Electron microscopic analysis of HIBCPP TJ structure.

<p>HIBCPP were grown on transwell filter supports in the standard (<b>A</b>, <b>C</b>) and the inverted (<b>B</b>, <b>D</b>) culture system, respectively. Transmission electron microscopy studies (<b>A</b>, <b>B</b>) show that in both culture systems the cells are connected by TJs (arrows), which are located close to the apical side as indicated by the presence of microvilli. Examination of HIBCPP by freeze fracture electron microscopy (<b>C</b>, <b>D</b>) revealed a broad band of closely meshed TJ strands. The diameter of meshes were in the magnitude of 0.2 to 0.4 µm.</p

Double immunofluorescence microscopy of adherence and invasion of HIBCPP standard cultures infected with <i>S. suis</i> and <i>N. meningitidis</i>, respectively.

<p>HIBCPP grown in the standard culture system were inoculated with the indicated bacteria (MOI 10) and after 4 h subjected to double immunofluorescence microscopy to detect intracellular (green) and extracellular (yellow) bacteria. Cell nuclei were 4,6-diamidino-2-phenylindole (blue)-stained. The actin cytoskeleton was visualized with phalloidin (purple/magenta). Apotom images: bottom of each panel is an <i>xy</i> en face view of HIBCPP shown in a maximum-intensity projection through the <i>z</i>-axis of selected slices; top and side of each panel is a cross-section through the <i>z</i>-plane of multiple optical slices. The apical side of HIBCPP is oriented towards the top or the right side, respectively, of the top and side images of each panel. <i>S. suis</i> strains 10 (<b>A</b>) and 10 cpsΔEF (<b>B</b>) show adherence to the apical membrane but no invasion. <i>N. meningitidis</i> serogroup B strain MC58 (<b>C</b>) and its isogenic acapsular mutant (<b>D</b>) display strong adhesion, but only rare invasion events (arrows). Similarly, <i>N. meningitidis</i> serogroup C strain WUE2120 (<b>E</b>) and its derivatives WUE2517 (siaD⁻) (<b>F</b>), WUE4345 (opcA⁺) (<b>G</b>) and WUE4346 (siaD⁻ opcA⁺) (<b>H</b>) adhere strongly to the apical membrane; invasion is rarely detected. Shown are representative examples of four independent experiments, which gave similar results.</p

High TEER values correlate with low FITC-inulin flux through HIBCPP-layers.

<p>HIBCPP were grown until a TEER above 70Ω×cm² was measured (day 0) and subsequently cultured in 15%, 1%, or 0% FCS, respectively, as indicated. At the indicated days TEER (<b>A</b>) and the FITC-inulin flux (<b>B</b>) were determined. Cells were grown in the standard transwell filter system (1×10⁵ cells; left panels) or the inverted transwell filter system (4×10⁴ cells; right panels). Shown is the mean+/−SD of eight experiments performed in triplicates.</p

RT-PCR analysis of the expression of the genes encoding junctional proteins and of the choroid plexus markers Transthyretin, IGF2 and FOXJ1 in HIBCPP.

<p>HIBCPP were grown in 6well plates until confluency and subsequently cultured for 1 day in medium containing 15, 1 or 0% FCS as indicated at the top of the lanes. The expression of the genes indicated at the right was analysed by RT-PCR. For comparison, RNA isolated from HeLa and Jurkat cells was analysed as well. Expression of the GAPDH gene served as control. The results shown are a typical example from three independently performed experiments.</p

HIBCPP display continuous tight junction strands.

<p>HIBCPP grown in the inverted transwell filter system were stained for detection of ZO-1 (<b>A</b>), Occludin (<b>B</b>) and Claudin-1 (<b>C</b>). Pictures presented are Apotome-generated images; <i>bottom</i> of each panel is an <i>xy</i> en face view of a cell culture monolayer shown in a maximum-intensity projection through the z-axis; <i>top and side</i> of each panel is a cross section through the z-plane of multiple optical slices. The apical side of HIBCPP is oriented towards the top or the right side, respectively, of the top and side images of each panel. In A and B the actin cytoskeleton was in parallel stained with phalloidin-FITC. Since Claudin-1 samples were fixed with methanol we could not observe a qualitatively sufficient actin staining. In all samples nuclei were stained with DAPI. The images shown are representative example of multiple stainings.</p

HIBCPP develop high TEER in standard and inverted transwell filter systems.

<p>Throughout this study cells were grown either on the upper side (standard transwell filter system) or the lower side (inverted transwell filter system) of the filter supports (<b>A</b>; schematic representation). For experiments (<b>B</b>, <b>C</b>) HIBCPP were seeded on transwell filters in the amounts indicated in the legend. Cells were cultivated either in the standard transwell filter system (<b>B</b>) or the inverted transwell filter system (<b>C</b>). TEER was measured over time at the days after seeding of the cells as indicated on the x-axis. Shown is the mean+/−SD of four (standard culture) or five (inverted culture) experiments, respectively, each performed in triplicates.</p

Polar invasion of bacteria into HIBCPP.

<p>Invasion of <i>S. suis</i> and <i>N. meningitidis</i> strains was analysed by double immunofluorescence. Standard (white bars) and inverted (black bars) transwell filter cultures were infected with the indicated strains for 4 h before immunofluorescence staining and quantification of invasion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030069#s4" target="_blank">Experimental procedures</a>. Data shown are the mean+SD of a minimum of four independent experiments each performed at least in duplicates. ** (highly significant; p<0.01), *** (extremely significant; p<0.001); when invasion in the inverted filter system compared to invasion in the standard filter system. # (significant; p<0.05), ### (extremely significant; p<0.001); when invasion of the unencapsulated strain compared to invasion of the capsulated strain in the standard or inverted filter system, respectively. §§ (highly significant; p<0.01); when invasion of the Opc complemented strain compared to the respective not complemented strain.</p

Analysis of transmigrated <i>N. meningitidis</i> attached to the apical membrane of HIBCPP.

<p>HIBCPP grown in the inverted system were infected with <i>N. meningitidis</i> strain WUE4346 (siaD⁻ opcA⁺) at an MOI of 100 (<b>A</b>) or 10 (<b>B</b>) for 4 h and subsequently analysed by scanning electron microscopy. Single bacteria (<b>A</b>) and microcolonies (<b>A</b>, <b>B</b>) are located at the apical membrane, which can be identified by the presence of microvilli.</p

Double immunofluorescence microscopy of adherence and invasion of HIBCPP inverted cultures infected with <i>S. suis</i> and <i>N. meningitidis</i>, respectively.

<p>HIBCPP grown in the inverted culture system were inoculated with the indicated bacteria and analysed for intracellular (green) and extracellular (yellow) bacteria as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030069#pone-0030069-g007" target="_blank">Fig. 7</a>. <i>S. suis</i> strains 10 (<b>A</b>) and 10 cpsΔEF (<b>B</b>) invade HIBCPP from the basolateral side. Similarly, basolateral invasion is observed for <i>N. meningitidis</i> serogoup B strain MC58 (<b>C</b>) and its isogenic acapsular mutant (<b>D</b>). <i>N. meningitidis</i> serogroup C strain WUE2120 (<b>E</b>) and its derivatives WUE2517 (siaD⁻) (<b>F</b>), WUE4345 (opcA⁺) (<b>G</b>) and WUE4346 (siaD⁻ opcA⁺) (<b>H</b>) invade HIBCPP from the basolateral side and form microcolonies at the apical membrane. Arrows indicate invaded bacteria directly below apically adhered microcolonies. Shown are representative examples of four independent experiments, which gave similar results.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p