Implications fonctionnelles des progéniteurs endothéliaux circulants dans la fibrose pulmonaire idiopathique

Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial pneumonia. It is characterized by progressive destruction of lung alveolar architecture and its replacement by fibrotic tissue. Consequences are pulmonary functions alterations. IPF is constantly associated to an important lung vascular remodelling associating an abnormal neoangiogenesis close to fibrotic areas and a major vascular rarefaction in fibrotic areas. An hypothesis that may explain this vascular remodelling is based on Endothelial colony-forming Cells (ECFCs) involvement. They are a subset of Endothelial Progenitor Cells (EPCs). ECFCs are vasculogenic cells coming from bone marrow or vessels and they are considered as a liquid biopsy of the pulmonary endothelial compartment. All EPCs subtypes have been described to be reduced in IPF but their modified phenotype could be involved in abnormal angiogenic process and/or fibrogenesis. Besides, ECFCs may represent a population of interest as a cell therapy product of IPF: normalization of lung vasculature during fibrogenesis should improve gas exchange by genesis of new stable vessels. This work is divided in two parts. First, we characterized ECFCs isolated from peripheral blood of patients with IPF (IPF-ECFCs) to clarify their potential involvement in the pathophysiology of the disease. Second, we injected ECFCs isolated from cord-blood or peripheral blood of IPF patients to study their ability to modulate lung fibrosis in a murine model of bleomycin-induced lung fibrosis. We confirmed the endothelial phenotype of IPF-ECFCs and show that their vasculogenic properties are similar to those of ECFCs isolated from cord-blood (CB-ECFCs) or peripheral blood of adult controls (PB-ECFCs). However, senescence and apoptotic states were increased in IPF-ECFCs compared to controls. Study of the three cell types secretome, focusing on angiogenesis and « senescent associated-secretory phenotype » (SASP) mediators, has shown a significant increase of interleukin-8 (IL-8) in conditioned medium of IPF-ECFCs compared to PB-ECFCs. We demonstrated that conditioned medium of IPF-ECFCs, depending on IL-8, triggers neutrophils migration in two models in vitro and in vivo. Last, there is an increase of infiltration by neutrophils in lung biopsies of patients with IPF compared to control biopsies. Finally, in a cohort of IPF patients, we have shown that an increased level of neutrophils at inclusion was associated to a poor prognosis, confirming potential clinical relevance of IL-8 overexpression in IPF. In the second part of this work, we injected CB-ECFCs in a murine model of bleomycin-induced lung fibrosis in immunodepressed lung mice. We studied ECFC involvement in fibrogenesis and also in constituted fibrosis. We did not found any effect of CB-ECFCs on fibrotic or angiogenic processes in this model evaluated by mortality rate, weight evolution, CT scan, histomorphometry measure of lung fibrosis and microvascular density. Injection of IPF-ECFCs led to similar results. Thus, IPF-ECFCs seems to play a paracrine role in IPF, similar to what has been descried in other diseases. Increased secretion of IL-8, reflect of enhanced senescence, could participate to the recruitment of neutrophils into the lung.La Fibrose pulmonaire idiopathique (FPI) est la plus courante des pneumopathies interstitielles. Elle est caractérisée par une destruction progressive de l'architecture alvéolaire pulmonaire et son remplacement par du tissu fibrotique. La FPI est constamment associée à un important remodelage vasculaire pulmonaire associant une néoangiogenèse aberrante en bordure des zones de fibrose et une raréfaction vasculaire majeure dans les zones fibrotiques. Une des hypothèses pouvant expliquer ce remodelage vasculaire repose sur l'implication des ECFCs ou Endothelial colony-forming Cells, un sous-groupe des progéniteurs endothéliaux circulants (PECs). Les ECFCs sont des cellules vasculogéniques originaires de la moelle osseuse ou des parois vasculaires. Les différents types de PECs sont diminués au cours de la FPI mais ils pourraient présenter un phénotype modifié qui, en interférant avec la vasculogenèse et/ou le microenvironnement pulmonaire, pourrait constituer un processus actif modulant la fibrogenèse. De plus, des ECFCs pourraient représenter une population d'intérêt dans un contexte de thérapie cellulaire de la FPI : la normalisation de la vascularisation au cours de la fibrogenèse pourrait permettre une amélioration des échanges gazeux par la genèse de nouveaux vaisseaux sains et stables. Ce travail se découpe en deux axes principaux. D'une part, la caractérisation d'ECFCs isolées de prélèvements sanguins de patients FPI (IPF-ECFCs), considérées comme une biopsie liquide du compartiment endothélial pulmonaire, afin d'éclaircir leur potentiel rôle dans la physiopathologie de la FPI. De l'autre, l'injection d'ECFCs isolées de sang de cordon ou de prélèvements sanguins de patients atteints de FPI afin d'étudier leur aptitude à moduler la fibrose pulmonaire dans un modèle murin induit par la bléomycine. Nous avons ainsi confirmé le caractère endothélial des IPF-ECFCs et montré que leurs propriétés vasculogéniques sont similaires à celles d'ECFCs témoins isolées de sang de cordon (CB-ECFCs) ou de sang périphérique de témoins adultes (PB-ECFCs). En revanche, nous avons mis en évidence une augmentation de la sénescence et de l'apoptose des IPF-ECFCs par rapport aux contrôles. L'étude du sécrétome des trois types cellulaires, en nous focalisant sur les médiateurs de l'angiogenèse et du « senescent associated-secretory phenotype » (SASP) ,a montré une augmentation significative du taux d'interleukine-8 (IL-8) dans le milieu conditionné d'IPF-ECFCs par rapport aux PB-ECFCs. Le milieu conditionné d'IPF-ECFCs induit la migration des polynucléaires neutrophiles (PNNs) de manière IL-8 dépendante dans deux modèles in vitro et in vivo. De plus, une augmentation de l'infiltration par les PNNs a été mise en évidence dans des biopsies pulmonaires de patients atteints de FPI par rapport à des biopsies contrôles. Enfin, dans une cohorte de patients FPI, il a été montré qu'une augmentation du taux de PNNs à l'inclusion était associée à un pronostic défavorable, ce qui confirme la pertinence de la surexpression de l'IL-8 dans la FPI. Dans la seconde partie de ce travail, nous avons injecté des CB-ECFCs dans un modèle murin de fibrose pulmonaire induite par la bléomycine chez la souris immunodéprimée Nude en nous intéressant à la fois à la formation de la fibrose et à la fibrose constituée. Nous n'avons pas mis en évidence de modulation des processus fibrotiques ou angiogéniques par les CB-ECFCs dans ce modèle évalué via le taux de mortalité, l'évolution du poids, la mesure de la fibrose pulmonaire par microCT et histomorphométrie et la densité microbvasculaire. L'injection d'IPF-ECFCs a conduit à des résultats similaires. Ainsi, le phénotype modifié des IPF-ECFCs pourrait être le reflet du rôle paracrine possiblement joué par ces cellules dans la physiopathologie de la FPI. La sécrétion augmentée d'IL-8, reflet d'une sénescence accrue, participerait au recrutement des PNNs au niveau pulmonaire

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies

International audienceSignificance There is growing evidence that preexisting autoantibodies neutralizing type I interferons (IFNs) are strong determinants of life-threatening COVID-19 pneumonia. It is important to estimate their quantitative impact on COVID-19 mortality upon SARS-CoV-2 infection, by age and sex, as both the prevalence of these autoantibodies and the risk of COVID-19 death increase with age and are higher in men. Using an unvaccinated sample of 1,261 deceased patients and 34,159 individuals from the general population, we found that autoantibodies against type I IFNs strongly increased the SARS-CoV-2 infection fatality rate at all ages, in both men and women. Autoantibodies against type I IFNs are strong and common predictors of life-threatening COVID-19. Testing for these autoantibodies should be considered in the general population

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies

International audienceSignificance There is growing evidence that preexisting autoantibodies neutralizing type I interferons (IFNs) are strong determinants of life-threatening COVID-19 pneumonia. It is important to estimate their quantitative impact on COVID-19 mortality upon SARS-CoV-2 infection, by age and sex, as both the prevalence of these autoantibodies and the risk of COVID-19 death increase with age and are higher in men. Using an unvaccinated sample of 1,261 deceased patients and 34,159 individuals from the general population, we found that autoantibodies against type I IFNs strongly increased the SARS-CoV-2 infection fatality rate at all ages, in both men and women. Autoantibodies against type I IFNs are strong and common predictors of life-threatening COVID-19. Testing for these autoantibodies should be considered in the general population