Bacteria-driven peribronchial lymphoid neogenesis in mice : effects of an immunomodulation with monoclonal antibodies or antibiotic and non-antibiotic macrolides.

Introduction. Les structures lymphoïdes tertiaires (SLT) sont retrouvées dans les poumons de patients atteints de mucoviscidose ou de dilatation des bronches alors qu'ils sont absents du poumon normal. L'infection bronchique persistante à Staphylococcus aureus (SA) ou Pseudomonas aeruginosa (PA), les deux bactéries les plus fréquemment retrouvées dans les voies aériennes des patients mucoviscidosiques, induit la formation de SLT péribronchiques en 14 jours chez la souris. Le rôle et les mécanismes de formation des SLT en réponse à l'infection ne sont pas parfaitement établis. Objectifs. Nos objectifs ont été : (1) d'étudier les effets d'une déplétion en lymphocytes B et/ou T sur la néogénèse lymphoïde et la charge bactérienne intrapulmonaire dans un modèle murin d'infection bronchique chronique à SA ; (2) d'évaluer les effets de l'azithromycine (AZM) - un antibiotique possédant des propriétés immunomodulatrices, et d'un analogue non-antibiotique de l'AZM sur le développement des TLS induit par l'infection bronchique chronique à PA ; (3) d'analyser l'impact d'un traitement par AZM et un analogue non antibiotique sur le microbiote digestif et respiratoire de souris. Méthodes. La déplétion lymphocytaire a été obtenue en traitant des souris C57Bl/6 avec (1) un anticorps monoclonal (AcM) anti-CD20 (déplétion B) ou (2) un AcM anti-CD4 et/ou anti-CD8 (Déplétion T), ou (3) une association d'AcM anti-CD20, anti-CD4 et anti-CD8 (Déplétion combinée CD4/CD8). Après induction de la déplétion lymphocytaire, les souris ont été infectées par une instillation intra-trachéale unique de billes d'agarose contenant du SA. Quatorze jours après l'instillation, les poumons ont été mis en culture pour évaluation de la charge bactérienne et inclus en paraffine pour analyse immunohistochimique. L'AZM et son analogue non antibiotique ont été donnés par voie orale à des souris 1 jour avant l'instillation intratrachéale de billes d'agarose contenant PA, et poursuivi pendant 1, 4 ou 14 jours avant sacrifice des souris et étude de la néogénèse lymphoïde par immunohistochimie. Les effets de l'AZM et de son analogue non antibiotique sur le microbiote respiratoire et digestif des souris a été étudié. Résultats. Quatorze jours après l'instillation des billes contenant SA, la charge bactérienne était comparable chez les souris contrôle et les souris déplétées (déplétion en lymphocytes B CD20+, T CD4+ et CD8+ ou déplétion B et T combinée). Alors qu'on observait le développement de TLS dans les poumons de souris traitées avec les AcM contrôle, ces structures étaient désorganisées dans les poumons de souris déplétées. L'absence de lymphocytes B CD20+ n'a pas d'effet sur le recrutement de lymphocytes T CD3+, alors que la déplétion en lymphocytes T CD4+/ CD8+ diminue le recrutement des lymphocytes B dans les poumons de souris infectées à SA. La déplétion en lymphocytes T CD4+ ou en T CD8+ entraine une disparition des centres germinatifs mais avait peu d'effet sur la formation d'agrégats lymphocytaires B. Malgré des concentrations intrapulmonaires élevées, le traitement par AZM ou un analogue non antibiotique ne semble pas avoir d'effets sur la néogénèse lymphoïde induite par PA. Contrairement à son analogue non antibiotique, l'AZM entraine une diminution rapide et importante de la charge bactérienne fécale chez les souris traitées. La composition du microbiote digestif était profondément modifiée par l'AZM, alors que son analogue non antibiotique induit des modifications moins importantes. Conclusion. La désorganisation des SLT péribronchiques n'est pas associée à une augmentation de la charge bactérienne pulmonaire. Les lymphocytes T CD4+ et CD8+ sont nécessaires au recrutement des lymphocytes B CD20+ en réponse à l'infection bronchique persistante à SA. Les macrolides ne semblent pas influer sur la néogénèse lymphoïde induite par PA. Les macrolides non antibiotiques pourraient présenter une alternative intéressante dans le cadre des traitements au long cours.Introduction. Tertiary lymphoid structures (TLS) are found in the lungs of patients with cystic fibrosis (CF) or with bronchiectasis while they are absent in normal lungs. Persistent bronchopulmonary infection with Staphylococcus aureus (SA) or Pseudomonas aeruginosa (PA), two of the major bacteria infecting CF airways, induces the development of peribronchial tertiary lymphoid structure (TLS) in mice in 14 days. The role of TLS and their formation mechanisms in response to infection are not totally elucidated. Aims of the study. Our objectives were: (1) to examine the effects of B and/or T-cell depletion on lymphoid neogenesis and bacterial infection in a mouse model of persistent SA lung infection; (2) to study the effects of azithromycin (AZM) - an antibiotic with immunomodulatory properties, and its non-antibiotic analogue on PA-induced TLS development; (3) to assess the effects of AZM and its non-antibiotic analogue on fecal and lung microbiota. Methods. Lymphocyte depletion was obtained by pretreating C57Bl/6 mice with (1) an anti-CD20 monoclonal antibody (mAb) (B-cell depletion) or (2) an anti-CD4 and/or an anti-CD8 mAbs (T-cell depletion) or (3) a combination of anti-CD20, anti-CD4 and anti-CD8 mAbs (combined B- and T-cell depletion). After lymphocyte depletion, mice were infected by intratracheal instillation of agarose beads containing SA. Fourteen days later, bacterial load and lung inflammatory cell infiltration were assessed by cultures of lung homogenates and immunohistochemistry, respectively. AZM and its non-antibiotic analogue were started one day prior to PA-induced chronic lung infection and were given orally for 1, 4 or 14 days after infection. Peribronchial lymphoid neogenesis was evaluated by immunohistochemistry. Finally, fecal and lung microbiota were studied in mice treated with AZM or its non-antibiotic analogue for 1, 4 and 14 days. Results. Fourteen days after SA-bead instillation, lung bacterial load was comparable between control and B-cell, T cell or B- and T-cell depleted mice. While TLS were observed in the lungs of persistently SA-infected mice pretreated with control mAbs, these structures were disrupted in the lungs of depleted mice. The absence of CD20+ B-lymphocytes has no effect on CD3+ T-lymphocyte recruitment, whereas CD4+/CD8+ T-cell depletion reduces CD20+ B-cell recruitment within the lungs of SA-infected mice. Depletion of CD4+ or CD8+ T-cells separately lead to germinal centre disruption but had only few effects on B-cell aggregate formation. Despite very high intrapulmonary concentration, treatment with AZM or its non-antibiotic analogue had no effect on PA-induced peribronchial lymphoid neogenesis. As opposed to its non-antibiotic analogue, treatment AZM induced a rapid and significant decrease in caecal bacterial load. Fecal microbiota composition was highly affected by AZM. AZM non-antibiotic analogue seemed to induce only minimal change in fecal microbiota. Conclusion. Lymphoid neogenesis disruption is not associated with an increased lung bacterial load in mice persistently infected with S. aureus. CD4+ and CD8+ T-cell are required for B-cell recruitment and TLS formation in response to persistent bronchopulmonary SA infection. Macrolides do not seem to affect PA-induced lymphoid neogenesis. Non-antibiotic macrolides could be an interesting alternative for long term treatment

Caractérisation des atteintes aspergillaires bronchopulmonaires chez les adultes mucoviscidosiques

Aspergillus fumigatus (A.f) is the most common fungi isolated in adult cystic fibrosis (CF) patients. A wide range of clinico-immunological presentations have been reported, from asymptomatic colonization to allergic bronchopulmonary aspergillosis (ABPA). Most available published data on pulmonary aspergillosis in CF patients come from pediatric cohort. Our objectives were (1) to determine the prevalence of pulmonary aspergillosis manifestations and their impact on lung function in adults with CF, (2) to evaluate the performance of sputum galactomannan (GM) and real-time PCR to detect A.f, and (3) to assess the clinical relevance of a new classification built with cluster analysis. We conducted a monocentric prospective study including 134 adults with CF. A.f sensitization was highly prevalent (35.8%) and ABPA was present in 16.4% of patients. Both conditions were associated with worse lung function decline. A.f colonization was not associated with accelerated lung function decline. Fungal culture was overall positive in 47% of patients whereas sputum A.f. PCR was positive in only 35.8% of patients. Sputum GM was positive in 76.1% patients and was not correlated to a specific aspergillosis-related manifestation. Among the 5 phenotypes identified with cluster analysis, those with high A.f. hypersensitivity markers had worse lung function decline. These findings confirm that A. fumigatus-related and ABPA occur frequently and are associated with worse lung function in adults with CF. They also suggest that A.f colonization has no impact on lung function in adults with CF.Aspergillus fumigatus (A.f.) est le champignon le plus fréquemment isolé dans les bronches des patients mucoviscidosiques. Il peut entrainer des atteintes respiratoires allant de la colonisation àl’aspergillose broncho-pulmonaire allergique (ABPA). Les données publiées portent essentiellement sur des populations pédiatriques. Les objectifs de cette étude étaient : (1) déterminer la prévalence des atteintes aspergillaires et leur impact sur la fonction respiratoire chez les adultes mucoviscidosiques, (2) étudier les performances du galactomannane (GM) et de la PCR A.f. dans les expectorations et (3) évaluer la pertinence clinique d’une nouvelle classification issue d’une analyse en clusters. Nous avons inclus 134 patients dans cette cohorte prospective. La sensibilisation aspergillaire était l’atteinte la plus fréquente (35,8%), devant l’ABPA qui était retrouvée chez 16,4% des patients. Toutes deux semblaient associées à un déclin plus rapide de la fonction respiratoire. La colonisation à A.f. n’était pas associée à une accélération du déclin de la fonction respiratoire. La culture fongique était positive pour A.f. chez 47% des patients alors que la PCR n’était positive que dans 35,8% des cas. Le dosage du GM dans les expectorations était positif chez 76,1% des patients et n’était pas spécifique d’une atteinte aspergillaire. Parmi les cinq phénotypes aspergillaires identifiés par l’analyse en clusters, ceux présentant des marqueurs d’hypersensibilité aspergillaire semblaient avoir un pronostic respiratoire plus défavorable. Ces résultats montrent que l’hypersensibilité induite par A.f serait un facteur de comorbidité chez les patients adultes mucoviscidosiques. Ils suggèrent que la colonisation à A.f. n’aurait pas d’impact sur la fonction respiratoire chez ces patients

CFTR Modulators in People with Cystic Fibrosis: Real-World Evidence in France

Cystic fibrosis (CF) is a rare genetic multisystemic disease, the manifestations of which are due to mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein and can lead to respiratory insufficiency and premature death. CFTR modulators, which were developed in the past decade, partially restore CFTR protein function. Their clinical efficacy has been demonstrated in phase 3 clinical trials, particularly in terms of lung function and pulmonary exacerbations, nutritional status, and quality of life in people with gating mutations (ivacaftor), homozygous for the F508del mutation (lumacaftor/ivacaftor and tezacaftor/ivacaftor), and in those with at least one F508del mutation (elexacaftor/tezacaftor/ivacaftor). However, many questions remain regarding their long-term safety and effectiveness, particularly in patients with advanced lung disease, liver disease, renal insufficiency, or problematic bacterial colonization. The impact of CFTR modulators on other important outcomes such as concurrent treatments, lung transplantation, chest imaging, or pregnancies also warrants further investigation. The French CF Reference Network includes 47 CF centers that contribute patient data to the comprehensive French CF Registry and have conducted nationwide real-world studies on CFTR modulators. This review seeks to summarize the results of these real-world studies and examine their findings against those of randomized control trials

CFTR Modulators in People with Cystic Fibrosis: Real-World Evidence in France

Cystic fibrosis (CF) is a rare genetic multisystemic disease, the manifestations of which are due to mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein and can lead to respiratory insufficiency and premature death. CFTR modulators, which were developed in the past decade, partially restore CFTR protein function. Their clinical efficacy has been demonstrated in phase 3 clinical trials, particularly in terms of lung function and pulmonary exacerbations, nutritional status, and quality of life in people with gating mutations (ivacaftor), homozygous for the F508del mutation (lumacaftor/ivacaftor and tezacaftor/ivacaftor), and in those with at least one F508del mutation (elexacaftor/tezacaftor/ivacaftor). However, many questions remain regarding their long-term safety and effectiveness, particularly in patients with advanced lung disease, liver disease, renal insufficiency, or problematic bacterial colonization. The impact of CFTR modulators on other important outcomes such as concurrent treatments, lung transplantation, chest imaging, or pregnancies also warrants further investigation. The French CF Reference Network includes 47 CF centers that contribute patient data to the comprehensive French CF Registry and have conducted nationwide real-world studies on CFTR modulators. This review seeks to summarize the results of these real-world studies and examine their findings against those of randomized control trials

Exploring the Role of Tertiary Lymphoid Structures Using a Mouse Model of Bacteria-Infected Lungs

International audienceAnimal models can be helpful tools for deciphering the generation, maintenance, and role of tertiary lymphoid structures (TLS) during infections or tumor development. We describe here the establishment of a persistent lung infection in immune-competent mice by intratracheal instillation of agarose beads containing Pseudomonas aeruginosa or Staphylococcus aureus bacteria. After instillation, animals develop a chronic pulmonary infection, marked by the presence of TLS. This experimental setting allows the study of the function of TLS induced by bacteria encountered in patients with cystic fibrosis (CF) as P. aeruginosa and S. aureus are the two main bacterial strains that infect bronchi of adult CF patients. Additionally, we describe also how to manipulate the immune response in these infected animals by targeting immune cells involved in TLS function. Overall, this approach makes it possible to explore the role of chronic inflammation in the induction and maintenance of TLS in infected tissues

Effective control of Staphylococcus aureus lung infection despite tertiary lymphoid structure disorganisation

Background: Tertiary lymphoid structures (TLS) are triggered by persistent bronchopulmonary infection with Staphylococcus aureus, but their roles remain elusive. The present study sought to examine the effects of B- and/or T-cell depletion on S. aureus infection and TLS development (lymphoid neogenesis) in mice.Methods: C57Bl/6 mice were pre-treated with 1) an anti-CD20 monoclonal antibody (mAb) (B-cell depletion) or 2) an anti-CD4 and/or an anti-CD8 mAb (T-cell depletion) or 3) a combination of anti-CD20, anti-CD4 and anti-CD8 mAbs (combined B- and T-cell depletion) or 4) isotype control mAbs. After lymphocyte depletion, mice were infected by intratracheal instillation of agarose beads containing S. aureus (106 CFU per mouse). 14 days later, bacterial load and lung inflammatory cell infiltration were assessed by cultures and immunohistochemistry, respectively.Results: 14 days after S. aureus-bead instillation, lung bacterial load was comparable between control and lymphocyte-depleted mice. While TLS were observed in the lungs of infected mice pre-treated with control mAbs, these structures were disorganised or abolished in the lungs of lymphocyte-depleted mice. The absence of CD20+ B-lymphocytes had no effect on CD3+ T-lymphocyte infiltration, whereas CD4+/CD8+ T-cell depletion markedly reduced CD20+ B-cell infiltration. Depletion of CD4+ or CD8+ T-cells separately had limited effect on B-cell infiltration, but led to the absence of germinal centres.Conclusion: TLS disorganisation is not associated with loss of infection control in mice persistently infected with S. aureus

Lung immunoglobulin A immunity dysregulation in cystic fibrosis.

In cystic fibrosis (CF), recurrent infections suggest impaired mucosal immunity but whether production of secretory immunoglobulin A (S-IgA) is impaired remains elusive. S-IgA is generated following polymeric immunoglobulin receptor (pIgR)-mediated transepithelial transport of dimeric (d-)IgA and represents a major defence through neutralisation of inhaled pathogens like Pseudomonas aeruginosa (Pa). Human lung tissue (n = 74), human sputum (n = 118), primary human bronchial epithelial cells (HBEC) (cultured in air-liquid interface) (n = 19) and mouse lung tissue and bronchoalveolar lavage were studied for pIgR expression, IgA secretion and regulation. Increased epithelial pIgR immunostaining was observed in CF lung explants, associated with more IgA-producing plasma cells, sputum and serum IgA, especially Pa-specific IgA. In contrast, pIgR and IgA transport were downregulated in F508del mice, CFTR-inhibited HBEC, and CF HBEC. Moreover, the unfolded protein response (UPR) due to F508del mutation, inhibited IgA transport in Calu-3 cells. Conversely, pIgR expression and IgA secretion were strongly upregulated following Pa lung infection in control and F508del mice, through an inflammatory host response involving interleukin-17. A complex regulation of IgA secretion occurs in the CF lung, UPR induced by CFTR mutation/dysfunction inhibiting d-IgA transcytosis, and Pa infection unexpectedly unleashing this secretory defence mechanism. This work was supported by the Forton's grant of the King Baudouin's Foundation, Belgium, the Fondazione Ricerca Fibrosi Cistica, Italy, and the Fonds National de la Recherche Scientifique, Belgium

D-Dimer Level and Neutrophils Count as Predictive and Prognostic Factors of Pulmonary Embolism in Severe Non-ICU COVID-19 Patients

International audienceThe incidence of pulmonary embolism (PE) is high during severe Coronavirus Disease 2019 (COVID-19). We aimed to identify predictive and prognostic factors of PE in non-ICU hospitalized COVID-19 patients. In the retrospective multicenter observational CLOTVID cohort, we enrolled patients with confirmed RT-PCR COVID-19 who were hospitalized in a medicine ward and also underwent a CT pulmonary angiography for a PE suspicion. Baseline data, laboratory biomarkers, treatments, and outcomes were collected. Predictive and prognostics factors of PE were identified by using logistic multivariate and by Cox regression models, respectively. A total of 174 patients were enrolled, among whom 86 (median [IQR] age of 66 years [55-77]) had post-admission PE suspicion, with 30/86 (34.9%) PE being confirmed. PE occurrence was independently associated with the lack of long-term anticoagulation or thromboprophylaxis (OR [95%CI], 72.3 [3.6-4384.8]) D-dimers ≥ 2000 ng/mL (26.3 [4.1-537.8]) and neutrophils ≥ 7.0 G/L (5.8 [1.4-29.5]). The presence of these two biomarkers was associated with a higher risk of PE (p = 0.0002) and death or ICU transfer (HR [95%CI], 12.9 [2.5-67.8], p < 0.01). In hospitalized non-ICU severe COVID-19 patients with clinical PE suspicion, the lack of anticoagulation, D-dimers ≥ 2000 ng/mL, neutrophils ≥ 7.0 G/L, and these two biomarkers combined might be useful predictive markers of PE and prognosis, respectively