Development of a Novel ex vivo Nasal Epithelial Cell Model Supporting Colonization With Human Nasal Microbiota

Abstract

The nasal mucosa provides first line defense against inhaled pathogens while creating a unique microenvironment for bacterial communities. Studying the impact of microbiota in the nasal cavity has been difficult due to limitations with current models including explant cultures, primary cells, or neoplastic cell lines. Most notably, none have been shown to support reproducible colonization by bacterial communities from human donors. Therefore, to conduct controlled studies of the human nasal ecosystem, we have developed a novel ex vivo mucosal model that supports bacterial colonization of a cultured host mucosa created by immortalized human nasal epithelial cells (NEC). For this model, immortalized NEC established from 5 male and 5 female donors were cultured with an air-interfaced, apical surface on a porous transwell membrane. NEC were grown from nasal turbinate tissues harvested from willed bodies or from discarded tissue collected during sinonasal procedures. Immortalized cells were evaluated through molecular verification of cell type, histological confirmation of tissue differentiation including formation of tight junctions, NEC multilayer viability, metabolism, physiology and imaging of the luminal surface by scanning electron microscopy. Results showed proper differentiation and multilayer formation at seven to 10 days after air interface that was maintained for up to 3 weeks. The optimized mucosal cultures created an environment necessary to sustain colonization by nasal microbiomes (NMBs) that were collected from healthy volunteers, cryogenically preserved and characterized with customized quantitative polymerase chain reaction (qPCR) arrays. Polymicrobial communities of nasal bacteria associated with healthy and inflamed states were consistently reproduced in matured NEC co-cultures by transplant of NMBs from multiple community types. The cultured NMBs were stable after an initial period of bacterial replication and equilibration. This novel ex vivo culture system is the first model that supports controlled cultivation of NMBs, allowing for lab-based causation studies and further experimentation to explore the complexities of host-microbe and microbe-microbe interactions.

Lacticaseibacillus casei AMBR2 modulates the epithelial barrier function and immune response in a donor-derived nasal microbiota manner

“…(without AMBR2) or Lactobacillus sp. (with AMBR2), with the exception of donor 3, which had a community dominated by unclassified Enterobacteriaceae, regardless of presence of AMBR2, after 24 h. In our, and other similar, more complex model systems 57,58 , a consistent increase of Staphylococcus sp. in co-cultured communities compared to the original inoculum has been observed.…”

“…It should however be noted that Calu-3 cells are limited in reflecting the responses to inflammatory stimulation observed in primary cells 70 . Recently, advances have been made in the immortalisation and differentiation of primary sinonasal epithelial cells 57 and in the generation of upper respiratory tract cells from pluripotent stem cells 87 . These cell types can be more representative than the cancer cell line used in this model system.…”

“…In order to better predict the efficacy and responders of LBPs, it is important to assess the potency of novel LBPs in a microbial community that is more representative for the one that will be encountered upon administration, including interindividual variability. Few in vitro host-microbe interaction models of the sinonasal cavities include natural nasal microbiota 57,58 .…”

Dual and Triple Epithelial Coculture Model Systems with Donor-Derived Microbiota and THP-1 Macrophages To Mimic Host-Microbe Interactions in the Human Sinonasal Cavities

“…Together with the mucus present on the differentiated Calu-3 cells, this limits the profound and immediate cytotoxicity that is typically observed when exposing cells to high levels of microbiota. For example, wild-type S. aureus at 10 8 cells ml Ϫ1 reduced epithelial integrity in direct-contact cocultures with differentiated Calu-3 cell layers within 4 h (25), and treatment with conditioned medium from this bacterium resulted in 90 to 100% cytotoxicity in differentiated Calu-3 cells after 24 h. This is in sharp contrast with our coculture assay, where Calu-3 cells were in contact with bacteria and yet remained viable for more than 48 h. In the coculture setup of Charles et al (29), consisting of differentiated primary nasal epithelial cell layers apically inoculated with 5 ϫ 10 2 methicillin-resistant S. aureus organisms, unconstrained growth of this bacterium was observed after 48 h, and yet the authors of that study observed that the colonization appeared to remain limited to the apical surface, without cell layer invasion. This is consistent with our observation that severe damage to epithelial cell layers cocultured with S. aureus was not observed after 48 h. Calu-3 cells exposed for 72 h with S. aureus displayed cytotoxic stress, concomitantly with a decrease in epithelial integrity and lower production of anti-inflammatory cytokine IL-10.…”

“…Although not experimentally proven, this may explain why we did not observe such cytotoxic stress from the nasal microbiota, despite the presumed presence of S. aureus. Scanning electron microscopy images of differentiated immortalized primary nasal epithelial cells cocultured with natural nasal communities for 48 h did not show indications of a negative impact on the cell layer (29), indicating that this type of model system can sustain a natural nasal community without detrimental effects to the cell layer. With respect to epithelial barrier function, endogenous microbiota has also been shown to support tight-junction functionality.…”