Genome-Wide and Phenotypic Evaluation of Stem Cell Progenitors Derived From Gprc5a-Deficient Murine Lung Adenocarcinoma With Somatic Kras Mutations

Lung adenocarcinomas (LUADs) with somatic mutations in the KRAS oncogene comprise the most common molecular subtype of lung cancer in smokers and present with overall dismal prognosis and resistance to most therapies. Our group recently demonstrated that tobacco carcinogen-exposed mice with knockout of the airway lineage G-protein coupled receptor, Gprc5a, develop LUADs with somatic mutations in Kras. Earlier work has suggested that cancer stem cells (CSCs) play crucial roles in clonal evolution of tumors and in therapy resistance. To date, our understanding of CSCs in LUADs with somatic Kras mutations remains lagging. Here we derived CSCs (as spheres in 3D cultures) with self-renewal properties from a murine Kras-mutant LUAD cell line we previously established from a tobacco carcinogen-exposed Gprc5a−/− mouse. Using syngeneic Gprc5a−/− models, we found that these CSCs, compared to their parental isoforms, exhibited increased tumorigenic potential in vivo, particularly in female animals. Using whole-transcriptome sequencing coupled with pathways analysis and confirmatory PCR, we identified gene features (n = 2,600) differentially expressed in the CSCs compared to parental cells and that were enriched with functional modules associated with an augmented malignant phenotype including stemness, tumor-promoting inflammation and anti-oxidant responses. Further, based on in silico predicted activation of GSK3β in CSCs, we found that tideglusib, an irreversible inhibitor of the kinase, exhibited marked anti-growth effects in the cultured CSCs. Our study underscores molecular cues in the pathogenesis of Kras-mutant LUAD and presents new models to study the evolution, and thus high-potential targets, of this aggressive malignancy

Study of IL-17-producing gamma-delta T cells in the context of respiratory pneumococcal infection

Le développement de la réponse immunitaire innée de l’hôte au cours des infections respiratoires nécessite la mise en place rapide d'un réseau moléculaire et cellulaire relativement complexe ayant pour but de contrôler la croissance microbienne ainsi que permettre son éradication. Dans certaines circonstances, et malgré l’existence de vaccins et d'antibiotiques efficaces, l’infection par Streptococcus pneumoniae peut aboutir à des pathologies graves telles qu'une pneumonie, une méningite et/ou une septicémie. Ainsi, à l'heure actuelle, les maladies associées au pneumocoque sont encore loin d'être sous contrôle. Dans ce contexte, une meilleure compréhension de la réponse immunitaire innée de l’hôte contre ce pathogène est nécessaire.Mes travaux de thèse ont permis pour la première fois de mettre en évidence la fonctionnalité et la relevance biologique de l’inflammasome NLRP3 au sein des neutrophiles pulmonaires in vivo dans un modèle d’infection respiratoire par S. pneumoniae.Ainsi, de façon inattendue, les neutrophiles jouent un rôle accessoire original à des temps précoces de l’infection via leur capacité à produire de l’IL-1β. Cette synthèse protéique est possible grâce à la combinaison de 2 signaux à la fois dérivés de l’hôte (TNF-α des macrophages alvéolaires) et de la bactérie (toxine). Ces deux signaux permettent l’assemblage et l’activation de l’inflammasome NLRP3 neutrophilique. D’un point de vue translationnel, nous avons été capables de démontrer un mécanisme similaire avec des neutrophiles humains.Cette production d’IL-1β par les neutrophiles participe à l’activation des lymphocytes T γδ producteurs d’IL-17; une cytokine essentielle dans le contrôle de l’infection bactérienne via sa capacité à induire rapidement le recrutement d’une deuxième vague de neutrophiles participant directement à l’élimination et la clairance bactérienne.Sur la base de ces travaux fondamentaux, nous avons émis l’hypothèse qu’une augmentation du pool de cellules innées sécrétrices d’IL-17A pourrait avoir un effet bénéfique sur le contrôle d’une infection respiratoire à pneumocoque. Ainsi via l’administration prophylactique et locale d’IL-7, nous avons été capables d’augmenter la fréquence et le nombre de lymphocytes innés producteurs d’IL-17A résultant en un meilleur contrôle de la charge bactérienne pathogène associée à une augmentation du recrutement neutrophilique. A ce stade, ces résultats encourageants, nous pousse à mieux comprendre les mécanismes moléculaires et cellulaires associés à cet effet dans l’éventualité de proposer à terme une nouvelle approche thérapeutique dans le contrôle des infections respiratoires pulmonaires basée sur la manipulation de la biologie de l’IL-7.The mounting of an appropriate host innate immune response in the lungs requires the rapid establishment of a complex cellular and molecular networking that allows the containment and clearance of pathogens during respiratory infections. Both neutrophils and γδT cells are central players in the host response during mucosal infections. Using a model of invasive pneumococcal disease, we illustrated a role for Interleukin-17A in controlling neutrophil recruitment, bacterial loads and survival. Following Streptococcus pneumoniae infection, we defined pulmonary γδT cells, especially the lung resident Vγ6Vδ1+ subset, as the primary source of IL-17A in an IL-23/IL- 1β-dependent manner. Using gene-targeted mice, we demonstrated that γδT cells largely contributed to neutrophilia and to the control of the pathology. Furthermore, we now defined a second and unexpected early role for neutrophils as accessory cells in γδT17 cell activation through IL-1β secretion. Neutrophil-derived IL-1β was dependent on NLRP3 inflammasome activity and required alveolar macrophage-secreted TNF-α for priming and bacterial pneumolysin for NLRP3- dependent caspase-1 activation. This report thus brings to light the sequential molecular/cellular events leading to γδT17 cell activation and highlights the existence of a biologically relevant and fully functional NLRP3 inflammasome in pulmonary neutrophils that regulates a key immune axis in the development of protective innate response to respiratory bacterial infection.Based on these observations, we hypothesized that an increase in the pool of IL-17A-producing innate-like T lymphocytes might play a protective role during pneumococcal infection. As recently suggested, we demonstrated that intranasal IL-7/M25 complex administration into naïve mice allowed the expansion of the cellular pool of innate immune cells presenting a Th17-like phenotype in the lungs especially T cells. Moreover, we showed during S. pneumoniae infection that prophylactic IL-7/M25 treatment increased the capacity of Vγ6Vδ1+ T cells to produce IL-17A. Interestingly, this phenotype led to higher neutrophil recruitment and a better control of bacterial burden in the lungs as well as systemic dissemination. Thus, we report a critical role of IL-7 in creating an IL-17-enriched microenvironment which improves the early development of host innate immune response to respiratory bacterial infection. This observation might pave the way to the development of future innovative cytokine/cell-based strategies against Streptococcus pneumoniae

Etude des lymphocytes T gamma-delta producteurs d'interleukine-17 au cours des infections respiratoires

The mounting of an appropriate host innate immune response in the lungs requires the rapid establishment of a complex cellular and molecular networking that allows the containment and clearance of pathogens during respiratory infections. Both neutrophils and γδT cells are central players in the host response during mucosal infections. Using a model of invasive pneumococcal disease, we illustrated a role for Interleukin-17A in controlling neutrophil recruitment, bacterial loads and survival. Following Streptococcus pneumoniae infection, we defined pulmonary γδT cells, especially the lung resident Vγ6Vδ1+ subset, as the primary source of IL-17A in an IL-23/IL- 1β-dependent manner. Using gene-targeted mice, we demonstrated that γδT cells largely contributed to neutrophilia and to the control of the pathology. Furthermore, we now defined a second and unexpected early role for neutrophils as accessory cells in γδT17 cell activation through IL-1β secretion. Neutrophil-derived IL-1β was dependent on NLRP3 inflammasome activity and required alveolar macrophage-secreted TNF-α for priming and bacterial pneumolysin for NLRP3- dependent caspase-1 activation. This report thus brings to light the sequential molecular/cellular events leading to γδT17 cell activation and highlights the existence of a biologically relevant and fully functional NLRP3 inflammasome in pulmonary neutrophils that regulates a key immune axis in the development of protective innate response to respiratory bacterial infection.Based on these observations, we hypothesized that an increase in the pool of IL-17A-producing innate-like T lymphocytes might play a protective role during pneumococcal infection. As recently suggested, we demonstrated that intranasal IL-7/M25 complex administration into naïve mice allowed the expansion of the cellular pool of innate immune cells presenting a Th17-like phenotype in the lungs especially T cells. Moreover, we showed during S. pneumoniae infection that prophylactic IL-7/M25 treatment increased the capacity of Vγ6Vδ1+ T cells to produce IL-17A. Interestingly, this phenotype led to higher neutrophil recruitment and a better control of bacterial burden in the lungs as well as systemic dissemination. Thus, we report a critical role of IL-7 in creating an IL-17-enriched microenvironment which improves the early development of host innate immune response to respiratory bacterial infection. This observation might pave the way to the development of future innovative cytokine/cell-based strategies against Streptococcus pneumoniae.Le développement de la réponse immunitaire innée de l’hôte au cours des infections respiratoires nécessite la mise en place rapide d'un réseau moléculaire et cellulaire relativement complexe ayant pour but de contrôler la croissance microbienne ainsi que permettre son éradication. Dans certaines circonstances, et malgré l’existence de vaccins et d'antibiotiques efficaces, l’infection par Streptococcus pneumoniae peut aboutir à des pathologies graves telles qu'une pneumonie, une méningite et/ou une septicémie. Ainsi, à l'heure actuelle, les maladies associées au pneumocoque sont encore loin d'être sous contrôle. Dans ce contexte, une meilleure compréhension de la réponse immunitaire innée de l’hôte contre ce pathogène est nécessaire.Mes travaux de thèse ont permis pour la première fois de mettre en évidence la fonctionnalité et la relevance biologique de l’inflammasome NLRP3 au sein des neutrophiles pulmonaires in vivo dans un modèle d’infection respiratoire par S. pneumoniae.Ainsi, de façon inattendue, les neutrophiles jouent un rôle accessoire original à des temps précoces de l’infection via leur capacité à produire de l’IL-1β. Cette synthèse protéique est possible grâce à la combinaison de 2 signaux à la fois dérivés de l’hôte (TNF-α des macrophages alvéolaires) et de la bactérie (toxine). Ces deux signaux permettent l’assemblage et l’activation de l’inflammasome NLRP3 neutrophilique. D’un point de vue translationnel, nous avons été capables de démontrer un mécanisme similaire avec des neutrophiles humains.Cette production d’IL-1β par les neutrophiles participe à l’activation des lymphocytes T γδ producteurs d’IL-17; une cytokine essentielle dans le contrôle de l’infection bactérienne via sa capacité à induire rapidement le recrutement d’une deuxième vague de neutrophiles participant directement à l’élimination et la clairance bactérienne.Sur la base de ces travaux fondamentaux, nous avons émis l’hypothèse qu’une augmentation du pool de cellules innées sécrétrices d’IL-17A pourrait avoir un effet bénéfique sur le contrôle d’une infection respiratoire à pneumocoque. Ainsi via l’administration prophylactique et locale d’IL-7, nous avons été capables d’augmenter la fréquence et le nombre de lymphocytes innés producteurs d’IL-17A résultant en un meilleur contrôle de la charge bactérienne pathogène associée à une augmentation du recrutement neutrophilique. A ce stade, ces résultats encourageants, nous pousse à mieux comprendre les mécanismes moléculaires et cellulaires associés à cet effet dans l’éventualité de proposer à terme une nouvelle approche thérapeutique dans le contrôle des infections respiratoires pulmonaires basée sur la manipulation de la biologie de l’IL-7

Thymic Program Directing the Functional Development of γδT17 Cells

International audienceγδT cells comprise a unique T cell sublineage endowed with a wide functional repertoire, which allow them to play important-sometimes opposite-roles in many immune responses associated with infection, cancer, and inflammatory processes. This is largely dependent on the existence of pre-programmed discrete functional subsets that differentiate within the thymus at specific temporal windows of life. Since they represent a major early source of interleukin-17A in many models of immune responses, the γδT17 cell population has recently gained considerable interest. Thus, a better dissection of the developmental program of this effector γδT subset appears critical in understanding their associated immune functions. Several recent reports have provided new exciting insights into the developmental mechanisms that control γδT cell lineage commitment and differentiation. Here, we review the importance of thymic cues and intrinsic factors that shape the developmental program of γδT17 cells. We also discuss the potential future areas of research in γδT17 cell development especially in regards to the recently provided data from deep RNA sequencing technology. Pursuing our understanding into this complex mechanism will undoubtedly provide important clues into the biology of this particular T cell sublineage

Type I IFN Receptor Signaling Controls IL7-Dependent Accumulation and Activity of Protumoral IL17A-Producing γδT Cells in Breast Cancer

International audienceThe protumoral activity of γδT17 cells has recently emerged in a wide variety of solid malignancies, including breast cancer. These cells exert their detrimental functions by promoting tumor growth, angiogenesis, and subsequent metastasis development. However, the intratumoral factors that regulate the biology of γδT17cells within the tumor microenvironment are less well understood. Here, using two experimental models of breast cancer, we reinforced the concept that tumor-infiltrating γδT17 cells are endowed with protumoral functions, which promote tumor progression and metastasis development. More importantly, we demonstrated a critical role for type I IFN signaling in controlling the preferential accumulation in the tumor bed of a peculiar subset of γδT17 cells displaying a CD27- CD3bright phenotype (previously associated with the invariant Vγ6Vδ1+ TCR). Interestingly, this effect was indirect and partially relied on the IFNAR1-dependent control of IL7 secretion, a factor that triggers proliferation and activating functions of deleterious γδT17 cells. Our work therefore identifies a key role of the type I IFN/IL7 axis in the regulation of intratumoral γδT17-cell functions and in the development of primary breast tumor growth and metastasis.Significance: Tumor-derived IL7 can represent a therapeutic target to prevent accumulation of immune cells endowed with potent protumoral activities

Exogenous Activation of Invariant Natural Killer T Cells by α-Galactosylceramide Reduces Pneumococcal Outgrowth and Dissemination Postinfluenza

International audienceInfluenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103+ dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections.IMPORTANCE:Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today's treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)-a potent agonist of invariant NKT cells that is currently in clinical development-in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia

Ozanimod-mediated remission in experimental autoimmune encephalomyelitis is associated with enhanced activity of CNS CD27low/- NK cell subset

BackgroundOzanimod (RPC1063) is an immunomodulator that has been recently approved by the FDA (2020) for the treatment of relapsing-remitting multiple sclerosis (RRMS). It is a selective agonist of the sphingosine-1-phophate receptors 1 and 5, expressed on naïve and central memory T and B cells, as well as natural killer (NK) cells, and is involved in lymphocyte trafficking. Oral administration of ozanimod was reported to result in rapid and reversible reduction in circulating lymphocytes in multiple sclerosis (MS) patients, however, only minimal effect on NK cells was observed. In this study, we sought to investigate the effect of ozanimod on NK cells and assess whether they play any role in ozanimod-induced remission in experimental autoimmune encephalomyelitis (EAE), the animal model of MS.MethodsActive EAE induction was done in C57BL/6 female mice, followed by daily oral treatment with ozanimod (0.6mg/kg) starting at disease onset (score 1). Flow cytometry of blood and CNS was performed 24 hours after the last oral dose of ozanimod treatment in diseased mice. Histological analysis of lumbar spinal cord was performed for evaluating the level of inflammation and demyelination. Depletion of peripheral NK cells was done using anti-NK1.1 mouse antibody (mAb) at day 5 post-EAE induction.ResultsOzanimod was effective in reducing the clinical severity of EAE and reducing the percentage of autoreactive CD4+ and CD8+ T cells along with significant inhibition of lymphocyte infiltration into the spinal cord, accompanied by reversed demyelination. Furthermore, ozanimod treatment resulted in a significant increase in the frequency of total NK cells in the blood and CNS along with upregulation of the activating receptor NKG2D on CD27low/- NK cell subset in the CNS. The effectiveness of ozanimod treatment in inhibiting the progression of the disease was reduced when NK cells were depleted using anti-NK1.1 mAb.ConclusionThe current study demonstrated that ozanimod treatment significantly improved clinical symptoms in EAE mice. Ozanimod and anti-NK1.1 mAb appear to function in opposition to one another. Collectively, our data suggest that ozanimod-mediated remission is associated with an increased percentage of total NK cells and CD27low/- NK cells expressing the activating receptor, NKG2D in the CNS

Clinical Course and Risk Factors for Infection in Severe Forms of Alcohol-Associated Liver Disease

Background and Aims: Infection is a major driver of mortality in patients with advanced alcohol-associated liver disease (ALD). The epidemiology and clinical course of patients infected with life-threatening forms of ALD, including severe alcohol-associated hepatitis (sAH) and decompensated alcohol-associated cirrhosis (DAC), and specific risk factors for infection remain mostly unknown. Approach and Results: In this observational study, we assessed all infectious episodes occurring within a 90-day period from diagnosis in all consecutive patients with biopsy-proven sAH (modified Maddrey’s discriminant function ≥ 32, Model for End-Stage Liver Disease [MELD] ≥ 18) and DAC (MELD ≥ 18) without alcohol-associated hepatitis in our tertiary hospital between 2003 and 2016. A total of 207 patients were included: 139 with sAH and 68 with DAC. One hundred seventeen (84%) patients with sAH and 41 (60%) patients with DAC experienced at least one infection episode at 90 days (P < 0.001). In multivariable analysis, factors associated with the development of infection were the presence of sAH and baseline MELD score. Bacterial infections represented the most common infection in the two groups, and only the MELD score was independently associated with the occurrence of bacterial infection. In both groups, pneumonia was the most prevalent bacterial infection, and gram-negative bacilli were the main pathogens. Invasive fungal infections (IFI) occurred in 20 (14.5%) patients with sAH and 3 (4.5%) with patients with DAC (P < 0.05). Multivariable regression showed that younger age, higher MELD, and corticosteroid therapy were independently associated with IFI. The 90-day cumulative incidence of death in patients infected with sAH and patients infected with DAC was 46% and 41.5%, respectively (P = 0.43). Conclusions: Patients with sAH are more susceptible to develop infection than those with DAC. In life-threatening forms of ALD, patients who were infected share a similar mortality rate. Corticosteroid treatment, not sAH, seems to be the main risk factor triggering IFI.SCOPUS: ar.jinfo:eu-repo/semantics/publishe