Complex urban atmosphere alters alveolar stem cells niche properties and drives lung fibrosis.

There is growing evidence suggesting that urban pollution has adverse effects on lung health. However, how urban pollution affects alveolar mesenchymal and epithelial stem cell niches remains unknown. This study aimed to determine how complex representative urban atmospheres alter alveolar stem cell niche properties. Mice were placed in an innovative chamber realistically simulating the atmosphere of a megalopolis, or "clean air," for 7 days. Lungs were collected, and fibroblasts and epithelial cells (EpCAM+) were isolated. Proliferative capacities of fibroblasts were tested by population doubling levels (PDL), and microarray analyses were performed. Fibroblasts and EpCAM+ cells from exposed, nonexposed, or naive mice were cocultured in organoid assays to assess the stem cell properties. Collagen deposition (Sirius red), lipofibroblasts (ADRP, COL1A1), myofibroblasts (αSMA), alveolar type 2 cells (AT2, SFTPC+), and alveolar differentiation intermediate cell [ADI, keratin-8-positive (KRT8+)/claudin-4-positive (CLDN4+)] markers were quantified in the lungs. Fibroblasts obtained from mice exposed to urban atmosphere had lower PDL and survival and produced fewer and smaller organoids. Microarray analysis showed a decrease of adipogenesis and an increase of genes associated with fibrosis, suggesting a lipofibroblast to myofibroblast transition. Collagen deposition and myofibroblast number increased in the lungs of urban atmosphere-exposed mice. AT2 number was reduced and associated with an increase in ADI cells KRT8+/CLDN4+. Furthermore, EpCAM+ cells from exposed mice also produced fewer and smaller organoids. In conclusion, urban atmosphere alters alveolar mesenchymal stem cell niche properties by inducing a lipofibroblast to myofibroblast shift. It also results in alveolar epithelial dysfunction and a fibrotic-like phenotype.NEW & NOTEWORTHY Urban pollution is known to have major adverse effects on lung health. To assess the effect of pollution on alveolar regeneration, we exposed adult mice to a simulated high-pollution urban atmosphere, using an innovative CESAM simulation chamber (Multiphase Atmospheric Experimental Simulation Chamber, https://cesam.cnrs.fr/). We demonstrated that urban atmosphere alters alveolar mesenchymal stem cell niche properties by inducing a lipofibroblast to myofibroblast shift and induces alveolar epithelial dysfunction.

Macrophage autophagy protects mice from cerium oxide nanoparticle-induced lung fibrosis.

BACKGROUND: Cerium (Ce) is a rare earth element, rapidly oxidizing to form CeO2, and currently used in numerous commercial applications, especially as nanoparticles (NP). The potential health effects of Ce remain uncertain, but literature indicates the development of rare earth pneumoconiosis accompanied with granuloma formation, interstitial fibrosis and inflammation. The exact underlying mechanisms are not yet completely understood, and we propose that autophagy could be an interesting target to study, particularly in macrophages. Therefore, the objective of our study was to investigate the role of macrophagic autophagy after pulmonary exposure to CeO2 NP in mice. Mice lacking the early autophagy gene Atg5 in their myeloid lineage and their wildtype counterparts were exposed to CeO2 NP by single oropharyngeal administration and sacrificed up to 1 month after. At that time, lung remodeling was thoroughly characterized (inflammatory cells infiltration, expression of fibrotic markers such as αSMA, TGFß1, total and type I and III collagen deposition), as well as macrophage infiltration (quantification and M1/M2 phenotype). RESULTS: Such pulmonary exposure to CeO2 NP induces a progressive and dose-dependent lung fibrosis in the bronchiolar and alveolar walls, together with the activation of autophagy. Blockage of macrophagic autophagy protects from alveolar but not bronchiolar fibrosis, via the modulation of macrophage polarization towards M2 phenotype. CONCLUSION: In conclusion, our findings bring novel insight on the role of macrophagic autophagy in lung fibrogenesis, and add to the current awareness of pulmonary macrophages as important players in the disease.

Targeting p16INK4a Promotes Lipofibroblasts and Alveolar Regeneration after Early-Life Injury.

