The innate immune system is the first line of host defense against external exposures. During these initial encounters, antigen presenting cells - specifically monocytes and macrophages - modulate further inflammatory responses. Macrophages exist along a spectrum of phenotypic programs; on the inflammatory M1 end they enhance immune activity while on the anti-inflammatory M2 end they suppress further immune activation. Furthermore, within M2 macrophages there exist many subpopulations, namely M2a and M2d, each with specific roles during infection or exposure. We sought to compare the epigenetic profiles of these subpopulations of macrophages to determine key regulatory gene networks and factors that could be exploited for therapeutic benefit.While traditionally viewed as primitive and nonspecific, a growing body of clinical and experimental evidence argues the innate immune system develops memory as a result of previous exposures, allowing the innate system to respond with enhanced and broad immunological protection upon exposure to a secondary stimulus. This biological process of innate immunity has been termed trained immunity. Trained immunity shares many phenotypic and epigenetic characteristics with adaptive immune memory; however, one of the starkest distinctions is the propensity of trained immunity to develop against heterologous stimuli. Innate memory is not antigen specific, frequently protecting the host against unrelated organisms.

In the version of this article initially published, author Raphael Duivenvoorden’s last name was spelt incorrectly as Duivenwoorden. The error has been corrected in the HTML and PDF versions of the article.

Microglia, the innate immune cells of the central nervous system, feature adaptive immune memory with implications for brain homeostasis and pathologies. However, factors involved in the emergence and regulation of these opposing responses in microglia have not been fully addressed. Recently, we showed that microglia from the newborn brain display features of trained immunity and immune tolerance after repeated contact with pathogens in a dose-dependent manner. Here, we evaluate the impact of developmental stage on adaptive immune responses of brain microglia after repeated challenge with ultra-low (1 fg/ml) and high (100 ng/ml) doses of the endotoxin LPS in vitro. We find that priming of naïve microglia derived from newborn but not mature and aged murine brain with ultra-low LPS significantly increased levels of pro-inflammatory mediators TNF-α, IL-6, IL-1β, MMP-9, and iNOS as well as neurotrophic factors indicating induction of trained immunity (p < 0.05). In contrast, stimulation with high doses of LPS led to a robust downregulation of pro-inflammatory cytokines and iNOS independent of the developmental state, indicating induced immune tolerance. Furthermore, high-dose priming with LPS upregulated anti-inflammatory mediators IL-10, Arg-1, TGF- β, MSR1, and IL-4 in newborn microglia (p < 0.05). Our data indicate pronounced plasticity of the immune response of neonate microglia compared with microglia derived from mature and aged mouse brain. Induced trained immunity after priming with ultra-low LPS doses may be responsible for enhanced neuro-inflammatory susceptibility of immature brain. In contrast, the immunosuppressed phenotype following high-dose LPS priming might be prone to attenuate excessive damage after recurrent systemic inflammation.

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

Le clade des invertébrés cristallise en 2017 de grandes problématiques sociétales à la fois économiques et sanitaires. En effet un certain nombre des organismes présent dans ce groupe phylétique sont des vecteurs des grandes pandémies infectieuses telles que le paludisme (Anopheles sp), Zika, Chinkungunya, Fièvre jaune, etc (Aedes sp), Chagas (Triatoma sp) ou encore la bilharziose (Biomphalaria sp, Bulinus sp). La compréhension du système immunitaire de ces organismes vecteurs doit aider la communauté scientifique à proposer des solutions pour réduire la transmission de toutes ces maladies sur le terrain. Biomphalaria glabrata est le vecteur unique de la Bilharziose intestinale (Schistosomamansoni) en Amérique Latine. Depuis un premier cas de résistance induite par une première infection en 1998, de nombreux travaux ont exploré la réponse immunitaire mémoire innée de cet escargot tropical d’eau douce. Dans le cadre de ce travail de thèse, différents aspects de cette immunité (également appelé priming, résistance acquise) ont été explorés, de la mise en place phénotypiques, aux bases moléculaires et cellulaires. En premier lieu,nous avons pu démontrer qu’elle était dépendante d’une bascule phénotypique (d’une réponse cellulaire d’encapsulation à une réponse humorale) et transcriptomique qui lui permet de mieux répondre lors d’une seconde infection. La spécificité de cette réponse est portée par la production de répertoire complexe de récepteurs et d’effecteurs immunitaire spécifiques qui sont capables de différencier jusqu’aux différents stades de développement parasitaire d’une même espèce de parasite. Nous avons également pu montrer que cette interaction dépendait de microARN circulants ainsi que de Biomphalysines, des ß-PFT acquises par transferts horizontaux depuis le monde bactérien. Enfin, cette résistance semble posséder une proximité avec l’immunité mémoire entraînée des cellules immunitaires innées des vertébrés en particulier sur la base des mécanismes moléculaires sous-jacent qui seraient liés chez Biomphalaria comme chez les Vertébrés à unereprogrammation épigénétique des cellules du système immunitaire innée. / Invertebrates focus in 2017 among the major economical and societal issuesacross Earth. Some members are vectors of important infectious pandemic as malaria(Anopheles sp), Zika, Chinkungunya, Yellow fever, etc (Aedes sp), Chagas (Triatoma sp) andtrematodes (Biomphalaria sp, Bulinus sp). Comprehension of immune system of thesevectors has to help scientist to decrease transmission on endemic area. Biomphalariaexposed first failure to be reinfected following first infection as soon as 1998. In my thesiswe explore this immune priming (innate immune memory) and describe an immune shiftfrom cellular to humoral immune response both in phenotype and transcriptomic response.A specificity is handle by specific immune receptor and effector repertoire to distinguish upto different developmental stage of same parasite species. This interaction is alsodependent of mRNAs and Biomphalysin, a ß-PFT coming from bacterial kingdom. Finally,this resistance seems to look alike the trained immune memory of innate cells in vertebrates.

