Airspace Diameter Map-A Quantitative Measurement of All Pulmonary Airspaces to Characterize Structural Lung Diseases.

(1) Background: Stereological estimations significantly contributed to our understanding of lung anatomy and physiology. Taking stereology fully 3-dimensional facilitates the estimation of novel parameters. (2) Methods: We developed a protocol for the analysis of all airspaces of an entire lung. It includes (i) high-resolution synchrotron radiation-based X-ray tomographic microscopy, (ii) image segmentation using the free machine-learning tool Ilastik and ImageJ, and (iii) calculation of the airspace diameter distribution using a diameter map function. To evaluate the new pipeline, lungs from adult mice with cystic fibrosis (CF)-like lung disease (βENaC-transgenic mice) or mice with elastase-induced emphysema were compared to healthy controls. (3) Results: We were able to show the distribution of airspace diameters throughout the entire lung, as well as separately for the conducting airways and the gas exchange area. In the pathobiological context, we observed an irregular widening of parenchymal airspaces in mice with CF-like lung disease and elastase-induced emphysema. Comparable results were obtained when analyzing lungs imaged with μCT, sugges-ting that our pipeline is applicable to different kinds of imaging modalities. (4) Conclusions: We conclude that the airspace diameter map is well suited for a detailed analysis of unevenly distri-buted structural alterations in chronic muco-obstructive lung diseases such as cystic fibrosis and COPD

Palmitoylation, pathogens and their host

S-Palmitoylation, the only reversible post-translational lipid modification, confers unique biochemical and functional properties to proteins. Although it has long been known that viral proteins are palmitoylated, recent studies reveal that this modification plays a critical role for pathogens of all kinds and at multiple steps of their life cycle. The present review examines the involvement of S-palmitoylation in infection by viruses, bacteria and parasites and illustrates how pathogens have evolved to manipulate the host palmitoylation machinery

What does S-palmitoylation do to membrane proteins?

S-Palmitoylation is post-translational modification, which consists in the addition of a C16 acyl chain to cytosolic cysteines and which is unique amongst lipid modifications in that it is reversible. It can thus, as phosphory lation or ubiquitination, act as a switch. While palmitoylation of soluble proteins allows them to interact with membranes, the consequences of palmitoylation for transmembrane proteins are more enigmatic. We will briefly review the current knowledge regarding the enzymes responsible for palmitate addition and removal. We will then describe various observed consequences of membrane protein palmitoylation. We propose that the direct effects of palmitoylation on transmembrane proteins might however be limited to four non-mutually exclusive mechanistic consequences: alterations in the conformation of transmembrane domains, association with specific membrane domains, controlled interactions with other protein s and controlled interplay with other post;translational modifications

Mechanistic effects of protein palmitoylation and the cellular consequences thereof

S-palmitoylation involves the attachment of a 16-carbon long fatty acid chain to the cysteine residues of proteins. The process is enzymatic and dynamic with DHHC enzymes mediating palmitoylation and acyl-protein thioesterases reverting the reaction. Proteins that undergo this modification span almost all cellular functions. While the increase in hydrophobicity generated by palmitoylation has the obvious consequence of triggering membrane association, the effects on transmembrane proteins are less intuitive and span a vast range. We review here the current knowledge on palmitoylating and depalmitoylating enzymes, the methods that allow the study of this lipid modification and which drugs can affect it, and finally we focus on four cellular processes for which recent studies reveal an involvement of palmitoylation: endocytosis, reproduction and cell growth, fat and sugar homeostasis and signal transduction at the synapse

Gestation and lactation exposure to nicotine induces transient postnatal changes in lung alveolar development.

