Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells.

BACKGROUND: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. METHODS: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. RESULTS: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. CONCLUSIONS: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

Alpha-1 Antitrypsin Mitigates the Inhibition of Airway Epithelial Cell Repair by Neutrophil Elastase.

Neutrophil elastase (NE) activity is associated with many destructive lung diseases and is a predictor for structural lung damage in early cystic fibrosis (CF), which suggests normal maintenance of airway epithelium is prevented by uninhibited NE. However, limited data exist on how the NE activity in airways of very young children with CF affects function of the epithelia. The aim of this study was to determine if NE activity could inhibit epithelial homeostasis and repair and whether any functional effect was reversible by antiprotease alpha-1 antitrypsin (α1AT) treatment. Viability, inflammation, apoptosis, and proliferation were assessed in healthy non-CF and CF pediatric primary airway epithelial cells (pAECnon-CF and pAECCF, respectively) during exposure to physiologically relevant NE. The effect of NE activity on pAECCF wound repair was also assessed. We report that viability after 48 hours was significantly decreased by 100 nM NE in pAECnon-CF and pAECCF owing to rapid cellular detachment that was accompanied by inflammatory cytokine release. Furthermore, both phenotypes initiated an apoptotic response to 100 nM NE, whereas ≥ 50 nM NE activity significantly inhibited the proliferative capacity of cultures. Similar concentrations of NE also significantly inhibited wound repair of pAECCF, but this effect was reversed by the addition of α1AT. Collectively, our results demonstrate free NE activity is deleterious for epithelial homeostasis and support the hypothesis that proteases in the airway contribute directly to CF structural lung disease. Our results also highlight the need to investigate antiprotease therapies in early CF disease in more detail.

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

RATIONALE: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. AIM OF THE STUDY: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. MATERIALS AND METHODS: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID50) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. RESULTS: HRV-1B infection affected viability that was both time and TCID50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID50, while a significant decrease in all three TJ protein expressions occurred at higher TCID50. Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. CONCLUSION: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze.

Abnormal wound repair has been observed in the airway epithelium of patients with chronic respiratory diseases, including asthma. Therapies focusing on repairing vulnerable airways, particularly in early life, present a potentially novel treatment strategy. We report defective lower airway epithelial cell repair to strongly associate with common pre-school-aged and school-aged wheezing phenotypes, characterized by aberrant migration patterns and reduced integrin α5ß1 expression. Next generation sequencing identified the PI3K/Akt pathway as the top upstream transcriptional regulator of integrin α5ß1, where Akt activation enhanced repair and integrin α5ß1 expression in primary cultures from children with wheeze. Conversely, inhibition of PI3K/Akt signaling in primary cultures from children without wheeze reduced α5ß1 expression and attenuated repair. Importantly, the FDA-approved drug celecoxib - and its non-COX2-inhibiting analogue, dimethyl-celecoxib - stimulated the PI3K/Akt-integrin α5ß1 axis and restored airway epithelial repair in cells from children with wheeze. When compared with published clinical data sets, the identified transcriptomic signature was also associated with viral-induced wheeze exacerbations highlighting the clinical potential of such therapy. Collectively, these results identify airway epithelial restitution via targeting the PI3K-integrin α5ß1 axis as a potentially novel therapeutic avenue for childhood wheeze and asthma. We propose that the next step in the therapeutic development process should be a proof-of-concept clinical trial, since relevant animal models to test the crucial underlying premise are unavailable.

Vitamin D supplementation of initially vitamin D-deficient mice diminishes lung inflammation with limited effects on pulmonary epithelial integrity.

In disease settings, vitamin D may be important for maintaining optimal lung epithelial integrity and suppressing inflammation, but less is known of its effects prior to disease onset. Female BALB/c dams were fed a vitamin D3-supplemented (2280 IU/kg, VitD+) or nonsupplemented (0 IU/kg, VitD-) diet from 3 weeks of age, and mated at 8 weeks of age. Male offspring were fed the same diet as their mother. Some offspring initially fed the VitD- diet were switched to a VitD+ diet from 8 weeks of age (VitD-/+). At 12 weeks of age, signs of low-level inflammation were observed in the bronchoalveolar lavage fluid (BALF) of VitD- mice (more macrophages and neutrophils), which were suppressed by subsequent supplementation with vitamin D3 There was no difference in the level of expression of the tight junction proteins occludin or claudin-1 in lung epithelial cells of VitD+ mice compared to VitD- mice; however, claudin-1 levels were reduced when initially vitamin D-deficient mice were fed the vitamin D3-containing diet (VitD-/+). Reduced total IgM levels were detected in BALF and serum of VitD-/+ mice compared to VitD+ mice. Lung mRNA levels of the vitamin D receptor (VDR) were greatest in VitD-/+ mice. Total IgG levels in BALF were greater in mice fed the vitamin D3-containing diet, which may be explained by increased activation of B cells in airway-draining lymph nodes. These findings suggest that supplementation of initially vitamin D-deficient mice with vitamin D3 suppresses signs of lung inflammation but has limited effects on the epithelial integrity of the lungs.

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

Rhenium tetrazolato complexes coordinated to thioalkyl-functionalised phenanthroline ligands: synthesis, photophysical characterisation, and incubation in live HeLa cells.

Three new complexes of formulation fac-[Re(CO)3(diim)L], where diim is either 1,10-phenanthroline or 1,10-phenanthroline functionalised at position 5 by a thioalkyl chain, and L is either a chloro or aryltetrazolato ancillary ligand, were synthesised and photophysically characterised. The complexes exhibit phosphorescent emission with maxima around 600 nm, originating from triplet metal-to-ligand charge transfer states with partially mixed ligand-to-ligand charge transfer character. The emission is relatively long-lived, within the 200-400 ns range, and with quantum yields of 2-4%. The complexes were trialed as cellular markers in live HeLa cells, along with two previously reported rhenium tetrazolato complexes bound to unsubstituted 1,10-phenanthroline. All five complexes exhibit good cellular uptake and non-specific perinuclear localisation. Upon excitation at 405 nm, the emission from the rhenium complexes could be clearly distinguished from autofluorescence, as demonstrated by spectral detection within the live cells. Four of the complexes did not appear to be toxic, however prolonged excitation could result in membrane blebbing. No major sign of photobleaching was detected upon multiple imaging on the same cell sample.