Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa

Abstract

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

Long‐term expanding human airway organoids for disease modeling

“…Enzymelinked immunosorbent assays confirmed the secretion of significant quantities of cytokines such as IP-10 and RANTES from RSVinfected AOs (Fig 4I). Of note, Yonker et al (2017) have described co-culture of neutrophils with RSV-infected human airway epithelium grown in 2D ALI culture. Of note, Yonker et al (2017) have described co-culture of neutrophils with RSV-infected human airway epithelium grown in 2D ALI culture.…”

Expansion of Airway Basal Cells and Generation of Polarized Epithelium

“…With this newly discovered ability to expand airway basal stem cells, patient-specific cells can be isolated and preserved from small biopsies of the airway, generating a virtually limitless supply of patient-specific airway epithelium in vitro (Mou et al , 2016). When differentiated on air-liquid interface, airway basal stem cell-derived epithelium forms a polarized mucociliary layer that exhibits proper epithelial architecture, physiology and response to clinically relevant pharmacologic agents (Mou et al , 2016; Yonker et al , 2017a and 2017b). Further, airway disease can be studied by generating basal stem cell-derived epithelium from individuals with airway disease, such as cystic fibrosis (CF), a genetic disease caused by a mutation in the CFTR gene, resulting in progressive respiratory failure.…”

“…A better understanding of cellular and molecular mechanisms mediating neutrophil trans-epithelial migration has the potential to inform and improve the efficacy of anti-inflammatory therapeutic strategies critically needed to treat inflammation mediated lung damage associated with CF. Recently, we have applied our advanced culture method in a co-culture system with neutrophils to investigate neutrophil-airway epithelium interaction and P. aeruginosa -induced neutrophil trans-epithelial migration (Yonker et al , 2017a and 2017b), Our findings from cultured human airway basal stem cells differentiated on ALI are consistent with prior molecular mechanistic studies implicating epithelial-derived neutrophil chemoattractant hepoxilin A3, an arachidonic acid metabolite synthesized by 12-lipoxygenase, as key in driving P. aeruginosa -induced trans-epithelial migration (Yonker et al. , 2017a and 2017b).…”

“…Neutrophils applied to the basolateral aspect of the infected airway mucosal ALI organize in clusters before migrating through the mucosal barrier in response to epithelial infection. These organized clusters of neutrophils persist along the apical surface following trans-epithelial migration before individual neutrophils begin detaching from clusters into the airspace compartment (Yonker et al , 2017a). These results demonstrate that our expandable airway basal stem cell culturing and ALI differentiation system has tremendous potential to be exploited in a variety of ways to better understand micro-anatomy, physiology, interaction with pathogens, and innate immunity at the airway mucosa.…”

Advances and Remaining Challenges in the Study of Influenza and Anthrax Infection in Lung Cell Culture

“…A triple co-culture system using lung cells, dendritic cells, and macrophages has also been constructed by Swiss researchers [44]. Yonker et al [45] have recently created an inflamed airway mucosa co-culture model that is comprised of neutrophils and primary lung cells that were studied in the context of Pseudomonas aeruginosa infection. As already mentioned, there are lung co-cultures commercially available through Epithelix (MucilAir™-HF) or MatTek (EpiAirwayFT) ( Table 1).…”