A responsive human triple-culture model of the air-blood barrier: incorporation of different macrophage phenotypes

Abstract

Current pulmonary research underlines the relevance of the alveolar macrophage (AM) integrated in multicellular co-culture-systems of the respiratory tract to unravel, for example, the mechanisms of tissue regeneration. AMs demonstrate a specific functionality, as they inhabit a unique microenvironment with high oxygen levels and exposure to external hazards. Healthy AMs display an antiinflammatory phenotype, prevent hypersensitivity to normally innocuous contaminants and maintain tissue homeostasis in the alveolus. To mirror the actual physiological function of the AM, we developed three different polarized [classically activated (M1) and alternatively activated (M2 wh , wound-healing; M2 reg , regulatory)] macrophage models using a mixture of differentiation mediators, as described in the current literature. To test their immunological impact, these distinct macrophage phenotypes were seeded on to the epithelial layer of an established in vitro air-blood barrier co-culture, consisting of alveolar epithelial cells A549 or H441 and microvascular endothelial cells ISO-HAS-1 on the opposite side of a Transwell filter-membrane. IL-8 and sICAM release were measured as functionality parameters after LPS challenge. The M1 model itself already provoked a severe inflammatory-like response of the air-blood barrier co-culture, thus demonstrating its potential as a useful in vitro model for inflammatory lung diseases. The two M2 models represent a 'non-inflammatory' phenotype but still showed the ability to trigger inflammation following LPS challenge. Hence, the latter could be used to establish a quiescent, physiological in vitro air-blood model. Thus, the more complex differentiation protocol developed in the present study provides a responsive in vitro triple-culture model of the air-blood-barrier that mimics AM features as they occur in vivo.

In vitro exposure of a 3D-tetraculture representative for the alveolar barrier at the air-liquid interface to silver particles and nanowires

“…Moreover, the basal inflammatory state of the tetra-culture by using rested macrophages was lower than in the previously described model [22]. The secretion of IL-4, IL-10 and IL-13, three interleukins which are mainly representative for an anti-inflammatory or regulatory response, increased in the same manner as the other interleukins with a pro-inflammatory function [70]. This auto-regulatory nature of the inflammatory process is further supported by the detailed gene expression of pro-inflammatory mediators observed.…”

High density of CD68+ tumor-associated macrophages predicts a poor prognosis in gastric cancer mediated by IL-6 expression

“…Subsequent to 1 day of incubation at 37°C, the cells fixed with 3.7% paraformaldehyde in CS buffer, 0.1 M piperazine-N,N'-bis (2-ethanesulfonic acid; PIPES), 1 mM EGTA, 4% polyethylene glycol 800 and 0.1 M NaOH, for 20 min at room temperature for subsequent crystal violet staining. The number of MGC-803 cells that penetrated the micropores were then counted at magnification ×200 with a light-microscope (Nikon Corporation, Tokyo, Japan) ( 14 ).…”

Autologous Co-culture of Primary Human Alveolar Macrophages and Epithelial Cells for Investigating Aerosol Medicines. Part II: Evaluation of IL-10-loaded Microparticles for the Treatment of Lung Inflammation

“…However, it is possible that other cellular models based on cell lines and/or cell types not linked to lung cells, could show a similar behaviour to the primary cell model. For example, in a recently published article, Kasper et al (39) compared various co-culture models addressing various macrophage phenotypes, which might serve as similar tools. The work of Herzog et al (40) investigated the triple co-culture model, which is based on A549 epithelial cells, human peripheral blood monocyte-derived dendritic cells and macrophages.…”