Optimization of Primary Human Bronchial Epithelial 3D Cell Culture with Donor-Matched Fibroblasts and Comparison of Two Different Culture Media.

In vitro airway models are increasingly important for pathomechanistic analyses of respiratory diseases. Existing models are limited in their validity by their incomplete cellular complexity. We therefore aimed to generate a more complex and meaningful three-dimensional (3D) airway model. Primary human bronchial epithelial cells (hbEC) were propagated in airway epithelial cell growth (AECG) or PneumaCult ExPlus medium. Generating 3D models, hbEC were airlifted and cultured on a collagen matrix with donor-matched bronchial fibroblasts for 21 days comparing two media (AECG or PneumaCult ALI (PC ALI)). 3D models were characterized by histology and immunofluorescence staining. The epithelial barrier function was quantified by transepithelial electrical resistance (TEER) measurements. The presence and function of ciliated epithelium were determined by Western blot and microscopy with high-speed camera. In 2D cultures, an increased number of cytokeratin 14-positive hbEC was present with AECG medium. In 3D models, AECG medium accounted for high proliferation, resulting in hypertrophic epithelium and fluctuating TEER values. Models cultured with PC ALI medium developed a functional ciliated epithelium with a stable epithelial barrier. Here, we established a 3D model with high in vivo-in vitro correlation, which has the potential to close the translational gap for investigations of the human respiratory epithelium in pharmacological, infectiological, and inflammatory research.

Human Metapneumovirus Infection in Chronic Obstructive Pulmonary Disease: Impact of Glucocorticosteroids and Interferon.

Background: Human metapneumovirus (hMPV) infection is implicated in exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Research into the pathogenesis of infection is restricted to animal models, and information about hMPV replication and inflammatory and immune responses in human disease is limited. Methods: Human primary bronchial epithelial cells (PBECs) from healthy and asthmatic subjects and those with COPD were infected with hMPV, with or without glucocorticosteroid (GCS) exposure. Viral replication, inflammatory and immune responses, and apoptosis were analyzed. We also determined whether adjuvant interferon (IFN) can blunt hMPV infection in vitro and in a murine model. Results: hMPV infected human PBECs and viral replication was enhanced in cells from patients with COPD. The virus induced gene expression of IFN-stimulated gene 56 (ISG56) and IFN-ß, as well as IFN-γ-inducible protein 10 (IP-10) and regulated on activation, normal T cell expressed and secreted (RANTES), and more so in cells from patients with COPD. GCS exposure enhanced hMPV replication despite increased IFN expression. Augmented virus replication associated with GCS was mediated by reduced apoptosis via induction of antiapoptotic genes. Adjuvant IFN treatment suppressed hMPV replication in PBECs and reduced hMPV viral titers and inflammation in vivo. Conclusions: hMPV infects human PBECs, eliciting innate and inflammatory responses. Replication is enhanced by GCS and adjuvant IFN is an effective treatment, restricting virus replication and proinflammatory consequences of hMPV infections.

Dysregulated mitochondrial metabolism upon cigarette smoke exposure in various human bronchial epithelial cell models.

Exposure to cigarette smoke (CS) is the primary risk factor for developing chronic obstructive pulmonary disease. The impact of CS exposure on the molecular mechanisms involved in mitochondrial quality control in airway epithelial cells is incompletely understood. Undifferentiated or differentiated primary bronchial epithelial cells were acutely/chronically exposed to whole CS (WCS) or CS extract (CSE) in submerged or air-liquid interface conditions. Abundance of key regulators controlling mitochondrial biogenesis, mitophagy and mitochondrial dynamics was assessed. Acute exposure to WCS or CSE increased the abundance of components of autophagy and receptor-mediated mitophagy in all models. Although mitochondrial content and dynamics appeared to be unaltered in response to CS, changes in both the molecular control of mitochondrial biogenesis and a shift toward an increased glycolytic metabolism were observed in particular in differentiated cultures. These alterations persisted, at least in part, after chronic exposure to WCS during differentiation and upon subsequent discontinuation of WCS exposure. In conclusion, smoke exposure alters the regulation of mitochondrial metabolism in airway epithelial cells, but observed alterations may differ between various culture models used. This article has an associated First Person interview with the joint first authors of the paper.

Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air-Liquid Interface Cultured Human Primary Bronchial Epithelial Cells.

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air-liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment.