PLoS ONE. 2016 Apr; 11(4):e0152794.
Porphyromonas gingivalis gingipain-dependently enhances IL-33 production in human gingival epithelial cells.
Tada H, Matsuyama T, Nishioka T, Hagiwara M, Kiyoura Y, Shimauchi H, Matsushita K.
Department of Oral Microbiology, Tohoku University Graduate School of Dentistry, Sendai, Miyagi 980-8575, JAPAN.
Epithelial cells work not only as a physical barrier to pathogens, but also play a pivotal role in initiating innate immune responses to microbes. Interleukin (IL)-33, a member of the IL-1 family, is constitutively expressed in epithelial cells and amplifies innate and adaptive Th2-type immune responses. We investigated the involvement of a periodontal pathogen, Porphyromonas gingivalis, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and P. gingivalis strongly increased IL-33 expression in human gingival epithelial cells. P. gingivalis-induced IL-33 was localized in the cytoplasm of the cells. However, fimbriae, lipopeptide, and lipopolysaccharide derived from P. gingivalis were not active in this respect. Protease inhibitors specific for gingipains efficiently inhibited the induction of IL-33 mRNA by P. gingivalis inoculation. Furthermore, P. gingivalis KDP136, a gingipains-null mutant, failed to increase IL-33 expression. A small interference RNA for protease-activated receptor-2 decreased P. gingivalis-induced IL-33 expression. Increased expression of IL-33 by P. gingivalis inoculation was inhibited in the presence of inhibitors to phospholipase C, p38, and NF-kB. These results indicate that increased expression of IL-33 in gingival epithelial cells might be involved in the pathogenesis of periodontal diseases.
- PMID: 27058037; DOI: 10.1371/journal.pone.0152794
In mucosal immune systems, including the oral mucosa, epithelial cells work not only as a physical barrier to bacterial pathogens, but also play a pivotal role in initiating and amplifying Th2-type immune responses in response to bacterial components. Epithelial cells produce Interleukin (IL)-33, IL-25, and thymic stromal lymphopoietin, which may be involved in the development and regulation of Th2-type inflammatory responses. IL-33 is a member of the IL-1 cytokine family, and is constitutively expressed in epithelial cells, endothelial cells and fibroblasts. IL-33 consists of two domains: a non-classical homeodomain-like helix-turn-helix DNA-binding domain, which consists of a chromatin-binding motif (CBM) and a nuclear localization sequence (NLS), and an IL-1-like domain (Fig. 1a). IL-33 localizes in the nuclei of resting epithelial cells and active form of IL-33 acts as an alarmin when released from necrotic cells (Fig. 1b). IL-33 has a protective role in inflammatory bowel disease and in the initiation of Toxoplasma infection that polarizes adaptive responses towards a Th2-biased response, which is protective in this disease. In contrast, a lot of evidence suggests that IL-33 is also involved in the development of chronic inflammatory diseases such as arthritis. IL-33 signals through the IL-33 receptor (IL-33R), which consists of heterodimers of ST2 and IL-1 receptor accessory protein (IL-1RAcP) (Fig. 2). IL-33 upon binding to ST2 induces the recruitment of IL-1RAcP and myeloid differentiation primary-response protein 88 (MyD88) to the Toll/IL-1R (TIR) domain in the cytoplasmic region of ST2. The MyD88 and TRAF6 complex activates NF-kB- and MAP kinase-mediated signaling pathways.
Expression of IL-33 in periodontal tissues from chronic periodontitis patients. Porphyromonas gingivalis is a periodontopathic pathogen in chronic periodontitis and has a variety of virulence factors that induce proinflammatory cytokines leading to chronic inflammation, resulting in destruction of periodontal tissues. TNF-a induces IL-33 expression in human gingival fibroblasts, although whether IL-33 expression is increased in the periodontal tissues in chronic periodontitis patients and the functions of IL-33 in the modulation of chronic periodontitis remain unelucidated. We first examined whether inflamed gingival tissues from chronic periodontitis patients expressed IL-33 by immunohistochemical studies. As expected, IL-33 was strongly expressed in the cytoplasm of the inflamed gingival epithelium from chronic periodontitis patients, but only weakly detected in the normal gingival epithelium from healthy individuals. Unlike the expression of IL-33 in the gingival epithelium, IL-33 expression in the lamina propria of gingival tissues was only weakly observed. IL-33 expression is possibly upregulated in epithelial, mesenchymal, and myeloid cells in response to proinflammatory stimuli, pathogen-associated molecular patterns, and pathogens. These findings suggest that gingival epithelial cells are capable of inducing IL-33 expression upon infection with periodontal pathogens.
p.gingivalis induces IL-33 mRNA expression in human gingival epithelial cells in culture. As P. gingivalis is implicated as a major pathogenic bacteria for chronic periodontitis, we examined the effect of P. gingivalis infection on IL-33 mRNA expression in human gingival epithelial cells in culture. The IL-33 mRNA expression was increased after stimulation with P. gingivalis W83 in human gingival epithelial cell line. Pretreatment of the cells with cycloheximide, a protein synthesis inhibitor, blocked the induction of IL-33 mRNA levels, suggesting that de novo protein synthesis was required for P. gingivalis-mediated IL-33 mRNA induction. P. gingivalis produces two types of arginine-specific cysteine proteinases (Arg-gingipains, RgpA and RgpB) and a lysine-specific cysteine proteinase (Lys-gingipain, Kgp). Gingipains are localized to a cell-associated form, a soluble form, and released as outer membrane blebs. We next examined whether enzymatic activities of gingipains are involved in the IL-33-inducing capacity in human gingival epithelial cells and primary oral epithelial cells. The induction of IL-33 mRNA expression by P. gingivalis W83 in the two types was completely and significantly inhibited by FPR-cmk (Rgp inhibitor) and KYT-36 (Kgp inhibitor), repectively. Furthermore, P. gingivalis KDP136, a gingipains-null mutant, unlike P. gingivalis ATCC 33277, a wild-type parent strain of KDP136, did not induce IL-33 mRNA expression in either cell type. P. gingivalis-induced IL-33 mRNA expression was abolished by heat treatment to inactivate enzymatic activities of P. gingivalis. We confirmed intact proteolytic activities for Rgps and Kgp in the whole cells of P. gingivalis W83 and ATCC 33277, but not in those of P. gingivalis KDP136. These observations suggest that the proteolytic activity of gingipains is essential for the induction of IL-33 mRNA expression by P. gingivalis in human gingival epithelial cells.
In this study, we revealed that P. gingivalis induced IL-33 via PAR-2-PLC-p38/NF-kB signaling pathways in human gingival epithelial cells (Fig. 3). Further studies are necessary to elucidate the role of intracellular IL-33 in maintaining host defense mechanisms in gingival epithelial cells against periodontal diseases.
Tada H, Matsuyama T, Nishioka T, Hagiwara M, Kiyoura Y, Shimauchi H, Matsushita K. Porphyromonas gingivalis gingipain-dependently enhances IL-33 production in human gingival epithelial cells. PLoS ONE. 2016 Apr; 11(4):e0152794.
Acknowledgements: We express our sincere thanks to M. Naito and K. Nakayama (Nagasaki University) for providing various mutant P. gingivalis strains, which enabled us to investigate the role of gingipains. We also thank R. Isoda and K. Kobayashi (National Center for Geriatrics and Gerontology), K. Nagano and F. Yoshimura (Aichi Gakuin University), S. Kawabata (Osaka University) and R. Tamai (Ohu University) for technical support. We are grateful to H. Takada (Tohoku University) for support and critical reading of the manuscript.
Hiroyuki Tada, D.D.S., Ph.D.
Department of Oral Microbiology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Sendai, Miyagi 980-8575, JAPAN.