Tenascin-C inactivation impacts lung structure and function beyond lung development

Tenascin-C (TNC) is an extracellular matrix protein expressed at high levels during lung organogenesis. Later, TNC is only transiently de novo expressed to orchestrate tissue repair in pathological situations. We previously showed that TNC inactivation affects lung development and thus evaluated here the implications on lung function in newborn/adult mice. Respiratory function parameters were measured in anesthetized and mechanically ventilated wild-type (WT) and TNC-deficient mice at 5 (P5) and 90 (P90) days of age under basal conditions, as well as following high tidal volume (HTV) ventilation. At P5, TNC-deficient mice showed an increased static compliance (Cst) and inspiratory capacity (IC) relative to WT at baseline and throughout HTV. At P90, however, Cst and IC were only elevated at baseline. Control non-ventilated newborn and adult TNC-deficient mice showed similar lung morphology, but less alpha smooth muscle actin (α-SMA) around small airways. SMA + cells were decreased by 50% in adult TNC-deficient lungs and collagen layer thickened around small airways. Increased surfactant protein C (SP-C) and altered TGFβ and TLR4 signaling pathways were also detected. Thus, TNC inactivation-related defects during organogenesis led to persisting functional impairment in adulthood. This might be of interest in the context of pulmonary diseases with thickened airway smooth muscle layer or ventilation heterogeneity, like asthma and COPD

A novel technique to determine the cell type specific response within an in vitro co-culture model via multi-colour flow cytometry

Determination of the cell type specific response is essential towards understanding the cellular mechanisms associated with disease states as well as assessing cell-based targeting of effective therapeutic agents. Recently, there have been increased calls for advanced in vitro multi-cellular models that provide reliable and valuable tools correlative to in vivo. In this pursuit the ability to assess the cell type specific response is imperative. Herein, we report a novel approach towards resolving each specific cell type of a multi-cellular model representing the human lung epithelial tissue barrier via multi-colour flow cytometry (FACS). We proved via ≤ five-colour FACS that the manipulation of this in vitro model allowed each cell type to be resolved with no impact upon cell viability. Subsequently, four-colour FACS verified the ability to determine the biochemical effect (e.g. oxidative stress) of each specific cell type. This technique will be vital in gaining information upon cellular mechanics when using next-level, multi- cellular in vitro strategies

Cellular uptake and cell-to-cell transfer of polyelectrolyte microcapsules within a triple co-culture system representing parts of the respiratory tract

Polyelectrolyte multilayer microcapsules around 3.4 micrometers in diameter were added to epithelial cells, monocyte-derived macrophages, and dendritic cells in vitro and their uptake kinetics were quantified. All three cell types were combined in a triple co-culture model, mimicking the human epithelial alveolar barrier. Hereby, macrophages were separated in a three-dimensional model from dendritic cells by a monolayer of epithelial cells. While passing of small nanoparticles has been demonstrated from macrophages to dendritic cells across the epithelial barrier in previous studies, for the micrometer-sized capsules, this process could not be observed in a significant amount. Thus, this barrier is a limiting factor for cell-to-cell transfer of micrometer-sized particles

Distribution of polymer-coated gold nanoparticles in a 3D lung model and indication of apoptosis after repeated exposure

Aim: The distribution and impact of aerosol-delivered gold nanoparticles (AuNPs) functionalized with a mixture of aminated-polyvinyl alcohol and amino-PEG ([polyvinyl alcohol/PEG]-NH2) upon repeated administration onto a 3D lung model were explored. Materials & methods: AuNPs were aerosolized and uptake and epithelial translocation was assessed by inductively coupled plasma optical-emission spectroscopy, flow cytometry and electron microscopy. In addition, cytotoxicity, apoptosis and proinflammation were evaluated. Results: Repeated AuNP aerosolization resulted in NP accumulation in macrophages and epithelial cells. Dendritic cells demonstrated substantial NP internalization after single administration which was reduced in later time points. No cytotoxicity or proinflammation was observed but after 96 h significant apoptosis was induced by the polymer coating. Conclusion: These results indicate the importance of repeated exposures in addressing potential effects of NPs

Anti-Fibrotic Effect of SDF-1β Overexpression in Bleomycin-Injured Rat Lung.

