDLr-/- entraîne une augmentation de la calcification intimale des lésions par rapport aux souris LDLr-/-Rev-erbα+/+, tandis que l'absence de Rev-erb-α dans les CMLprimaires augmente leur différenciation en ostéoblastes et la formation d'un dépôt calcique invitro. De manière intéressante, cet effet de la délétion de Rev-erbα observé sur le dépôt calciquein vitro est exacerbé lorsque les CML primaires sont stimulées en culture avec de l’IL1B. Reverb-α ne jouerait donc pas seulement un rôle dans la production d’IL1B par les macrophagesmais également dans la réception et l’interprétation de ce signal par les CML. L’activation deRev-erb-α par son ligand inhibe ces effets. Ces travaux mettent en évidence l’importance deRev-erb-α dans la régulation circadienne de la réponse inflammatoire et les phénomènesassociés au développement des maladies cardiovasculaires, identifiant Rev-erb-α comme unenouvelle cible thérapeutique qui permettrait de cibler avec une seule molécule plusieurs aspectsde la pathologie.Atherosclerosis is chronic inflammatory disease of the vascular wall, which consists inthe accumulation of Low Density Lipoproteins (LDL) into the vascular wall triggering theinternalization of leucocytes. Recruited monocytes differentiate into macrophages to removeLDL through the so-called reverse cholesterol transport pathway. However, when flux isimpaired, lipid-laden macrophages become foam cells leading to their apoptosis or necrosis.Their lipid content and cellular debris are then released in the extracellular matrix, thus formingthe necrotic core. In addition, laden-cholesterol crystals trigger lysosomal damage inmacrophage, which activates the NLRP3 inflammasome complex. NLRP3 inflammasome isinvolved in the maturation of the pro-caspase 1, which subsequently cleaves the pro- IL1B andpro-IL18 into mature IL1B and IL18. These interleukins are then released and stimulate thesecretion of interferon γ by CD4+ T cells, leading to the proliferation and the migration ofSmooth Muscle Cells (SMC) toward the necrotic core to form the fibrous cap. In addition tothe numerous risk factors of cardiovascular diseases such as diabetes or smoking, disruption ofdaily rhythms, as seen in shiftwork, increases the incidence of these diseases. Beyond its majorrole in metabolism and inflammation control, the nuclear receptor Rev-erb-α is also a corecomponent of the molecular clock. Altogether, it thus represents a putative therapeutic target inthe treatment of such multifactorial diseases related to clock impairment. My work shows thatthe deletion of Rev-erbα in a murine model of atherosclerosis (LDLr-/-) leads to an increase inplasma lipid levels and accelerates atherogenesis. In addition, Rev-erb-α regulates the circadianexpression of the NLRP3 pathway components as well as the subsequent secretion of IL1B andIL18 in primary human and mouse macrophages. At the molecular level, Rev-erb-α directlyrepresses the NLRP3 pathway, whereas Rev-erbα deficiency enhances its activation. Thismechanism may regulate the circadian susceptibility to inflammatory stimuli depending on thetime of challenge in acute inflammation models such as fulminant hepatitis or peritonitis. Theabsence of Rev-erbα also induces the expression of Nlrp3, Il1β and Il18 in models of chronicinflammation such as atherosclerosis as well as the expression of NLRP3 in lesionalmacrophages. In addition, IL1B and IL18 stimulate the differentiation of SMCs into osteoblastlikecells that form intimal calcification. It is noteworthy that Rev-erbα deletion in LDLr-/- miceis associated to an increase in lesion calcification in vivo, while the absence of Rev-erb-α inprimary SMCs increases their differentiation into osteoblasts and the formation of calciumdeposit in vitro. Interestingly, this differential effect observed on calcium deposition in vitro isexacerbated when primary SMCs are stimulated in culture with IL1B. Therefore, Rev-erb-α may be involved not only in macrophage-mediated IL1B production but also in the sensing ofsuch signal by SMCs. Conversely, treatment with Rev-erb ligands inhibits these effects. Thiswork emphasizes the key role of Rev-erb-α in the circadian regulation of the inflammatoryresponse and in the development of cardiovascular diseases, thus identifying Rev-erb-α as anew therapeutic target that act on several aspects of the pathology

Circadian control of metabolism and pathological consequences of clock perturbations.

International audienceMost organisms have developed an autonomous time-keeping system that generates self-sustained daily fluctuations in behavior and physiological processes. These biological clocks are reset every day by light to adjust physiology to the day/night cycle generated by the rotation of the Earth. Clocks present in organs involved in glucose and lipid metabolism such as the liver, muscle, adipose tissue and pancreas are also reset by feeding cues which permits the local integration of systemic and nutritional signals to switch fuel production and utilization according to the feeding/fasting cycle. However, derangements in this finely tuned system can be induced by extending light exposure, 24/7 food availability and altered food intake patterns, repeated jet-lag and shift-working, promoting metabolic imbalances ranging from body weight gain to the development of insulin resistance and liver diseases. Here, we review recent findings on the link between the clock and metabolic fluxes to maintain whole-body homeostasis, and what clock disruption in mice has revealed about the role of the clock in metabolic regulation

Post-LGM coastline evolution of the NW Sicilian Channel: Comparing high-resolution geophysical data with Glacial Isostatic Adjustment modeling

Since about 20,000 years ago, the geography of the Earth has been profoundly modified by the gradual sea-level rise caused by the melting of continental ice sheets. Flat areas and regions characterized by very low gradients experienced, more than others, rapid flooding, with the progressive disappearance of vast coastal territories. Here we present a recon- struction of the late Quaternary coastline evolution of the north-western sector of the Sicilian Channel, constrained by high-resolution seismic profiles where the marker of the post-Last Glacial Maximum (LGM) marine transgression has been clearly identified and mapped. The locations of the post-LGM seismic horizon have been compared with predictions of a Glacial Isostatic Adjustment (GIA) model, which accounts for the migration of the shorelines in response to sea-level rise and for Earth’s rotational and deformational effects associated with deglaciation. We have verified that most of the points mapped through seismic data interpretation fall along the palaeo-coastline that the GIA model predicts for the 21 kyrs B.P. time frame. However, the model shows a misfit in the marine sector between Mazara del Vallo and Sciacca, where the available data indicate a Quaternary tectonic uplift. The analy- sis of the seismic profiles provides useful constraints to current GIA models. These add on existing histories of relative sea level in the Mediterranean Sea, allowing to gain new insight into the evolution of the palaeo-geography of the region of study and of the whole Sicilian Channel since the LGM, even in areas where direct geophysical observations are not avail- able yet. In this respect, one of the most attractive implications of the ancient coastline evo- lution is linked with the underwater archaeology. The sea-level rise heavily impacted the distribution of human settlements, possibly forcing site abandonment and migrations, and this is particularly relevant in the Mediterranean basin, the cradle of the western civilization. The underwater traces left by these ancient populations represent the fundamental proofs to reconstruct the early history of our precursors

Nuclear Receptor Subfamily 1 Group D Member 1 Regulates Circadian Activity Of NLRP3 Inflammasome to Reduce the Severity of Fulminant Hepatitis in Mice

International audienceBACKGROUND & AIMS:The innate immune system responds not only to bacterial signals, but also to non-infectious danger-associated molecular patterns that activate the NLRP3 inflammasome complex after tissue injury. Immune functions vary over the course of the day, but it is not clear whether these changes affect the activity of the NLRP3 inflammasome. We investigated whether the core clock component nuclear receptor subfamily 1 group D member 1 (NR1D1, also called Rev-erbα) regulates expression, activity of the NLRP3 inflammasome, and its signaling pathway.METHODS:We collected naïve peritoneal macrophages and plasma, at multiple times of day, from Nr1d1-/- mice and their Nr1d1+/+ littermates (controls) and analyzed expression of NLR family pyrin domain containing 3 (NLRP3), interleukin 1 beta (IL1B, in plasma), and IL18 (in plasma). We also collected bone marrow-derived primary macrophages from these mice. Levels of NR1D1 were knocked down with small hairpin RNAs in human primary macrophages. Bone marrow-derived primary macrophages from mice and human primary macrophages were incubated with lipopolysaccharide (LPS) to induce expression of NLRP3, IL1B and IL18; cells were incubated with LPS and ATP to activate the NLRP3 complex. We analyzed caspase 1 activity and cytokine secretion. NR1D1 was activated in primary mouse and human macrophages by incubation with SR9009; some of the cells were also incubated with an NLRP3 inhibitor or inhibitors of caspase 1. Nr1d1-/- mice and control mice were given intraperitoneal injections of LPS to induce peritoneal inflammation; plasma samples were isolated and levels of cytokines were measured. Nr1d1-/- mice, control mice, and control mice given injections of SR9009 were given LPS and galactosamine to induce fulminant hepatitis and MCC950 to specifically inhibit NLRP3; plasma was collected to measure cytokines and a marker of liver failure (ALAT); liver tissues were collected and analyzed by quantitative PCR, immunohistochemistry, and flow cytometry.RESULTS:In peritoneal macrophages, expression of NLRP3 and activation of its complex varied with time of day (circadian rhythm)-this regulation required NR1D1. Primary macrophages from Nr1d1-/- mice and human macrophages with knockdown of NR1D1 had altered expression patterns of NLRP3, compared to macrophages that expressed NR1D1, and altered patterns of IL1B and 1L18 production. Mice with disruption of Nr1d1 developed more-severe acute peritoneal inflammation and fulminant hepatitis than control mice. Incubation of macrophage with the NR1D1 activator SR9009 reduced expression of NLRP3 and secretion of cytokines. Mice given SR9009 developed less-severe liver failure and had longer survival times than mice given saline (control).CONCLUSIONS:In studies of Nr1d1-/- mice and human macrophages with pharmacologic activation of NR1D1, we found NR1D1 to regulate the timing of NLRP3 expression and production of inflammatory cytokines by macrophages. Activation of NR1D1 reduced the severity of peritoneal inflammation and fulminant hepatitis in mice

Glycogen Dynamics Drives Lipid Droplet Biogenesis during Brown Adipocyte Differentiation

International audienceBrowning induction or transplantation of brown adipose tissue (BAT) or brown/beige adipocytes derived from progenitor or induced pluripotent stem cells (iPSCs) can represent a powerful strategy to treat metabolic diseases. However, our poor understanding of the mechanisms that govern the differentiation and activation of brown adipocytes limits the development of such therapy. Various genetic factors controlling the differentiation of brown adipocytes have been identified, although most studies have been performed using in vitro cultured pre-adipocytes. We investigate here the differentiation of brown adipocytes from adipose progenitors in the mouse embryo. We demonstrate that the formation of multiple lipid droplets (LDs) is initiated within clusters of glycogen, which is degraded through glycophagy to provide the metabolic substrates essential for de novo lipogenesis and LD formation. Therefore, this study uncovers the role of glycogen in the generation of LDs

NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression

International audienceThe sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase-dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy