Club and goblet secretory cells are both present in the epithelial space of the BEM.

<p>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.</p

3D Reconstruction of the Human Airway Mucosa <i>In Vitro</i> as an Experimental Model to Study NTHi Infections

<div><p>We have established an <i>in vitro</i> 3D system which recapitulates the human tracheo-bronchial mucosa comprehensive of the pseudostratified epithelium and the underlying stromal tissue. In particular, we reported that the mature model, entirely constituted of primary cells of human origin, develops key markers proper of the native tissue such as the mucociliary differentiation of the epithelial sheet and the formation of the basement membrane. The infection of the pseudo-tissue with a strain of NonTypeable <i>Haemophilus influenzae</i> results in bacteria association and crossing of the mucus layer leading to an apparent targeting of the stromal space where they release large amounts of vesicles and form macro-structures. In summary, we propose our <i>in vitro</i> model as a reliable and potentially customizable system to study mid/long term host-pathogen processes.</p></div

Mucociliary differentiation of NHBE cells grown on a BEM.

<p>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.</p

Characterization of epithelial markers by confocal microscopy.

<p>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.</p

Different morphology of NHBE cells grown on BEM or transwell.

<p>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.</p

BEM cytokine secretion profile.

<p>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.</p

In vivo SAP protein expression is modulated by the presence of α-glucans.

<p>(A) RT-PCR and WB analysis of SAP expression in COH1 wt strain and COH1Δsap strain grown in the presence of different sugars. Peptidoglycan-associated protein fraction was separated by 10% (w/v) SDS-PAGE. Blots were overlaid with a mouse anti-SAP polyclonal antibody and stained with HRP-conjugated antibody. (B) Immunogold electron microscopy and confocal imaging of SAP expression in COH1 wild type strain and COH1Δsap strain grown as in (A). For IEM, fixed bacteria were incubated with an anti-SAP serum and then labeled with secondary antibody conjugated to 10-nm gold particles. Scale bars 200 nm. In confocal imaging experiments, bacteria were stained with mouse polyclonal anti-capsular type III antibodies (red) and the SAP protein with rabbit polyclonal anti-SAP antibodies (green). Magnification, ×100.</p