Patients with cystic fibrosis (pwCF) are highly susceptible to chronic pulmonary infections due to mutations in the CFTR gene. From early childhood, pwCF experience repeated lung infections and often develop chronic bacterial and/or fungal colonization. Among the most clinically relevant pathogens, Pseudomonas aeruginosa and Aspergillus fumigatus frequently co-infect and are associated with worse outcomes, including excessive IL-1β-driven inflammation and accelerated lung function decline. Here we investigated the mechanisms underlying inflammasome overactivation during super-infection. We found that inflammasome hyperactivation occurred across macrophage populations, was independent of exogenous priming, and required live co-infection with both pathogens. P. aeruginosa and A. fumigatus cooperatively activated the NLRP3 inflammasome, and this response required both caspase-1 and caspase-8. Unexpectedly, gasdermin D was dispensable for IL-1β release. Bacterial flagellin, type IV pili and the type III secretion system, as well as the fungal polysaccharide galactosaminogalactan (GAG), were each required for overactivation. Mechanistically, P. aeruginosa activated the MyD88–TLR pathway, enhancing macrophage responses and promoting ITGAM (CD11b) expression. Under fungal super-infection, macrophages secreted complement component C3, which may bound fungal surface and engaged the complement receptor C3R (CD11b/CD18). Downstream SYK and ERK signaling amplified inflammasome activation and IL-1β release. Single-cell transcriptomic analysis of pwCF broncho-alveolar lavage and lung samples supported coordinated upregulation of complement and inflammasome pathways during bacterial-fungal infection. Together, these findings identify a complement–inflammasome signaling axis that drives pathological inflammation during bacterial-fungal co-infection in airways of pwCF and may represent a therapeutic target.

STING gain-of-function (GOF) mutations cause STING-Associated Vasculopathy with onset in Infancy (SAVI), a severe autoinflammatory disease. Mice carrying STING GOF V154M mutation develop profound T cell lymphopenia, partly due to impaired thymic development. To investigate the mechanisms of peripheral T cell dysfunctions, we analyzed transcriptomic and phenotypic profiles of splenic T cells from these mice. We found a terminally exhausted T cell phenotype, established early in life upon entry into the periphery, independent of type I interferons and intrinsic STING activation in T cells or stromal cells. Mechanistically, naive T cells in the lymphopenic periphery experienced heightened stimulation of the IL-7 receptor and TCR, including NFAT pathway, a key factor in T cell exhaustion. Transplantation of STING GOF hematopoietic stem cells with wild-type bone marrow prevented exhaustion in this non-lymphopenic context, placing lymphopenia as a key driver. T cell exhaustion was also observed in lymphopenic mice carrying Rag1 hypomorphic mutations. In conclusion, our results highlight T cell exhaustion induced by lymphopenia and could have important implications for the management of patients with severe immune deficiencies.

Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis meanwhile caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalisation from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells, despite mitochondrial outer membrane permeabilization, during pyroptosis and apoptosis. Gasdermin targeting the plasma membrane promotes a fast-mitochondrial collapse with the initial accumulation of mitochondrial DNA in the cytosol and then facilitate its release from the cell upon plasma membrane rupture. These findings demonstrate a critical role of gasdermin action on the plasma membrane facilitating the release of mitochondrial DNA as a damage-associated molecular pattern.

Exposure to ambient Ozone (O3) air pollution directly causes by its oxidative properties, respiratory epithelial cell injury, and cell death, which promote inflammation and hyperreactivity, posing a significant public health concern. Recent clinical and experimental studies have made strides in elucidating the mechanisms underlying O3-induced epithelial cell injury, inflammation, and airway hyperreactivity, which are discussed herein. The current data suggest that O3-induced oxidative stress is a central event-inducing oxeiptotic cell death pathway. O3-induced epithelial barrier damage and cell death, triggering the release of alarmins and damage-associated molecular patterns (DAMPs), with subsequent endogenous activation of Toll-like receptors (TLRs), DNA sensing pathways, and inflammasomes, activating interleukin-1-Myd88 inflammatory pathway with the production of a range of chemokines and cytokines. This cascade orchestrates lung tissue-resident cell activation in response to O3 in leukocyte and non-leukocyte populations, driving sterile innate immune response. Chronic inflammatory response to O3, by repeated exposures, supports a mixed phenotype combining asthma and emphysema, in which their exacerbation by other particulate pollutants potentially culminates in respiratory failure. We use data from lung single-cell transcriptomics to map genes of O3-damage sensing and signaling pathways to lung cells and thereby highlight potential hotspots of O3 responses. Deeper insights into these pathological pathways might be helpful for the identification of novel therapeutic targets and strategies.

