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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)

International Journal of Oral Biology Vol.46 No.4 pp.190-199
DOI : https://doi.org/10.11620/IJOB.2021.46.4.190

Two Sjogren syndrome-associated oral bacteria, Prevotella melaninogenica and Rothia mucilaginosa, induce the upregulation of major histocompatibility complex class I and hypoxia-associated cell death, respectively, in human salivary gland cells

Jaewon Lee, Sumin Jeon, Youngnim Choi*

Department of Immunology and Molecular Microbiology, School of Dentistry and Dental Research Institute, Seoul National University,
Seoul 03080, Republic of Korea

†

Jaewon Lee and Sumin Jeon contributed equally to this work and share first authorship.

^*Correspondence to: Youngnim Choi, E-mail: youngnim@snu.ac.kr

Received December 6, 2021 Review Accepted December 14, 2021

Abstract

Despite evidence that bacteria-sensing Toll-like receptors (TLRs) are activated in salivary gland tissues of Sjogren syndrome (SS) patients, the role of oral bacteria in SS etiopathogenesis is unclear. We previously reported that two SS-associated oral bacteria, Prevotella melaninogenica (Pm) and Rothia mucilagenosa (Rm), oppositely regulate the expression of major histocompatibility complex class I (MHC I) in human salivary gland (HSG) cells. Here, we elucidated the mechanisms underlying the differential regulation of MHC I expression by these bacteria. The ability of Pm and Rm to activate TLR2, TLR4, and TLR9 was examined using TLR reporter cells. HSG cells were stimulated by the TLR ligands, Pm, and Rm. The levels of MHC I expression, bacterial invasion, and viability of HSG cells were examined by flow cytometry. The hypoxic status of HSG cells was examined using Hypoxia Green. HSG cells upregulated MHC I expression in response to TLR2, TLR4, and TLR9 activation. Both Pm and Rm activated TLR2 and TLR9 but not TLR4. Rm-induced downregulation of MHC I strongly correlated with bacterial invasion and cell death. Rm-induced cell death was not rescued by inhibitors of the diverse cell death pathways but was associated with hypoxia. In conclusion, Pm upregulated MHC I likely through TLR2 and TLR9 activation, while Rm-induced hypoxia-associated cell death and the downregulation of MHC I, despite its ability to activate TLR2 and TLR9. These findings may provide new insight into how oral dysbiosis can contribute to salivary gland tissue damage in SS.

Key Words : Sjogren's syndrome , Salivary glands , Major histocompatibility complex , Bacteria

초록

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Introduction

Sjogren syndrome (SS) is a chronic autoimmune disease in which the immune system mistakenly attacks the salivary and lacrimal glands, leading to sicca symptoms [1]. One of the key characteristics of SS is the lymphocytic infiltration of the exocrine glands [2,3].

Although the etiopathogenesis of SS is poorly understood, accumulating evidence supports the view that the inflammatory change in salivary gland epithelial cells (SGECs), termed “epithelitis”, is the first pathogenic event in SS [4]. SGECs in SS patients often present the phenotype of antigen-presenting cells characterized by increased expression of major histocompatibility complex class I (MHC I) and class Ⅱ and the presence of costimulatory molecules CD80, CD86, and CD40, indicating the possibility for antigen presentation to the infiltrated T cells [5].

Toll-like receptors (TLRs) are regarded as potential mediators of epithelitis in SS [6]. The expression of TLR2, TLR4, TLR6, TLR7, TLR8, and TLR9 is increased in the salivary gland tissue of SS patients, in which TLRs are detected in the ductal epithelial cells and infiltrated immune cells [7-10]. Stimulation of SGECs with ligands for TLR2, TLR3, and TLR4 results in increased expression of MHC I and CD40 [11].

Although TLR1, TLR2, TLR4, TLR6, and TLR9 recognize bacterial components, only endogenous ligands, i.e., damage-associated molecular patterns (DAMPs) to these TLRs have been considered in the context of SS [6]. Recently, we reported the bacterial infection of ductal cells in the labial salivary glands from SS patients [12]. When human submandibular gland tumor (HSG) cells were challenged with selected SS-associated oral bacterial species in a previous study, Prevotella melaninogenica (Pm) upregulated the expression of MHC I, MHC II, and CD80, and Rothia mucilaginosa (Rm) downregulated MHC I and CD86 [12]. The aim of this study was to elucidate the mechanisms underlying the differential regulation of MHC I expression by Pm and Rm in HSG cells.

