Mesenteric lymph node cells from neonates present a prominent IL-12 response to CpG oligodeoxynucleotide via an IL-15 feedback loop of amplification

At birth, the immune system is still in development making neonates more susceptible to infections. The recognition of microbial ligands is a key step in the initiation of immune responses. It can be mimicked to stimulate the immune system by the use of synthetic ligands recognising pattern recognition receptors. In human and mouse, it has been found that neonatal cytokine responses to toll-like receptor (TLR) ligands differ in many ways from those of adults but the relevant studies have been limited to cord blood and spleen cells. In this study, we compared the responses in neonate and adult sheep to CpG oligodeoxynucleotides (ODN), a TLR9 ligand, in both a mucosal and a systemic organ. We observed that in response to CpG-ODN more IL-12 was produced by neonatal than adult sheep cells from mesenteric lymph nodes (MLN) and spleen. This higher IL-12 response was limited to the first 20 days after birth for MLN cells but persisted for a longer period for spleen cells. The major IL-12-producing cells were identified as CD14+CD11b+. These cells were poor producers of IL-12 in response to direct stimulation with CpG-ODN and required the cooperation of other MLN cells. The difference in response to CpG-ODN between neonates and adults can be attributed to both a higher proportion of CD14+CD11b+ cells in neonate lambs and their higher capacity to produce IL-15. The IL-15 increases IL-12 production by an amplifying feedback loop involving CD40

Méthodes alternatives in vitro pour l’étude des interactions hôte-pathogène du poumon

Les maladies respiratoires, qu’elles touchent les animaux et/ou les hommes, ont un impact sanitaire et économique considérable sur notre société. Pouvoir mieux les contrôler, les traiter et les prédire, nécessite de pouvoir les étudier. Pour cela des modèles d’études pertinents, reproductibles, efficaces aisés d’utilisation, et alternatifs à l’expérimentation animale doivent être proposés. D’énormes progrès méthodologiques ont été réalisés ces dernières années avec l’émergence de modèles in vitro qui miment le poumon en reproduisant la diversité des types cellulaires, l’architecture du tissu et certaines de ses fonctionnalités (activité ciliaire, sécrétion). Cette revue présente les avancées dans la génération de ces modèles chez le bovin : les organoïdes, les cultures Air-liquide-interface (ALI) et les coupes fines de poumon (PCLS). Ils sont utilisés pour mieux décrire et comprendre les processus physiopathologiques induits par des infections (virus, bactérie, parasite) respiratoires et permettent de tester des approches prophylactiques ou curatives

Paradigms of Lung Microbiota Functions in Health and Disease, Particularly, in Asthma

Improvements in our knowledge of the gut microbiota have broadened our vision of the microbes associated with the intestine. These microbes are essential actors and protectors of digestive and extra-digestive health and, by extension, crucial for human physiology. Similar reconsiderations are currently underway concerning the endogenous microbes of the lungs, with a shift in focus away from their involvement in infections toward a role in physiology. The discovery of the lung microbiota was delayed by the long-held view that the lungs of healthy individuals were sterile and by sampling difficulties. The lung microbiota has a low density, and the maintenance of small numbers of bacteria seems to be a critical determinant of good health. This review aims to highlight how knowledge about the lung microbiota can change our conception of lung physiology and respiratory health. We provide support for this point of view with knowledge acquired about the gut microbiota and intestinal physiology. We describe the main characteristics of the lung microbiota and its functional impact on lung physiology, particularly in healthy individuals, after birth, but also in asthma. We describe some of the physiological features of the respiratory tract potentially favoring the installation of a dysbiotic microbiota. The gut microbiota feeds and matures the intestinal epithelium and is involved in immunity, when the principal role of the lung microbiota seems to be the orientation and balance of aspects of immune and epithelial responsiveness. This implies that the local and remote effects of bacterial communities are likely to be determinant in many respiratory diseases caused by viruses, allergens or genetic deficiency. Finally, we discuss the reciprocal connections between the gut and lungs that render these two compartments inseparable

Cellular and Molecular Mechanisms Underlying the Strong Neonatal IL-12 Response of Lamb Mesenteric Lymph Node Cells to R-848

International audienceabsen

Bacteria isolated from lung modulate asthma susceptibility in mice

Asthma is a chronic, non-curable, multifactorial disease with increasing incidence in industrial countries. This study evaluates the direct contribution of lung microbial components in allergic asthma in mice. Germ-Free and Specific-Pathogen-Free mice display similar susceptibilities to House Dust Mice-induced allergic asthma, indicating that the absence of bacteria confers no protection or increased risk to aeroallergens. In early life, allergic asthma changes the pattern of lung microbiota, and lung bacteria reciprocally modulate aeroallergen responsiveness. Primo-colonizing cultivable strains were screened for their immunoregulatory properties following their isolation from neonatal lungs. Intranasal inoculation of lung bacteria influenced the outcome of allergic asthma development: the strain CNCM I 4970 exacerbated some asthma features whereas the pro-Th1 strain CNCM I 4969 had protective effects. Thus, we confirm that appropriate bacterial lung stimuli during early life are critical for susceptibility to allergic asthma in young adults

Nucleoprotein Nanostructures Combined with Adjuvants Adapted to the Neonatal Immune Context: A Candidate Mucosal RSV Vaccine

BACKGROUND: The human respiratory syncytial virus (hRSV) is the leading cause of severe bronchiolitis in infants worldwide. The most severe RSV diseases occur between 2 and 6 months-of-age, so pediatric vaccination will have to be started within the first weeks after birth, when the immune system is prone to Th2 responses that may turn deleterious upon exposure to the virus. So far, the high risk to prime for immunopathological responses in infants has hampered the development of vaccine. In the present study we investigated the safety and efficacy of ring-nanostructures formed by the recombinant nucleoprotein N of hRSV (N(SRS)) as a mucosal vaccine candidate against RSV in BALB/c neonates, which are highly sensitive to immunopathological Th2 imprinting. METHODOLOGY AND PRINCIPAL FINDINGS: A single intranasal administration of N(SRS) with detoxified E. coli enterotoxin LT(R192G) to 5-7 day old neonates provided a significant reduction of the viral load after an RSV challenge at five weeks of age. However, neonatal vaccination also generated an enhanced lung infiltration by neutrophils and eosinophils following the RSV challenge. Analysis of antibody subclasses and cytokines produced after an RSV challenge or a boost administration of the vaccine suggested that neonatal vaccination induced a Th2 biased local immune memory. This Th2 bias and the eosinophilic reaction could be prevented by adding CpG to the vaccine formulation, which, however did not prevent pulmonary inflammation and neutrophil infiltration upon viral challenge. CONCLUSIONS/SIGNIFICANCE: In conclusion, protective vaccination against RSV can be achieved in neonates but requires an appropriate combination of adjuvants to prevent harmful Th2 imprinting

Immune lung features related to the higher severity of respiratory syncytial virus infection in neonates

Le virus respiratoire syncytial (VRS) est la cause majeure des bronchiolites du nourrisson. Mon travail de thèse a porté sur les réponses immunitaires et inflammatoires propres au poumon en période néonatale, à l’homéostasie, lors d’une infection par le VRS et en réponse à un vaccin.A l’homéostasie, l’environnement pulmonaire du souriceau BALB/c de 6 jours est pauvre en cellules dendritiques (DC) plasmacytoïdes et conventionnelles mais riches en lymphocytes exprimant le facteur GATA3 qui semblent à l’origine du biais Th2 des réponses néonatales. Les évènements précoces mis en jeu au niveau pulmonaire lors de l’infection VRS ont été appréhendés par une étude transcriptomique comparant le souriceau nouveau-né à l’adulte. J’ai ainsi montré que le recrutement des DC et les voies interférons (IFN) sont déficients chez le souriceau infecté. Le traitement des souriceaux par un facteur d’expansion des DC, le Flt3-L, avant une primo-infection par le VRS, augmente le nombre de DC pulmonaires, restaure les voies IFN et prévient l’exacerbation de la pathologie pulmonaire lors d’une ré-infection à l’âge adulte. Enfin, j’ai évalué le potentiel protecteur d’un vaccin contre le VRS basé sur l’administration nasale de nanostructures formées par la nucléoprotéine virale. J’ai montré que la nature des adjuvants combinés à notre antigène est un élément critique pour générer des réponses immunitaires antivirales protectrices sans risque d’exacerbation de l’inflammation pulmonaire.En conclusion, l’étude des réponses pulmonaires du nouveau-né a montré leurs spécificités par rapport à celles de l’adulte et m’a permis de proposer des pistes pour adapter les traitements à cet âge.The respiratory syncytial virus (RSV) is the main causative agent of bronchiolitis during infancy. During my thesis, I have characterized the immunological and inflammatory responses in the lung during the neonatal period, at steady-state, during an RSV infection and in response to a vaccine.At steady-state, the lung environment of 6-days-old BALB/c pups is poor in plasmacytoid and conventional dendritic cells (DC), but rich in T lymphocytes expressing GATA-3, suggesting that the Th2 bias of immune responses is an intrinsic feature of the neonatal T lymphocytes. Then, I used a global transcriptomic approach to gain more insight into early events to RSV infection, by comparing pups and adult mice lung responses. I showed that DC recruitment and interferon (IFN) pathways were deficient in infected neonates. Pups treated with the widely used DC growth factor, Flt3-L, prior to RSV infection, have an increased lung DC number and their IFN pathways were partially restored. They were protected against enhanced weight loss and airway inflammation upon reinfection at adult age. Finally, I evaluated a nasal vaccine against RSV (recombinant viral nucleoproteins) in BALB/c pups. I showed that the nature of adjuvants combined with our antigen is a critical component to generate antiviral immune responses that protect against virus replication without exacerbating lung inflammation.Thus, the study of neonatal lung immune responses showed specificities in comparison with those of adults, and allowed me to propose new strategies to prevent RSV disease at this age

Neutrophils and close relatives in the hypoxic environment of the tuberculous granuloma: New avenues for host-directed therapies?

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is one of the most prevalent lung infections of humans and kills ~1.7 million people each year. TB pathophysiology is complex with a central role played by granuloma where a delicate balance takes place to both constrain bacilli and prevent excessive inflammation that may destroy lung functions. Neutrophils reach the lung in waves following first encounter with bacilli and contribute both to early Mtb elimination and late deleterious inflammation. The hypoxic milieu where cells and bacilli cohabit inside the granuloma favors metabolism changes and the impact on TB infection needs to be more thoroughly understood. At the cellular level while the key role of the alveolar macrophage has long been established, behavior of neutrophils in the hypoxic granuloma remains poorly explored. This review will bring to the front new questions that are now emerging regarding neutrophils activity in TB. Are different neutrophil subsets involved in Mtb infection and how? How do neutrophils and close relatives contribute to shaping the granuloma immune environment? What is the role of hypoxia and hypoxia induced factors inside granuloma on neutrophil fate and functions and TB pathophysiology? Addressing these questions is key to the development of innovative host-directed therapies to fight TB