Caractérisation des réponses inflammatoires dans les PBMC grâce au séquençage en cellules uniques

Type I Interferons (IFNs) act as potent initiators of the immune response. Lack of response to IFNs can result in severe viral infections. By contrast, uncontrolled production of type I IFNs leads to chronic inflammation. The cGAS-STING pathway is an important DNA sensing pathway which can trigger a robust immune response by initiating the production of type I IFNs. Gain-of-function mutations in STING1, which codes for the Stimulator of Interferon Gene (STING), induce a constitutive activation of STING and result in a severe autoinflammatory disease termed STING-associated vasculopathy with onset in infancy (SAVI). SAVI is a rare genetic disease which typically includes pulmonary and cutaneous inflammation. Although high IFN production is thought to be the main cause of the symptoms observed in patients, STING can activate other pathways that are cell type specific, and their role in the onset and severity of SAVI remain to be elucidated. To address this question, we have gathered a cohort of 5 SAVI patients and 7 controls and have explored the transcriptome of their peripheral blood mononuclear cells (PBMCs) at the single cell level. This dataset combines samples of patients before and after treatment, which has allowed us to evaluate the effect of the treatment both at cellular and molecular levels. To better understand which molecular characteristics of SAVI are a direct consequence of type I IFNs constitutive production, we generated a dataset of PBMCs treated with IFN-β and profiled them at the single cell level. Comparing the SAVI dataset and the IFN-β-stimulated PBMCs revealed drastic decrease of effector T cells and loss of MAIT cells in SAVI patients. We show that T cells of SAVI patients are in a constant activated state, associated with senescence and apoptosis. We highlight a group of pathologic monocytes in SAVI patients, which are highly inflammatory and express a strong integrated stress response (ISR) signature. Prediction of cell-cell communication suggest that pathogenic monocytes may drive the T cell hyperactivation. These results provide a deeper understanding on the molecular pathways underlying SAVI and could help improve patients' treatment. Additionally, we leverage the use of scRNA-seq data to pinpoint a SAVI patient with low ISR who has a second germline mutation in EIF2AK3, which was recently shown to be activated by STING to trigger the ISR. A second axis of my thesis concerned Multisystem Inflammatory Syndrome in Children (MIS-C). Severe COVID-19 patients present with a pulmonary inflammation and vascular defects reminiscent of what is observed in SAVI. This observation led us to get implicated in research against COVID-19 pandemic. SARS-CoV-2 infection was initially thought to induce only mild disease in children; however, it was soon revealed that it can provoke MIS-C, a severe pediatric post-acute inflammation, a few weeks after initial infection. This rare disease is characterized by multiorgan inflammation. Severe forms are further aggravated by myocarditis, but the mechanisms governing these severe forms are yet to be explored at the cellular level. We gathered a cohort of MIS-C patients with and without myocarditis to explore the mechanisms underlying the pathogenesis of the most severe forms. We used a multi-omics approach and combined proteomics, bulk, and single-cell transcriptomics to analyze the blood immune cells of 56 patients and age-matched controls. This strategy led to the identification of a molecular signature of monocytes and dendritic cells which allows the stratification of patients with severe myocarditis. We highlight an exacerbation of TNF-α signaling due to dampening of NF-kB inhibitors and show that patients with myocarditis have low response to type I IFNs despite presence of IFN-α/β in the blood. Overall, this thesis explores how to leverage single-cell transcriptomics to uncover molecular mechanisms and cell populations involved in inflammatory pediatric diseases.Les interférons (IFN) de type I agissent comme des initiateurs de la réponse immunitaire. L'absence de réponse aux IFN peut entrainer des infections virales sévères. A l'inverse, la production incontrôlée d'IFN entraîne des inflammations chroniques. La voie cGAS-STING joue un rôle important dans la détection des fragments d'ADN cytoplasmiques et déclenche une réponse immunitaire en initiant la production d'IFN. Des mutations gain-de-fonction dans STING1, qui code pour le Stimulator of Interferon Gene (STING), induisent une activation constitutive de STING qui conduit à une maladie auto-inflammatoire sévère appelée STING-associated vasculopathy with onset in infancy (SAVI). C'est une maladie génétique rare caractérisée par une atteinte pulmonaire et cutanée. Bien que la production d'IFN de type I soit considérée comme la cause principale des symptômes observés chez les patients, STING induit aussi des fonctions qui sont spécifiques à certains types cellulaires, et leur rôle dans le SAVI n'a pas encore été adressé. Nous avons collecté une cohorte de 5 patients atteints de SAVI et 7 contrôles, et avons exploré le transcriptome de leurs cellules mononuclées du sang (PBMC) en cellules uniques. Des échantillons avant et après traitement ont aussi été collectés, nous permettant d'évaluer l'effet du traitement aux niveaux cellulaire et moléculaire. Afin de mieux comprendre les caractéristiques du SAVI qui résultent directement de la production constitutive d'IFN de type I, nous avons stimulé des PBMC de donneurs sains avec de l'IFN-β. La comparaison de ces deux jeux de données a révélé une diminution des lymphocytes T effecteurs et des MAIT chez les patients SAVI. Nous montrons que les lymphocytes T des patients sont dans un état constant d'activation précoce, avec senescence et apoptose. Nous mettons en évidence un groupe de monocytes pathogéniques hautement inflammatoires qui expriment une forte signature de la voie de stress eIF2-ATF4 (ISR). La prédiction des communications intercellulaires suggère que ces monocytes sont responsables de l'hyperactivation des lymphocytes T. Ces résultats étendent la compréhension du SAVI et pourraient aider à améliorer les traitements. Nous identifions également un patient présentant une réponse au stress diminuée, et montrons une deuxième mutation germinale dans EIF2AK3, qui est activé par STING pour déclencher l'ISR. Un deuxième axe de ma thèse a porté sur le Multisystem Inflammatory Syndrome in Children (MIS-C). Les patients atteints de COVID-19 sévères présentent des symptômes qui évoquent ceux du SAVI. Cette observation nous a mené à nous impliquer dans la recherche contre la pandémie de SARS-CoV-2. Les infections pédiatriques étaient initialement considérées comme n'entrainant que peu de symptômes. Néanmoins, il a rapidement été montré que le SARS-CoV-2 pouvait entrainer un MIS-C, une inflammation systémique post-aigue se déclenchant quelques semaines après l'infection initiale. Les formes sévères sont compliquées par des myocardites, mais les mécanismes qui gouvernent leur apparition n'ont pas encore été explorés. Nous avons collecté une cohorte de patients MIS-C avec ou sans myocardites, et exploré leurs PBMC grâce à une approche multi-omique combinant des données de protéomique, et de transcriptomique aux niveaux global et unicellulaire. Cette stratégique nous a conduit à identifier, dans les monocytes et cellules dendritiques, une signature moléculaire permettant la stratification des patients avec et sans myocardites. Nous montrons une exacerbation de la voie du TNF-α due à une diminution des inhibiteurs de NF-kB, et montrons que les patients avec myocardites ont une réponse faible à l'IFN de type I malgré la présence d'IFN-α/β dans le sang. Cette thèse explore comment les données de transcriptomes en cellules uniques peuvent être utilisées pour dévoiler des mécanismes moléculaires et des populations cellulaires impliqués dans les maladies pédiatriques inflammatoires

Caractérisation des réponses inflammatoires dans les PBMC grâce au séquençage en cellules uniques

Les interférons (IFN) de type I agissent comme des initiateurs de la réponse immunitaire. L'absence de réponse aux IFN peut entrainer des infections virales sévères. A l'inverse, la production incontrôlée d'IFN entraîne des inflammations chroniques. La voie cGAS-STING joue un rôle important dans la détection des fragments d'ADN cytoplasmiques et déclenche une réponse immunitaire en initiant la production d'IFN. Des mutations gain-de-fonction dans STING1, qui code pour le Stimulator of Interferon Gene (STING), induisent une activation constitutive de STING qui conduit à une maladie auto-inflammatoire sévère appelée STING-associated vasculopathy with onset in infancy (SAVI). C'est une maladie génétique rare caractérisée par une atteinte pulmonaire et cutanée. Bien que la production d'IFN de type I soit considérée comme la cause principale des symptômes observés chez les patients, STING induit aussi des fonctions qui sont spécifiques à certains types cellulaires, et leur rôle dans le SAVI n'a pas encore été adressé. Nous avons collecté une cohorte de 5 patients atteints de SAVI et 7 contrôles, et avons exploré le transcriptome de leurs cellules mononuclées du sang (PBMC) en cellules uniques. Des échantillons avant et après traitement ont aussi été collectés, nous permettant d'évaluer l'effet du traitement aux niveaux cellulaire et moléculaire. Afin de mieux comprendre les caractéristiques du SAVI qui résultent directement de la production constitutive d'IFN de type I, nous avons stimulé des PBMC de donneurs sains avec de l'IFN-β. La comparaison de ces deux jeux de données a révélé une diminution des lymphocytes T effecteurs et des MAIT chez les patients SAVI. Nous montrons que les lymphocytes T des patients sont dans un état constant d'activation précoce, avec senescence et apoptose. Nous mettons en évidence un groupe de monocytes pathogéniques hautement inflammatoires qui expriment une forte signature de la voie de stress eIF2-ATF4 (ISR). La prédiction des communications intercellulaires suggère que ces monocytes sont responsables de l'hyperactivation des lymphocytes T. Ces résultats étendent la compréhension du SAVI et pourraient aider à améliorer les traitements. Nous identifions également un patient présentant une réponse au stress diminuée, et montrons une deuxième mutation germinale dans EIF2AK3, qui est activé par STING pour déclencher l'ISR. Un deuxième axe de ma thèse a porté sur le Multisystem Inflammatory Syndrome in Children (MIS-C). Les patients atteints de COVID-19 sévères présentent des symptômes qui évoquent ceux du SAVI. Cette observation nous a mené à nous impliquer dans la recherche contre la pandémie de SARS-CoV-2. Les infections pédiatriques étaient initialement considérées comme n'entrainant que peu de symptômes. Néanmoins, il a rapidement été montré que le SARS-CoV-2 pouvait entrainer un MIS-C, une inflammation systémique post-aigue se déclenchant quelques semaines après l'infection initiale. Les formes sévères sont compliquées par des myocardites, mais les mécanismes qui gouvernent leur apparition n'ont pas encore été explorés. Nous avons collecté une cohorte de patients MIS-C avec ou sans myocardites, et exploré leurs PBMC grâce à une approche multi-omique combinant des données de protéomique, et de transcriptomique aux niveaux global et unicellulaire. Cette stratégique nous a conduit à identifier, dans les monocytes et cellules dendritiques, une signature moléculaire permettant la stratification des patients avec et sans myocardites. Nous montrons une exacerbation de la voie du TNF-α due à une diminution des inhibiteurs de NF-kB, et montrons que les patients avec myocardites ont une réponse faible à l'IFN de type I malgré la présence d'IFN-α/β dans le sang. Cette thèse explore comment les données de transcriptomes en cellules uniques peuvent être utilisées pour dévoiler des mécanismes moléculaires et des populations cellulaires impliqués dans les maladies pédiatriques inflammatoires.Type I Interferons (IFNs) act as potent initiators of the immune response. Lack of response to IFNs can result in severe viral infections. By contrast, uncontrolled production of type I IFNs leads to chronic inflammation. The cGAS-STING pathway is an important DNA sensing pathway which can trigger a robust immune response by initiating the production of type I IFNs. Gain-of-function mutations in STING1, which codes for the Stimulator of Interferon Gene (STING), induce a constitutive activation of STING and result in a severe autoinflammatory disease termed STING-associated vasculopathy with onset in infancy (SAVI). SAVI is a rare genetic disease which typically includes pulmonary and cutaneous inflammation. Although high IFN production is thought to be the main cause of the symptoms observed in patients, STING can activate other pathways that are cell type specific, and their role in the onset and severity of SAVI remain to be elucidated. To address this question, we have gathered a cohort of 5 SAVI patients and 7 controls and have explored the transcriptome of their peripheral blood mononuclear cells (PBMCs) at the single cell level. This dataset combines samples of patients before and after treatment, which has allowed us to evaluate the effect of the treatment both at cellular and molecular levels. To better understand which molecular characteristics of SAVI are a direct consequence of type I IFNs constitutive production, we generated a dataset of PBMCs treated with IFN-β and profiled them at the single cell level. Comparing the SAVI dataset and the IFN-β-stimulated PBMCs revealed drastic decrease of effector T cells and loss of MAIT cells in SAVI patients. We show that T cells of SAVI patients are in a constant activated state, associated with senescence and apoptosis. We highlight a group of pathologic monocytes in SAVI patients, which are highly inflammatory and express a strong integrated stress response (ISR) signature. Prediction of cell-cell communication suggest that pathogenic monocytes may drive the T cell hyperactivation. These results provide a deeper understanding on the molecular pathways underlying SAVI and could help improve patients' treatment. Additionally, we leverage the use of scRNA-seq data to pinpoint a SAVI patient with low ISR who has a second germline mutation in EIF2AK3, which was recently shown to be activated by STING to trigger the ISR. A second axis of my thesis concerned Multisystem Inflammatory Syndrome in Children (MIS-C). Severe COVID-19 patients present with a pulmonary inflammation and vascular defects reminiscent of what is observed in SAVI. This observation led us to get implicated in research against COVID-19 pandemic. SARS-CoV-2 infection was initially thought to induce only mild disease in children; however, it was soon revealed that it can provoke MIS-C, a severe pediatric post-acute inflammation, a few weeks after initial infection. This rare disease is characterized by multiorgan inflammation. Severe forms are further aggravated by myocarditis, but the mechanisms governing these severe forms are yet to be explored at the cellular level. We gathered a cohort of MIS-C patients with and without myocarditis to explore the mechanisms underlying the pathogenesis of the most severe forms. We used a multi-omics approach and combined proteomics, bulk, and single-cell transcriptomics to analyze the blood immune cells of 56 patients and age-matched controls. This strategy led to the identification of a molecular signature of monocytes and dendritic cells which allows the stratification of patients with severe myocarditis. We highlight an exacerbation of TNF-α signaling due to dampening of NF-kB inhibitors and show that patients with myocarditis have low response to type I IFNs despite presence of IFN-α/β in the blood. Overall, this thesis explores how to leverage single-cell transcriptomics to uncover molecular mechanisms and cell populations involved in inflammatory pediatric diseases

Dissecting human population variation in single-cell responses to SARS-CoV-2

International audienceHumans display substantial interindividual clinical variability after SARS-CoV-2 infection 1–3 , the genetic and immunological basis of which has begun to be deciphered 4 . However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells—from 222 healthy donors of diverse ancestries—that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk

Environmental and genetic drivers of population differences in SARS-CoV-2 immune responses

The RNA sequencing data generated and analyzed in this study have been deposited in the Institut Pasteur data repository, OWEY, which can be accessed via the following link: https://doi.org/XXXX. The genome-wide genotyping data generated or used in this study have been deposited in OWEY and can be accessed at the following URL: https://doi.org/XXXX. Data access and use is restricted to academic research related to the variability of the human immune response.Humans display vast clinical variability upon SARS-CoV-2 infection 1–3 , partly due to genetic and immunological factors 4 . However, the magnitude of population differences in immune responses to SARS-CoV-2 and the mechanisms underlying such variation remain unknown. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells from 222 healthy donors of various ancestries stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces a weaker, but more heterogeneous interferon-stimulated gene activity than influenza A virus, and a unique pro-inflammatory signature in myeloid cells. We observe marked population differences in transcriptional responses to viral exposure that reflect environmentally induced cellular heterogeneity, as illustrated by higher rates of cytomegalovirus infection, affecting lymphoid cells, in African-descent individuals. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell proportions on population differences in immune responses, with genetic variants having a narrower but stronger effect on specific loci. Additionally, natural selection has increased immune response differentiation across populations, particularly for variants associated with SARS-CoV-2 responses in East Asians. We document the cellular and molecular mechanisms through which Neanderthal introgression has altered immune functions, such as its impact on the myeloid response in Europeans. Finally, colocalization analyses reveal an overlap between the genetic architecture of immune responses to SARS-CoV-2 and COVID-19 severity. Collectively, these findings suggest that adaptive evolution targeting immunity has also contributed to current disparities in COVID-19 risk

Single-cell RNA-sequencing of PBMCs from SAVI patients reveals disease-associated monocytes with elevated integrated stress response

International audienceGain-of-function mutations in stimulator of interferon gene 1 (STING1) result in STING-associated vasculopathy with onset in infancy (SAVI), a severe autoinflammatory disease. Although elevated type I interferon (IFN) production is thought to be the leading cause of the symptoms observed in patients, STING can induce a set of pathways, which have roles in the onset and severity of SAVI and remain to be elucidated. To this end, we performed a multi-omics comparative analysis of peripheral blood mononuclear cells (PBMCs) and plasma from SAVI patients and healthy controls, combined with a dataset of healthy PBMCs treated with IFN-β. Our data reveal a subset of disease-associated monocyte, expressing elevated CCL3, CCL4, and IL-6, as well as a strong integrated stress response, which we suggest is the result of direct PERK activation by STING. Cell-to-cell communication inference indicates that these monocytes lead to T cell early activation, resulting in their senescence and apoptosis. Last, we propose a transcriptomic signature of STING activation, independent of type I IFN response

NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells

Abstract Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease

A monocyte/dendritic cell molecular signature of SARS-CoV-2-related multisystem inflammatory syndrome in children with severe myocarditis

International audienceBackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is generally milder than in adults, but a proportion of cases result in hyperinflammatory conditions often including myocarditis.MethodsTo better understand these cases, we applied a multiparametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. Plasma cytokine and chemokine levels and blood cellular composition were measured, alongside gene expression at the bulk and single-cell levels.FindingsThe most severe forms of multisystem inflammatory syndrome in children (MIS-C) related to SARS-CoV-2 that resulted in myocarditis were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomics analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis characterized by sustained nuclear factor κB (NF-κB) activity and tumor necrosis factor alpha (TNF-α) signaling and associated with decreased gene expression of NF-κB inhibitors. We also found a weak response to type I and type II interferons, hyperinflammation, and response to oxidative stress related to increased HIF-1α and Vascular endothelial growth factor (VEGF) signaling.ConclusionsThese results provide potential for a better understanding of disease pathophysiology

A monocyte/dendritic cell molecular signature of SARS-CoV2-related multisystem inflammatory syndrome in children (MIS-C) with severe myocarditis

Posté sur bioRxiv le 23/02/2021SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-κB activity, TNF-α signaling, associated with decreased gene expression of NF-κB inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1α and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology