The development and characterization of in vivo-like three-dimensional models of bronchial epithelial cell lines.

In vitro models of differentiated respiratory epithelium that allow high-throughput screening are an important tool to explore new therapeutics for chronic respiratory diseases. In the present study, we developed in vivo-like three-dimensional (3-D) models of bronchial epithelial cell lines that are commonly used to study chronic lung disease (16HBE14o-, CFBE41o- and CFBE41o- 6.2 WT-CFTR). To this end, cells were cultured on porous microcarrier beads in the rotating wall vessel (RWV) bioreactor, an optimized suspension culture method that allows higher throughput experimentation than other physiologically relevant models. Cell differentiation was compared to conventional two-dimensional (2-D) monolayer cultures and to the current gold standard in the respiratory field, i.e. air-liquid interface (ALI) cultures. Cellular differentiation was assessed in the three model systems by evaluating the expression and localization of markers that reflect the formation of tight junctions (zonula occludens 1), cell polarity (intercellular adhesion molecule 1 at the apical side and collagen IV expression at the basal cell side), multicellular complexity (acetylated α-tubulin for ciliated cells, CC10 for club cells, keratin-5 for basal cells) and mucus production (MUC5AC) through immunostaining and confocal laser scanning microscopy. Results were validated using Western Blot analysis. We found that tight junctions were expressed in 2-D monolayers, ALI cultures and 3-D models for all three cell lines. All tested bronchial epithelial cell lines showed polarization in ALI and 3-D cultures, but not in 2-D monolayers. Mucus secreting goblet-like cells were present in ALI and 3-D cultures of CFBE41o- and CFBE41o- 6.2 WT-CFTR cells, but not in 16HBE14o- cells. For all cell lines, there were no ciliated cells, basal cells, or club cells found in any of the model systems. In conclusion, we developed RWV-derived 3-D models of commonly used bronchial epithelial cell lines and showed that these models are a valuable alternative to ALI cultures, as they recapitulate similar key aspects of the in vivo parental tissue.

Commensal bacteria of the lung microbiota synergistically inhibit inflammation in a three-dimensional epithelial cell model.

Patients with chronic lung disease suffer from persistent inflammation and are typically colonized by pro-inflammatory pathogenic bacteria. Besides these pathogens, a wide variety of commensal species is present in the lower airways but their role in inflammation is unclear. Here, we show that the lung microbiota contains several species able to inhibit activation of the pro-inflammatory NF-κB pathway and production of interleukin 8 (IL-8), triggered by lipopolysaccharide (LPS) or H2O2, in a physiologically relevant three-dimensional (3D) lung epithelial cell model. We demonstrate that the minimal dose needed for anti-inflammatory activity differs between species (with the lowest dose needed for Rothia mucilaginosa), and depends on the type of pro-inflammatory stimulus and read out. Furthermore, we evaluated synergistic activity between pairs of anti-inflammatory bacteria on the inhibition of the NF-κB pathway and IL-8 secretion. Synergistic anti-inflammatory activity was observed for 4/10 tested consortia. These findings indicate that various microbiota members can influence lung inflammation either alone or as a consortium. This information can contribute to a better understanding of the lung microbiota in chronic lung disease development and process, and could open up new avenues for treatment.

R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease.

BACKGROUND: Chronic airway inflammation is the main driver of pathogenesis in respiratory diseases such as severe asthma, chronic obstructive pulmonary disease, cystic fibrosis (CF) and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognised, the influence of other microbiota members is still poorly understood. METHODS: We hypothesised that the lung microbiota contains bacteria with immunomodulatory activity which modulate net levels of immune activation by key respiratory pathogens. Therefore, we assessed the immunomodulatory effect of several members of the lung microbiota frequently reported as present in CF lower respiratory tract samples. RESULTS: We show that Rothia mucilaginosa, a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- or lipopolysaccharide-induced pro-inflammatory responses, in vitro (three-dimensional cell culture model) and in vivo (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of Rothia species was negatively correlated with pro-inflammatory markers (interleukin (IL)-8 and IL-1ß) and matrix metalloproteinase (MMP)-1, MMP-8 and MMP-9 in sputum. Mechanistic studies revealed that R. mucilaginosa inhibits NF-κB pathway activation by reducing the phosphorylation of IκBα and consequently the expression of NF-κB target genes. CONCLUSIONS: These findings indicate that the presence of R. mucilaginosa in the lower airways potentially mitigates inflammation, which could in turn influence the severity and progression of chronic respiratory disorders.

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics.

Antibiotic susceptibility of bacterial pathogens is typically evaluated using in vitro assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy in vitro and in vivo, with some antibiotics being effective in vitro but not in vivo or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen Pseudomonas aeruginosa, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an in vivo-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against P. aeruginosa, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics in vitro and in vivo may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.

Influence of three-dimensional lung epithelial cells and interspecies interactions on antibiotic efficacy against Mycobacterium abscessus and Pseudomonas aeruginosa.

Mycobacterium abscessus lung infection is a major health problem for cystic fibrosis (CF) patients. Understanding the in vivo factors that influence the outcome of therapy may help addressing the poor correlation between in vitro and in vivo antibiotic efficacy. We evaluated the influence of interspecies interactions and lung epithelial cells on antibiotic efficacy. Therefore, single and dual-species biofilms of M. abscessus and a major CF pathogen (Pseudomonas aeruginosa) were cultured on a plastic surface or on in vivo-like three-dimensional (3-D) lung epithelial cells, and the activity of antibiotics (colistin, amikacin, clarithromycin, ceftazidime) in inhibiting biofilm formation was evaluated. Using the most physiologically relevant model (dual-species biofilms on 3-D cells), we observed that treatment with antibiotics during biofilm development inhibited P. aeruginosa but not M. abscessus biofilms, resulting in a competitive advantage for the latter. Clarithromycin efficacy against P. aeruginosa was inhibited by 3-D lung cells. In addition, biofilm induction of M. abscessus was observed by certain antibiotics on plastic but not on 3-D cells. Pseudomonas aeruginosa influenced the efficacy of certain antibiotics against M. abscessus, but not vice versa. In conclusion, these results suggest a role of host cells and interspecies interactions in bacterial responses to antimicrobials.