Lung on Chip: In vitro HGF effects on injured alveolar A549 epithelial cells in microfluidic system

Microfluidic systems have become competitive tools in the invitro modelling of diseases and promising alternatives to animal studies. They allow obtaining more invivo like conditions for cellular assays. Research in idiopathic pulmonary fibrosis could benefit from this novel methodological approach to understand the pathophysiology of the disease & develop efficient therapies. The use of hepatocyte growth factor (HGF) for alveolar reepithelisation is a promising approach. In this study, we show a new microfluidic system to analyse the effects of HGF on injured alveolar epithelial cells. Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells (10,000 cells) were seeded and studied in these microfluidic systems with media perfusion (1μl/30min). Injury tests were made on the cells by the perfusion with media containing H2O2 or bleomycin. The degree of injury was then assessed by a metabolic and an apoptotic assays. Wound assays were also performed with a central laminar flow of trypsin. Monitoring of wound closure with HGF vs control media was assessed. The alveolar A549 epithelial cells grew and proliferated in the microfluidic system. In the wound closure assay, the degree of wound closure after 5 hours was (53.3±1.3%) with HGF compared to (9.8±2.4%) without HGF (P <0.001). We present a novel microfluidic model that allows culture, injury and wounding of A549 epithelial cells and represents the first step towards the development of an invitro reconstitution of the alveolar-capillary interface. We were also able to confirm that HGF increased alveolar epithelial repair in this system.

Phosphoinositide 3-kinase C2β regulates RhoA and the actin cytoskeleton through an interaction with Dbl

The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2β. Dbl was co-immunoprecipitated with PI3KC2β in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2β in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2β strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2β over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant-negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal-regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2β expression. Furthermore, increased expression of PI3KC2β protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2β modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA.

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.

A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

BACKGROUND: Staphylococcus aureus, a leading cause of chronic or acute infections, is traditionally considered an extracellular pathogen despite repeated reports of S. aureus internalization by a variety of non-myeloid cells in vitro. This property potentially contributes to bacterial persistence, protection from antibiotics and evasion of immune defenses. Mechanisms contributing to internalization have been partly elucidated, but bacterial processes triggered intracellularly are largely unknown. RESULTS: We have developed an in vitro model using human lung epithelial cells that shows intracellular bacterial persistence for up to 2 weeks. Using an original approach we successfully collected and amplified low amounts of bacterial RNA recovered from infected eukaryotic cells. Transcriptomic analysis using an oligoarray covering the whole S. aureus genome was performed at two post-internalization times and compared to gene expression of non-internalized bacteria. No signs of cellular death were observed after prolonged internalization of Staphylococcus aureus 6850 in epithelial cells. Following internalization, extensive alterations of bacterial gene expression were observed. Whereas major metabolic pathways including cell division, nutrient transport and regulatory processes were drastically down-regulated, numerous genes involved in iron scavenging and virulence were up-regulated. This initial adaptation was followed by a transcriptional increase in several metabolic functions. However, expression of several toxin genes known to affect host cell integrity appeared strictly limited. CONCLUSION: These molecular insights correlated with phenotypic observations and demonstrated that S. aureus modulates gene expression at early times post infection to promote survival. Staphylococcus aureus appears adapted to intracellular survival in non-phagocytic cells.

Cell cycle-dependent regulation of extra-adrenal glucocorticoid synthesis in murine intestinal epithelial cells

Glucocorticoids are anti-inflammatory steroids with important applications in the treatment of inflammatory diseases. Endogenous glucocorticoids are mainly produced by the adrenal glands, although there is increasing evidence for extra-adrenal sources. Recent findings show that intestinal crypt cells produce glucocorticoids, which contribute to the maintenance of intestinal immune homeostasis. Intestinal glucocorticoid synthesis is critically regulated by the transcription factor liver receptor homologue-1 (LRH-1). As expression of steroidogenic enzymes and LRH-1 is restricted to the proliferating cells of the crypts, we aimed to investigate the role of the cell cycle in the regulation of LRH-1 activity and intestinal glucocorticoid synthesis. We here show that either pharmacological or molecular modulation of cell cycle progression significantly inhibited expression of steroidogenic enzymes and synthesis of glucocorticoids in intestinal epithelial cells. Synchronization of intestinal epithelial cells in the cell cycle revealed that expression of steroidogenic enzymes is preferentially induced at the G(1)/S stage. Differentiation of immature intestinal epithelial cells to mature nonproliferating cells also resulted in reduced expression of steroidogenic enzymes. This cell cycle-related effect on intestinal steroidogenesis was found to be mediated through the regulation of LRH-1 transcriptional activity. This mechanism may restrict intestinal glucocorticoid synthesis to the proliferating cells of the crypts.

Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro

Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

Physiologic cold shock increases adherence of Moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26 degrees C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26 degrees C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26 degrees C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulence.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Targeted gene transfer of hepatocyte growth factor to alveolar type II epithelial cells reduces lung fibrosis in rats

Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-β(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells.

Bovine colostrogenesis is distinguished by the specific transfer of IgG1 from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E2 ) and progesterone (P4 ) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta-2-Microglobulin (β2M). We hypothesized that steroid hormone treatments (E2 and P4 ) of bovine mammary epithelial cells in vitro would induce up-regulation of IgG1 transcytosis candidate gene mRNA expression suggesting involvement in IgG1 transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up-regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG1 transcytosis in bovine mammary cells.

Effects of induced energy deficiency on lactoferrin concentration in milk and the lactoferrin reaction of primary bovine mammary epithelial cells in vitro.

A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid-lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control-fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat-inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme-linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post-partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up-regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk-extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non-invasive milk-extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.

Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.