Materials and Methods

1. Cells

HSG cells [13] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin. Chinese hamster ovary (CHO)/CD14/TLR2 and CHO/CD14/ TLR4 cells [14] were maintained as previously described [15]. Human embryonic kidney (HEK)-Blue^TM human TLR9 (hTLR9) cells (InvivoGen, Toulouse, France) were maintained according to the manufacturer’s protocols.

2. Bacteria

Pm KCTC 5457 (Korean Collection for Type Cultures, Jeongeup, Korea), and Rm KCTC 19862 were cultured as previously described [12]. To examine bacterial invasion of HSG cells, bacteria were used after staining with 1 μM pHrodo^TM Red succinimidyl ester (Thermo Fisher Scientific, Skokie, IL, USA).

3. Immunofluorescence

HSG cells (6 × 10⁴ cells/well) were plated on coverslips (12 mm-diameter) in 24-well plates. To examine the expression of TLRs, the cells were fixed with 4% paraformaldehyde and then subjected to heat-induced antigen retrieval by the citrate buffer method. After blocking with 5% bovine serum albumin in phosphate-buffered saline, the cells were incubated with anti-TLR2 (BD Bioscience, Franklin Lake, NJ, USA), anti-TLR4 (BioLegend, San Diego, CA, USA), or anti-TLR9 (BioLegend) antibodies, followed by either Alexa Fluor 488–conjugated rabbit anti-mouse Immunoglobulin G (IgG) or FITC-conjugated rabbit anti-rat IgG. To examine the hypoxic status of cells, HSG cells were stained with 2.5 μM Hypoxia Green (Thermo Fisher Scientific). After staining with Hoechst 33342, the cells were examined by confocal microscopy (Carl Zeiss, Oberkochen, Germany).

4. Stimulation of HSG cells with TLR ligands or bacteria

HSG cells (6 × 10⁴ cells/well) were seeded into 24-well plates one day before challenge. The HSG cells were incubated with fresh antibiotic-free medium containing the indicated concentrations of Pam₃CSK₄ (InvivoGen), lipopolysaccharide (LPS) isolated from Escherichia coli (Sigma, St. Louis, MO, USA), CpG-containing oligonucleotide (ODN) 2006 (InvivoGen), Pm, or Rm for 24–72 hours. To prevent the overgrowth of Rm in the culture medium, gentamicin (500 μg/mL) was added to the medium in the sets using bacteria. When cells were infected with Rm in the presence of various cell death inhibitors, including 3-methyladenine (3-MA, Sigma), z-WEHD-fmk (R&D Systems, Minneapolis, MN, USA), z-DEVD-fmk (R&D Systems), and necrosulfonamide (Sigma), they were pretreated with each inhibitor for 1 hour, infected with Rm, and further cultured for 24 hours.

5. Isolation of genomic DNA and removal of endotoxins

Genomic DNA was extracted from Pm, Rm, and HSG cells using a Blood & Cell Culture DNA Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For Rm, 0.15 mm garnet beads (Qiagen) were applied to disrupt the peptidoglycan cell wall before DNA extraction. The isolated DNA was subjected to three rounds of endotoxin removal using Triton X-114 (Sigma), which was confirmed using a LAL chromogenic endotoxin quantitation kit (Thermo Fisher Scientific).

6. TLR reporter cell assays

CHO/CD14/TLR2 and CHO/CD14/TLR4 cells (6 × 10⁴ cells/ well) were seeded into 24-well plates. After an overnight culture, cells were stimulated with the indicated number of live or heat-killed bacteria for 16 hours in the presence of gentamicin. The levels of CD25, which is expressed as a result of TLR2- or TLR4-mediated nuclear factor-κB translocation, was analyzed by flow cytometry.

HEK-blue_hTLR9 cells (8 × 10⁴ cells/well) were seeded into 96-well plates. After an overnight culture, the cells were transfected with the indicated concentrations of bacterial or HSG genomic DNA mixed with Lipofectamine (Thermo Fisher Scientific). The next day, the medium was changed to detection medium for the secreted alkaline phosphatase (InvivoGen). After incubation at 37℃ for 16 hours, the optical density was measured with a spectrophotometer at 620 nm.

7. Flow cytometry

HSG cells were stained with FITC-conjugated anti-human TLR2 antibody clone TL2.1 (BioLegend), PE-conjugated antihuman TLR4 antibody clone TF901 (BD Biosciences), or PEconjugated anti-human TLR9 antibody clone eB72–1665 (BD Biosciences) with or without fixation/permeabilization. MHC I expression on HSG cells was examined by staining with the PerCP-conjugated anti-human HLA-A, B, and C antibody clone W6/32 (BioLegend). The viability of the HSG cells infected with bacteria were determined by staining with 5 μM SYTOX^TM Green (Invitrogen, Waltham, MA, USA). CHO/CD14/ TLR2 and CHO/CD14/TLR4 cells were stained with the FITCconjugated mouse anti-human CD25 antibody clone M-A251 (BD Biosciences). All stained cells were analyzed using a FACSCalibur (BD Biosciences) equipped with the CellQuest software.

8. Cell Counting Kit-8 assay

The viability of Rm in the presence of gentamicin was determined using Cell Counting Kit-8 (CCK-8) reagent (Dojindo Molecular Technologies Inc., Rockville, MD, USA) according to the manufacturer’s protocols.

9. Statistical analysis

All data are presented as the mean ± standard error of the mean of repeated experiments. The difference between the control and experimental groups was analyzed by a t-test. Correlation was determined by Spearman correlation analysis. Significance was set at p ＜ 0.05.

Results

1. HSG cells express bacteria sensing TLRs and upregulate MHC I expression in response to their ligands

The expression of bacteria sensing TLRs was examined in HSG cells. Because neither Pm nor Rm has flagella, the expression of TLR5 was not examined. When the expression of TLR proteins was examined by flow cytometry, not only TLR9 but also TLR2 and TLR4 were detected intracellularly but not on the cell surface (Fig. 1A). Confocal microscopy confirmed the expression of the three TLR proteins in the HSG cells (Fig. 1B). Stimulation of the HSG cells with Pam₃CSK₄, LPS, and CpGODN 2006, which were chosen as ligands for TLR2, TLR4, and TLR9, respectively, upregulated MHC I expression in a dosedependent manner 48 hours after treatment, but MHC I expression returned to the basal level at 72 hours (Fig. 1C). These results indicate that bacteria-sensing TLRs expressed in HSG cells can upregulate MHC I expression.

2. Pm and Rm activate TLR2 and TLR9 but not TLR4

Next, the abilities of Pm and Rm to activate TLR2, TLR4, and TLR9 were examined using reporter cells. Both Pm and Rm activated TLR2 at similar levels but did not activate TLR4 (Fig. 2A and 2B). Both Pm and Rm DNA activated TLR9 in a dosedependent manner, but HSG DNA did not (Fig. 2C). These results suggest that both Pm and Rm have the potential to upregulate MHC I via TLR2 and TLR9.

3. Downregulation of MHC I expression by Rm correlates with bacterial invasion and cell death

Because Rm-induced downregulation of MHC I expression remained a puzzle, changes in cell viability, bacterial invasion, and MHC I expression were simultaneously examined by flow cytometry at 24, 48, and 72 hours post infection with either Pm or Rm (Fig. 3A). While Pm did not affect the viability of the HSG cells, Rm substantially decreased the viability in a dosedependent manner at 24 hours post infection, which recovered over time (Fig. 3B). Both Pm and Rm invaded the HSG cells in a dose-dependent manner, and the pHrodo Red positive cells decreased over time, suggesting intracellular degradation of the bacteria (Fig. 3C). Pm upregulated, but Rm downregulated MHC I expression, as previously shown (Fig. 3D). Interestingly, the Rm-induced downregulation of MHC I expression by the multiplicity of infection 500 was most evident at 24 hours and recovered over time, similar to the cell viability. Indeed, the levels of MHC I expression in the Rm-infected cells had a strong positive correlation with the cell viability, although MHC I expression was analyzed after gating on the live cells (Fig. 3A and 3E). In addition, the degree of Rm invasion had negative correlations with both the viability and MHC I expression level of the host cells (Fig. 3F and 3G). These results indicate that the cell death caused by Rm invasion may be associated with MHC I downregulation.