Malarial infection in naïve individuals induces a robust innate immune response, but our understanding of the mechanisms by which the innate immune system recognizes malaria and regulates its response remain incomplete. Our group previously showed that stimulation of macrophages with Plasmodium falciparum genomic DNA (gDNA) and AT-rich oligodeoxynucleotides (ODNs) derived from this gDNA induces the production of type I interferons (IFN-I) through a STING/TBK1/IRF3-dependent pathway; however, the identity of the upstream cytosolic DNA receptor remained elusive. Here, we demonstrate that this IFN-I response is dependent on cyclic GMP-AMP synthase (cGAS). cGAS produced the cyclic dinucleotide 2’3’-cGAMP in response to P. falciparum gDNA and AT-rich ODNs, inducing IRF3 phosphorylation and IFNB transcription. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed “training”) or hyporespond (“tolerance”) to subsequent immune challenge. Previous work in mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with P. falciparum-infected red blood cells (iRBCs) or the malaria crystal hemozoin (Hz) induced human adherent peripheral blood mononuclear cells (PBMCs) to hyperrespond to subsequent Toll-like receptor (TLR) challenge. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in monocytes from Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that malaria-induced trained immunity may occur via previously unrecognized mechanism(s).

The global population is rapidly aging. It is estimated that over the next thirty years, the number of individuals >60 years of age will increase by over a billion, and the number of individuals over age 80 may increase by 300 million. As humans age, our immune system becomes progressively weaker through a process called immune senescence. This age-related decrease in immune function increases susceptibility to infection and chronic diseases. Sepsis is a leading cause of death worldwide. Over the past two decades, there has been an increased incidence of sepsis which is due, in part, to our aging population and immune senescence. The gut microbiome, which plays an essential role in health and disease, is altered in aging and sepsis. Specifically, the commensal microorganisms of the gut microbiota are replaced with potentially pathogenic bacteria. This contributes to immune dysfunction and worsened outcomes in critical illness. The innate immune system can be “trained” to respond more effectively to pathogens. We examined trained immunity as an approach to modulating immunosenescence and microbiome diversity in aging. We investigated the effect of trained immunity on: i) immune cells from healthy aging subjects and sepsis patients and ii) the diversity of the microbiome in aging and sepsis. Our results indicate that trained immunity is effective in combatting age-related immunosenescence. We found that β-glucan induced trained immunity enhances monocyte metabolism, increases functionality as well as alters the transcriptome and epigenome in aging individuals and sepsis patients. We also found that trained immunity induced the expansion of a unique population of myeloid cells in sepsis. These cells are defined as FSChi, CD11b+, GR-1hi and express high levels of immunosuppressive PD-L1. In addition, we found that trained immunity reversed age-related changes to the microbiome and prevented alterations to the microbiome in septic mice. We found that the Firmicutes/Bacteroidetes ratio increased in aging; however, trained immunity reversed this increase and increased Clostridia in aged mice. In sepsis, trained immunity prevented expansion of Proteobacteria observed in control mice. Thus, our results indicate that trained immunity may be effective in modulating immune senescence and the microbiome in aging and sepsis.

Chronic kidney disease (CKD) affects 13.4% of adults in America; and 38% in people aged 65 years or older[1]. In addition, cardiovascular disease (CVD) is the leading cause of death in CKD patients with end-stage kidney disease. CKD is associated with chronic inflammation, which contributes to the progression of CVD[2]. Furthermore, CKD alter apolipoprotein profile and elevate plasma lipid levels. It has been reported that 68.8% of CKD patients are associated with hyperlipidemia[3]. Therefore, hyperlipidemia is the critical risk factor for cardiovascular morbidity and mortality in CKD patients [4, 5]. In addition, trained immunity has been shown to play a critical role in chronic inflammatory diseases[6]. However, whether trained immunity promotes the inflammation in hyperlipidemia-CKD remains unclear. Circulating lipopolysaccharide (LPS) is significantly increased in atherosclerotic and CKD patients[7]. Clinical data indicates that circulating LPS is positively associated with the progression of CKD, and its levels even higher in patients with hemodialysis or dialysis[8]. Studies found that circulating LPS is delivered into cytosol for caspase-4/11 activation[9]. The Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) involving 10,061 patients found that targeting interleukin-1β (IL-1β) innate immunity pathway is significantly lowered the rate of recurrent cardiovascular events independent of lipid-level lowering[10]. Therefore, inhibiting the secretion of proinflammatory cytokine IL-1β has high potential to future development of novel therapeutics for hyperlipidemia-CKD accelerated CVD. Gasdermin D (GSDMD) is cleaved by inflammatory caspase-1 and caspase-4. N-terminal GSDMD binds to plasma membrane forming protein channel [11] and mediates the secretion of IL-1β[12, 13]. We found that caspase-1 activation was significantly decreased in caspase-4/11 deficient high-fat diet (HFD)-CKD mice, indicating that caspase-4 could regulate caspase-1 activation in HFD-CKD. Whether increased cytosolic LPS contribute to the increased vascular inflammation via caspase-4/11-GSDMD-IL-1β pathway remains unknown. In this study, we used HFD fed 5/6 nephrectomy CKD mice in vivo and cytosolic LPS stimulation in human aortic endothelial cell (HAECs) in vitro. We made the following results: 1) Inflammatory pathways are significantly increased in the aorta of HFD-CKD compared to HFD-Sham, normal diet (ND)-CKD, and ND-Sham. 2) Expression levels of endothelial cell activation markers (ICAM1 and VCAM1) are significantly increased in the aorta of HFD-CKD mice compared to HFD-Sham, ND-CKD, and ND-Sham. 3) Caspase-4 activation and N-GSDMD cleavage are significantly increased in the aorta of HFD-CKD mice compared to HFD-Sham, ND-CKD, and ND-Sham and in cytosolic LPS stimulated HAECs. 4) The increased inflammatory pathways and increased expression of adhesion molecules are decreased in the deficiency of caspase-4 in vivo and in the presence of caspase-4 inhibitor and N-terminal GSDMD cleavage inhibitor in vitro. 5) The increased mitochondrial ROS promote endothelial cell activation via caspase-4-GSDMD axis. Taken together, the caspase-4/11-GSDMD axis mediates endothelial cell activation and vascular inflammation in the aorta of HFD-CKD mice compared to controls. Furthermore, the increased endothelial cell activation and vascular inflammation are restored by caspase-4/11 deficiency in the aorta of HFD-CKD mice. These evidence indicate that inhibiting caspase-4/11-GSDMD axis could be a potential therapeutic target for inhibiting vascular inflammation associated with hyperlipidemia-CKD. / Biomedical Sciences

Les ß-glucanes (BG) sont les polysaccharides les plus abondants de la paroi de Saccharomyces cerevisiae. Depuis des millénaires, ils sont utilisés pour leurs propriétés immunostimulantes et leurs potentiels thérapeutiques. L'objectif de ce travail était de caractériser la réponse immunitaire induite par les BG et de comprendre leurs modes d'action sur les macrophages murins en contexte infectieux. Nous avons montré que (i) les extraits de paroi enrichis en BG n'induisent qu'une faible production de cytokines par les macrophages contrairement aux extraits bruts, (ii) la réponse inflammatoire médiée par les extraits bruts résulte de la signalisation des TLRs et non de Dectin-1 et (iii) les BG stimulent la synthèse tardive de GM-CSF via Dectin-1. En conditions infectieuses, les BG enrichis confèrent une forte signature inflammatoire aux macrophages prétraités conduisant à l'amplification de la production cytokinique, à la synthèse de ROS et l'optimisation de la clairance bactérienne. En conclusion, cette étude souligne les enjeux de l'utilisation des BG enrichis comme adjuvants dans l'amélioration de la résistance des individus aux infections. / ß-glucans (BG) are the most abundant polysaccharides of the Saccharomyces cerevisiae cell wall. For decades, they have been extensively used because of their immuno-modulatory properties and their potential therapeutic effects. The aim of this study was to characterize the immune response induced by BG and to understand their mechanisms of action on murine macrophages occurring upon bacterial infections. We demonstrated that (i) BG-enriched extracts trigger low amounts of cytokine production in contrast with crude products, (ii) the immune response mediated by crude extracts results from TLRs and not from Dectin-1 signaling and (iii) BG-enriched compounds stimulate the late and strong induction of GM-CSF in a Dectin-1-dependent manner. Upon bacteria exposure, BG-enriched extracts confer a strong inflammatory to pretreated macrophages leading to synergistic increase of cytokine release, ROS production and better clearance of pathogens. Altogether, our findings emphasize the relevancy of using BG-enriched extracts for the design of novel adjuvant formulations contributing to individuals' resistance to infections.

La vaccination est l'un des plus grands progrès réalisés en santé publique. Toutefois, malgré de nombreuses connaissances sur le système immunitaire, de nombreux pans d’ombre empêchent la conception de vaccins contre des pathogènes complexes. Pour pallier ce problème, une meilleure compréhension des modes d'action des vaccins est requise. En particulier, la plupart des vaccins nécessitent plusieurs immunisations pour induire une mémoire immunitaire adaptative au long terme, mais l'impact du délai entre primo-vaccination, induisant une mémoire primaire, et rappel(s) la restimulant pour générer une mémoire secondaire, est peu défini. De plus, la réponse immunitaire innée, induite à chaque immunisation et façonnant l'immunité adaptative, reste peu caractérisée dans ce contexte vaccinal. En vaccinant des macaques cynomolgus avec le virus de la vaccine modifiée Ankara, selon un schéma de primo-vaccination suivie d’un rappel homologue à deux mois, et en utilisant la cytométrie de masse couplée à des analyses bio-informatiques, nous avons caractérisé la réponse innée induite par chaque immunisation. Les réponses innées diffèrent entre primo-vaccination et rappel, avec induction par la primo-vaccination d’une modification phénotypique tardive des cellules innées, suggérant une meilleure capacité à répondre au rappel. De surcroît, la réduction à deux semaines du délai entre primo-vaccination et rappel abroge la mobilisation de ces cellules innées phénotypiquement modifiées et altère la qualité de la réponse humorale. En définitive, en plus de la réponse innée précoce, ce projet a mis en évidence l'induction par la primo-vaccination d'un vraisemblable entraînement inné tardif, un concept émergent traduisant la capacité de mémorisation des cellules innées via des modifications épigénétiques. Ce vraisemblable entraînement, non seulement des monocytes et cellules tueuses naturelles, mais aussi des cellules dendritiques et surprenamment des neutrophiles, est corrélé à la qualité de la mémoire immunitaire adaptative, de manière hautement dépendante du délai entre primo-vaccination et rappel. Ces résultats contribuent à ouvrir la voie vers l’optimisation rationnelle des futurs vaccins, via l'optimisation des calendriers vaccinaux et la valorisation de l'entraînement inné. / Vaccination is one of the best achievements made in public health. However, designing vaccines against complex pathogens is currently challenging. The immune system is indeed uncompletely characterized, despite large amount of accumulated knowledges. A better understanding of vaccine-induced immunity is then required to optimize vaccine design. In particular, while most vaccines require several immunizations to induce a long-lasting adaptive immune memory, little is known on the impact of the delay beween the prime inducing a primary memory and the boost restimulating it to induce a secondary memory. Also, the innate immunity induced by each immunization and shaping the adaptative immunity is poorly characterized in this vaccine context.We studied the innate immune responses in cynomolgus macaques immunized with the modified vaccinia virus Ankara, following an homologous prime-boost vaccination at two months apart. We applied mass cytometry and bioinformatic analyses to characterize the innate response induced by each immunization. We showed that prime and boost vaccination triggered distinct innate responses. Actually, prime induced late phenotypic modifications of innate cells. These phenotypic changes suggest a stronger ability to react to the boost. Moreover, reducing the delay between prime and boost to two weeks impeded the mobilization of these phenotypically modified innate cells, and qualitatively altered humoral response.In conclusion, beyond the early innate responses, these results highlight the late induction by the prime of "likely trained" innate cells. This emerging concept corresponds to the ability of innate cells to display memory features based on epigenetic modifications. This "likely training" occured not only on monocytes and NK cells, but also on dendritic cells and strikingly on neutrophils. It was deeply connected with adaptive immune memory establishment, in a prime-boost delay dependant fashion. These findings contribute to pave the way towards to the rationale design of future vaccines, via vaccine schedule optimization and harnessment of innate training.