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. 2020 Jul 2;8(3):1804249.
doi: 10.1080/21688370.2020.1804249. Epub 2020 Aug 23.

IL-1β strengthens the physical barrier in gingival epithelial cells

Kim Natalie Stolte  1   2 , Carsten Pelz  1   2 , Cynthia V Yapto  1   2 , Jan-Dirk Raguse  1   3 , Henrik Dommisch  1   4   5 , Kerstin Danker  1   2
Affiliations

IL-1β strengthens the physical barrier in gingival epithelial cells

Kim Natalie Stolte et al. Tissue Barriers. .

Abstract

Periodontitis is one of the most common oral diseases worldwide and is caused by a variety of interactions between oral bacteria and the host. Here, pathogens induce inflammatory host responses that cause the secretion of proinflammatory cytokines such as IL-1β, IL-6, and IL-8 by oral epithelial cells. In various systems, it has been shown that inflammation compromises physical barriers, which enables bacteria to invade the tissue. In this study, we investigated the barrier properties of the oral mucosa under physiological and inflamed conditions. For this purpose, we assessed the influence of IL-1β on the transepithelial electrical resistance and in particular on tight junctions in vitro in human stratified squamous epithelium models. Indirect immunofluorescence and western blot analyses were performed to investigate localization and expression of tight junction proteins in primary gingival cells, immortalized gingival cells and native gingiva. Furthermore, the TEER of gingival keratinocytes was assessed. The results showed that IL-1β led to strengthening of the gingival keratinocyte barrier. This was demonstrated by an increase in TEER, the upregulation of TJ proteins, and an increase in the formation of TJ strands. The IL-1β-mediated upregulation of occludin was prevented by the NF-κB inhibitor BAY 11-7085. These observations provide insights into host responses in the early stages of periodontal disease and offer information about TJ formation in human gingival epithelial cells under physiological and inflammatory conditions. Comprehensive knowledge of the physical barrier during inflammation may help in developing strategies to effectively _target the inflammatory barrier to improve the bioavailability of drugs for the treatment of periodontitis.

Keywords: Gingiva; IL1beta; TEER; oral mucosa; tight junctions.

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Figures

Figure 1.
Figure 1.
IL-1β induces the secretion of the proinflammatory cytokines IL-6 and IL-8 in gingival keratinocytes.
Figure 2.
Figure 2.
IL-1β does not influence the metabolic activity or proliferation of OKG4 cells.
Figure 3.
Figure 3.
IL-1β increases transepithelial resistance in a human 3D epithelial model.
Figure 4.
Figure 4.
Expression and localization of CLDN1, ZO-1 and OCLN in healthy human gingiva.
Figure 5.
Figure 5.
IL-1β increases the expression of OCLN and enhances the formation of TJ stands in gingival keratinocytes.
Figure 6.
Figure 6.
IL-1β enhances the subapical localization of OCLN in TJs.
Figure 7.
Figure 7.
Inhibition of NF-κB activation prevents IL-1β-induced upregulation of OCLN.
Figure A1.
Figure A1.
Secretion of proinflammatory cytokines in the presence and absence of IL-1β. IL-8 signals obtained by the cytokine array (Figure 1(a)) were quantified. Error bars represent the standard deviation and * indicates significant difference between the test and control group (p = .04).
Figure A2.
Figure A2.
Influence of IL-1β on the morphology of OKG4 cells. Visualization of fluorescence-labeled wheat germ agglutinin (WGA) in OKG4 cells (above) and GECs (below) in control and IL-1β-treated cells. 1.2 × 104 OKG4 cells and GECs were cultured in 8-well chamber slides and stimulated with IL-1β or left untreated for 24 h, respectively. Subsequently, cells were fixed. Membranes were visualized with WGA and cell nuclei were stained with Hoechst 33342. Image processing was performed with ZEN and ImageJ, scale bar, 20 µm.
Figure A3.
Figure A3.
Costaining of ZO-1 and occludin in adult human gingiva.
Figure A4.
Figure A4.
TJ strands are not formed in oral keratinocytes in the presence of a low Ca2+ concentration. 1.2 × 104 cells cultivated in the presence of 60 µM Ca2+ were fixed and stained for claudin-1, ZO-1 and occludin. Image processing was performed with ZEN and ImageJ. Scale bar, 50 µm.
Figure A5.
Figure A5.
IL-1β increases the expression of occludin and enhances the formation of TJ strands in OKG4 cells. 1.2 × 104 OKG4 cells treated with IL-1β or left untreated for 24 h were fixed and stained for claudin-1, ZO-1 and occludin. Image processing was performed with ZEN and ImageJ. Scale bar, 50 µm.
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Grants and funding

This work was supported by the Sonnenfeld-Stiftung (Antje Bürgel grant to Kim N. Stolte) and the Deutsche Forschungsgemeinschaft under grant DA310/8-1 and grant DO 1375/2-1.
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