Senescence is a cellular aging-related process triggered by different stresses and characterized by the secretion of various inflammatory factors referred to as the senescence-associated secretory phenotype (SASP). Here, we present evidence that the inflammasome sensor, NLRP1, is a key mediator of senescence induced by irradiation both in vitro and in vivo. The NLRP1 inflammasome promotes senescence by regulating the expression of p16, p21, p53, and SASP in Gasdermin D (GSDMD)-dependent manner as these responses are reduced in conditions of NLRP1 insufficiency or GSDMD inhibition. Mechanistically, the NLRP1 inflammasome is activated downstream of the cytosolic DNA sensor cGMP-AMP (cGAMP) synthase (cGAS) in response to genomic damage. These findings provide a rationale for inhibiting the NLRP1 inflammasome-GSDMD axis to treat senescence-driven disorders.

An uncontrolled activity of neutrophil serine proteases (NSPs) contributes to inflammatory diseases. Cathepsin C (CatC) is known to activate NSPs during neutrophilic differentiation and represents a promising pharmacological target in NSP-mediated diseases. In humans, Papillon-Lefèvre syndrome (PLS) patients have mutations in theirCTSC gene, resulting in the complete absence of CatC activity. Despite this, low residual NSP activities are detected in PLS neutrophils (<10% vs healthy individuals), suggesting the involvement of CatC-independent proteolytic pathway(s) in the activation of proNSPs. This prompted us to characterize CatC-independent NSP activation pathways by blocking proCatC maturation. In this study, we show that inhibition of intracellular CatS almost completely blocked CatC maturation in human promyeloid HL-60 cells. Despite this, NSP activation was not significantly reduced, confirming the presence of a CatC-independent activation pathway involving a CatC-like protease that we termed NSPs-AAP-1. Similarly, when human CD34+ progenitor cells were treated with CatS inhibitors during neutrophilic differentiation in vitro, CatC activity was nearly abrogated but ∼30% NSP activities remained, further supporting the existence of NSPs-AAP-1. Our data indicate that NSPs-AAP-1 is a cysteine protease that is inhibited by reversible nitrile compounds designed for CatC inhibition. We further established a proof of concept for the indirect, although incomplete, inhibition of NSPs by pharmacological targeting of CatC maturation using CatS inhibitors. This emphasizes the potential of CatS as a therapeutic target for inflammatory diseases. Thus, preventing proNSP maturation using a CatS inhibitor, alone or in combination with a CatC/NSPs-AAP-1 inhibitor, represents a promising approach to efficiently control the extent of tissue injury in neutrophil-mediated inflammatory diseases.

Environmental air pollutants including ozone cause severe irritation and respiratory diseases. Here, we report that 6week's ozone exposure in mice (1.5ppm, 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.

IntroductionGround-level ozone is an important gaseous constituent of air pollution that contributes to lung disease progression and mortality. Ozone exposure in mice causes pulmonary inflammation evolving into lung emphysema and/or fibrotic patterns but the mechanisms are not well understood [1]. We investigated the role of the poorly characterized innate receptor nucleotide-binding domain and leucine-rich repeat containing protein 6 (NLRP6) involved in inflammasome scaffold [2], in ozone exposure-induced in the context of immunogenic cell death [3].MethodsUsing a chronic ozone exposure model of chronic obstructive pulmonary disease (COPD) in mice, we investigated the role of the NLRP6 receptor in pulmonary inflammation, emphysema and fibrosis by exposing wild-type, Nlrp6 deficient mice and mice deficient for Nlrp6 specifically in lung epithelial cells. In addition, we analyzed NLRP6 expression in lung using NLRP6 FLAG-tagged mice. NLRP6-dependent expression and/or activation of proteins characteristic of immunological cell deaths such as pyroptosis, apoptosis and necroptosis were analyzed by western blotting and immunofluorescence.ResultsWe observed that mouse chronic ozone exposure increased NLRP6 expression in bronchial and alveolar epithelial cells and in a lesser extend in airway macrophages. Interestingly Nlrp6 deficiency dampened pulmonary inflammation and alveolar damage with reduced neutrophil and eosinophil influxes, attenuated chemokine/cytokine and remodeling factor production, collagen deposition and lung fibrosis. Chronic ozone-induced a loss of alveolar type 1 pneumocytes that was attenuated in Nlrp6 deficient mice. Mechanistically, we report that chronic ozone exposure promoted NLRP6-dependent caspase-1, caspase-11 and gasdermin D activation in alveolar type 1 pneumocytes. Chronic ozone exposure also induced NLRP6-dependent expression of apoptotic and necroptotic markers in lung tissue.ConclusionWe identified NLRP6 as a new innate sensor of chronic ozone-induced lung injury promoting pulmonary inflammation leading to emphysema and fibrosis in mice. Our results suggest that chronic ozone induces pulmonary inflammation through NLRP6 inflammasome-dependent pyroptosis and necroptosis of alveolar type 1 pneumocytes. Understanding the mechanisms of pollutant-induced alveolar cell death, lung inflammation and repair might help fight COPD and lung fibrosis.

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.

Introduction:The 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.Methods:We 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.Results:We 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.Discussion:Together, 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.

