Fig 1.
Schematic representation of the bronchial equivalent model.
a) Schematic representation of the basic BEM, consisting of NHLFs and NHBE cells. b) Toluidine blue staining of a mature standard BEM.
Fig 2.
Different morphology of NHBE cells grown on BEM or transwell.
Haematoxylin-eosin-alcian blue staining of NHBE cells differentiated on a BEM (a) or on a transwell system (b). The arrow indicates the basal layer that forms when NHBE cells are grown on a BEM. Images are representative of 4 different experiments.
Fig 3.
Mucociliary differentiation of NHBE cells grown on a BEM.
Mucus granules are visible by SEM (a) (indicated by arrows), TEM (b) and confocal analysis (c) (blue is DNA, green is MUC5AC, red is F-actin). Cilia are visible by SEM (d), TEM (e) and confocal analysis (f) (blue is DNA, white is B-tubulin, red is F-actin). Images are representative of 3 independent experiments.
Fig 4.
Characterization of differentiation markers of the BEM epithelial barrier.
Immunofluorescence staining for a) ZO-1 (green, blue is DNA) and b) F-actin (red). c) Immunofluorescence staining of laminin and integrin alpha-6 (ITGA6) (blue is DNA; red is laminin; green is ITGA6). d) IHC staining of laminin. The arrow indicates the presence of a continuous layer of laminin at the interface between the epithelial sheet and the stromal compartment; antibody isotype control is represented in (e). Images are representative of 4 different experiments.
Fig 5.
Characterization of epithelial markers by confocal microscopy.
a) Immunofluorescence staining of cytokeratin 5 (blue is DNA; green is CK5; white is transmitted light). b) Immunofluorescence staining of nerve growth factor receptor and integrin alpha-6 (blue is DNA; red is NGFR; green is ITGA6; white is transmitted light). c) Immunofluorescence double staining for cytokeratin 5 and nerve growth factor receptor (blue is DNA; red is NGFR; green is CK5). d) Differential expression of basal cells CK14 and p63 markers (blue is DNA; red is CK14; green is p63). e) Enlargement of an area represented in d); the yellow arrow indicates positivity for both p63 and CK14. Images are representative of 4 different experiments.
Fig 6.
Club and goblet secretory cells are both present in the epithelial space of the BEM.
a) Immunofluorescence staining of ClCs (positive for CCSP, red). b) Immunofluorescence staining of ClCs (positive for CCSP, red) and goblet cells (positive for MUC5AC, green). c) Immunofluorescence staining reveals the presence of cells positive for both Club cells and goblet cells markers (blue is DNA; red is CCSP; green is MUC5AC). White dotted lines separate the epithelium from the scaffold. Images are representative of 4 different experiments.
Fig 7.
BEM cytokine secretion profile.
Histogram plot representing cytokine concentration (pg/mL) in culture basal supernatants collected after 7 days from the model assembly before ALI transition (pre-ALI) and after 28 days from the model assembly at the end of the differentiation protocol (post-ALI). Data are represented as mean values (+ SD) of 3 biological replicates. Multiple t-test was performed to determine statistical significance. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.
Fig 8.
Immunofluorescence characterization of NTHi-infected BEM.
a) Bacteria (red), revealed by using polyclonal antibodies against the whole bacterium, are embedded into a thick mucus layer (white). The contour of epithelial cells is delineated by phalloidin staining (green). b) Bacteria (red) associated with and traversing the epithelial layer (phalloidin, green; DNA, blue). c) NTHi bacteria (red) colonizing the stromal compartment (transmitted light, grey; DNA, blue). d) Outer Membrane Vesicles (red) detected within the bottom side of the BEM stromal compartment (transmitted light, grey; DNA, blue) and bacteria (red), highlighted by asterisks.