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

Intérêt du valganciclovir dans le traitement des infections à herpèsvirus équin 1 chez les équidés.

National audienceÀ ce jour, neuf herpèsvirus (HVE) ont été décrits chez les équidés. L'HVE-1, responsable des formes cliniques les plus graves, est un virus à ADN double brin d'environ 150kb qui contient 80 cadres de lecture ouverts codant 76 gènes uniques. Il se transmet par inhalation ou, après un avortement, par contact direct avec des éléments infectés comme le fœtus ou les annexes fœtales. En l'absence d'anticorps neutralisants dans le mucus, les cellules épithéliales des muqueuses nasale et nasopharyngienne sont infectées. La dissémination de l'HVE-1, via la circulation sanguine, jusqu'au tractus génital de la jument conduit généralement à un avortement. La dissémination du virus au niveau des tissus nerveux peut conduire à la forme nerveuse de la maladie nommée myéloencéphalopathie (EHM) nécessitant trop souvent l'euthanasie de l'animal.Si la vaccination contre l'HVE-1 existe, elle présente aujourd'hui des limitations ce qui conduit les praticiens vétérinaires à se tourner vers l'utilisation de traitements antiviraux pour les formes graves. Bien que son efficacité ne soit pas clairement démontrée, l'aciclovir demeure la molécule la plus utilisée sur le terrain.Notre travail a consisté à cribler in vitro 2891 molécules issues de différentes chimiothèques par des techniques de Real-Time Cell Analysis couplées à des méthodes de qPCR afin d'identifier des hits présentant une efficacité supérieure à celle de l'aciclovir. Ces travaux nous ont conduit à sélectionner 8 molécules parmi lesquelles le valganciclovir (VGCV) qui présentait une activité antivirale in vitro 10 fois supérieure à celle de l'aciclovir (Thieulent et al. 2020). Seul le valganciclovir avait fait l'objet d'études pharmacocinétiques préalables ce qui laissait entrevoir les perspectives d'un essai terrain (Charmichael et al., 2013). Notre étude démontre l'efficacité in vivo du valganciclovir après administration orale chez des poneys infectés par le nouveau variant HVE-1 FR-56628 (C2254) (Sutton et al. ; 2020).Lors de l'étude expérimentale, réalisée au sein de la PFIE (UE 1277 de l'INRAE de Nouzilly ; CEEA VdL, Comité éthique N° 19 ; autorisation APAFiS -22708), l'infection de 8 poneys Welsh mâles âgés de 10 mois a été réalisée par voie intranasale. Quatre poneys ont été traités avec une dose de 6,5 mg/kg de VGCV. Les signes cliniques, l'excrétion virale et la virémie ont été suivis pendant 3 semaines après infection. L'excrétion virale et la virémie ont été mesurées par culture cellulaire et par qPCR.L'ensemble des chevaux infectés ont présenté à des degrés divers des signes cliniques caractéristiques des infections respiratoires à HVE-1 (fièvre > 38,5°C ; jetage nasal ; toux, ganglions et secrétions oculaire). L'excrétion du virus HVE-1 mesurée à partir des écouvillons nasopharyngés est réduite de manière significative dans le groupe traité entre J+1 et J+12. Une diminution significative de la virémie dans le groupe traité a pu être mesurée. Le dosage du VGCV sérique a démontré la présence d'une concentration supérieure à 0.153µg/ml (EC50 déterminée lors de nos expériences in vitro) chez les 4 poneys traités.L'administration orale de valganciclovir réduit les signes cliniques, le jetage nasal et la virémie chez les chevaux infectés par la souche d'HVE1 FR-56628 (C2254)

Oral administration of valganciclovir reduces clinical signs, virus shedding and cell‐associated viraemia in ponies experimentally infected with equid herpesvirus‐1

International audienceBackground: Equid alphaherpesvirus-1 (EHV-1) is a frequent respiratory pathogen of the horse, causing mild disease and occasionally myeloencephalopathy (EHM) or abortion. Current vaccines reduce the nasopharyngeal excretion and dissemination of the virus and therefore the extent of an epizooty, but their efficacy against secondary forms of diseases (abortion and EHM) is either limited or remains untested respectively. Several antiviral compounds are active against EHV-1 in vitro but no pharmaceuticals are licenced for in vivo treatment to date.Objectives: To measure the in vivo efficacy of antiviral compounds, starting on the day of experimental infection of the target species with EHV-1 (C2254), as assessed by any reduction of clinical signs, virus shedding and viraemia.Study design: Randomised semi-blinded experiment.Methods: Four ponies were treated with valganciclovir (VGCV, the oral prodrug of ganciclovir [GCV]) at 6.5 mg/kg bodyweight, three times on day 1 and twice daily until day 14 inclusive. Four other ponies received a placebo. All ponies were experimentally infected with a field EHV-1 strain (5e07 TCID50/pony). Clinical signs of disease, virus shedding and blood/cell associated viraemia were recorded and measured for 3 weeks.Results: Serum GCV concentration was maintained above the EC50 (0.153 µg/mL) for at least 15 days. The overall cumulative clinical score was significantly reduced in VGCV treated ponies when compared with controls (p<0.009; pyrexia duration, nasal discharge and coughing). Infectious EHV-1 shedding measured on RK13 cells was significantly reduced in the VGCV treated group when compared with the control group between D+1 and D+12 (p=0.006). Blood and cell-associated viraemia were also both significantly reduced in the VGCV treated group (p=0.02 and 0.03, respectively). All ponies seroconverted after infection

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

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

Development of automated measures of indicators of wellbeing and health of poultry: special case of avian infectious bronchitis

International audienc

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

WOS:000467333800001International audienceSwine 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 M 8 to their murine counterparts: subcapsular sinus M 8, medullary cord M 8 and medullary sinus M 8. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of M 8, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (M 8). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN M 8 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

Développement d’un modèle porcin d’infection intra-osseuse à <em>Staphylococcus aureus</em> suite à la pose chirurgicale d’implants en titane

National audienceLes infections relatives à la pose d’implant en chirurgie humaine sont des affections cliniques sérieuses engendrant un taux de mortalité élevé. Le traitement de ces infections requiert de façon quasi systématique l’association d’un traitement antibiotique de longue durée et d’un traitement chirurgical concomitant, le remplacement de l’implant. Cependant, les protocoles de traitement antibiotique standards présentent une faible efficacité pour ce genre d’infection et l’intervention chirurgicale est souvent complexe. Ces pratiques représentent un risque élevé pour le patient et un coût non négligeable pour la société. Il est nécessaire de trouver des solutions alternatives, comme par exemple, d’utiliser un traitement de surface sur titane permettant de délivrer des antibiotiques localement seulement en cas d’infection. Pour répondre à cet objectif, il est donc indispensable de développer un modèle animal de pose chirurgicale d’implants en titane avec infection osseuse. L’objectif de cette étude est de développer un modèle chirurgical de pose d’implants au niveau du tibia chez le porc associé à une infection intra-osseuse à Staphylococcus aureus concomitante. Nous avons défini la taille et la forme des implants en titane (cylindres de 4 et 8 mm de longueur pour 4 mm de diamètre) à utiliser. L’intervention chirurgicale d’ostéotomie partielle double et bilatérale du tibia porcin a été validée. En parallèle et afin d’induire une infection locale osseuse, nous avons testé deux souches de S. aureus : ATCC 25923 et SAHOS (isolat clinique osseux humain fourni par le CNR de Lyon) à deux concentrations différentes (102 CFU et 106 CFU). Les résultats cliniques et mes analyses bactériologiques ont permis de sélectionner la souche de S. aureus SAHOS pour induire une infection osseuse locale aux deux concentrations testées que la souche ATCC. Dans les deux cas, une inflammation et une infection de la zone osseuse d’implantation sont observées des 7 jours post-infection, grâce au suivi clinique et à l’imagerie médicale (IRM) de la zone d’implantation et confirmer par l’augmentation des cytokines inflammatoires (CRP, IL-6 et TNFa). Ce modèle porcin est donc valide et ouvre la possibilité de tester l’efficacité thérapeutique d’implants biomédicaux contre les infections intra-osseuses postopératoires aigues ou chroniques, en vue d’une utilisation humaine