Deregulation of salivary glandular epithelial cells by oral bacteria

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치의학대학원 치의과학과
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서울대학교 대학원
Sjogren's syndrome
학위논문 (석사)-- 서울대학교 대학원 : 치의과학과, 2017. 2. 최영님.
Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease characterized by dryness of mouth. Lymphocytic infiltrates develop around the epithelial cells in salivary and lacrimal glands, leading to dysfunction of exocrine glands. The pathogenesis of SS involves many components, including environmental and hormonal factors together with genetic background. Especially, the dysregulated salivary glandular epithelial cells (SGECs) as a non-professional antigen presenting cells (APCs) play a key role in the activation and recruitment of lymphocytes as well as in the perpetuation of the inflammatory process. However, the potential roles of oral bacteria in the induction of deregulation of SGECs were not fully understood. Here, we hypothesized that specific oral bacteria may be responsible for induction of APC marker expression and production of SS-related molecules in SGECs.

Previously, the communities of oral bacteria collected by mouth rinse from SS patients (SS, n = 25), healthy controls (HC, n = 15), and patients with dry mouth due to medication as sicca controls (SC, n = 10) were analyzed by pyrosequencing. Based on the results of microbiota analysis, Streptococcus salivarius, Streptococcus oralis, Rothia mucilaginosa, Fusobacterium nucleatum, Prevotella melaninogenica, and P. histicola were selected for further study. In particular, an increase in P. melaninogenica was associated with SS risk by logistic regression analysis (odd ratio 5.4, p = 0.003).
Human salivary gland (HSG) cells were infected with the selected bacteria in the absence or presence of IFNfor 3 days. To prevent the outgrowth of bacteria, gentamicin was added to the culture 6 h after infection with the bacteria. Expression of major histocompatibility complex class I (MHC I), MHC class II (MHC II), CD80, and CD86 was then examined by flow cytometry. The amounts of IL-6, IP-10, and IFNλ secreted into the culture supernatant were also measured by ELISA.
To investigate mechanisms for immune modulation by oral bacteria, invasion ability and location of bacteria within HSG cells were analyzed by flow cytometry and confocal microscopy. The expression of Toll-like receptors (TLRs) in HSG cells and induction of immunoactive molecules in stimulated HSG cells with various ligands to pattern recognition receptors (PRRs) were also examined by flow cytometry and ELISA.
To assess the influence of inflammatory cytokines and oral bacteria on physical epithelial barrier function, transepithelial electrical resistance (TER) value of stimulated HSG cells was measured for 3 days after a confluent monolayer of HSG cells was formed.

The expression of APC markers such as MHC class I, class II, and costimulatory molecules on HSG cells was induced by IFN, a major cytokine present in the salivary gland of SS. HSG cells expressed very low levels of CD80 and CD86. These molecules were slightly upregulated by IFN.
F. nucleatum, a highly immune stimulatory species used as a positive control, and P. melaninogenica upregulated MHC class I and CD86 in the absence of IFN and further upregulated the IFN–induced expression of MHC class I and CD80. Interestingly, P. melaninogenica and F. nucleatum upregulated the IFN–induced expression of MHC class II. They also induced the production of IL-6 and IP-10 both in the absence or presence of IFN. In contrast, S. salivarius, the most abundant commensal species, and two species S. oralis and R. mucilaginosa that were increased in sicca condition compared to HC showed suppressive effects on the expression of APC markers and IP-10. Surprisingly, both P. melaninogenica and F. nucleatum significantly increased the secretion levels of IFNλ compared to control. However, R. mucilaginosa and P. histicola downregulated the expression of this molecule in the absence of IFN.
Three bacteria P. melaninogenica, F. nucleatum, and R. mucilaginosa, which induced deregulation of HSG cells, highly invaded into HSG cells within few hours, and they were detected in the endosomal maturation process. This finding suggested oral bacteria inside the cells could differentially modulate the HSG cells by signaling pathway through TLRs and/or NOD-like receptors (NLRs).
The basal expression levels of TLR2, -4, and -9 were confirmed, and those were further upregulated by IFN. Deregulation of HSG cells was induced by stimulation with various PRR ligands. Bacterial components may potentially play a role in the deregulation of SGECs.
Treatment of pro-inflammatory cytokines and the SS-associated species decreased the TER value of HSG cells, suggesting that inflammatory meiliu made by active immune response in target tissue and specific oral bacteria could disrupt the physical epithelial barrier.

Changes in the oral microbiota associated with SS patients may potentially induce deregulation of SGECs and contribute to the pathogenesis of SS.
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College of Dentistry/School of Dentistry (치과대학/치의학대학원)Dept. of Dental Science(치의과학과)Theses (Master's Degree_치의과학과)
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