Lung inflammation and interstitial fibrosis by targeted alveolar epithelial type I cell death

IntroductionThe pathogenesis of chronic lung diseases is multifaceted with a major role of recurrent micro-injuries of the epithelium. While several reports clearly indicated a prominent role for surfactant-producing alveolar epithelial type 2 (AT2) cells, the contribution of gas exchange-permissive alveolar epithelial type 1 (AT1) cells has not been addressed yet. Here, we investigated whether repeated injury of AT1 cells leads to inflammation and interstitial fibrosis.MethodsWe chose an inducible model of AT1 cell depletion following local diphtheria toxin (DT) administration using an iDTR flox/flox (idTRfl/fl) X Aquaporin 5CRE (Aqp5CRE) transgenic mouse strain.ResultsWe investigated repeated doses and intervals of DT to induce cell death of AT1 cells causing inflammation and interstitial fibrosis. We found that repeated DT administrations at 1ng in iDTRfl/fl X Aqp5CRE mice cause AT1 cell death leading to inflammation, increased tissue repair markers and interstitial pulmonary fibrosis.DiscussionTogether, we demonstrate that depletion of AT1 cells using repeated injury represents a novel approach to investigate chronic lung inflammatory diseases and to identify new therapeutic targets

NLRP6 controls pulmonary inflammation from cigarette smoke in a gut microbiota-dependent manner

Chronic obstructive pulmonary disease (COPD) is a major health issue primarily caused by cigarette smoke (CS) and characterized by breathlessness and repeated airway inflammation. NLRP6 is a cytosolic innate receptor controlling intestinal inflammation and orchestrating the colonic host–microbial interface. However, its roles in the lungs remain largely unexplored. Using CS exposure models, our data show that airway inflammation is strongly impaired in Nlrp6-deficient mice with drastically fewer recruited neutrophils, a key cell subset in inflammation and COPD. We found that NLRP6 expression in lung epithelial cells is important to control airway and lung tissue inflammation in an inflammasome-dependent manner. Since gut-derived metabolites regulate NLRP6 inflammasome activation in intestinal epithelial cells, we investigated the link between NLRP6, CS-driven lung inflammation, and gut microbiota composition. We report that acute CS exposure alters gut microbiota in both wild-type (WT) and Nlrp6-deficient mice and that antibiotic treatment decreases CS-induced lung inflammation. In addition, gut microbiota transfer from dysbiotic Nlrp6-deficient mice to WT mice decreased airway lung inflammation in WT mice, highlighting an NLRP6-dependent gut-to-lung axis controlling pulmonary inflammation

Etude de l’implication des récepteurs NLRP3 et NLRP6 ainsi que de la Gasdermine D dans la réponse inflammatoire pulmonaire induite par l’exposition à la fumée de cigarette ou à l’ozone

In our project, we studied NOD-like receptor family, pyrin domain containing 3 and 6 (NLRP3 and NLRP6) and Gasdermin D implications in smoke cigarette or ozone induced pulmonary inflammation in mice. First, we showed that NLRP3 is involved in lung inflammation induced by smoke cigarette exposure and deficient mice (Nlrp3⁻/⁻) or wild type treated with NLRP3 inhibitor display a reduced inflammation.Gasdermin D activation in bronchial epithelial cells and airway recruited macrophages depends on NLRP3. In a second part, we highlighted NLRP6 is involved in lung inflammation induced in by ozone exposure and deficient mice ( (Nlrp6⁻/⁻) display a decrease of inflammation, fibrotic foci and emphysema. Gasdermin D activation in type 1 pneumocytes and a reduced proportion of airway recruited macrophages depends on NLRP6 and caspase 11 activation.Dans le cadre de notre projet, nous avons étudié l'implication des récepteurs NOD-like receptor family, pyrin domain containing 3 et 6 (NLRP3 et NLRP6) et de la Gasdermine D dans l'inflammation pulmonaire induite par une exposition à la fumée de cigarette d'une part et à l'ozone d'autre part, chez la souris. Dans une première partie, nous avons montré que l'inflammation pulmonaire induite par une exposition à la fumée de cigarette était dépendante de NLRP3 puisque les souris déficientes (Nlrp3⁻/⁻) et sauvages traitées avec un inhibiteur de NLRP3 ont une inflammation réduite. L'activation de la Gasdermine D dans les cellules épithéliales bronchiques et les macrophages recrutés au niveau de l'espace bronchoalvéolaire dépend de NLRP3. Dans une seconde partie, nous avons mis en évidence que l'inflammation pulmonaire induite par une exposition à l'ozone était dépendante de NLRP6 puisque les souris déficientes (Nlrp6⁻/⁻) ont une diminution de cette inflammation, des paramètres de fibrose et d'emphysème. L'activation de la Gasdermine D dans les pneumocytes de type 1 et une proportion réduite de macrophages recrutés dans l'espace bronchoalvéolaire dépend de NLRP6 et de l'activation de la caspase 11

Etude de l’implication des récepteurs NLRP3 et NLRP6 ainsi que de la Gasdermine D dans la réponse inflammatoire pulmonaire induite par l’exposition à la fumée de cigarette ou à l’ozone

In our project, we studied NOD-like receptor family, pyrin domain containing 3 and 6 (NLRP3 and NLRP6) and Gasdermin D implications in smoke cigarette or ozone induced pulmonary inflammation in mice. First, we showed that NLRP3 is involved in lung inflammation induced by smoke cigarette exposure and deficient mice (Nlrp3⁻/⁻) or wild type treated with NLRP3 inhibitor display a reduced inflammation.Gasdermin D activation in bronchial epithelial cells and airway recruited macrophages depends on NLRP3. In a second part, we highlighted NLRP6 is involved in lung inflammation induced in by ozone exposure and deficient mice ( (Nlrp6⁻/⁻) display a decrease of inflammation, fibrotic foci and emphysema. Gasdermin D activation in type 1 pneumocytes and a reduced proportion of airway recruited macrophages depends on NLRP6 and caspase 11 activation.Dans le cadre de notre projet, nous avons étudié l'implication des récepteurs NOD-like receptor family, pyrin domain containing 3 et 6 (NLRP3 et NLRP6) et de la Gasdermine D dans l'inflammation pulmonaire induite par une exposition à la fumée de cigarette d'une part et à l'ozone d'autre part, chez la souris. Dans une première partie, nous avons montré que l'inflammation pulmonaire induite par une exposition à la fumée de cigarette était dépendante de NLRP3 puisque les souris déficientes (Nlrp3⁻/⁻) et sauvages traitées avec un inhibiteur de NLRP3 ont une inflammation réduite. L'activation de la Gasdermine D dans les cellules épithéliales bronchiques et les macrophages recrutés au niveau de l'espace bronchoalvéolaire dépend de NLRP3. Dans une seconde partie, nous avons mis en évidence que l'inflammation pulmonaire induite par une exposition à l'ozone était dépendante de NLRP6 puisque les souris déficientes (Nlrp6⁻/⁻) ont une diminution de cette inflammation, des paramètres de fibrose et d'emphysème. L'activation de la Gasdermine D dans les pneumocytes de type 1 et une proportion réduite de macrophages recrutés dans l'espace bronchoalvéolaire dépend de NLRP6 et de l'activation de la caspase 11

Study of NLRP3 and NLRP6 receptors as well as Gasdermin D involvement in smoke cigarette or ozone induced pulmonary inflammation

Dans le cadre de notre projet, nous avons étudié l'implication des récepteurs NOD-like receptor family, pyrin domain containing 3 et 6 (NLRP3 et NLRP6) et de la Gasdermine D dans l'inflammation pulmonaire induite par une exposition à la fumée de cigarette d'une part et à l'ozone d'autre part, chez la souris. Dans une première partie, nous avons montré que l'inflammation pulmonaire induite par une exposition à la fumée de cigarette était dépendante de NLRP3 puisque les souris déficientes (Nlrp3⁻/⁻) et sauvages traitées avec un inhibiteur de NLRP3 ont une inflammation réduite. L'activation de la Gasdermine D dans les cellules épithéliales bronchiques et les macrophages recrutés au niveau de l'espace bronchoalvéolaire dépend de NLRP3. Dans une seconde partie, nous avons mis en évidence que l'inflammation pulmonaire induite par une exposition à l'ozone était dépendante de NLRP6 puisque les souris déficientes (Nlrp6⁻/⁻) ont une diminution de cette inflammation, des paramètres de fibrose et d'emphysème. L'activation de la Gasdermine D dans les pneumocytes de type 1 et une proportion réduite de macrophages recrutés dans l'espace bronchoalvéolaire dépend de NLRP6 et de l'activation de la caspase 11.In our project, we studied NOD-like receptor family, pyrin domain containing 3 and 6 (NLRP3 and NLRP6) and Gasdermin D implications in smoke cigarette or ozone induced pulmonary inflammation in mice. First, we showed that NLRP3 is involved in lung inflammation induced by smoke cigarette exposure and deficient mice (Nlrp3⁻/⁻) or wild type treated with NLRP3 inhibitor display a reduced inflammation.Gasdermin D activation in bronchial epithelial cells and airway recruited macrophages depends on NLRP3. In a second part, we highlighted NLRP6 is involved in lung inflammation induced in by ozone exposure and deficient mice ( (Nlrp6⁻/⁻) display a decrease of inflammation, fibrotic foci and emphysema. Gasdermin D activation in type 1 pneumocytes and a reduced proportion of airway recruited macrophages depends on NLRP6 and caspase 11 activation

Protective role of the nucleic acid sensor STING in pulmonary fibrosis Running title: STING protects against IPF

International audienceIdiopathic pulmonary fibrosis (IPF) is the most common and severe type of interstitial lung disease for which current treatments display limited efficacy. IPF is largely driven by host-derived danger signals released upon recurrent local tissue damage. Here we explored the roles of self-DNA and stimulator of interferon genes (STING), a protein belonging to an intracellular DNA sensing pathway that leads to type I and/or type III interferon (IFN) production upon activation. Using a mouse model of IPF, we report that STING deficiency leads to exacerbated pulmonary fibrosis with increased collagen deposition in the lungs and excessive remodeling factors expression. We further show that STING-mediated protection does not rely on type I IFN signaling nor on IL-17A or TGF-β modulation but is associated with dysregulated neutrophils. Together, our data support an unprecedented immunoregulatory function of STING in lung fibrosis

Protective Role of the Nucleic Acid Sensor STING in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common and severe type of interstitial lung disease for which current treatments display limited efficacy. IPF is largely driven by host-derived danger signals released upon recurrent local tissue damage. Here we explored the roles of self-DNA and stimulator of interferon genes (STING), a protein belonging to an intracellular DNA sensing pathway that leads to type I and/or type III interferon (IFN) production upon activation. Using a mouse model of IPF, we report that STING deficiency leads to exacerbated pulmonary fibrosis with increased collagen deposition in the lungs and excessive remodeling factors expression. We further show that STING-mediated protection does not rely on type I IFN signaling nor on IL-17A or TGF-β modulation but is associated with dysregulated neutrophils. Together, our data support an unprecedented immunoregulatory function of STING in lung fibrosis.</jats:p

Cigarette smoke-induced gasdermin D activation in bronchoalveolar macrophages and bronchial epithelial cells dependently on NLRP3.

Chronic pulmonary inflammation and chronic obstructive pulmonary disease (COPD) are major health issues largely due to air pollution and cigarette smoke (CS) exposure. The role of the innate receptor NLRP3 (nucleotide-binding domain and leucine-rich repeat containing protein 3) orchestrating inflammation through formation of an inflammasome complex in CS-induced inflammation or COPD remains controversial. Using acute and subchronic CS exposure models, we found that Nlrp3-deficient mice or wild-type mice treated with the NLRP3 inhibitor MCC950 presented an important reduction of inflammatory cells recruited into the bronchoalveolar space and of pulmonary inflammation with decreased chemokines and cytokines production, in particular IL-1β demonstrating the key role of NLRP3. Furthermore, mice deficient for Caspase-1/Caspase-11 presented also decreased inflammation parameters, suggesting a role for the NLRP3 inflammasome. Importantly we showed that acute CS-exposure promotes NLRP3-dependent cleavage of gasdermin D in macrophages present in the bronchoalveolar space and in bronchial airway epithelial cells. Finally, Gsdmd-deficiency reduced acute CS-induced lung and bronchoalveolar space inflammation and IL-1β secretion. Thus, we demonstrated in our model that NLRP3 and gasdermin D are key players in CS-induced pulmonary inflammation and IL-1β release potentially through gasdermin D forming-pore and/or pyroptoctic cell death

PPP3CB overexpression mediates EGFR TKI resistance in lung tumors via calcineurin/MEK/ERK signaling

International audienceDespite initial high response rates to first-line EGFR TKI, all non–small-cell lung cancer (NSCLC) with EGFR-activating mutation will ultimately develop resistance to treatment. Identification of resistance mechanisms is critical to adapt treatment and improve patient outcomes. Here, we show that a PPP3CB transcript that encodes full-length catalytic subunit 2B of calcineurin accumulates in EGFR-mutant NSCLC cells with acquired resistance against different EGFR TKIs and in post-progression biopsies of NSCLC patients treated with EGFR TKIs. Neutralization of PPP3CB by siRNA or inactivation of calcineurin by cyclosporin A induces apoptosis in resistant cells treated with EGFR TKIs. Mechanistically, EGFR TKIs increase the cytosolic level of calcium and trigger activation of a calcineurin/MEK/ERK pathway that prevents apoptosis. Combining EGFR, calcineurin, and MEK inhibitors overcomes resistance to EGFR TKI in both in vitro and in vivo models. Our results identify PPP3CB overexpression as a new mechanism of acquired resistance to EGFR TKIs, and provide a promising therapeutic approach for NSCLC patients that progress under TKI treatment