Déborah Rousseau

In Vivo Adjuvant-Induced Mobilization and Maturation of Gut Dendritic Cells after Oral Administration of Cholera Toxin

Although dendritic cells (DCs) regulate immune responses, they exhibit functional heterogeneity depending on their anatomical location. We examined the functional properties of intestinal DCs after oral administration of cholera toxin (CT), the most potent mucosal adjuvant. Two CD11c+ DC subsets were identified both in Peyer’s patches and mesenteric lymph nodes (MLN) based on the expression of CD8α (CD8+ and CD8− DCs, respectively). A third subset of CD11c+CD8int was found exclusively in MLN. Feeding mice with CT induced a rapid and transient mobilization of a new CD11c+CD8− DC subset near the intestinal epithelium. This recruitment was associated with an increased production of the chemokine CCL20 in the small intestine and was followed by a massive accumulation of CD8int DCs in MLN. MLN DCs from CT-treated mice were more potent activators of naive T cells than DCs from control mice and induced a Th2 response. This increase in immunostimulating properties was accounted for by CD8int and CD8− DCs, whereas CD8+ DCs remained insensitive to CT treatment. Consistently, the CD8int and CD8− subsets expressed higher levels of costimulatory molecules than CD8+ and corresponding control DCs. Adoptive transfer experiments showed that these two DC subsets, unlike CD8+ DCs, were able to present Ags orally coadministered with CT in an immunostimulating manner. The ability of CT to mobilize immature DCs in the intestinal epithelium and to promote their emigration and differentiation in draining lymph nodes may explain the exceptional adjuvant properties of this toxin on mucosal immune responses.

Transcutaneous immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective induction of Th1 immune responses.

Topical application of cholera toxin (CT) onto mouse skin can induce a humoral immune response to CT as well as to coadministered Ags. In this study, we examined the nontoxic cell-binding B subunit of CT (CTB) as a potential adjuvant for cutaneous immune responses when coadministered with the prototype protein Ag, OVA. CTB applied onto skin induced serum Ab responses to itself with magnitudes comparable to those evoked by CT but was poorly efficient at promoting systemic Ab responses to coadministered OVA. However, transcutaneous immunization (TCI) with either CT or CTB and OVA led to vigorous OVA-specific T cell proliferative responses. Furthermore, CTB potentiated Th1-driven responses (IFN-γ production) whereas CT induced both Th1 and Th2 cytokine production. Coadministration of the toxic subunit CTA, together with CTB and OVA Ag, led to enhanced Th1 and Th2 responses. Moreover, whereas TCI with CT enhanced serum IgE responses to coadministered OVA, CTB suppressed these responses. TCI with either CT or CTB led to an increased accumulation of dendritic cells in the exposed epidermis and the underlying dermis. Thus, in contrast to CT, CTB appears to behave very differently when given by the transcutaneous as opposed to a mucosal route and the results suggest that the adjuvanticity of CT on Th1- and Th2-dependent responses induced by TCI involves two distinct moieties, the B and the A subunits, respectively.

ER stress induces NLRP3 inflammasome activation and hepatocyte death

The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. However, the causes and mechanisms of inflammation-mediated liver damage remain poorly understood. Endoplasmic reticulum (ER) stress is an initiator of cell death and inflammatory mechanisms. Although obesity induces ER stress, the interplay between hepatic ER stress, NLRP3 inflammasome activation and hepatocyte death signaling has not yet been explored during the etiology of chronic liver diseases. Steatosis is a common disorder affecting obese patients; moreover, 25% of these patients develop steatohepatitis with an inherent risk for progression to hepatocarcinoma. Increased plasma LPS levels have been detected in the serum of patients with steatohepatitis. We hypothesized that, as a consequence of increased plasma LPS, ER stress could be induced and lead to NLRP3 inflammasome activation and hepatocyte death associated with steatohepatitis progression. In livers from obese mice, administration of LPS or tunicamycin results in IRE1α and PERK activation, leading to the overexpression of CHOP. This, in turn, activates the NLRP3 inflammasome, subsequently initiating hepatocyte pyroptosis (caspase-1, -11, interleukin-1β secretion) and apoptosis (caspase-3, BH3-only proteins). In contrast, the LPS challenge is blocked by the ER stress inhibitor TUDCA, resulting in: CHOP downregulation, reduced caspase-1, caspase-11, caspase-3 activities, lowered interleukin-1β secretion and rescue from cell death. The central role of CHOP in mediating the activation of proinflammatory caspases and cell death was characterized by performing knockdown experiments in primary mouse hepatocytes. Finally, the analysis of human steatohepatitis liver biopsies showed a correlation between the upregulation of inflammasome and ER stress markers, as well as liver injury. We demonstrate here that ER stress leads to hepatic NLRP3 inflammasome pyroptotic death, thus contributing as a novel mechanism of inflammation-mediated liver injury in chronic liver diseases. Inhibition of ER-dependent inflammasome activation and cell death pathways may represent a potential therapeutic approach in chronic liver diseases.

Dendritic Cell-Mediated Induction of Mucosal Cytotoxic Responses following Intravaginal Immunization with the Nontoxic B Subunit of Cholera Toxin

The use of the nontoxic B subunit of cholera toxin (CTB) as mucosal adjuvant and carrier-delivery system for inducing secretory Ab responses has been documented previously with different soluble Ags. In this study, we have evaluated this approach for inducing CTL responses against a prototype Ag, OVA, in the female genital mucosa. We report here the ability of an immunogen comprised of CTB conjugated to OVA (CTB-OVA) given by intravaginal (ivag) route to induce genital OVA-specific CTLs in mice. Using adoptive transfer models, we demonstrate that ivag application of CTB-OVA activates OVA-specific IFN-γ-producing CD4 and CD8 T cells in draining lymph nodes (DLN). Moreover, ivag CTB induces an expansion of IFN-γ-secreting CD8+ T cells in DLN and genital mucosa and promotes Ab responses to OVA. In contrast, ivag administration of OVA alone or coadministered with CTB failed to induce such responses. Importantly, we demonstrate that ivag CTB-OVA generates OVA-specific CTLs in DLN and the genital mucosa. Furthermore, genital CD11b+CD11c+ dendritic cells (DCs), but not CD8+CD11c+ or CD11c− APCs, present MHC class I epitopes acquired after ivag CTB-OVA, suggesting a critical role of this DC subset in the priming of genital CTLs. Inhibition studies indicate that the presentation of OVA MHC class I epitopes by DCs conditioned with CTB-OVA involves a proteasome-dependent and chloroquine-sensitive mechanism. These results demonstrate that CTB is an efficient adjuvant-delivery system for DC-mediated induction of genital CTL responses and may have implications for the design of vaccines against sexually transmitted infections.

Immunisation with DNA encoding Leishmania infantum protein papLe22 decreases the frequency of parasitemic episodes in infected hamsters.

We tested in outbred golden hamsters the protective potential of highly immunogenic Leishmania infantum protein papLe22 which we recently identified. Immunisation was performed using papLe22 cDNA, administered as a single intramuscular injection. The level of antibodies directed against total leishmanial antigens was significantly decreased in the vaccinated hamsters as compared with the controls, indicating that the administration of papLe22 cDNA downregulated the Th2 type response and suggesting that the immune response was reoriented toward the cell-mediated type. The presence of the parasite kDNA in the peripheral blood was systematically detected as early as 3 weeks post infection in all mock-vaccinated hamsters. By contrast, in the vaccinated animals the occurrence of the episodes of Leishmania circulation was reduced by 50%. The immunisation presenting efficacy in this highly susceptible species which develop VL similar in gravity to human and canine disease should prove also efficient in naturally infected hosts. The marked decrease of the frequency of parasite circulation induced by papLe22 cDNA immunisation appears therefore important and potentially able to reduce transmission and thus to control the spread of the disease.

CD44 is a key player in non-alcoholic steatohepatitis

BACKGROUND & AIMS Cluster of differentiation (CD)44 regulates adipose tissue inflammation in obesity and hepatic leukocyte recruitment in a lithogenic context. However, its role in hepatic inflammation in a mouse model of steatohepatitis and its relevance in humans have not yet been investigated. We aimed to evaluated the contribution of CD44 to non-alcoholic steatohepatitis (NASH) development and liver injury in mouse models and in patients at various stages of non-alcoholic fatty liver disease (NAFLD) progression. METHODS The role of CD44 was evaluated in CD44-/- mice and after injections of an αCD44 antibody in wild-type mice challenged with a methionine- and choline-deficient diet (MCDD). In obese patients, hepatic CD44 (n=30 and 5 NASH patients with a second liver biopsy after bariatric surgery) and serum sCD44 (n=64) were evaluated. RESULTS Liver inflammation (including inflammatory foci number, macrophage and neutrophil infiltration and CCL2/CCR2 levels), liver injury and fibrosis strongly decreased in CD44-/- mice compared to wild-type mice on MCDD. CD44 deficiency enhanced the M2 polarization and strongly decreased the activation of macrophages by lipopolysaccharide (LPS), hepatocyte damage-associated molecular patterns (DAMPs) and saturated fatty acids. Neutralization of CD44 in mice with steatohepatitis strongly decreased the macrophage infiltration and chemokine ligand (CCL)2 expression with a partial correction of liver inflammation and injury. In obese patients, hepatic CD44 was strongly upregulated in NASH patients (p=0.0008) and correlated with NAFLD activity score (NAS) (p=0.001), ballooning (p=0.003), alanine transaminase (p=0.005) and hepatic CCL2 (p<0.001) and macrophage marker CD68 (p<0.001) expression. Correction of NASH was associated with a strong decrease in liver CD44+ cells. Finally, the soluble form of CD44 increased with severe steatosis (p=0.0005) and NASH (p=0.007). CONCLUSION Human and experimental data suggest that CD44 is a marker and key player of hepatic inflammation and its targeting partially corrects NASH. LAY SUMMARY Human and experimental data suggest that CD44, a cellular protein mainly expressed in immune cells, is a marker and key player of non-alcoholic steatohepatitis (NASH). Indeed, CD44 enhances the non-alcoholic fatty liver (NAFL) (hepatic steatosis) to NASH progression by regulating hepatic macrophage polarization (pro-inflammatory phenotype) and infiltration (macrophage motility and the MCP1/CCL2/CCR2 system). Targeting CD44 partially corrects NASH, making it a potential therapeutic strategy.

Bax inhibitor‐1 protects from nonalcoholic steatohepatitis by limiting inositol‐requiring enzyme 1 alpha signaling in mice

Endoplasmic reticulum (ER) stress is activated in nonalcoholic fatty liver disease (NAFLD), raising the possibility that ER stress‐dependent metabolic dysfunction, inflammation, and cell death underlie the transition from steatosis to steatohepatitis (nonalcoholic steatohepatitis; NASH). B‐cell lymphoma 2 (BCL2)‐associated X protein (Bax) inhibitor‐1 (BI‐1), a negative regulator of the ER stress sensor, inositol‐requiring enzyme 1 alpha (IRE1α), has yet to be explored in NAFLD as a hepatoprotective agent. We hypothesized that the genetic ablation of BI‐1 would render the liver vulnerable to NASH because of unrestrained IRE1α signaling. ER stress was induced in wild‐type and BI‐1–/– mice acutely by tunicamycin (TM) injection (1 mg/kg) or chronically by high‐fat diet (HFD) feeding to determine NAFLD phenotype. Livers of TM‐treated BI‐1–/– mice showed IRE1α‐dependent NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, hepatocyte death, fibrosis, and dysregulated lipid homeostasis that led to liver failure within a week. The analysis of human NAFLD liver biopsies revealed BI‐1 down‐regulation parallel to the up‐regulation of IRE1α endoribonuclease (RNase) signaling. In HFD‐fed BI‐1–/– mice that presented NASH and type 2 diabetes, exaggerated hepatic IRE1α, X‐box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP) expression was linked to activated NLRP3 inflammasome and caspase‐1/‐11. Rises in interleukin (IL)‐1β, IL‐6, monocyte chemoattractant protein 1 (MCP1), chemokine (C‐X‐C motif) ligand 1 (CXCL1), and alanine transaminase (ALT)/aspartate transaminase (AST) levels revealed significant inflammation and injury, respectively. Pharmacological inhibition of IRE1α RNase activity with the small molecules, STF‐083010 or 4μ8c, was evaluated in HFD‐induced NAFLD. In BI‐1–/– mice, either treatment effectively counteracted IRE1α RNase activity, improving glucose tolerance and rescuing from NASH. The hepatocyte‐specific role of IRE1α RNase activity in mediating NLRP3 inflammasome activation and cell death was confirmed in primary mouse hepatocytes by IRE1α axis knockdown or its inhibition with STF‐083010 or 4μ8c. Conclusion: Targeting IRE1α‐dependent NLRP3 inflammasome signaling with pharmacological agents or by BI‐1 may represent a tangible therapeutic strategy for NASH. (Hepatology 2018).France; Centre Hospitalier Universitaire Nice, Hôpital l’Archet, Département Digestif, 06200 Nice, France; Université Paris Descartes, Sorbonne Paris Cité; Paris, France; Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers; Paris, France; INSERM, U1138; Paris, France; Université Pierre et Marie Curie, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus; Villejuif, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP; Paris, France; Department of Womens and Childrens Health,

PO24 Le stress du réticulum endoplasmique dialogue avec l’inflammasome dans la pathophysiologie des stéatophaties métaboliques

Introduction Les complications hepatiques associees a l’obesite vont de la steatose simple, dont le pronostic est excellent, a la steatohepatite (NASH), plus severe, qui augmente le risque de developper des stades deleteres (fibrose, hepatocarcinome). La voie du stress du reticulum endoplasmique (RE) peut conduire a l’apoptose, l’inflammation et a la resistance a l’insuline des mecanismes cruciaux dans la pathophysiologie de la NASH. Nos objectifs sont 1) d’etudier les processus d’apoptose et d’inflammation liees au stress du RE dans un modele animal de NASH et 2) d’analyser les mecanismes moleculaires impliques apres traitement avec un inhibiteur de ce stress : l’acide tauro-ursodeoxycholate (TUDCA). Materiels et methodes Les souris genetiquement obeses (obob) qui developpent une steatose severe ont ete utilisees comme modele d’etude animal. Les souris ont ete traitees 5 jours avec l’inhibiteur du stress du RE (TUDCA) puis l’inflammation hepatique a ete induite par une injection au LPS (lipopolysaccharides). Des etudes, in vitro, au niveau d’hepatocytes de souris en culture primaire ont aussi ete realisees. Resultats Nous avons observe (i) une elevation des transaminases serique, (ii) au niveau histologique une augmentation de la souffrance hepatique correlant a une augmentation du nombre d’hepatocytes en apoptose (TUNEL), (iii) une mobilisation de l’inflammasome (activation caspase-1, caspase-11, production accrue d’IL1b) (iv) une activation soutenue de la proteine du stress du RE, IRE1a et une augmentation de l’expression de CHOP. Au contraire, le pre-traitement des souris avec le TUDCA (avant l’injection au LPS) diminue les transaminases (75 %), reduit le pourcentage d’hepatocytes en apoptose (75 %), le nombre de foyers inflammatoires (75 %) et l’activite des caspases inflammatoires (50 %) en comparaison avec les souris ob/ob injectees au LPS. En accord avec ces changements, nous analysons une forte diminution de l’expression d’XBP1s, le substrat d’IRE1a et de CHOP. Conclusion Ces resultats ouvrent des perspectives therapeutiques encourageantes. En effet, aucun traitement n’existe pour la NASH. Il semblerait que l’inhibition du stress du RE puisse s’averer etre une strategie attractive dans son traitement.