Introduction
The endoplasmic reticulum (ER) is a crucial organelle in eukaryotic cells, responsible for the proper folding, modification, and transport of proteins [1]. Under normal conditions, the ER maintains cellular homeostasis by ensuring that proteins are correctly folded and functional [2,3]. However, various stressors can disrupt this balance, leading to an accumulation of misfolded or unfolded proteins, a condition known as ER stress [4]. To mitigate this, cells activate a signaling network called the unfolded protein response (UPR), which aims to restore ER function by halting protein translation, degrading misfolded proteins, and activating signaling pathways that increase the production of molecular chaperones such as glucose-regulated protein 78 (GRP78) [5,6].
ER stress is implicated in numerous diseases, including diabetes, neurodegeneration, and cancer [7]. Recent studies have also highlighted its role in inflammatory responses and immune regulation [8,9]. One of the key triggers of ER stress in immune cells, such as macrophages, is bacterial infection [10]. Pathogenic bacteria can exploit the host’s cellular machinery, leading to increased protein load in the ER and subsequent UPR activation [11].
Oral bacteria, in particular, are known to play a significant role in oral and systemic diseases through mechanisms involving inflammation and immune modulation [12]. However, the specific impact of different oral bacteria on ER stress pathways in immune cells remains less explored. This study aims to bridge this gap by examining the ER stress response induced by various oral bacteria in THP-1-derived macrophages. By focusing on key ER stress-related molecules such as GRP78, ATF6, IRE1α, XBP1, PERK, ATF4, and CHOP, this research seeks to elucidate the differential activation of ER stress pathways by distinct bacterial species.
To this end, we investigated the mRNA expression of ER stress markers and the protein levels of IRE1α, eIF2α, and CHOP in macrophages infected with four common oral bacteria: Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter actinomycetemcomitans , and Streptococcus oralis. Additionally, we measured the production of pro-inflammatory cytokines, TNF-α and IL-1β, to assess the inflammatory response associated with ER stress activation. Our findings provide insights into how different oral bacteria modulate ER stress and inflammation, potentially contributing to the pathogenesis of oral diseases and offering new targets for therapeutic intervention.
By comparing the ER stress responses elicited by these bacterial species, we aim to enhance our understanding of their role in oral and systemic disease progression. This research underscores the importance of targeting ER stress pathways in developing novel strategies for managing infections and associated inflammatory conditions.
Materials and Methods
1. Bacterial culture
P. gingivalis (strain 33277; Korean Collection for Type Cultures) and P. intermedia (strain 15032; Korean Collection for Type Cultures) were grown under anaerobic conditions at 37℃ in Gifu anaerobic medium (Nissui Seiyaku) supplemented with 5 μg/mL of hemin (Sigma Aldrich) and 0.5 mg/mL of vitamin K1 (Sigma Aldrich) to an optical density (OD) of 1.0 (at 650 nm), which was estimated to contain 109 colony-forming units (CFU)/mL. A. actinomycetemcomitans (strain 33384; Korean Collection for Type Cultures) was grown in tryptic soy broth (Becton Dickinson) with 1% yeast (LPS Solution) at 37℃ in a 5% CO2 incubator. An OD of 0.25 (650 nm) was determined to correlate to 1 × 109 CFU/mL. S. oralis (strain 55229; Korean Collection for Type Cultures) was grown aerobically in brain heart infusion at 37℃ in a 5% CO2 atmosphere up to an OD of 1.0 (at 650 nm), which was estimated to contain 108 CFU/mL. All bacterial species were harvested by centrifugation at 5,000 rpm for 5 minutes, resuspended in Roswell Park Memorial Institute (RPMI) medium (WelGENE) before being used to infect THP-1-derived macrophages at a multiplicity of infection (MOI) of 50.
2. Cell culture
THP-1 cells (Korean Cell Line Bank) were cultured in RPMI 1,640 medium (WelGENE) supplemented with 10% heatinactivated fetal bovine serum (WelGENE). Cells were differentiated into macrophages by treating them with 50 μg/mL of phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich) for 24 hours.
3. MTT assay
Cell viability was determined using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit (Sigma Aldrich). Briefly, 5 × 104 cells were seeded and differentiated into macrophages by treating them with PMA in 96-well plates overnight and then infected with various MOIs of oral bacterial. After incubation for 4 hours, the medium was replaced with a serum-free medium containing 500 μg/mL MTT reagents for 4 hours. The conversion of MTT reagent into chromogenic formazan was determined using a microplate reader at 570 nm.
4. Real-time polymerase chain reaction
Total RNA was extracted using the RNeasy mini kit (Qiagen), and cDNA was synthesized with a reverse transcription system (Bioneer). cDNA was subjected to real-time polymerase chain reaction (PCR) using SYBR Green PCR Master Mix (Applied Biosystems) and an ABI 7500 real-time PCR system (Qiagen). The primer sequences were as follows: human GAPDH, 5′-ACAACTTTGGTATCGTGGAAGG′ (forward) and 5′-GCCATCACGCCACAGTTTC-3′ (reverse); human GRP78, 5′-CATCACGCCGTCCTATGTCG-3′ (forward) and 5′-CGTCAAAGACCGTGTTCTCG-3′ (reverse); human ATF6, 5′-GACAGTACCAACGCTTATGCC-3′ (forward) and 5′-CTGGCCTTTAGTGGGTGCAG-3′ (reverse); human IRE1α, 5′-AGAGAAGCAGCAGACTTTGTC-3′ (forward) and 5′-GTTTTGGTGTCGTACATGGTGA-3′ (reverse); human XBP1, 5′-CCCTCCAGAACATCTCCCCAT-3′ (forward) and 5′-ACATGACTGGGTCCAAGTTGT-3′ (reverse); human PERK, 5′-GGAAACGAGAGCCGGATTTATT-3′ (forward) and 5′-ACTATGTCCATTATGGCAGCTTC-3′ (reverse); human ATF4, 5′-ATGACCGAAATGAGCTTCCTG-3′ (forward) and 5′ -GCTGGAGAACCCATGAGGT-3′ (reverse); human CHOP, 5′ -GCTCCTGCCTTTCACCTTGG-3′ (forward) and 5′-GGTTTTTGATTCTTCCTCTTC- 3′ (reverse). In this study, ‘n = 3’ indicates that each experimental condition was tested three times independently to ensure reliability and reproducibility.
5. Western blotting
Cells (THP-1-derived macrophages) were lysed in radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology) supplemented with complete ethylene-diamine-tetraacetic acid (EDTA)-free protease inhibitor (Sigma Aldrich) and a phosphatase inhibitor cocktail (Genedepot). Cell lysates, containing equal amounts of protein (20 μg), were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore), which were then immunoblotted using the following specific antibodies: CHOP (Cell Signaling Technology), eIF2α (Cell Signaling Technology), phosphorylated eIF2α (p-eIF2α) (Cell Signaling Technology), IRE1α (Santa Cruz), XBP1 (Santa Cruz), and GAPDH (Santa Cruz). Signal detection was performed using enhanced chemiluminescence, and resulting signals were visualized using the Super Signal West Femto Maximum Sensitivity Substrate (Pierce) and a LAS-4000 Immuno-Image Analyzer (Fuji Film). Band intensities were quantified using Image J software (National Institute of Health) and normalized versus GAPDH. In this study, ‘n = 3’ indicates that each experimental condition was tested three times independently to ensure reliability and reproducibility.
6. Cytokine analysis
After peptide pretreatment and P. gingivalis infection, culture supernatant was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (Biolegend) to determine cytokine (IL-1β and TNF-α) levels. Absorbances were read using a microplate reader (Allsheng). In this study, ‘n = 3’ indicates that each experimental condition was tested three times independently to ensure reliability and reproducibility.
7. Statistics
Statistically significant difference between two groups was analyzed with an unpaired Student’s t-test. The data are shown as the mean ± standard deviation (SD). p ＜ 0.05 was considered statistically significant.
Results
1. MTT assay confirms viability at MOI 50 for bacterial infections
To determine the appropriate MOI for our experiments, we performed an MTT assay to assess cell viability following bacterial infection. THP-1-derived macrophages were infected with P. gingivalis, P. intermedia, A. actinomycetemcomitans , and S. oralis at various MOIs. Infection with an MOI of 50 for 4 hours showed cell viability above 90% across four all bacterial species, confirming that this MOI was suitable for subsequent experiments without causing significant cytotoxicity (Fig. 1). Thus, we proceeded with an MOI of 50 for all bacterial infections in our study to ensure reliable and consistent results.
2. Differential mRNA expression of ER stress markers induced by oral bacterial infections
The mRNA expression levels of key ER stress-related molecules, including GRP78, ATF6, IRE1α, XBP1, PERK, ATF4, and CHOP, were analyzed in THP-1-derived macrophages infected with P. gingivalis, P. intermedia, A. actinomycetemcomitans , and S. oralis at an MOI of 50 for 4 hours (Fig. 2). P. gingivalis infection led to a significant upregulation of all examined ER stress markers compared to the control group, with particularly high expression levels of XBP1 and PERK. A. actinomycetemcomitans infection also resulted in increased expression of all ER stress markers, with the highest gene expression observed for GRP78, ATF6, IRE1α, ATF4, and CHOP. P. intermedia infection showed notably high expression levels for PERK, while the expression levels of other markers were only slightly elevated compared to the control group. S. oralis infection induced elevated expression levels of GRP78, PERK, ATF4, and CHOP. These results indicate that different oral bacteria induce distinct ER stress responses in THP-1-derived macrophages at the mRNA level, with variations in the activation profiles of specific ER stress-related genes.
3. Western blot analysis of ER stress-related protein expression following bacterial infections
The protein expression levels of key ER stress markers, including IRE1α, p-eIF2α, and CHOP, were examined in THP- 1-derived macrophages infected with P. gingivalis, P. intermedia, A. actinomycetemcomitans , and S. oralis at a MOI of 50 for 4 hours using western blot analysis (Fig. 3). S. oralis infection resulted in the highest phosphorylation levels of eIF2α and IRE1α among the tested bacteria, indicating a strong activation of the ER stress response. P. intermedia infection led to the highest expression of CHOP protein, suggesting a significant induction of ER stress-associated apoptosis. P. gingivalis and A. actinomycetemcomitans infections also increased the expression levels of these ER stress proteins compared to the control, but to a lesser extent than S. oralis and P. intermedia. These results demonstrate that different oral bacteria induce varying levels of ER stress-related protein expression in THP- 1-derived macrophages, reflecting distinct activation patterns of the ER stress response at the protein level.
4. ELISA analysis of pro-inflammatory cytokine production following bacterial infections
The production of pro-inflammatory cytokines such as TNF-α and IL-1β was measured in the supernatants of THP- 1-derived macrophages infected with P. gingivalis, P. intermedia, A. actinomycetemcomitans, and S. oralis at a MOI of 50 for 4 hours using ELISA (Fig. 4). P. intermedia infection induced the highest levels of TNF-α. This was followed by P. gingivalis, S. oralis, and A. actinomycetemcomitans in descending order of TNF-α production. P. gingivalis infection resulted in the highest IL-1β expression. This was followed by P. intermedia, S. oralis, and A. actinomycetemcomitans. These results indicate that different oral bacteria induce distinct pro-inflammatory cytokine responses in THP-1-derived macrophages, with variations in the levels of TNF-α and IL-1β production. This highlights the diverse inflammatory potential of these bacterial species and their possible contributions to the pathogenesis of oral infectious diseases.
Discussion
This study provides a comprehensive analysis of the differential activation of ER stress pathways by various oral bacteria in THP-1-derived macrophages. Our findings reveal distinct patterns of ER stress marker expression and pro-inflammatory cytokine production in response to infections with P. gingivalis, P. intermedia, A. actinomycetemcomitans, and S. oralis. These differences underscore the complex interactions between host cells and bacterial pathogens, highlighting the potential role of ER stress in the pathogenesis of oral diseases.
