Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus

Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning

CIMAC. Chirurgie - Imagerie médicale - Anesthésie en Confinement

Présentation du poster en 180 sec de l'activité des équipes expérimentation / élevage de la PFIENational audienc

Établissement d'un modèle d'anse intestinale chez l'agneau nouveau-né afin d'évaluer de nouvelles méthodes de lutte contre les maladies entériques et de réduire l'utilisation d'animaux de laboratoire

National audienceDans le but d'évaluer des alternatives naturelles pour contrôler la cryptosporidiose, infection entérique parasitaire affectant principalement les jeunes ruminants d'espèce de rente mais également l'être humain (zoonose), nous utilisons les agneaux comme espèce cible et comme modèle pour les animaux de plus grande taille comme les veaux. Nous avons pour objectif de stimuler les réponses immunitaires des animaux dès la naissance avec du colostrum supplémenté avec des produits naturels tels que des produits dérivés de levures qui contiennent des ligands des récepteurs de l'immunité innée. Cependant, la tolérance immunitaire intestinale s'installe rapidement après la naissance en réponse à la colonisation microbienne débutant dès la mise-bas par voie naturelle et par la suite via le colostrum, le lait et les multiples contacts avec l'environnement, et se caractérise par une hypo-réactivité aux antigènes microbiens (cf. modèles murins). Nous avons donc développé le modèle de chirurgie des anses intestinales sur agneaux nouveau-nés mis au monde par césarienne (et donc sans flore commensale) adapté à l'étude des interactions hôte-agent pathogène dans un environnement contrôlé et à l'évaluation de nouveaux composés antiparasitaires naturels in vivo. La possibilité de comparer différents produits naturels simultanément ainsi que des effets doses sur ce modèle, permet de réduire considérablement le nombre d’animaux expérimentaux à utiliser.Pour faciliter la réalisation de ces protocoles expérimentaux, nous utilisons notre troupeau d'ovins qui synchronise les mises-bas.Le jour de la chirurgie, la brebis est transférée dans la salle de chirurgie (confinement de niveau 2). Une césarienne est pratiquée et les agneaux sont réanimés. Après une courte phase de récupération, un agneau est choisi et transféré en salle de chirurgie. Il est installé sur une table de réanimation néonatale.Le protocole d'anesthésie / analgésie comprend : induction au masque (isoflurane), pose d'une sonde endotrachéale sous anesthésie locale (xylocaïne), maintien sous isoflurane (ventilation contrôlée), analgésie parentérale (buprénorphine), pose d'un cathéter veineux et mise sous perfusion, anesthésie locale traçante (lidocaïne).Pendant toute la procédure, les paramètres suivants sont surveillés : fréquences cardiaque et respiratoire, température rectale, SaO2, ETCO2.Une chirurgie à 4 mains est ensuite réalisée :- Extériorisation de la jonction iléo-caecale (IC) et de la portion distale de l'intestin grêle (IG)) ;- Ligatures avec création en général de triplicats - séparés par un inter-segment vide de "sécurité", y compris en amont et en aval de l'anse - (9 triplicats pour une anse de 50 cm environ) ;- Entérotomie en aval de l'anse ainsi créée, à proximité de la jonction IC, suture de l'intestin du côté de l'anse, puis idem en amont ;- Anastomose termino-terminale ;- Injection des préparations d'intérêt dans chaque segment.Pendant la chirurgie, une irrigation régulière de la portion externalisée de l'intestin est réalisée au sérum physiologique tiédi

Establishment of a newborn lamb gut-loop model to evaluate new methods of enteric disease control and reduce experimental animal use

International audienceEnteric infectious diseases are not all well controlled, which leads to animal suffering and sometimes death in the most severe cases, in addition to economic losses for farmers. Typical symptoms of enteric infections include watery diarrhea, stomach cramps or pain, dehydration, nausea, vomiting, fever and weight loss. Evaluation of new control methods against enteric infections requires the use of many animals. We aimed to develop a new method for an initial in vivo screen of promising compounds against neonatal diseases such as cryptosporidiosis while limiting experimental animal use. We therefore adapted an in vivo method of multiple consecutive but independent intestinal loops to newborn lambs delivered by cesarean section, in which endotoxin responsiveness is retained. This new method allowed for the screening of natural yeast fractions for their ability to stimulate immune responses and to limit early Cryptosporidium parvum development. This model may also be used to investigate host-pathogen interactions and immune responses in a neonatal controlled environment

Novel approaches boosting innate immunity against <em>Pseudomonas aeruginosa</em>

National audiencePseudomonas aeruginosa, an opportunistic gram-negative bacterium that rarely infects human lungs unless the host immune system has been impaired, is one of the main pathogens found in cystic fibrosis (CF) patients. P. aeruginosa infections in CF patients are difficult to treat, becoming chronic and contributing to exacerbated lung inflammation and respiratory failure. Modulation of innate immunity has been proposed as an alternative to improve defence against infections. This approach is particularly attractive in CF since exacerbated immune response is central to the pathogenesis of CF lung disease. Innate immunity in epithelial and immune cells can be stimulated through activation of Toll-like receptors (TLRs), the main family of pattern recognition receptors. Stimulation of TLR5 through flagellin-based interventions have demonstrated protective activity against several gram-negative bacteria (Salmonella sp., Burkholderia cepacia, Yersinia pseudotuberculosis), restoring immune-competence and promoting tissue repair processes. Here, we aimed to determine the effect of flagellin stimulation on the innate immune response against P. aeruginosa using an experimental pig model of lung infection. The pig model presents several advantages since swine and human lungs are similar in terms of anatomical, histological, biochemical, and physiological features. This is especially true in CF, where pigs lacking CFTR present a similar phenotype to what is typically observed in human patients. P. aeruginosa-infected pigs showed an acute neutrophilic response with an exacerbated release of neutrophil serine proteases that peaked 3-6 h post-infection (p.i.) leading to lung destruction and tissue hepatisation 24h p.i. When animals were pre-treated with flagellin 24h before the experimental infections, we observed a significant decrease in the expression of pro-inflammatory markers. In addition, a better lung status was observed on infected animals that had been pre-treated with flagellin compared to infected controls. The effect of flagellin pre-treatment on immune response to P. aeruginosa infection was confirmed using ex-vivo and in vitro models of lung epithelium from CFTR-/- pigs. In conclusion, our data point to a modulatory role of flagellin pre-treatment on the immune response to P. aeruginosa. This approach may have a therapeutic potential to improve inflammatory manifestations of CF

Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets

International audienceTetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy

Novel approaches boosting innate immunity against <em>Pseudomonas aeruginosa</em>

National audiencePseudomonas aeruginosa, an opportunistic gram-negative bacterium that rarely infects human lungs unless the host immune system has been impaired, is one of the main pathogens found in cystic fibrosis (CF) patients. P. aeruginosa infections in CF patients are difficult to treat, becoming chronic and contributing to exacerbated lung inflammation and respiratory failure. Modulation of innate immunity has been proposed as an alternative to improve defence against infections. This approach is particularly attractive in CF since exacerbated immune response is central to the pathogenesis of CF lung disease. Innate immunity in epithelial and immune cells can be stimulated through activation of Toll-like receptors (TLRs), the main family of pattern recognition receptors. Stimulation of TLR5 through flagellin-based interventions have demonstrated protective activity against several gram-negative bacteria (Salmonella sp., Burkholderia cepacia, Yersinia pseudotuberculosis), restoring immune-competence and promoting tissue repair processes. Here, we aimed to determine the effect of flagellin stimulation on the innate immune response against P. aeruginosa using an experimental pig model of lung infection. The pig model presents several advantages since swine and human lungs are similar in terms of anatomical, histological, biochemical, and physiological features. This is especially true in CF, where pigs lacking CFTR present a similar phenotype to what is typically observed in human patients. P. aeruginosa-infected pigs showed an acute neutrophilic response with an exacerbated release of neutrophil serine proteases that peaked 3-6 h post-infection (p.i.) leading to lung destruction and tissue hepatisation 24h p.i. When animals were pre-treated with flagellin 24h before the experimental infections, we observed a significant decrease in the expression of pro-inflammatory markers. In addition, a better lung status was observed on infected animals that had been pre-treated with flagellin compared to infected controls. The effect of flagellin pre-treatment on immune response to P. aeruginosa infection was confirmed using ex-vivo and in vitro models of lung epithelium from CFTR-/- pigs. In conclusion, our data point to a modulatory role of flagellin pre-treatment on the immune response to P. aeruginosa. This approach may have a therapeutic potential to improve inflammatory manifestations of CF

Modeling Hepatitis E infection in immunocompromised patients using a new pig model

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A new immunosuppressed pig model to study chronic Hepatitis E infection and its impact on host innate immune responses

International audienceHepatitis E virus (HEV) is associated with chronic hepatitis and cirrhosis in immunocompromised patients1. Chronic HEV pathogenesis remains unknow. To this aim, we developed a new pig model to characterize the HEV genetic changes associated with chronicity, and the impact of HEV infection on the host innate immune responses.Twenty piglets were separated into control (n=5), immunocompromised (n=5) and immunocompromisedinfected group (IC+HEV, n=10). Pigs were HEV-3 infected after two weeks of treatment with immunosuppressive drugs (tacrolimus, mycophenolic acid and prednisolone). Pigs were treated and monitored weekly until 11 weeks post infection (wpi). HEV infection and viral diversity were followed-up by qRT-PCR and NGS analysis, respectively. Host innate immune responses were followed-up using DNA array. A unique and relevant animal model of chronic HEV infection was successfully established, where 9/10 pigs progressed to chronicity (> 8 wpi) with inflammatory infiltrates in the liver. Extrahepatic replication in the colon and a HEV compartmentalization between sera and feces were observed. Mutation Y590C (ORF1) was detected (>20%) during the chronic phase in the feces of 6/9 pigs, but not in sera nor in the initial inoculum.During the chronic phase of HEV infection, host innate immune responses (PRR, NF-kB and type I IFN pathways) were activated in the blood but were down regulated in the liver. This suggested a possible inhibition of the host innate immune responses induced by HEV replication.This model could contribute to a better understanding of the pathogenesis of chronic HEV infection and the development of effective therapies

A new immunosuppressed pig model to study chronic Hepatitis E infection and the genetic viral diversity

Session : Virologie : Infections virales de l'immunodépriméInternational audienceIntroduction and Objectives: Hepatitis E virus (HEV) is a zoonosis associated with chronic hepatitis and cirrhosis in immunocompromised patients. Robust animal models are needed for a better understanding of the pathogenesis of chronic HEV infection. To this aim, we developed a new pig model to characterize the HEV genetic changes associated with chronicity, and the impact of HEV infection on the host innate immune responses. Material and Methods: Twenty piglets were separated into control (n=5), immunocompromised (IC, n=5) and immunocompromised and HEV infected group (IC+HEV, n=10). Pigs were intravenously infected with HEV-3 after two weeks of treatment with immunosuppressive drugs used in kidney transplant recipients (tacrolimus, mycophenolic acid and prednisolone). Pigs were treated and monitored weekly until 11 weeks post infection (wpi). Tacrolimus through concentrations were monitored by HPLCMS/MS (Waters). HEV infection and viral diversity were followed-up by qRT-PCR and NGS analysis (MiSeq, Illumina), respectively. Host innate immune responses were followed-up using DNA array (Fluidigm).Results, Discussion and Conclusion: A unique and relevant animal model of chronic HEV infection was successfully established where 9/10 pigs progressed to chronicity (> 8 wpi) with an inflammatory infiltrate in the liver. Extrahepatic replication in the colon and a unique HEV compartmentalization between sera and feces were observed. One mutation, Y590C (ORF1), was detected (>20%) during the chronic phase in the feces of 6/9 pigs, but not in sera nor in the initial inoculum. Three other mutations were detected (>50%) during the chronic phase: H662L (ORF1, feces), V871A (ORF1, sera) and A639V (ORF2, sera), each in one pig. A pro-inflammatory response and activation of the host innate immune responses (PRR, NF-kB and type I IFN pathways) was observed in blood at acute (4 wpi) and chronic (10 wpi) phases of HEV infection. This systemic inflammation during chronic hepatitis E could be associated with disease progression and/or extrahepatic replication. Contrary, a down-regulation of these pathways was observed in the liver. This suggested a possible inhibition of the host innate immune responses induced by HEV replication. This model could contribute to better understand the pathogenesis of chronic HEV infection and the development of effective therapies