Un modèle macrophagique humain pour étudier la dynamique d’activation de l’inflammasome

Les inflammasomes sont des complexes protéiques impliqués dans l’immunité innée, qui sont activés par de multiples signaux de danger. Des mutations héréditaires des protéines de l’inflammasome peuvent être responsables de son activation excessive et in fine de la survenue de pathologies auto-inflammatoires chez l’être humain. À l’heure actuelle, aucun modèle cellulaire ne permet d’étudier spécifiquement la dynamique d’activation des inflammasomes et de préciser les conséquences des mutations activatrices sur celles-ci. J’ai donc généré un modèle humain macrophagique exprimant une protéine recombinante FLAG3x-ASC endogène, commune aux différents inflammasomes dans la lignée cellulaire humaine monocytaire/macrophagique THP-1. Cette lignée a été générée par édition génique par la technologie CRISPR-Cas9 en utilisant un substrat de recombinaison permettant d’insérer la séquence codant pour le FLAG3X in frame du locus PYCARD codant pour ASC. J’ai pu générer 6 clones FLAG3x-ASC dans la lignée THP-1. Les clones générés ont été validés en confirmant l’expression et la fonctionnalité de la protéine recombinante FLAG3x-ASC et en vérifiant l’absence de mutations indésirables hors-cible générée par la nucléase Cas9. Une fois ce modèle généré, j’ai pu également reproduire un variant génétique du gène NLRC4, protéine sensor de l’inflammasome du même nom, retrouvé chez un patient présentant une maladie auto-inflammatoire. La validation des clones mutant pour NLRC4 est en cours. Ce projet permettra la caractérisation de la dynamique d’activation de l’inflammasome dans un modèle physiologique et pathologique. Ceci permettra une avancée importante dans la compréhension de l’inflammasome et son agrégation ainsi que la régulation de ce complexe face aux signaux de danger.Inflammasomes are multiproteic complexes that are involved in innate immunity and are activated by multiple signals of dangers. Hereditary mutations in inflammasome components lead to its excessive activation that is responsible for human auto-inflammatory disease. While these mutations are supposed to alter the dynamic of inflammasome activation, there is no current human model allowing the dynamic study of this complex. I generated a human cellular model expressing an endogenous FLAG3x ASC protein, an adaptator protein common to several inflammasomes, in the human monocytic/macrophagic THP-1 cell line. This model was created through CRISPR-Cas9 genome engineering using a recombination template allowing the in frame integration of the sequence encoding the FLAG3X peptide at the PYCARD locus encoding ASC. I generated and validated the expression and the functionality of 6 FLAG3x-ASC THP-1 cell lines. Furthermore, these cell lines are devoided of CRISPR-Cas9 off-target effect. In this model, I further reproduced a genetic variant of the inflammasome component NLRC4 observed in a patient presenting with autoinflammatory manifestation. The functional validation of the FLAG3x-ASC THP-1 harboring the NLRC4 variant is on-going. This project will allow to study the dynamic of the activation of the inflammasome in healthy and pathological conditions. Those results will help refine our comprehension of inflammasome complexation and regulation in response to danger signals

Rapamycin as an Adjunctive Therapy for NLRC4 Associated Macrophage Activation Syndrome

Gain of function (GOF) mutations affecting the inflammasome component NLRC4 are known to cause early-onset macrophage activation syndrome (MAS) and neonatal enterocolitis. Here we report a patient with a NLRC4 GOF mutation presenting with neonatal MAS efficiently treated with a combination of anakinra and rapamycin. Through in vitro studies, we show that rapamycin reduces both IL-1β and IL-18 secretion by the patient's phagocytic cells. The reduction of cytokine secretion is associated with a reduction of caspase-1 activation regardless of the pathogen- or danger-associated molecular patterns triggering the activation of the inflammasome. This study suggests that patients with inherited auto-inflammatory disorders could benefit from an adjunctive therapy with rapamycin

Reconstruction of a polyclonal ADCC antibody repertoire from an HIV-1 non-transmitting mother

Summary: Human natural history and vaccine studies support a protective role of antibody dependent cellular cytotoxicity (ADCC) activity against many infectious diseases. One setting where this has consistently been observed is in HIV-1 vertical transmission, where passively acquired ADCC activity in HIV-exposed infants has correlated with reduced acquisition risk and reduced pathogenesis in HIV+ infants. However, the characteristics of HIV-specific antibodies comprising a maternal plasma ADCC response are not well understood. Here, we reconstructed monoclonal antibodies (mAbs) from memory B cells from late pregnancy in mother MG540, who did not transmit HIV to her infant despite several high-risk factors. Twenty mAbs representing 14 clonal families were reconstructed, which mediated ADCC and recognized multiple HIV Envelope epitopes. In experiments using Fc-defective variants, only combinations of several mAbs accounted for the majority of plasma ADCC of MG540 and her infant. We present these mAbs as evidence of a polyclonal repertoire with potent HIV-directed ADCC activity

Temperature Influences the Interaction between SARS-CoV-2 Spike from Omicron Subvariants and Human ACE2

SARS-CoV-2 continues to infect millions of people worldwide. The subvariants arising from the variant-of-concern (VOC) Omicron include BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4, and BA.5. All possess multiple mutations in their Spike glycoprotein, notably in its immunogenic receptor-binding domain (RBD), and present enhanced viral transmission. The highly mutated Spike glycoproteins from these subvariants present different degrees of resistance to recognition and cross-neutralisation by plasma from previously infected and/or vaccinated individuals. We have recently shown that the temperature affects the interaction between the Spike and its receptor, the angiotensin converting enzyme 2 (ACE2). The affinity of RBD for ACE2 is significantly increased at lower temperatures. However, whether this is also observed with the Spike of Omicron and sub-lineages is not known. Here we show that, similar to other variants, Spikes from Omicron sub-lineages bind better the ACE2 receptor at lower temperatures. Whether this translates into enhanced transmission during the fall and winter seasons remains to be determined

Sustained IFN signaling is associated with delayed development of SARS-CoV-2-specific immunity

Abstract Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized patients with COVID-19. Integrated analysis using k-means reveals four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors segregate into high and low early antibody responders. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4+ T cell frequencies. These data suggest that the “Interferon paradox” previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity