Cellular senescence and pathophysiology of chronic lung diseases : application to chronic obstructive pulmonary disease (COPD)

L'accumulation de cellules sénescentes (CS) est considérée comme un processus pathologique majeur impliqué dans la physiopathologie de plusieurs maladies pulmonaires parmi lesquelles la bronchopneumopathie chronique obstructive (BPCO) et la fibrose pulmonaire. Les CS sont stables, elles peuvent survivre in vivo ou in vitro tout en perdant leur capacité de réparation, mais également en surexprimant certains médiateurs agissant sur les tissus environnants et appelé SASP (senescence associated secretory phenotype). Les CS pulmonaires contribuent ainsi au défaut de réparation tissulaire et, via leur SASP, à des altérations tissulaires aussi variées que à l'inflammation, l'emphysème, ou l'hypertension artérielle pulmonaire (HTAP). Limiter la sénescence cellulaire et le SASP représente ainsi un objectif thérapeutique majeur dans la BPCO.Au cours de ma thèse, j’ai exploré les mécanismes conduisant à la senescence cellulaire dans la BPCO en utilisant une double approche : études sur tissus et cellules humaines couplées à l’étude d’animaux transgéniques.La première étude à laquelle j’ai contribué porte sur le rôle de la voie de signalisation mTOR dans la sénescence cellulaire. Nous avons montré une activation des membres de cette voie dans les poumons et les cellules en culture (cellules musculaires lisses et cellules endothéliales) de patients BPCO en comparaison avec des sujets témoins, et montré que l’inhibition de mTOR par la rapamycine protège de la sénescence cellulaire. Nous avons évalué les conséquences de l'activation de mTOR, induite chez des souris transgéniques par la délétion inductible du régulateur négatif du complexe 1 de mTOR (TSC1) dans les cellules endothéliales et les cellules épithéliale alvéolaires. Ces souris développent un emphysème pulmonaire et une HTAP en 3 mois, montrant un lien étroit entre la voie mTOR, l’accumulation de CS et l’emphysème.La seconde étude porte sur le rôle du stress oxydant présent dans les poumons de patients BPCO, et associé à une réduction des facteurs de transcription junD et Nrf2. J’ai étudié des souris déficientes pour JunD (JunD-KO) présentant un stress oxydant chronique et une accumulation de CS en comparaison avec des souris témoins à l’âge de 4 mois et 12-18 mois, traitées ou non par l’antioxydant N-acétylcystéine (NAC). Nous avons observé une réduction du stress oxydant par le NAC, avec une diminution des dommages à l’ADN, du nombre de CS, et de l’emphysème pulmonaire. Ces effets, cependant, ont lieu de façon contemporaine à la survenue d’adénocarcinome pulmonaire chez 50% des souris JunD-KO et 10% des souris témoins à l’âge de 12-18 mois. Seules les souris traitées par le NAC développent un cancer. L’hypothèse est que le NAC crée un échappement à la sénescence cellulaire et favorise la transformation cellulaire, ce qui semble pouvoir être reproduit in vitro sur des fibroblastes embryonnaires de souris JunD-KO et témoins.Dans une troisième étude, j’ai montré que l’exposition de souris à l’hypoxie chronique induit une senescence pulmonaire qui s’accompagne d’une HTAP et un léger emphysème. Pour évaluer les effets des CS dans ce modèle, j’ai utilisé des souris permettant l’inactivation de p16 (délétion de p16 chez les souris homozygotes p16-luciférase) ou l’élimination des CS (souris p16-ATTAC dans lesquelles le gène suicide ATTAC est activable par le rapalog AP20187). Nos résultats montrent que l’inactivation de p16 et l’élimination des CS aggravent l’HTAP hypoxique malgré une réduction de l’inflammation pulmonaire. Une analyse RNAseq est en cours pour comprendre le mécanisme.Ces résultats montrent que les CS jouent un rôle majeur dans les altérations structurales des vaisseaux et du parenchyme pulmonaire au cours de la BPCO. Limiter la sénescence cellulaire peut être bénéfique, mais peut également, dans le cas du NAC, favoriser l’émergence cancer pulmonaire.Accumulation of senescent cells (SC) is considered as a major pathological process in the pathogenesis of various lung diseases including chronic obstructive pulmonary disease (COPD) and lung fibrosis. SCs are stable, can survive in vitro and in vivo while losing their reparative capacity. They also overexpress mediators acting on the surrounding tissue, a phenomenon called SASP (senescence-associated secretory phenotype). Lung SCs contribute to the loss of tissue repair and, through their SASP, to tissue alteration including inflammation, emphysema or pulmonary arterial hypertension (PAH). Limiting cellular senescence and the SASP thus represents a major therapeutic possibility in COPD.During my PhD I explored mechanisms leading to cellular senescence in COPD using human tissue and cells, coupled with studies of transgenic mice.The first study focused on the role of the mTOR pathway in cellular senescence. We have shown an activation of this pathway in lungs and cells in culture (smooth muscle and endothelial cells) from COPD patients compared to control subjects, and showed that inhibition of mTOR by Rapamycin protects from cellular senescence. Specifically, we assessed the consequences of mTOR activation in mice with the deletion of mTOR negative regulator TSC1 in endothelial and epithelial cells. These mice develop lung emphysema and PAH within 3 months, showing a tight link between mTOR pathway, accumulation of SCs and emphysema.The second study focused on oxidative stress in lungs of COPD patients. This oxidative stress was associated with downregulation of JunD and NRF2 transcription factors. I studied mice lacking JunD (JunD-KO) exerting a chronic oxidative stress and SCs accumulation, at 4 as well as 12-18 months of age, treated with the antioxidant N-acetylcyteine (NAC) or vehicle. We observed a reduction in oxidative stress by NAC treatment, along with a diminution of DNA damage, SCs accumulation and emphysema. However NAC treatment also induced the development of lung adenocarcinoma in 50% of JunD-KO and 10% in control mice at the age of 12-18 month. Remarkably, only mice treated with NAC developed cancer. Based on these observations we speculated that NAC may lead to an escape from cellular senescence, a finding conformed in embryonic fibroblasts obtained from JunD-KO and control mice in vitro.In the third study, I showed that exposure of mice to chronic hypoxia leads to pulmonary senescence along with PAH and mild emphysema. To study the role of SCs in this model I used mice allowing p16 inactivation (p16 deletion in p16-luciferase homozygous mice) or elimination of SCs (p16-ATTAC mice harboring the ATTAC suicide gene, inducible with rapalog AP20187). Our results showed that both inactivation of p16 and elimination of SCs worsened hypoxic PAH despite a reduction in pulmonary inflammation. An RNA-Seq study is ongoing to understand the underlying mechanism.Collectively, these results demonstrated that SCs play a critical role in vascular and parenchymal structural remodeling in COPD. Limiting cellular senescence can be beneficial, but also, in the case of NAC, favors the emergence of lung cancer

Le traitement antioxydant protège contre l’emphysème mais favorise la survenue et la progression du cancer du poumon

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Role of interleukin-1 receptor 1/MyD88 signalling in the development and progression of pulmonary hypertension

Pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation are key components of pulmonary arterial hypertension (PAH). Interleukin (IL)-1β binds to IL-1 receptor (R)1, thereby recruiting the molecular adaptor myeloid differentiation primary response protein 88 (MyD88) (involved in IL-1R1 and Toll-like receptor signal transduction) and inducing IL-1, IL-6 and tumour necrosis factor-α synthesis through nuclear factor-κB activation.We investigated the IL-1R1/MyD88 pathway in the pathogenesis of pulmonary hypertension.Marked IL-1R1 and MyD88 expression with predominant PA-SMC immunostaining was found in lungs from patients with idiopathic PAH, mice with hypoxia-induced pulmonary hypertension and SM22-5-HTT(+) mice. Elevations in lung IL-1β, IL-1R1, MyD88 and IL-6 preceded pulmonary hypertension in hypoxic mice. IL-1R1(-/-), MyD88(-/-) and control mice given the IL-1R1 antagonist anakinra were protected similarly against hypoxic pulmonary hypertension and perivascular macrophage recruitment. Anakinra reversed pulmonary hypertension partially in SM22-5-HTT(+) mice and markedly in monocrotaline-treated rats. IL-1β-mediated stimulation of mouse PA-SMC growth was abolished by anakinra and absent in IL-1R1(-/-) and MyD88(-/-) mice. Gene deletion confined to the myeloid lineage (M.lys-Cre MyD88(fl/fl) mice) decreased pulmonary hypertension severity versus controls, suggesting IL-1β-mediated effects on PA-SMCs and macrophages. The growth-promoting effect of media conditioned by M1 or M2 macrophages from M.lys-Cre MyD88(fl/fl) mice was attenuated.Pulmonary vessel remodelling and inflammation during pulmonary hypertension require IL-1R1/MyD88 signalling. Targeting the IL-1β/IL-1R1 pathway may hold promise for treating human PAH.status: publishe

CCR2/CCR5-mediated macrophage-smooth muscle cell crosstalk in pulmonary hypertension

Macrophages are major players in the pathogenesis of pulmonary arterial hypertension (PAH).To investigate whether lung macrophages and pulmonary-artery smooth muscle cells (PASMCs) collaborate to stimulate PASMC growth and whether the CCL2-CCR2 and CCL5-CCR5 pathways inhibited macrophage-PASMC interactions and PAH development, we used human CCR5-knock-in mice and PASMCs from patients with PAH and controls.Conditioned media from murine M1 or M2 macrophages stimulated PASMC growth. This effect was markedly amplified with conditioned media from M2 macrophage/PASMC co-cultures. CCR2, CCR5, CCL2 and CCL5 were upregulated in macrophage/PASMC co-cultures. Compared to inhibiting either receptor, dual CCR2 and CCR5 inhibition more strongly attenuated the growth-promoting effect of conditioned media from M2-macrophage/PASMC co-cultures. Deleting either CCR2 or CCR5 in macrophages or PASMCs attenuated the growth response. In mice with hypoxia- or SUGEN/hypoxia-induced PH, targeting both CCR2 and CCR5 prevented or reversed PH more efficiently than targeting either receptor alone. Patients with PAH exhibited CCR2 and CCR5 upregulation in PASMCs and perivascular macrophages compared to controls. The PASMC growth-promoting effect of conditioned media from M2-macrophage/PASMC co-cultures was greater when PASMCs from PAH patients were used in the co-cultures or as the target cells and was dependent on CCR2 and CCR5. PASMC migration toward M2-macrophages was greater with PASMCs from PAH patients and was attenuated by blocking CCR2 and CCR5.CCR2 and CCR5 are required for collaboration between macrophages and PASMCs to initiate and amplify PASMC migration and proliferation during PAH development. Dual targeting of CCR2 and CCR5 may hold promise for treating human PAH.status: publishe

CCR2/CCR5-mediated macrophage–smooth muscle cell crosstalk in pulmonary hypertension

International audienceMacrophages are major players in the pathogenesis of pulmonary arterial hypertension (PAH).To investigate whether lung macrophages and pulmonary-artery smooth muscle cells (PASMCs) collaborate to stimulate PASMC growth and whether the CCL2-CCR2 and CCL5-CCR5 pathways inhibited macrophage-PASMC interactions and PAH development, we used human CCR5-knock-in mice and PASMCs from patients with PAH and controls.Conditioned media from murine M1 or M2 macrophages stimulated PASMC growth. This effect was markedly amplified with conditioned media from M2 macrophage/PASMC co-cultures. CCR2, CCR5, CCL2 and CCL5 were upregulated in macrophage/PASMC co-cultures. Compared to inhibiting either receptor, dual CCR2 and CCR5 inhibition more strongly attenuated the growth-promoting effect of conditioned media from M2-macrophage/PASMC co-cultures. Deleting either CCR2 or CCR5 in macrophages or PASMCs attenuated the growth response. In mice with hypoxia- or SUGEN/hypoxia-induced PH, targeting both CCR2 and CCR5 prevented or reversed PH more efficiently than targeting either receptor alone. Patients with PAH exhibited CCR2 and CCR5 upregulation in PASMCs and perivascular macrophages compared to controls. The PASMC growth-promoting effect of conditioned media from M2-macrophage/PASMC co-cultures was greater when PASMCs from PAH patients were used in the co-cultures or as the target cells and was dependent on CCR2 and CCR5. PASMC migration toward M2-macrophages was greater with PASMCs from PAH patients and was attenuated by blocking CCR2 and CCR5.CCR2 and CCR5 are required for collaboration between macrophages and PASMCs to initiate and amplify PASMC migration and proliferation during PAH development. Dual targeting of CCR2 and CCR5 may hold promise for treating human PAH

The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice

International audienceOxidative stress is a major contributor to chronic lung diseases. Antioxidants such as N-acetylcysteine (NAC) are broadly viewed as protective molecules that prevent the mutagenic effects of reactive oxygen species. Antioxidants may, however, increase the risk of some forms of cancer and accelerate lung cancer progression in murine models. Here, we investigated chronic NAC treatment in aging mice displaying lung oxidative stress and cell senescence due to inactivation of the transcription factor JunD, which is downregulated in diseased human lungs. NAC treatment decreased lung oxidative damage and cell senescence and protected from lung emphysema but concomitantly induced the development of lung adenocarcinoma in 50% of JunD-deficient mice and 10% of aged control mice. This finding constitutes the first evidence to our knowledge of a carcinogenic effect of antioxidant therapy in the lungs of aged mice with chronic lung oxidative stress and warrants the utmost caution when considering the therapeutic use of antioxidants

Telomerase Prevents Emphysema in Old Mice by Sustaining Subpopulations of Endothelial and AT2 Cells

Abstract Accumulation of senescent cells has been causally linked to the development of age-related pathologies. Here, we characterized a new mouse model (p21 +/Tert ) whose telomerase (TERT) is expressed from the p21 promoter that can be activated in response to telomere dysfunction. Lung parenchyma from p21 +/Tert old mice accumulated fewer senescent cells with age and this correlated with a reduction in age-related alveolar space enlargement, a feature of pulmonary emphysema. This protection against emphysema depends on TERT catalytic activity and is associated with increased proliferation of pulmonary endothelial cells (EC) and capillary density. Single-cell RNA sequencing of lung cells revealed that TERT expression was associated with the enrichment of ECs expressing genes involved in vessel regeneration and in AT2 cells overexpressing S/G2M markers. These findings indicate that p21-promoter-dependent expression of catalytically active telomerase prevents emphysema by sustaining the proliferation of subclasses of EC and AT2 cells

Phospholipase A2 receptor 1 promotes lung cell senescence and emphysema in obstructive lung disease

International audienceBackground Cell senescence is a key process in age-associated dysfunction and diseases, notably chronic obstructive pulmonary disease (COPD). We previously identified phospholipase A2 receptor 1 (PLA2R1) as a positive regulator of cell senescence acting via Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling. Its role in pathology, however, remains unknown. Here, we assessed PLA2R1-induced senescence in COPD and lung emphysema pathogenesis. Methods We assessed cell senescence in lungs and cultured lung cells from patients with COPD and controls subjected to PLA2R1 knockdown, PLA2R1 gene transduction and treatment with the JAK1/2 inhibitor ruxolitinib. To assess whether PLA2R1 upregulation caused lung lesions, we developed transgenic mice overexpressing PLA2R1 ( PLA2R1 -TG) and intratracheally injected wild-type mice with a lentiviral vector carrying the Pla2r1 gene (LV- PLA2R1 mice). Results We found that PLA2R1 was overexpressed in various cell types exhibiting senescence characteristics in COPD lungs. PLA2R1 knockdown extended the population doubling capacity of these cells and inhibited their pro-inflammatory senescence-associated secretory phenotype (SASP). PLA2R1-mediated cell senescence in COPD was largely reversed by treatment with the potent JAK1/2 inhibitor ruxolitinib. Five-month-old PLA2R1 -TG mice exhibited lung cell senescence, and developed lung emphysema and lung fibrosis together with pulmonary hypertension. Treatment with ruxolitinib induced reversal of lung emphysema and fibrosis. LV- PLA2R1 -treated mice developed lung emphysema within 4 weeks and this was markedly attenuated by concomitant ruxolitinib treatment. Conclusions Our data support a major role for PLA2R1 activation in driving lung cell senescence and lung alterations in COPD. Targeting JAK1/2 may represent a promising therapeutic approach for COPD

Induction of telomerase in p21-positive cells counteracts capillaries rarefaction in aging mice lung

ABSTRACT Telomerase is required for long-term cell proliferation and linked to stem cells. This is evident in the lung where short telomeres are associated with lung dysfunction. We constructed a mouse model in which the telomerase ( Tert ) is expressed from the p21 Cdkn1a promoter. We found that this peculiar Tert expression curb age-related emphysema and pulmonary perivascular fibrosis in old mice. In old mice lungs, such Tert expression preferentially occurs in endothelial cells where it reduces the number of senescent endothelial cells. Remarkably, we report that Tert counteracts the age-related decline in capillary density. This was associated with an increased number of Cd34+ cells identified as a subclass of capillary cells with proliferative capacity. Expression of catalytically inactive Tert neither prevents the decline of capillary density in old mice nor protects against age-related emphysema and fibrosis. These findings reveal that telomerase decreases age-decline of pulmonary functions by sustaining microvasculature regeneration and outgrowth