Allies or enemies? The effect of regulatory T cells and related T lymphocytes on the profibrotic environment in bleomycin-injured lung mouse models.

Idiopathic pulmonary fibrosis (IPF) is characterized by permanent scarring of lung tissue and declining lung function, and is an incurable disease with increase in prevalence over the past decade. The current consensus is that aberrant wound healing following repeated injuries to the pulmonary epithelium is the most probable cause of IPF, with various immune inflammatory pathways having been reported to impact disease pathogenesis. While the role of immune cells, specifically T lymphocytes and regulatory T cells (Treg), in IPF pathogenesis has been reported and discussed recently, the pathogenic or beneficial roles of these cells in inducing or preventing lung fibrosis is still debated. This lack of understanding could be due in part to the difficulty in obtaining diseased human lung tissue for research purposes. For this reason, many animal models have been developed over the years to attempt to mimic the main clinical hallmarks of IPF: among these, inducing lung injury in rodents with the anti-cancer agent bleomycin has now become the most commonly studied animal model of IPF. Pulmonary fibrosis is the major side effect when bleomycin is administered for cancer treatment in human patients, and a similar effect can be observed after intra-tracheal administration of bleomycin to rodents. Despite many pathophysiological pathways of lung fibrosis having been investigated in bleomycin-injured animal models, one central facet still remains controversial, namely the involvement of specific T lymphocyte subsets, and in particular Treg, in disease pathogenesis. This review aims to summarize the major findings and conclusions regarding the involvement of immune cells and their receptors in the pathogenesis of IPF, and to elaborate on important parallels between animal models and the human disease. A more detailed understanding of the role of Treg and other immune cell subsets in lung injury and fibrosis derived from animal models is a critical basis for translating this knowledge to the development of new immune-based therapies for the treatment of human IPF

Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model

ABSTRACT: BACKGROUND: Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization. RESULTS: Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 mum) and nano-sized (0.078 mum) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 mum) and titanium dioxide (0.02-0.03 mum) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-alpha in the supernatants. We measured a 2-3 fold increase of tumour necrosis factor-alpha in the supernatants after applying 1 mum polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles. CONCLUSION: Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-alpha

Particles induce apical plasma membrane enlargement in epithelial lung cell line depending on particle surface area dose

<p>Abstract</p> <p>Background</p> <p>Airborne particles entering the respiratory tract may interact with the apical plasma membrane (APM) of epithelial cells and enter them. Differences in the entering mechanisms of fine (between 0.1 μm and 2.5 μm) and ultrafine ( ≤ 0.1 μm) particles may be associated with different effects on the APM. Therefore, we studied particle-induced changes in APM surface area in relation to applied and intracellular particle size, surface and number.</p> <p>Methods</p> <p>Human pulmonary epithelial cells (A549 cell line) were incubated with various concentrations of different sized fluorescent polystyrene spheres without surface charge (∅ fine – 1.062 μm, ultrafine – 0.041 μm) by submersed exposure for 24 h. APM surface area of A549 cells was estimated by design-based stereology and transmission electron microscopy. Intracellular particles were visualized and quantified by confocal laser scanning microscopy.</p> <p>Results</p> <p>Particle exposure induced an increase in APM surface area compared to negative control (p < 0.01) at the same surface area concentration of fine and ultrafine particles a finding not observed at low particle concentrations. Ultrafine particle entering was less pronounced than fine particle entering into epithelial cells, however, at the same particle surface area dose, the number of intracellular ultrafine particles was higher than that of fine particles. The number of intracellular particles showed a stronger increase for fine than for ultrafine particles at rising particle concentrations.</p> <p>Conclusion</p> <p>This study demonstrates a particle-induced enlargement of the APM surface area of a pulmonary epithelial cell line, depending on particle surface area dose. Particle uptake by epithelial cells does not seem to be responsible for this effect. We propose that direct interactions between particle surface area and cell membrane cause the enlargement of the APM.</p

Human Bronchial Epithelial Cells Induce CD141/CD123/DC-SIGN/FLT3 Monocytes That Promote Allogeneic Th17 Differentiation.

Little is known about monocyte differentiation in the lung mucosal environment and about how the epithelium shapes monocyte function. We studied the role of the soluble component of bronchial epithelial cells (BECs) obtained under basal culture conditions in innate and adaptive monocyte responses. Monocytes cultured in bronchial epithelial cell-conditioned media (BEC-CM) specifically upregulate CD141, CD123, and DC-SIGN surface levels and FLT3 expression, as well as the release of IL-1β, IL-6, and IL-10. BEC-conditioned monocytes stimulate naive T cells to produce IL-17 through IL-1β mechanism and also trigger IL-10 production by memory T cells. Furthermore, monocytes cultured in an inflammatory environment induced by the cytokines IL-6, IL-8, IL-1β, IL-15, TNF-α, and GM-CSF also upregulate CD123 and DC-SIGN expression. However, only inflammatory cytokines in the epithelial environment boost the expression of CD141. Interestingly, we identified a CD141/CD123/DC-SIGN triple positive population in the bronchoalveolar lavage fluid (BALF) from patients with different inflammatory conditions, demonstrating that this monocyte population exists in vivo. The frequency of this monocyte population was significantly increased in patients with sarcoidosis, suggesting a role in inflammatory mechanisms. Overall, these data highlight the specific role that the epithelium plays in shaping monocyte responses. Therefore, the unraveling of these mechanisms contributes to the understanding of the function that the epithelium may play in vivo

Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy

Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions

Toxic effects of brake wear particles on epithelial lung cells in vitro

Background: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. Results: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p &lt; 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. Conclusion: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress. [Authors]]]> Vehicle Emissions ; Particulate Matter ; Particle Size ; Epithelial Cells ; Toxicity Tests eng https://serval.unil.ch/resource/serval:BIB_834D29655A82.P001/REF.pdf http://nbn-resolving.org/urn/resolver.pl?urn=urn:nbn:ch:serval-BIB_834D29655A828 info:eu-repo/semantics/altIdentifier/urn/urn:nbn:ch:serval-BIB_834D29655A828 info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/openAccess Copying allowed only for non-profit organizations https://serval.unil.ch/disclaimer application/pdf oai:serval.unil.ch:BIB_834DC6847A73 2022-02-19T02:25:16Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_834DC6847A73 A liposomal peptide vaccine inducing CD8+ T cells in HLA-A2.1 transgenic mice, which recognise human cells encoding hepatitis C virus (HCV) proteins info:doi:10.1016/j.vaccine.2004.05.009 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.vaccine.2004.05.009 info:eu-repo/semantics/altIdentifier/pmid/15519708 Engler, O. B. Schwendener, R. A. Dai, W. J. Wolk, B. Pichler, W. Moradpour, D. Brunner, T. Cerny, A. info:eu-repo/semantics/article article 2004-11 Vaccine, vol. 23, no. 1, pp. 58-68 info:eu-repo/semantics/altIdentifier/pissn/0264-410X <![CDATA[Virus specific T cell responses play an important role in resolving acute hepatitis C virus (HCV) infections. Using the HLA-A2.1 transgenic mouse model we investigated the potential of a liposomal peptide vaccine to prime a CD8(+) T cell response against 10 different HCV epitopes, relevant for human applications. We were able to demonstrate the induction of strong cytotoxic T cell responses and high numbers of IFN-gamma-secreting cells, which persisted at high levels for at least 3 months. Co-integrating CpG oligonucleotides into liposomes further increased the number of IFN-gamma-secreting cells by 2-10-fold for most epitopes tested. The frequency of specific cells was further analysed with chimeric A2 tetramers bearing the NS31073-1081 epitope and was estimated at 2-23% of the CD8(+) T cell population. Importantly, mouse effector cells, specific for this epitope, were also capable of lysing a human target cell line expressing HCV proteins. This finding and the specific protection observed in challenge experiments with recombinant vaccinia virus expressing HCV sequences emphasise the biological relevance of the vaccine-induced immune response. In conclusion, such liposome formulations represent a safe and promising strategy to stimulate the CD8(+) T cell against HCV

Computer-Aided Histopathological Characterisation of Endometriosis Lesions.

Endometriosis is a common gynaecological condition characterised by the growth of endometrial tissue outside the uterus and is associated with pain and infertility. Currently, the gold standard for endometriosis diagnosis is laparoscopic excision and histological identification of endometrial epithelial and stromal cells. There is, however, currently no known association between the histological appearance, size, morphology, or subtype of endometriosis and disease prognosis. In this study, we used histopathological software to identify and quantify the number of endometrial epithelial and stromal cells within excised endometriotic lesions and assess the relationship between the cell contents and lesion subtypes. Prior to surgery for suspected endometriosis, patients provided menstrual and abdominal pain and dyspareunia scores. Endometriotic lesions removed during laparoscopic surgery were collected and prepared for immunohistochemistry from 26 patients. Endometrial epithelial and stromal cells were identified with Cytokeratin and CD10 antibodies, respectively. Whole slide sections were digitised and the QuPath software was trained to automatically detect and count epithelial and stromal cells across the whole section. Using this classifier, we identified a significantly larger number of strongly labelled CD10 stromal cells (p = 0.0477) in deeply infiltrating lesions (99,970 ± 2962) compared to superficial lesions (2456 ± 859). We found the ratio of epithelial to stromal cells was inverted in deeply infiltrating endometriosis lesions compared to superficial peritoneal and endometrioma lesions and we subsequently identified a correlation between total endometrial cells and abdominal pain (p = 0.0005) when counted via the automated software. Incorporating histological software into current standard diagnostic pipelines may improve endometriosis diagnosis and provide prognostic information in regards to severity and symptoms and eventually provide the potential to personalise adjuvant treatment decisions

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation

Introduction: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods: Bone marrow–derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4⁺ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results: The frequency of PS particle–positive CD11c⁺/CD11b⁺ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4⁺ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4⁺ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Surfactant Protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model

<p>Abstract</p> <p>Background</p> <p>Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated.</p> <p>Methods</p> <p>SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4⁺T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array.</p> <p>Results</p> <p>SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines.</p> <p>Conclusion</p> <p>These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.</p

Engineering an in vitro air-blood barrier by 3D bioprinting

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing