Revue des maladies respiratoires最新文献

Introduction

La bronchiolite est la principale infection respiratoire chez le nourrisson, dont l’agent étiologique principal est le virus respiratoire syncytial (VRS) contre lequel il n’existe pas de vaccins. La sensibilité du nourrisson à l’infection par le VRS est intrinsèquement liée aux caractéristiques de la muqueuse pulmonaire en période périnatale qui évoluent avec la mise en place de l’immunité et la colonisation du poumon par un microbiote bactérien. Bien que décrit pour le microbiote intestinal, le rôle du microbiote pulmonaire sur la maturation de l’immunité de la muqueuse pulmonaire, et donc sur la trajectoire de la santé respiratoire reste peu décrit. Notre étude vise à évaluer si l’absence de microbiote en période périnatale modifie la réplication virale et la réponse immunitaire pulmonaire déclenchée par l’infection VRS. La compréhension des interactions précoces entre le microbiote et la muqueuse pulmonaire pourrait contribuer au développement de nouvelles approches visant à renforcer la réponse immunitaire et ainsi limiter la sévérité de l’infection par le VRS en période néonatale.

Méthodes

(1) Infection de souriceaux C57BL/6 dépourvus de flore (animaux axéniques) ou issus de géniteurs colonisés par une flore bactérienne (animaux colonisés) par une souche de VRS recombinant exprimant la luciférase (VRS-Luc).

(2) Mesure du niveau de réplication virale (bioluminescence et qPCR) et de la réponse immunitaire pulmonaire (infiltration cellulaire dans les lavages bronchoalvéolaires [LBA] et qPCR sur les gènes dépendants des interférons de type 1 [ISG]) à j₁, j₂ et j₄ post-infection.

Résultats

Les animaux axéniques montrent une augmentation du niveau de réplication virale entre j₁ et j₄ post-infection par détection de bioluminescence et par qPCR. À l’inverse, les animaux colonisés présentent une diminution du signal entre j₁ et j₄ post-infection. L’infiltration cellulaire dans les LBA des animaux axéniques est augmentée par rapport à celle quantifiée dans les animaux colonisés. L’expression des ISG (IRF7, ISG15 ou OAS) analysée par qPCR est augmentée à j₄ post-infection chez les animaux colonisés, alors que celle des animaux colonisés est induite dès j₁ post-infection.

Conclusion

L’absence de flore bactérienne s’accompagne de différences chez le souriceau dans la cinétique de réplication virale et dans la mise en place de la réponse immunitaire lors d’une infection par le VRS. La caractérisation des cellules immunitaires de la muqueuse pulmonaire est prévue afin d’identifier un partenaire cellulaire important dans la défense antivirale influencé par la présence d’un microbiote en période néonatale.

Introduction

L’emphysème, une des composantes de la bronchopneumopathie chronique obstructive (BPCO), correspond à une destruction des alvéoles pulmonaires dont la physiopathologie est mal connue. Des modèles de culture de cellules épithéliales alvéolaires (AEC) en 3 dimensions (3D) dans du Matrigel permettent d’étudier les capacités de prolifération et de différenciation des AEC2, mais manquent de reproductibilité. L’objectif principal de ce travail est le développement pérenne d’un modèle d’alvéolosphères 3D à partir d’AEC2 humaines ainsi que la modélisation de l’emphysème par exposition à l’extrait de fumée de cigarettes (CSE).

Méthodes

Ce modèle est basé sur l’isolement d’AEC2 par tri immunomagnétique (HTII-280+) à partir de 18 échantillons de parenchymes issus de patients fumeurs et non-fumeurs. Ces cellules sont mises en culture 3D dans des micropuits d’hydrogel préformés (200 μm de diamètre) par photopolymérisation permettant une analyse morphologique (taille, lumière) et phénotypique (immunomarquages, qPCR, microscopie électronique [MET]) à j₁, 7, 14 et 21. L’impact de l’exposition à 5 jours de CSE 5 % est étudié par qPCR et immunomarquages sur les alvéolosphères. Enfin, les cytokines sécrétées par les sphères exposées au CSE sont analysées par cytokine array, secondairement confirmées par ELISA.

Résultats

Les alvéolosphères sont maintenues en culture pendant 21 jours et forment progressivement une lumière centrale, dès j₇. La présence d’une barrière épithéliale est confirmée par la mise en évidence de jonctions serrées et adhérentes par MET et immunomarquage ZO-1. Des qPCR à j₁, 7, 14 et 21 montrent une apparition progressive de marqueurs d’AEC1 (expression de p2xr4, pdpn) alors que les marqueurs d’AEC2 persistent (expression de abca3, sftpa, sftpc). Les organelles permettant la synthèse de surfactant sont visualisées en MET (corps lamellaires, corps lipidiques). Enfin, l’exposition à 5 jours de CSE 5 % entraine une tendance à une diminution de la viabilité cellulaire (calcéine), une augmentation des marqueurs de stress oxydant (expression de hmox, nqo1, srxn1 en qPCR) ainsi qu’un relargage des cytokines (MIF et IL8) dans le surnagent.

Conclusion

Ainsi, nous avons obtenu, à partir d’échantillons de patients fumeurs et non-fumeurs, un modèle reproductible d’alvéolosphères ayant une capacité d’auto organisation en 3D, répondant à la définition d’un d’organoïde et permettant l’étude de la physiopathologie de l’emphysème induite par l’exposition à l’extrait de fumée de cigarettes.

Environmental air pollutants including ozone cause severe irritation and respiratory diseases. Here, we report that 6 week's ozone exposure in mice (1.5 ppm, twice weekly) causes airway hyperreactivity, eosinophil and neutrophil recruitment, Th2 immune response, respiratory barrier disruption with inflammation, fibrosis and emphysema reminiscent of COPD, more rapidly than cigarette smoke exposure. This model features important aspects of asthma-COPD overlap syndrome (ACOS) as recently described in patients. Since Tiotropium (TTP), an anticholinergic receptor antagonist, blocks smooth muscle cell contraction and mucus secretion with a prolonged bronchodilator effect in patients with asthma or COPD, we asked whether its effect is limited to bronchodilation. We report here that Tiotropium not only reduced airways hyperreactivity, but also drastically diminished eosinophil recruitment, Th2 cell response and ozone-induced lung inflammatory pathology including emphysema. Therefore, chronic O₃-induced lung pathology in mice mimics ACOS in patients and is attenuated by TTP treatment. The mechanisms of TTP protective effect on respiratory barrier disruption and chronic inflammation need to be further explored.

Introduction

Emphysema is a respiratory disease characterized by chronic alveolar destruction. Lipofibroblasts (LIF) play a key role in the stem cell niche surrounding alveolar type II (AT2) cells and may contribute to alveolar regeneration. We have previously shown that senescent cell elimination induces alveolar regeneration, increased LIF numbers and activation of the sterol regulatory binding protein (SREBP) and peroxisome proliferator-activated receptor gamma (PPARG) pathways [1]. However, it remains unclear whether the activation of these pathways can increase fibroblast stem cell niche properties and promote alveolar regeneration during emphysema.

Methods

Human lung tissue slides were obtained from patients with or without emphysema and immunofluorescent staining was performed to identify LIF (ADRP+Vimentin+). Both human primary fibroblasts and primary AT2 cells were isolated from lobectomies through the explant method and magnetic sorting (HT2-280+) respectively. Fibroblasts were treated with Rosiglitazone and T0901317 for 72 hours. LIF phenotype acquisition was evaluated through IF staining, qPCR and lipidomic analysis. Stem cell niche properties were evaluated by performing alveolar organoid formation assay by co-culturing treated fibroblasts with H-441 cells or primary AT2 cells. Adult C57BL6 mice received intra-tracheal injection of either Elastase or PBS. From D₂₁ mice were treated by intraperitoneal injections of Rosiglitazone (5 μg/g/d), T0901317 (10 μg/g/d) or vehicule, 5/7 days. Lungs were collected at D₉₀. Left lung was fixated for morphological analysis.

Results

Our study showed a decrease in LIF populations among patients with emphysema compared to controls. Furthermore, Rosiglitazone, a PPARG agonist, and T0901317, a SREBP agonist, can induce lipogenic differentiation in human lung fibroblasts. Activation of both pathways increased the expression of ADRP and the activation of the SREBP pathway induced the accumulation of neutral lipids in the fibroblasts. Using an organoid model of alveolar regeneration, we show that activating these pathways increases the stem cell niche properties of fibroblasts and enhances the number of organoids formed with either H441 cells or primary AT2. Lastly, in a murine mode of elastase-induced emphysema, we show that Rosiglitazone partially reverts emphysema.

Conclusion

Activation of PPARG and SREBP pathways promotes lipogenic differentiation of fibroblasts, enhances human alveolar organoid formation and partially reverts emphysema in vivo. These results provide insight into potential therapeutic strategies for promoting alveolar regeneration in patients with emphysema.

Introduction

Lung fibrosis, including idiopathic pulmonary fibrosis (IPF), results from dysfunctional wound repair involving different cell types, including fibroblasts, epithelial cells and macrophages, which respond to multiple soluble and matrix factors. Fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of lung fibrosis, in particular in the regulation of fibroblast to myofibroblast transition (FMT), cell proliferation, and extracellular matrix production. However, individual FGF family members may exert pro- and anti-fibrotic effects, depending on the responding cell, the expression levels of the different FGF receptors (FGFR1-4) and the context of other signaling molecules, such as Transforming growth factor β (TGF-β). In order to better understand the complex functions of FGFs on pulmonary fibrosis, we evaluated the effect of a modified version of a FGFR3 decoy receptor [1] that specifically sequesters FGFR3 ligands including FGF1, FGF2 and FGF9 as a potential anti-fibrotic drug.

Methods

The effect of several FGFs in the presence or the absence of the FGFR3 ligand Trap was evaluated in vitro on human lung fibroblasts from healthy donors and IPF patients on various fibrotic parameters such as cell proliferation, cell contraction, production of extracellular matrix (ECM) and modulation of signaling pathways. The effect of the FGFR3 ligand trap was also assessed in vivo on the bleomycin mouse model, by monitoring mice body weight, Ashcroft score, hydroxyproline and soluble collagen content.

Results

Our results revealed that FGFs (mainly FGF2) stimulate fibroblast proliferation, contraction, ECM production and expression of various fibrotic markers such as chemokine ligand 2 (CCL2), connective tissue growth factor (CTGF), interleukin 6 (IL6), interleukin receptor 4 (IL4R) or ECM-related genes like fibronectin (FN1). The FGFR3 ligands Trap was able to reduce this FGF mediated pro-fibrotic phenotype and to desensitize the TGF-β canonical pathway in IPF cells. In the bleomycin lung fibrosis mouse model, the FGFR3 ligands Trap partially reversed lung fibrosis, as evidenced by a reduced body weight loss as well as diminution of the aschcroft score, hydroxyproline and soluble collagen content in lung samples.

Conclusion

Our data highlight the interplay between the TGF-β and the FGF signaling pathways in pulmonary fibrosis and demonstrate the potential of targeting FGFR3 signaling as a novel therapy for IPF.

Introduction

Cystic fibrosis (CF) is a genetic disorder that affects the respiratory and digestive systems. CF patients exhibit considerable variation in their symptoms and disease progression, suggesting complex genotype–phenotype relationships that may involve environmental factors. This study aimed to use unsupervised clustering analyses to identify distinct profiles and trajectories of CF patients, while also assessing their associations with various environmental factors.

Methods

Data from the French CF Registry, which covers 90% of CF patients in France and provides comprehensive health information for monitoring and research purposes, were utilized. By employing dimensionality reduction and clustering techniques, such as self-organizing maps (SOMs), reverse graph embedding (DDRTree algorithm, ClinTrajAn), and trajectory analyses (latent class analysis) based on longitudinal lung function tests, patients were grouped based on their clinical characteristics.

Results

Preliminary findings revealed the existence of different subgroups among CF children and adult patients, characterized by significant differences in overall health status, decline in lung function, comorbidities, incidence of infections, and exposure to environmental factors like passive smoking. Additionally, the study investigates the connections between CF profiles and air pollution at the geographic level of French departments.

Conclusion

Applying clustering techniques to large medical datasets reveals valuable insights into the impact of the environment on the physiological and pathological processes of CF. By uncovering distinct patient profiles, this approach can optimize treatment strategies and improve patient outcomes.

Introduction

One of the top global causes of death worldwide is respiratory viral infections. Among these, influenza virus-related infections cause deadly epidemics and pandemics. Each year, seasonal influenza infects more than 1 billion people (i.e. ∼20% of the world's population) and results in approximately 500,000 deaths. Vaccination efficacy can be impaired by viral intrinsic antigenic drift and the efficiency of drugs targeting directly influenza viruses is largely disputed. Hence, the development of innovative options is required to better treat influenza. In that regard, we recently demonstrated the antiviral activity of succinate (EMBO J., 2022) as well as of “C2”, two host metabolites. In this project, our objectives are to synthesize succinate-derived drugs against influenza viruses, select the best candidates and test them in vitro and in vivo, and finally formulate them into dry powders for direct lung delivery.

Methods

A series of compounds are synthesized by a structure-activity relationship strategy. Various chemical modifications will be introduced in the “hit” compound in the aim to identify analogues with high activity and good druggability.

Results

A preliminary screening of a short series of commercially available analogues using human bronchial epithelial cells led to the identification of three new active compounds (coined “S1”, “S10” and “S11”). All have an antiviral and anti-inflammatory effect more potent than the natural metabolites succinate and C2. In an in vivo model of influenza pneumonia, mice treated at day 2 post-infection with these analogues resisted better than non-treated or C2-treated animals to a lethal dose of influenza virus (survival rate: 85%, 0%, and 50% respectively).

Conclusion

Our study will pave the way for the development of appropriate “drug-and-devices” that will help to administer these succinate analogues directly into the respiratory tract. This project is funded through an ANR program.