Rationale: Promoting endogenous pulmonary regeneration is crucial after damage to restore normal lungs and prevent the onset of chronic adult lung diseases.Objectives: To investigate whether the cell-cycle inhibitor p16INK4a limits lung regeneration after newborn bronchopulmonary dysplasia (BPD), a condition characterized by the arrest of alveolar development, leading to adult sequelae.Methods: We exposed p16INK4a-/- and p16INK4aATTAC (apoptosis through targeted activation of caspase 8) transgenic mice to postnatal hyperoxia, followed by pneumonectomy of the p16INK4a-/- mice. We measured p16INK4a in blood mononuclear cells of preterm newborns, 7- to 15-year-old survivors of BPD, and the lungs of patients with BPD.Measurements and Main Results: p16INK4a concentrations increased in lung fibroblasts after hyperoxia-induced BPD in mice and persisted into adulthood. p16INK4a deficiency did not protect against hyperoxic lesions in newborn pups but promoted restoration of the lung architecture by adulthood. Curative clearance of p16INK4a-positive cells once hyperoxic lung lesions were established restored normal lungs by adulthood. p16INK4a deficiency increased neutral lipid synthesis and promoted lipofibroblast and alveolar type 2 (AT2) cell development within the stem-cell niche. Besides, lipofibroblasts support self-renewal of AT2 cells into alveolospheres. Induction with a PPARγ (peroxisome proliferator-activated receptor γ) agonist after hyperoxia also increased lipofibroblast and AT2 cell numbers and restored alveolar architecture in hyperoxia-exposed mice. After pneumonectomy, p16INK4a deficiency again led to an increase in lipofibroblast and AT2 cell numbers in the contralateral lung. Finally, we observed p16INK4a mRNA overexpression in the blood and lungs of preterm newborns, which persisted in the blood of older survivors of BPD.Conclusions: These data demonstrate the potential of targeting p16INK4a and promoting lipofibroblast development to stimulate alveolar regeneration from childhood to adulthood.

Smoking, telomere length and lung function decline: a longitudinal population-based study.

Telomere shortening is associated with COPD and impaired lung function in cross-sectional studies, but there is no longitudinal study. We used data from 448 participants recruited as part of the French follow-up of the European Community Respiratory Health Survey. We found no relationship between telomere length at baseline and FEV1 decline after 11 years of follow-up. However, heavy smoking was associated with an accelerated FEV1 decline in individuals with short telomeres, but not in subjects with longer telomeres (p for interaction p=0.08). Our findings suggest that short telomere length in peripheral leucocytes might be a marker for increased susceptibility to the effect of smoking.

Lack of Transcription Factor p53 Exacerbates Elastase-Induced Emphysema in Mice.

The transcription factor p53 is overexpressed in the lung of patients with emphysema, but it remains unclear if it has a deleterious or protective effect in disease progression. We investigated the role of p53 in the elastase-induced emphysema model and the molecular underlining mechanisms. Wild-type (WT) and p53(-/-) mice were instilled with pancreatic porcine elastase. We quantified emphysema (morphometric analysis), chemokine (C-C motif) ligand 2 (CCL2), and TNF-α in bronchoalveolar lavage (BAL) (ELISA), oxidative stress markers [heme oxygenase 1 (HO1), NAD(P)H dehydrogenase quinone 1 (NQO1), and quantitative RT-PCR], matrix metalloproteinase 12 (MMP12) expression, and macrophage apoptosis (cleaved caspase-3, immunofluorescence). p53 gene expression was up-regulated in the lung of elastase-instilled mice. p53 deletion aggravated elastase-induced emphysema severity, pulmonary inflammation (macrophage and neutrophil numbers and CCL2 and TNF-α levels in BAL), and lung oxidative stress. These findings, except for the increase in CCL2, were reproduced in WT mice transplanted with p53(-/-) bone marrow cells. The increased number of macrophages in p53(-/-) mice was not a consequence of reduced apoptosis or an excess of chemotaxis toward CCL2. Macrophage expression of MMP12 was higher in p53(-/-) mice compared with WT mice after elastase instillation. These findings provide evidence that p53(-/-) mice and WT mice grafted with p53(-/-) bone marrow cells are more prone to developing elastase-induced emphysema, supporting a protective role of p53, and more precisely p53 expressed in macrophages, against emphysema development. The pivotal role played by macrophages in this phenomenon may involve the MMP12-TNF-α pathway.

Role for telomerase in pulmonary hypertension.

BACKGROUND: Cells exhibiting dysregulated growth may express telomerase reverse transcriptase (TERT), the dual function of which consists of maintaining telomere length, in association with the RNA template molecule TERC, and controlling cell growth. Here, we investigated lung TERT in human and experimental pulmonary hypertension (PH) and its role in controlling pulmonary artery smooth muscle cell (PA-SMC) proliferation. METHODS AND RESULTS: Marked TERT expression or activity was found in lungs from patients with idiopathic PH and from mice with PH induced by hypoxia or serotonin-transporter overexpression (SM22-5HTT(+) mice), chiefly within PA-SMCs. In cultured mouse PA-SMCs, TERT was expressed on growth stimulation by serum. The TERT inhibitor imetelstat and the TERT activator TA65 abrogated and stimulated PA-SMC growth, respectively. PA-SMCs from PH mice showed a heightened proliferative phenotype associated with increased TERT expression, which was suppressed by imetelstat treatment. TERC(-/-) mice at generation 2 and TERT(-/-) mice at generations 2, 3, and 4 developed less severe PH than did wild-type mice exposed to chronic hypoxia, with less distal pulmonary artery muscularization and fewer Ki67-stained proliferating PA-SMCs. Telomere length differed between TERC(-/-) and TERT(-/-) mice, whereas PH severity was similar in the 2 strains and across generations. Chronic imetelstat treatment reduced hypoxia-induced PH in wild-type mice or partially reversed established PH in SM22-5HTT(+) mice while simultaneously decreasing TERT expression. Opposite effects occurred in mice treated with TA65. CONCLUSIONS: Telomerase exerts telomere-independent effects on PA-SMC growth in PH and may constitute a treatment target for PH.

Early signs of multi-walled carbon nanotbues degradation in macrophages, via an intracellular pH-dependent biological mechanism; importance of length and functionalization.

BACKGROUND: Carbon nanotubes (CNT) can interact with the biological environment, which could participate in their associated toxicity. We recently demonstrated that pH is an important player of CNT fate inside macrophages. We wanted to further characterize such process, and therefore designed a study dedicated to decipher CNT biodegradation by macrophages, as a function of two major physico-chemical properties in regard with nanotoxicology; length and degree of functionalization. To achieve our aim, we synthesized, following a single initial production process, four MWCNT differing in length and/or surface chemistry: S-CNT (short), SF-CNT (short functionalized), L-CNT (long) and LF-CNT (long functionalized). RESULTS: Raman spectroscopy analysis performed on CNT recovered after exposure of RAW 264.7 macrophages for 6, 24, or 48 h demonstrate that CNT show early signs of biodegradation over time inside macrophages. The modulation of CNT length and functionalization, resulting in the modification of iron accessibility, both represent critical determinants of the biodegradation process; short pristine CNT were more prone to biodegradation than long CNT (pristine or functionalized), while short functionalized CNT were protected. Incubation of cells with Concanamycin completely prevents CNT from being modified, demonstrating that this biodegradation process is dependent on an intracellular pH-dependent mechanism. Interestingly, and despite evidence of degradation via Raman spectroscopy, the CNT length and diameter were not altered during the course of the study. CONCLUSIONS: In conclusion, our results identify a new mechanism of CNT biodegradation inside macrophages. This could give new insights for the understanding of CNT-associated toxicity, and represent important tools to develop safe(r)-by-design nanomaterials.

Permanent culture of macrophages at physiological oxygen attenuates the antioxidant and immunomodulatory properties of dimethyl fumarate.

We hypothesized that O2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O2) or atmospheric (20% O2) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O2 exhibited a pro-oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O2. At 20% O2, DMF induced a stronger antioxidant response compared to 5% O2 as evidenced by a higher expression of heme oxygenase-1, NAD(P)H:quinone oxydoreductase-1 and superoxide dismutase-2. After challenge of macrophages with LPS, several pro-inflammatory (iNOS, TNF-α, MMP-2, MMP-9), anti-inflammatory (arginase-1, IL-10) and pro-angiogenic (VEGF-A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O2. This study brings new insights on the effects of O2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators.

P21-dependent protective effects of a carbon monoxide-releasing molecule-3 in pulmonary hypertension.

OBJECTIVE: Carbon monoxide-releasing molecules (CORMs) represent a pharmacological alternative to CO gas inhalation. Here, we questioned whether CORM-3, a well-characterized water-soluble CORM, could prevent and reverse pulmonary hypertension (PH) in chronically hypoxic mice and in smooth muscle promoter 22 serotonin transporter mice overexpressing the serotonin transporter in smooth muscle cells (SMCs). APPROACH AND RESULTS: Treatment with CORM-3 (50 mg/kg per day once daily) for 3 weeks prevented PH, right ventricular hypertrophy, and distal pulmonary artery muscularization in mice exposed to chronic hypoxia and partially reversed PH in smooth muscle promoter 22 serotonin transporter mice by reducing Ki67 dividing pulmonary artery SMCs (PA-SMCs). In these models, CORM-3 markedly increased lung p21 mRNA and protein levels and p21-stained PA-SMCs. These effects contrasted with the transient pulmonary vasodilatation and rise in lung cGMP levels induced by a single injection of CORM-3 in mice exposed to acute hypoxia. Studies in cultured rat PA-SMCs revealed that the inhibitory effects of CORM-3 on cell growth were independent of cGMP formation but associated with increased p21 mRNA and protein levels. Protection against PH by CORM-3 required increased lung expression of p21, as indicated by the inability of CORM-3 to prevent chronic hypoxia-induced PH in p21-deficient mice and to alter the growth of PA-SMCs derived from p21-deficient mice. CORM-3-induced p21 overexpression was linked to p53 activation as assessed by the inability of CORM-3 to prevent PH and induce p21 expression in p53-deficient mice and in PA-SMCs derived from p53-deficient mice. CONCLUSIONS: CORM-3 inhibits pulmonary vascular remodeling via p21, which may represent a useful approach for treating PH.

The role of p53 in lung macrophages following exposure to a panel of manufactured nanomaterials.

Manufactured nanomaterials (MNMs) have the potential to improve everyday life as they can be utilised in numerous medical applications and day-to-day consumer products. However, this increased use has led to concerns about the potential environmental and human health impacts. The protein p53 is a key transcription factor implicated in cellular defence and reparative responses to various stress factors. Additionally, p53 has been implicated in cellular responses following exposure to some MNMs. Here, the role of the MNM mediated p53 induction and activation and its downstream effects following exposure to five well-characterised materials [namely two types of TiO2, two carbon black (CB), and one single-walled carbon nanotube (SWCNT)] were investigated. MNM internalisation, cellular viability, p53 protein induction and activation, oxidative stress, inflammation and apoptosis were measured in murine cell line and primary pulmonary macrophage models. It was observed that p53 was implicated in the biological responses to MNMs, with oxidative stress associated with p53 activation (only following exposure to the SWCNT). We demonstrate that p53 acted as an antioxidant and anti-inflammatory in macrophage responses to SWCNT and CB NMs. However, p53 was neither involved in MNM-induced cellular toxicity, nor in the apoptosis induced by these MNMs. Moreover, the physicochemical characteristics of MNMs seemed to influence their biological effects-SWCNT the materials with the largest surface area and a fibrous shape were the most cytotoxic in this study and were capable of the induction and activation of p53.

Activation of lung p53 by Nutlin-3a prevents and reverses experimental pulmonary hypertension.

BACKGROUND: Induction of cellular senescence through activation of the p53 tumor suppressor protein is a new option for treating proliferative disorders. Nutlins prevent the ubiquitin ligase MDM2 (murine double minute 2), a negative p53 regulator, from interacting with p53. We hypothesized that cell senescence induced by Nutlin-3a exerted therapeutic effects in pulmonary hypertension (PH) by limiting the proliferation of pulmonary artery smooth muscle cells (PA-SMCs). METHODS AND RESULTS: Nutlin-3a treatment of cultured human PA-SMCs resulted in cell growth arrest with the induction of senescence but not apoptosis; increased phosphorylated p53 protein levels; and expression of p53 target genes including p21, Bax, BTG2, and MDM2. Daily intraperitoneal Nutlin-3a treatment for 3 weeks dose-dependently reduced PH, right ventricular hypertrophy, and distal pulmonary artery muscularization in mice exposed to chronic hypoxia or SU5416/hypoxia. Nutlin-3a treatment also partially reversed PH in chronically hypoxic or transgenic mice overexpressing the serotonin-transporter in SMCs (SM22-5HTT+ mice). In these mouse models of PH, Nutlin-3a markedly increased senescent p21-stained PA-SMCs; lung p53, p21, and MDM2 protein levels; and p21, Bax, PUMA, BTG2, and MDM2 mRNA levels; but induced only minor changes in control mice without PH. Marked MDM2 immunostaining was seen in both mouse and human remodeled pulmonary vessels, supporting the use of Nutlins as a PH-targeted therapy. PH prevention or reversal by Nutlin-3a required lung p53 stabilization and increased p21 expression, as indicated by the absence of Nutlin-3a effects in hypoxia-exposed p53(-/-) and p21(-/-) mice. CONCLUSIONS: Nutlin-3a may hold promise as a prosenescence treatment targeting PA-SMCs in PH.

Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders.

BACKGROUND: Although major concerns exist regarding the potential consequences of human exposure to nanoparticles (NP), no human toxicological data is currently available. To address this issue, we took welders, who present various adverse respiratory outcomes, as a model population of occupational exposure to NP.The aim of this study was to evaluate if welding fume-issued NP could be responsible, at least partially, in the lung alterations observed in welders. METHODS: A combination of imaging and material science techniques including ((scanning) transmission electron microscopy ((S)TEM), energy dispersive X-ray (EDX), and X-ray microfluorescence (µXRF)), was used to characterize NP content in lung tissue from 21 welders and 21 matched control patients. Representative NP were synthesized, and their effects on macrophage inflammatory secretome and migration were evaluated, together with the effect of this macrophage inflammatory secretome on human lung primary fibroblasts differentiation. RESULTS: Welding-related NP (Fe, Mn, Cr oxides essentially) were identified in lung tissue sections from welders, in macrophages present in the alveolar lumen and in fibrous regions. In vitro macrophage exposure to representative NP (Fe2O3, Fe3O4, MnFe2O4 and CrOOH) induced the production of a pro-inflammatory secretome (increased production of CXCL-8, IL-1ß, TNF-α, CCL-2, -3, -4, and to a lesser extent IL-6, CCL-7 and -22), and all but Fe3O4 NP induce an increased migration of macrophages (Boyden chamber). There was no effect of NP-exposed macrophage secretome on human primary lung fibroblasts differentiation. CONCLUSIONS: Altogether, the data reported here strongly suggest that welding-related NP could be responsible, at least in part, for the pulmonary inflammation observed in welders. These results provide therefore the first evidence of a link between human exposure to NP and long-term pulmonary effects.

The cyclooxygenase-2-prostaglandin E2 pathway maintains senescence of chronic obstructive pulmonary disease fibroblasts.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by the irreversible loss of replicative capacity associated with the secretion of inflammatory mediators. However, the mechanisms of this phenomenon remain poorly defined. OBJECTIVES: The aim of this study was to analyze the role of prostaglandin E2 (PGE2), a prostaglandin known to be increased in COPD lung fibroblasts, in inducing senescence and related inflammation in vitro in lung fibroblasts and in vivo in mice. METHODS: Fibroblasts were isolated from patients with COPD and from smoker and nonsmoker control subjects. Senescence markers and inflammatory mediators were investigated in fibroblasts and in mice. MEASUREMENTS AND MAIN RESULTS: Lung fibroblasts from patients with COPD exhibited higher expression of PGE2 receptors EP2 and EP4 as compared with nonsmoker and smoker control subjects. Compared with both nonsmoker and smoker control subjects, during long-term culture, COPD fibroblasts displayed increased senescent markers (increased senescence associated-ß galactosidase activity, p16, and p53 expression and lower proliferative capacity), and an increased PGE2, IL-6, IL-8, growth-regulated oncogene (GRO), CX3CL1, and matrix metalloproteinase-2 protein and cyclooxygenase-2 and mPGES-1 mRNA expression. Using in vitro pharmacologic approaches and in vivo experiments in wild-type and p53(-/-) mice we demonstrated that PGE2 produced by senescent COPD fibroblasts is responsible for the increased senescence and related inflammation. PGE2 acts either in a paracrine or autocrine fashion by a pathway involving EP2 and EP4 prostaglandin receptors, cyclooxygenase-2-dependent reactive oxygen species production and signaling, and consecutive p53 activation. CONCLUSIONS: PGE2 is a critical component of an amplifying and self-perpetuating circle inducing senescence and inflammation in COPD fibroblasts. Modulating the described PGE2 signaling pathway could provide a new basis to dampen senescence and senescence-associated inflammation in COPD.

Titanium dioxide nanoparticles induce matrix metalloprotease 1 in human pulmonary fibroblasts partly via an interleukin-1ß-dependent mechanism.

Exposure to titanium dioxide (TiO2) nanoparticles (NPs) is associated with lung remodeling, but the underlying mechanisms are unknown. Matrix metalloprotease (MMP)-1 is an important actor in matrix homeostasis and could therefore participate in TiO2 NP effects. Our aim was to evaluate the effects of TiO2 NPs on MMP-1 expression and activity in lung pulmonary fibroblasts and to understand the underlying mechanisms and assess the importance of the physicochemical characteristics of the particles in these effects. Human pulmonary fibroblasts (MRC-5 cell line and primary cells) were exposed to 10 or 100 µg/cm(2) TiO2 (two anatases, two anatase/rutile mix, one rutile NP, and one micrometric) and carbon black (CB) NPs for 6 to 48 hours. We examined cell viability, MMP-1 expression and activity, and the implication of oxidative stress, transforming growth factor (TGF)-ß, extracellular MMP inducer, and IL-1ß in MMP-1 expression. All TiO2 NPs induced MMP-1 (mRNA and protein expression), repression of procollagen-1, and α-actin expression, but only the two anatase/rutile mix induced MMP-1 activity. Micrometric TiO2 had smaller effects than TiO2 NPs, and CB NPs did not induce MMP-1. MMP-1 induction by TiO2 NPs was not related to TGF-ß, oxidative stress, or EMPRIN expression but was related to IL-1ß expression, which partly drives MMP-1 induction by two TiO2 NPs (one anatase/rutile mix and the rutile one). Taken together, our results show that TiO2 NPs are potent inducers and regulators of MMP-1 expression and activity, partly via an IL-1ß-dependent mechanism. This may explain TiO2 lung remodeling effects.

Cellular and molecular mechanisms of goblet cell metaplasia in the respiratory airways.

The mucociliary system, consisting of mucus-secreting goblet cells and ciliated cells, generates a constant overturning layer of protective mucus that lines the airway epithelium. Mucus hypersecretion and the pathophysiological changes associated are hallmarks of many pulmonary diseases including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Excessive mucus production leads to airway obstruction and, because there is currently no effective treatment, contributes to morbidity and mortality of many patients. Goblet cell differentiation and mucus production are subject to extensive control. An emerging concept is that not all goblet cells are phenotypically identical suggesting that specific molecular pathways orchestrate mucin overproduction. This paper attempts to describe the cellular and molecular mechanisms governing the differentiation of goblet cells in pulmonary diseases, a prerequisite for the development of new therapeutic agents.

Intracellular fate of carbon nanotubes inside murine macrophages: pH-dependent detachment of iron catalyst nanoparticles.

BACKGROUND: Carbon nanotubes (CNT) are a family of materials featuring a large range of length, diameter, numbers of walls and, quite often metallic impurities coming from the catalyst used for their synthesis. They exhibit unique physical properties, which have already led to an extensive development of CNT for numerous applications. Because of this development and the resulting potential increase of human exposure, an important body of literature has been published with the aim to evaluate the health impact of CNT. However, despite evidences of uptake and long-term persistence of CNT within macrophages and the central role of those cells in the CNT-induced pulmonary inflammatory response, a limited amount of data is available so far on the CNT fate inside macrophages. Therefore, the overall aim of our study was to investigate the fate of pristine single walled CNT (SWCNT) after their internalization by macrophages. METHODS: To achieve our aim, we used a broad range of techniques that aimed at getting a comprehensive characterization of the SWCNT and their catalyst residues before and after exposure of murine macrophages: X-ray diffraction (XRD), High Resolution (HR) Transmission Electron Microscopy (TEM), High Angle Annular Dark Field-Scanning TEM (HAADF-STEM) coupled to Electron Energy Loss Spectroscopy (EELS), as well as micro-X-ray fluorescence mapping (µXRF), using synchrotron radiation. RESULTS: We showed 1) the rapid detachment of part of the iron nanoparticles initially attached to SWCNT which appeared as free iron nanoparticles in the cytoplasm and nucleus of CNT-exposed murine macrophages, and 2) that blockade of intracellular lysosomal acidification prevented iron nanoparticles detachment from CNT bundles and protected cells from CNT downstream toxicity. CONCLUSIONS: The present results, while obtained with pristine SWCNT, could likely be extended to other catalyst-containing nanomaterials and surely open new ways in the interpretation and understanding of CNT toxicity.

Secreted phospholipase A2 XIIA triggers a mitochondrial damage-induced senescence in chronic obstructive pulmonary disease fibroblasts.

RATIONALE: Lung fibroblast senescence is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the mechanisms underlining this phenomenon are still poorly understood. Secreted phospholipases (sPLA2, a subclass of phospholipases) are secreted by senescent cells and can in turn induce senescence. However, their role in fibroblasts senescence in COPD is unknown. OBJECTIVES: The aim of this study was to analyze the role of sPLA2 in pulmonary fibroblast senescence. METHODS: Fibroblasts were isolated from patients with COPD and control subjects, and senescence markers and inflammatory profile was analyzed. sPLA2 levels were quantified in serum of COPD and controls. MAIN RESULTS: In comparison with non-smokers and smoker controls, senescent lung COPD fibroblasts exhibited a higher mRNA and protein expression of the sPLA2 isoform XIIA and of syndecan 4 (one of its receptors). sPLA2 XIIA induced in turn senescence of non-senescent pulmonary fibroblasts via a pathway involving consecutively syndecan 4, activation of MAPK and p-serine 727 STAT-3, increased mitochondrial ROS production, and activation of AMPK/p53. This pathway was associated with a specific inflammatory secretome (IL-10, IL-12 and TNFα), globally suggesting occurrence of a mitochondrial damage-induced senescence. COPD fibroblasts were more susceptible to this sPLA2 XIIA effect than cells from controls subjects. sPLA2 XIIA levels were significantly higher in serum from COPD patients as compared to controls. CONCLUSION: sPLA2 XIIA is involved in senescence in COPD and could be a potential target to dampen this process.

The telomerase activator TA-65 protects from cigarette smoke-induced small airway remodeling in mice through extra-telomeric effects.