Rational: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease and is associated with high mortality due to a lack of effective treatment. Excessive deposition of the extracellular matrix by activated myofibroblasts in the alveolar space leads to scar formation that hinders gas exchange. Therefore, selectively removing activated myofibroblasts with the aim to repair and remodel fibrotic lungs is a promising approach. Stromal-derived growth factor (SDF-1) is known to stimulate cellular signals which attract stem cells to the site of injury for tissue repair and remodeling. Here, we investigate the effect of overexpression of SDF-1β on lung structure using the bleomycin-injured rat lung model. Methods: Intratracheal administration of bleomycin was performed in adult male rats (F344). Seven days later, in vivo electroporation-mediated gene transfer of either SDF-1β or the empty vector was performed. Animals were sacrificed seven days after gene transfer and histology, design-based stereology, flow cytometry, and collagen measurement were performed on the tissue collected. For in vitro experiments, lung fibroblasts obtained from IPF patients were used. Results: Seven days after SDF-1β gene transfer to bleomycin-injured rat lungs, reduced total collagen, reduced collagen fibrils, improved histology and induced apoptosis of myofibroblasts were observed. Furthermore, it was revealed that TNF-α mediates SDF-1β-induced apoptosis of myofibroblasts; moreover, SDF-1β overexpression increased alveolar epithelial cell numbers and proliferation in vivo and also induced their migration in vitro. Conclusions: Our study demonstrates a new antifibrotic mechanism of SDF-1β overexpression and suggests SDF-1β as a potential new approach for the treatment of lung fibrosis

Interaction of biomedical nanoparticles with the pulmonary immune system.

Engineered nanoparticles (NPs) offer site-specific delivery, deposition and cellular uptake due to their unique physicochemical properties and were shown to modulate immune responses. The respiratory tract with its vast surface area is an attractive target organ for innovative immunomodulatory therapeutic applications by pulmonary administration of such NPs, enabling interactions with resident antigen-presenting cells (APCs), such as dendritic cells and macrophages. Depending on the respiratory tract compartment, e.g. conducting airways, lung parenchyma, or lung draining lymph nodes, APCs extensively vary in their number, morphology, phenotype, and function. Unique characteristics and plasticity render APC populations ideal targets for inhaled specific immunomodulators. Modulation of immune responses may operate in different steps of the immune cell-antigen interaction, i.e. antigen uptake, trafficking, processing, and presentation to T cells. Meticulous analysis of the immunomodulatory potential, as well as pharmacologic and biocompatibility testing of inhalable NPs is required to develop novel strategies for the treatment of respiratory disorders such as allergic asthma. The safe-by-design and characterization of such NPs requires well coordinated interdisciplinary research uniting engineers, chemists biologists and respiratory physicians. In this review we will focus on in vivo data available to facilitate the design of nanocarrier-based strategies using NPs to modulate pulmonary immune responses

Basal-Like Cell-Conditioned Medium Exerts Anti-Fibrotic Effects In Vitro and In Vivo.

In idiopathic pulmonary fibrosis (IPF), basal-like cells are atypically present in the alveolar region, where they may affect adjacent stromal cells by paracrine mechanisms. We here aimed to confirm the presence of basal-like cells in peripheral IPF lung tissue in vivo, to culture and characterize the cells in vitro, and to investigate their paracrine effects on IPF fibroblasts in vitro and in bleomycin-injured rats in vivo. Basal-like cells are mainly localized in areas of pathological bronchiolization or honeycomb cysts in peripheral IPF lung tissue. Single-cell RNA sequencing (scRNA-seq) demonstrated an overall homogeneity, the expression of the basal cell markers cytokeratin KRT5 and KRT17, and close transcriptomic similarities to basal cells in the majority of cells cultured in vitro. Basal-like cells secreted significant levels of prostaglandin E2 (PGE2), and their conditioned medium (CM) inhibited alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1) and upregulated matrix metalloproteinase-1 (MMP-1) and hepatocyte growth factor (HGF) by IPF fibroblasts in vitro. The instillation of CM in bleomycin-injured rat lungs resulted in reduced collagen content, improved lung architecture, and reduced α-SMA-positive cells. Our data suggested that basal-like cells may limit aberrant fibroblast activation and differentiation in IPF through paracrine mechanisms

Aerosol delivery of functionalized gold nanoparticles target and activate dendritic cells in a 3D lung cellular model

Nanocarrier design combined with pulmonary drug delivery holds great promise for the treatment of respiratory tract disorders. In particular, targeting of dendritic cells that are key immune cells to enhance or suppress an immune response in the lung is a promising approach for the treatment of allergic diseases. Fluorescently encoded poly(vinyl alcohol) (PVA)-coated gold nanoparticles, functionalized with either negative (−COO–) or positive (−NH3+) surface charges, were functionalized with a DC-SIGN antibody on the particle surface, enabling binding to a dendritic cell surface receptor. A 3D coculture model consisting of epithelial and immune cells (macrophages and dendritic cells) mimicking the human lung epithelial tissue barrier was employed to assess the effects of aerosolized AuNPs. PVA-NH2 AuNPs showed higher uptake compared to that of their −COOH counterparts, with the highest uptake recorded in macrophages, as shown by flow cytometry. None of the AuNPs induced cytotoxicity or necrosis or increased cytokine secretion, whereas only PVA-NH2 AuNPs induced higher apoptosis levels. DC-SIGN AuNPs showed significantly increased uptake by monocyte-derived dendritic cells (MDDCs) with subsequent activation compared to non-antibody-conjugated control AuNPs, independent of surface charge. Our results show that DC-SIGN conjugation to the AuNPs enhanced MDDC targeting and activation in a complex 3D lung cell model. These findings highlight the potential of immunoengineering approaches to the targeting and activation of immune cells in the lung by nanocarriers

Uptake efficiency of surface modified gold nanoparticles does not correlate with functional changes and cytokine secretion in human dendritic cells in vitro

Engineering nanoparticles (NPs) for immune modulation require a thorough understanding of their interaction(s) with cells. Gold NPs (AuNPs) were coated with polyethylene glycol (PEG), polyvinyl alcohol (PVA) or a mixture of both with either positive or negative surface charge to investigate uptake and cell response in monocyte-derived dendritic cells (MDDCs). Inductively coupled plasma optical emission spectrometry and transmission electron microscopy were used to confirm the presence of Au inside MDDCs. Cell viability, (pro-)inflammatory responses, MDDC phenotype, activation markers, antigen uptake and processing were analyzed. Cell death was only observed for PVA-NH2 AuNPs at the highest concentration. MDDCs internalize AuNPs, however, surface modification influenced uptake. Though limited uptake was observed for PEG-COOH AuNPs, a significant tumor necrosis factor-alpha release was induced. In contrast, (PEG + PVA)-NH2 and PVA-NH2 AuNPs were internalized to a higher extent and caused interleukin-1beta secretion. None of the AuNPs caused changes in MDDC phenotype, activation or immunological properties

Перспективи роботи з іноземними лікарями в системі післядипломної освіти

BACKGROUND: Polymer nanoparticles (PNP) are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce. RESULTS: Fluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm) and surface charges (positive and negative) were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP(45) (45 nm) showed a higher cytotoxicity compared to the positive bigger PNP(90) (90 nm) particles including reduction in mitochondrial membrane potential (ΔΨ(m)), induction of reactive oxygen species (ROS) production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨ(m)), uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM). Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP), caveolin (for negative PNP) and mannose receptors (for hydroxylated PNP) were found with smaller PNP(45) showing stronger interactions with the receptors than bigger PNP(90). CONCLUSIONS: The size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges) play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose) leading to cellular internalization were observed to depend on size and surface properties of the different PNP. These properties of the nanoparticles also dominate their cytotoxicity, which was analyzed for many factors. The effective reduction in the mitochondrial membrane potential (ΔΨ(m)), uncoupling of the electron transfer chain in mitochondria and resulting ATP depletion, induction of ROS and oxidative stress likely all play a role in the mechanisms behind the cytotoxicity of these PNP