4. Rm-induced cell death is not rescued by various cell death inhibitors

We tested if the inhibition of the Rm-induced cell death restores MHC I expression. Bacteria can induce the cell death of host cells via autophagy, apoptosis, pyroptosis, or necroptosis [16]. We chose 3-MA, the caspase-1 inhibitor z-WEHD, the caspase-3 inhibitor z-DEVD, and the mixed lineage kinase domain- like (MLKL) inhibitor necrosulfonamide to inhibit autophagy, apoptosis, pyroptosis, and necroptosis, respectively [17]. Inhibition of either autophagy or MLKL decreased the viability of the HSG cells in the absence of Rm infection. Unexpectedly, inhibition of any of the tested cell death pathways did not rescue the Rm-induced cell death (Fig. 4A). Interestingly, each of the cell death inhibitors positively or negatively regulated MHC I expression in the absence of Rm infection but did not restore the Rm-induced MHC I downregulation (Fig. 4B).

5. Rm-induced cell death is associated with hypoxia

Because none of the tested cell death pathways rescued the Rm-induced cell death, we hypothesized that Rm may consume oxygen inside the host cells and induce hypoxic cell death. HSG cells cultured under anaerobic conditions as a positive control were strongly stained with Hypoxia Green. Hypoxia Green staining was observed in a few cells after Pm infection but in most of the cells after Rm infection (Fig. 5A). Flow cytometric analysis confirmed that Rm induced hypoxia in a dose-dependent manner (Fig. 5B and 5C), and this tendency was negatively correlated with cell viability (Fig. 5D). When the viability of Rm in DMEM containing gentamicin was assessed, a substantial proportion of Rm maintained viability for 6 hours (Fig. 5E), the period of which is enough for the Rm invasion of HSG cells [12].

Discussion

This study showed that Pm upregulates MHC I probably via activation of TLR2 and TLR9, while Rm induces hypoxiaassociated cell death and the downregulation of MHC I despite its ability to activate TLR2 and TLR9.

The upregulation of MHC I expression by stimulation with ligands for TLR2 and TLR4 in HSG cells was consistent with a previous study [11]. TLR9 ligands also increased the expression of MHC I. These results indicate that bacteria can upregulate MHC I expression through activation of bacteriasensing TLRs in HSG cells. It has been shown in dendritic cells that TLR signaling induces the accumulation of MHC I within phagosomes containing microbial components which enhances cross-presentation of microbial antigens [18]. Similarly, TLR stimulation of SGECs by cell invading bacteria, such as Pm and Rm, may augment cross-presentation of bacterial antigens on MHC I, making these cells the target of CD8⁺ T cells.

Interestingly, Rm induced hypoxia in the host cells while hypoxia was not induced by the obligate anaerobic organism Pm. Rm is a facultative anaerobe that has superoxide dismutase and eight oxidases, including cytochrome d ubiquinol oxidase subunits I and II [19]. Although facultative anaerobes can survive/grow in the absence of oxygen, they preferentially utilize oxygen as a terminal electron acceptor [20]. Therefore, consumption of oxygen by intracellular Rm seemed to have led to hypoxia in HSG cells. Tissue hypoxia is a common feature observed at the site of inflammation during bacterial infection, which is the result of increased oxygen demands from microorganisms and recruited leukocytes but a reduced blood supply [21]. However, hypoxia induction in epithelial cells by infection with invading bacteria has not been reported. Of note, the mode of Rm-induced cell death seemed to be hypoxic necrosis because this cell death was not rescued by inhibition of any of the programmed cell death pathways, including those of autophagy, apoptosis, pyroptosis, and necroptosis. This finding suggests that infection of salivary gland cells with Rm may lead to hypoxic necrosis and the release of DAMPs and autoantigens. In addition, downregulation of MHC I by hypoxia and hypoxia-inducible factor 1α has been shown in tumor cells [22], which explains the Rm-induced downregulation of MHC I expression observed in this study.

Unfortunately, HSG cells have recently been reported to be a HeLa contaminant [23]. As long as Pm and Rm can invade into SGECs, the relevance of this study to SS would be maintained. Although we did not have a chance to test bacterial invasion of SGECs, we confirmed that both Pm and Rm invade immortalized human oral keratinocytes 16B cells and induce the same phenotypes observed in HSG cells (data not shown).

The lack of activation of TLR4 by Pm was different from our expectation. However, there are other examples of gram-negative bacteria that barely activate TLR4, such as Vibrio cholerae and the periodontal pathogen Porphyromonas gingivalis, because their LPS is a very weak agonist of TLR4 [24-26]. Likewise, the LPS of Pm may also have a weak agonistic function, which needs to be confirmed using purified LPS.

Despite the evidence that bacteria-sensing TLRs are activated in the labial salivary glands of SS patients, the role of bacteria has not been considered in the pathogenesis of SS. The levels of TLR2, TLR4, and TLR6 expression have been shown to correlate with the focus grades of focal lymphocytic sialadenitis in SS patients [7]. The role of a dysbiotic oral microbiota in the development of focal lymphocytic sialadenitis has been shown in IκB-ζ-deficient mice through a cohousing study [27], which supports the role of oral bacteria in the pathogenesis of SS.

Collectively, two invasive SS-associated oral bacteria, Pm and Rm, upregulated MHC I expression and hypoxic death in HSG cells, respectively. The hypoxic death of SGECs will contribute to the release of DAMPs and autoantigens, while the upregulated MHC I expression will contribute to increased presentation of autoantigens. This finding may provide new insights into how dysbiosis of the oral microbiome can contribute to the etiopathogenesis of SS.

Acknowledgements

This study was supported by the National Research Foundation of Korea (Daejeon, Korea) through the grants 2018R1A5A2024418 and 2020R1A2C2007038.

Figure

Fig. 1.

Expression of bacteria-sensing TLRs and upregulation of MHC I expression in response to the corresponding ligands in HSG cells. (A) HSG cells were stained with anti- TLR2, anti-TLR4, or anti-TLR9 antibodies without (surface) or with (intracellular) fixation/ permeabilization. The cells were analyzed by flow cytometry and overlaid on the cells stained with isotype control antibodies (gray line). (B) HSG cells cultured on coverslips were fixed, permeabilized, and then subjected to staining with anti-TLR2, anti-TLR4, or anti- TLR9 antibodies followed with Alexa Fluor 488- or FITC-conjugated secondary antibodies. Original magnification: ×400. (C) HSG cells were treated with the indicated concentrations of Pam₃CSK₄, LPS, or CpG-ODN for 24–72 hours. Subsequently, the expression of MHC I was analyzed by flow cytometry. Results are the summary of five independent experiments performed in duplicate.

TLRs, Toll-like receptors; Ab, antibody; MHC I, major histocompatibility complex class I; HSG, human submandibular gland tumor; Con, control; LPS, lipopolysaccharide; ODN, oligonucleotide; MFI, mean fluorescence intensity.

*p < 0.05, **p < 0.005, and ***p < 0.001 versus the control.

Fig. 2.

The abilities of Prevotella melaninogenica (Pm) and Rothia mucilaginosa (Rm) to activate bacteria-sensing TLRs. (A) CHO/CD14/TLR2 cells (6 × 10⁴ cells/well in 24-well plates) were stimulated with Pm or Rm at the indicated multiplicities of infection (MOIs) for 16 hours. Pam₃CSK₄ was used as a positive control. (B) CHO/CD14/TLR4 cells (6 × 10⁴ cells/well in 24-well plates) were stimulated with the indicated MOIs of Pm or Rm for 16 hours. LPS was used as a positive control. (C) HEK-blue_hTLR9 cells (8 × 10⁴ cells/well in 96 plates) were transfected with the indicated amounts of genomic DNA isolated from Pm, Rm, or HSG cells using Lipofectamine. As a positive control, cells were treated with CpG-ODN. After incubation for 16 hours, activation of TLR9 was determined by the activity of secreted alkaline phosphatase. Results are the summary of five independent experiments performed in duplicate.