Small airway remodeling (SAR) is a key phenomenon of airflow obstruction in smokers, leading to chronic obstructive pulmonary disease (COPD). SAR results in an increased thickness of small airway walls, with a combination of peribronchiolar fibrosis with increased fibrous tissue and accumulation of mesenchymal and epithelial cells. SAR pathogenesis is still unclear but recent data suggest that alterations in telomerase activity could represent a possible underlying mechanism of SAR. Our study was dedicated to identify a potential protective role of TA-65, a pharmacological telomerase activator, in a cigarette smoke (CS) model of SAR in mice, and to further precise if extra-telomeric effects of telomerase, involving oxidative stress modulation, could explain it. C57BL/6J mice were daily exposed to air or CS during 4 weeks with or without a concomitant administration of TA-65 starting 7 days before CS exposure. Morphological analyses were performed, and mucus production, myofibroblast differentiation, collagen deposition, as well as transforming growth factor-ß1 (TGF-ß1) expression in the small airway walls were examined. In addition, the effects of TA-65 treatment on TGF-ß expression, fibroblast-to-myofibroblast differentiation, reactive oxygen species (ROS) production and catalase expression and activity were evaluated in primary cultures of pulmonary fibroblasts and/or mouse embryonic fibroblasts in vitro. Exposure to CS during 4 weeks induced SAR in mice, characterized by small airway walls thickening and peribronchiolar fibrosis (increased deposition of collagen, expression of α-SMA in small airway walls), without mucus overproduction. Treatment of mice with TA-65 protected them from CS-induced SAR. This effect was associated with the prevention of CS-induced TGF-ß expression in vivo, the blockade of TGF-ß-induced myofibroblast differentiation, and the reduction of TGF-ß-induced ROS production that correlates with an increase of catalase expression and activity. Our findings demonstrate that telomerase is a critical player of SAR, probably through extra-telomeric anti-oxidant effects, and therefore provide new insights in the understanding and treatment of COPD pathogenesis.

Longitudinal and noninvasive assessment of emphysema evolution in a murine model using proton MRI.

Ultrashort echo time (550 µs) MR imaging was implemented to track the emphysema development in mice lung challenged with elastase. Two parameters, namely, signal intensity and T 2, were used to monitor the disease evolution. Nine mice were imaged before and at 24 h as well as at 3 and 8 weeks after elastase instillation. Five mice instilled with saline served as controls. At week 8, the mean normalized signal intensity ± SD was 0.89 ± 0.20 for healthy controls and 0.64 ± 0.10 for animals with emphysema. Similarly, a reduced value of T 2 (1.27 ± 0.35 ms vs 0.96 ± 0.18 ms) was found in the emphysema group. The mean signal intensity drop and the reduction of T 2 were prominent at 3 weeks following elastase instillation and stabilized between 3 and 8 weeks. The results indicated an excellent agreement between MR findings and histological morphometry (signal intensity, r = -0.78, P = 0.004; T 2, r = -0.78, P = 0.001). This result shows that proton MRI allows structural changes at alveolar level to be monitored longitudinally. This technique, applied routinely in preclinical trials will represent a valuable tool for assessment of drug therapy efficacy.

Critical role of surface chemical modifications induced by length shortening on multi-walled carbon nanotubes-induced toxicity.

Given the increasing use of carbon nanotubes (CNT) in composite materials and their possible expansion to new areas such as nanomedicine which will both lead to higher human exposure, a better understanding of their potential to cause adverse effects on human health is needed. Like other nanomaterials, the biological reactivity and toxicity of CNT were shown to depend on various physicochemical characteristics, and length has been suggested to play a critical role. We therefore designed a comprehensive study that aimed at comparing the effects on murine macrophages of two samples of multi-walled CNT (MWCNT) specifically synthesized following a similar production process (aerosol-assisted CVD), and used a soft ultrasonic treatment in water to modify the length of one of them. We showed that modification of the length of MWCNT leads, unavoidably, to accompanying structural (i.e. defects) and chemical (i.e. oxidation) modifications that affect both surface and residual catalyst iron nanoparticle content of CNT. The biological response of murine macrophages to the two different MWCNT samples was evaluated in terms of cell viability, pro-inflammatory cytokines secretion and oxidative stress. We showed that structural defects and oxidation both induced by the length reduction process are at least as responsible as the length reduction itself for the enhanced pro-inflammatory and pro-oxidative response observed with short (oxidized) compared to long (pristine) MWCNT. In conclusion, our results stress that surface properties should be considered, alongside the length, as essential parameters in CNT-induced inflammation, especially when dealing with a safe design of CNT, for application in nanomedicine for example.