Harmful consequences of cigarette smoke (CS) exposure during lung development can already manifest in infancy. In particular, early life exposure to nicotine, the main component of CS, was shown to affect lung development in animal models. We aimed to characterize the effect of nicotine on alveoli formation. We analyzed the kinetics of normal alveolar development during the alveolarization phase and then looked at the effect of nicotine in a mouse model of gestational and early life exposure. Immunohistochemical staining revealed that the wave of cell proliferation (i.e. vascular endothelial cells, alveolar epithelial cells (AEC) type II and mesenchymal cell) occurs at pnd8 in control and nicotine-exposed lungs. However, FACS analysis of individual epithelial alveolar cells revealed nicotine-induced transient increase of AEC type I proliferation and decrease of vascular endothelial cell proliferation at pnd8. Furthermore, nicotine increased the percentage of endothelial cells at pnd2. Transcriptomic data also showed significant changes in nicotine samples compared to the controls on cell cycle associated genes at pnd2, but not anymore at pnd16. Accordingly, the expression of survivin, involved in cell cycle regulation, also follows a different kinetics in nicotine lung extracts. These changes resulted in an increased lung size detected by stereology at pnd16, but no longer in adult age, suggesting that nicotine can act on the pace of lung maturation. Taken together, our results indicate that early life nicotine exposure could be harmful to alveolar development independently from other toxicants contained in CS

Early life exposure to nicotine modifies lung gene response after elastase-induced emphysema

Background: Chronic obstructive pulmonary disease (COPD) is among the top 5 causes of mortality in the world and can develop as a consequence of genetic and/or environmental factors. Current efforts are focused on identifying early life insults and how these contribute to COPD development. In line with this, our study focuses on the influence of early life nicotine exposure and its potential impact on (a) lung pulmonary functions, and (b) elastase-induced emphysema in adulthood.Methods: To address this hypothesis, we developed a model of 2 hits, delivered at different time points: mouse pups were first exposed to nicotine/placebo in utero and during lactation, and then subsequently received elastase/placebo at the age of 11 weeks. The effect of nicotine pretreatment and elastase instillation was assessed by (a) measurement of pulmonary function at post-elastase day (ped) 21, and (b) transcriptomic profiling at ped3 and 21, and complementary protein determination. Statistical significance was determined by 3- and 2-way ANOVA for pulmonary functions, and RNAseq results were analyzed using the R project.Results: We did not observe any impact of nicotine pre- and early post-natal exposure compared to control samples on lung pulmonary functions in adulthood, as measured by FLEXIVENT technology. After elastase instillation, substantial lung damage was detected by x-ray tomography and was accompanied by loss in body weight at ped3 as well as an increase in cell numbers, inflammatory markers in BAL and lung volume at ped21. Lung functions showed a decrease in elastance and an increase in deep inflation volume and pressure volume (pv) loop area in animals with emphysema at ped21. Nicotine had no effect on elastance and deep inflation volume, but did affect the pv loop area in animals with emphysema at ped21. Extensive transcriptomic changes were induced by elastase at ped3 both in the nicotine-pretreated and the control samples, with several pathways common to both groups, such as for cell cycle, DNA adhesion and DNA damage. Nicotine pretreatment affected the number of lymphocytes present in BAL after elastase instillation and some of the complement pathway related proteins, arguing for a slight modification of the immune response, as well as changes related to general body metabolism. The majority of elastase-induced transcriptomic changes detected at ped3 had disappeared at ped21. In addition, transcriptomic profiling singled out a common gene pool that was independently activated by nicotine and elastase.Conclusions: Our study reports a broad spectrum of transient transcriptomic changes in mouse emphysema and identifies nicotine as influencing the emphysema-associated immune system response.</p

Di-tyrosine crosslinking and NOX4 expression as oxidative pathological markers in the lungs of patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT andNOX4in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining andNOX4was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link betweenNOX4and DT was addressed in human fibroblasts deficient forNOX4activity (mutation in theCYBAgene). Induction ofNOX4by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient forNOX4activity. Our data indicate that DT is a histological marker of IPF and thatNOX4can generate a sufficient amount of H2O2for DT formation in vitro