TLRs, Toll-like receptors; CHO, Chinese hamster ovary; LPS, lipopolysaccharide; ODN, oligonucleotide; HSG, human submandibular gland tumor; NT, no treatment; SEAP, secreted alkaline phosphatase.

*p < 0.05, **p < 0.005, and ***p < 0.001 versus the control.

Fig. 3.

Changes in cell viability and MHC I expression in HSG cells in response to invasion of either Prevotella melaninogenica (Pm) or Rothia mucilaginosa (Rm). HSG cells (6 × 10⁴ cells/well) seeded into 24-well plates were infected with the indicated MOIs of pHrodo Red-labeled Pm or Rm and incubated for 24– 72 hours. Cells were stained with SYTOX Green and PerCP-conjugated anti-MHC I antibodies and analyzed by flow cytometry. (A) Live cells gated based on FSC and SYTOX Green staining were analyzed for the presence of intracellular bacteria and MHC I expression. (B) Percentages of live HSG cells infected with Pm or Rm over time. (C) Percentages of HSG cells with intracellular bacteria among live cells. (D) The levels of MHC I on live HSG cells in mean fluorescence intensity are expressed as percentages compared to control cells without bacterial infection. (B-D) Results are the summary of five independent experiments performed in triplicate. (E) A correlation plot between cell viability and MHC I expression in Rm-infected cells. (F) A correlation plot between bacterial invasion and cell viability in Rm-infected cells. (G) A correlation plot between bacterial invasion and MHC I expression in Rm-infected cells.

MHC I, major histocompatibility complex class I; HSG, human submandibular gland tumor; MOIs, multiplicities of infection; Con, control; FSC, forward scatter.

*p < 0.05, **p < 0.005, and ***p < 0.001 versus the control.

Fig. 4.

Effects of inhibiting diverse cell death pathways on Rothia mucilaginosa (Rm)-induced cell death and downregulation of MHC I expression. HSG cells (6 × 10⁴ cells/well) seeded into 24-well plates were infected with Rm (MOI 500) in the presence of the indicated concentrations of 3-methyladenine (3-MA), z- WEHD-fmk, z-DEVD-fmk, or necrosulfonamide (NSA) and incubated for 24 hours. The viability (A) and MHC I expression (B) of infected cells were analyzed by flow cytometry. Results are the summary of five independent experiments performed in triplicate.

MHC I, major histocompatibility complex class I; MHC I, major histocompatibility complex class I; HSG, human submandibular gland tumor; MOI, multiplicities of infection; MFI, mean fluorescence intensity.

*p < 0.05, **p < 0.005, and ***p < 0.001 compared with cells without inhibitor treatment (none). ^#p < 0.05, ^##p < 0.005, and ^###p < 0.001 compared with uninfected cells (- Rm).

Fig. 5.

Detection of hypoxic cells. (A–C) HSG cells were infected with either Prevotella melaninogenica (Pm) or Rothia mucilaginosa (Rm) for 24 hours. As a positive control, cells were also incubated under anaerobic conditions. The cells were stained with Hypoxia Green reagent for the last 3 hours and then examined by confocal microscopy (A) or flow cytometry (B, C). Original magnification: ×400. (B) A representative result obtained by flow cytometry. (C) Summary of two independent experiments performed in triplicate. (D) Parallel sets of experiments were performed with those described in C, and cell viability was determined by staining with trypan blue. (E) Rm was cultured with or without gentamicin in DMEM supplemented with 10% FBS. The viability of Rm at indicated hours was determined using the CCK-8 assay by incubating with the CCK-8 reagent for last 1 hour and expressed as a percentage to control Rm at 1 hour. Results are the summary of two independent experiments performed in quintuplicate.

HSG, human submandibular gland tumor; MOI, multiplicities of infection; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; CCK-8, Cell Counting Kit-8.

*p < 0.05, **p < 0.005, ***p < 0.001 compared with none.

Table

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