Chronic obstructive pulmonary disease (COPD) is a major health issue not only due to cigarette smoking but also to air pollution, in particular ozone exposure that is expected to increase in the context of global warming. COPD is characterized by progressive airway obstruction and impaired immune response to bacterial and viral infections. Pollutants induce pulmonary damage, chronic inflammation, and mucus hypersecretion leading to bronchial obstruction, alveolar wall destruction, and strongly impairing lung functions. The underlying mechanisms resulting in chronic inflammation, exacerbation, and impaired lung function remain poorly understood. However, oxidative stress and host-derived danger signals are common triggers leading to cigarette smoke (CS)- or ozone-induced lung injury and inflammation notably by activating inflammasome pathways. Inflammasomes are cytoplasmic multiprotein complexes involved in the regulation of homeostasis and inflammation. Their activation leads to maturation and secretion of major proinflammatory cytokines IL-1β and IL-18 as well as gasdermin D-mediated pore formation. Here we review recent studies analyzing the role of the inflammasomes in human and animal models of CS- or ozone-exposure in the development of pulmonary inflammation potentially leading to COPD and/or fibrosis.

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.

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

The cGAS–STING pathway displays important functions in the regulation of innate and adaptive immunity following the detection of microbial and host-derived DNA. Here, we briefly summarize biological functions of STING and review recent literature highlighting its important contribution in the context of respiratory diseases. Over the last years, tremendous progress has been made in our understanding of STING activation, which has favored the development of STING agonists or antagonists with potential therapeutic benefits. Antagonists might alleviate STING-associated chronic inflammation and autoimmunity. Furthermore, pharmacological activation of STING displays strong antiviral properties, as recently shown in the context of SARS-CoV-2 infection. STING agonists also elicit potent stimulatory activities when used as an adjuvant promoting antitumor responses and vaccines efficacy.

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.

Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis, whereas caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent, this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalization from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells during pyroptosis and apoptosis. Gasdermin targeted at the plasma membrane promotes a fast mitochondrial collapse along with the initial accumulation of mitochondrial DNA in the cytosol and then facilitates the DNA's release from the cell when the plasma membrane ruptures. These findings demonstrate that gasdermin action has a critical effect on the plasma membrane and facilitates the release of mitochondrial DNA as a damage-associated molecular pattern.

Cystic fibrosis is associated with chronic Pseudomonas aeruginosa colonization and inflammation. The role of MyD88, the shared adapter protein of the proinflammatory TLR and IL-1R families, in chronic P. aeruginosa biofilm lung infection is unknown. We report that chronic lung infection with the clinical P. aeruginosa RP73 strain is associated with uncontrolled lung infection in complete MyD88-deficient mice with epithelial damage, inflammation, and rapid death. Then, we investigated whether alveolar or myeloid cells contribute to heightened sensitivity to infection. Using cell-specific, MyD88-deficient mice, we uncover that the MyD88 pathway in myeloid or alveolar epithelial cells is dispensable, suggesting that other cell types may control the high sensitivity of MyD88-deficient mice. By contrast, IL-1R1–deficient mice control chronic P. aeruginosa RP73 infection and IL-1β Ab blockade did not reduce host resistance. Therefore, the IL-1R1/MyD88 pathway is not involved, but other IL-1R or TLR family members need to be investigated. Our data strongly suggest that IL-1 targeted neutralizing therapies used to treat inflammatory diseases in patients unlikely reduce host resistance to chronic P. aeruginosa infection.

Innate immunity is regulated by a broad set of evolutionary conserved receptors to finely probe the local environment and maintain host integrity. Besides pathogen recognition through conserved motifs, several of these receptors also sense aberrant or misplaced self-molecules as a sign of perturbed homeostasis. Among them, self-nucleic acid sensing by the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway alerts on the presence of both exogenous and endogenous DNA in the cytoplasm. We review recent literature demonstrating that self-nucleic acid detection through the STING pathway is central to numerous processes, from cell physiology to sterile injury, auto-immunity and cancer. We address the role of STING in autoimmune diseases linked to dysfunctional DNAse or related to mutations in DNA sensing pathways. We expose the role of the cGAS/STING pathway in inflammatory diseases, neurodegenerative conditions and cancer. Connections between STING in various cell processes including autophagy and cell death are developed. Finally, we review proposed mechanisms to explain the sources of cytoplasmic DNA.

The P2X7 receptor is an ATP-gated cation channel with a still ambiguous role in cancer progression, proposed to be either pro- or anti-cancerous, depending on the cancer or cell type in the tumour. Its role in mammary cancer progression is not yet defined. Here, we show that P2X7 receptor is functional in highly aggressive mammary cancer cells, and induces a change in cell morphology with fast F-actin reorganization and formation of filopodia, and promotes cancer cell invasiveness through both 2- and 3-dimensional extracellular matrices in vitro. Furthermore, P2X7 receptor sustains Cdc42 activity and the acquisition of a mesenchymal phenotype. In an immunocompetent mouse mammary cancer model, we reveal that the expression of P2X7 receptor in cancer cells, but not in the host mice, promotes tumour growth and metastasis development, which were reduced by treatment with specific P2X7 antagonists. Our results demonstrate that P2X7 receptor drives mammary tumour progression and represents a pertinent target for mammary cancer treatment.