Prior to investigating the ER stress response, we performed an MTT assay to determine the appropriate MOI for our experiments. The assay confirmed that a MOI of 50 for 4 hours maintained cell viability above 90% across all bacterial species, indicating that this MOI was suitable for subsequent experiments without causing significant cytotoxicity. This validation step ensured that the observed effects on ER stress markers and cytokine production were not due to cytotoxic effects but were attributable to the specific bacterial infections.
The mRNA analysis revealed that P. gingivalis infection induced the highest expression levels of XBP1 and PERK, suggesting a robust activation of the UPR pathway. This bacterium is a well-known periodontal pathogen associated with chronic inflammation and tissue destruction. The elevated expression of these markers indicates a significant ER stress response, which may contribute to the pathogen’s ability to evade host immune defenses and persist in the oral cavity. Additionally, the high levels of IL-1β produced in response to P. gingivalis infection further support its role in promoting inflammatory responses.
Similarly, A. actinomycetemcomitans , another key periodontal pathogen, showed increased expression of multiple ER stress markers, particularly GRP78, ATF6, IRE1α, ATF4, and CHOP. The activation of these pathways suggests that A. actinomycetemcomitans induces a broad ER stress response, which may facilitate its pathogenicity by disrupting normal cellular functions and promoting inflammation [13,14]. The elevated TNF-α levels observed with A. actinomycetemcomitans infection align with its known role in aggressive periodontitis and systemic inflammatory responses.
In contrast, P. intermedia exhibited a more selective activation of the ER stress response, with particularly high PERK expression but only modest increases in other markers. This bacterium is associated with various periodontal conditions, including acute necrotizing ulcerative gingivitis and pregnancyassociated gingivitis [15,16]. The moderate ER stress response, coupled with high CHOP protein expression and significant TNF-α and IL-1β production, indicates that P. intermedia can elicit a strong inflammatory response, potentially contributing to its pathogenic effects in the oral cavity.
S. oralis, a commensal bacterium, elevated the expression of GRP78, PERK, ATF4, and CHOP, with the highest phosphorylation levels of eIF2α and IRE1α among the bacteria tested. Despite being generally considered less pathogenic, S. oralis can cause opportunistic infections, particularly in immunocompromised individuals [17,18]. The robust activation of ER stress pathways observed in this study suggests that even commensal bacteria can induce significant cellular stress under certain conditions, potentially leading to inflammation and disease.
The differential induction of pro-inflammatory cytokines further illustrates the complex interplay between ER stress and inflammation. The highest levels of TNF-α were induced by P. intermedia , followed by P. gingivalis , S. oralis, and A. actinomycetemcomitans , while IL-1β expression was highest with P. gingivalis infection. These cytokines play crucial roles in mediating inflammatory responses and tissue damage in periodontal disease. The correlation between ER stress marker expression and cytokine production underscores the potential for targeting ER stress pathways as a therapeutic strategy to modulate inflammation and improve disease outcomes.
These findings underline the crucial role of ER stress as a modulator of inflammation, further suggesting that targeting these pathways could provide a therapeutic strategy for managing both pathogenic and opportunistic infections. By exploring how these bacteria induce ER stress and the resulting inflammatory responses, future research could lead to novel approaches for treating not only oral diseases but also broader systemic inflammatory conditions linked to bacterial infections. In addition, this study calls for continued investigation into the specific molecular mechanisms by which ER stress contributes to the progression of oral diseases. This research will provide a foundation for the development of targeted therapies aimed at mitigating inflammation and tissue damage.
In conclusion, our study demonstrates that different oral bacteria induce distinct ER stress responses in THP-1-derived macrophages, which are closely linked to their ability to provoke inflammatory reactions. These findings highlight the importance of considering ER stress in the pathogenesis of oral diseases and suggest that targeting ER stress pathways could be a promising approach for developing new treatments for oral bacterial infections and associated inflammatory conditions. Further research is needed to explore the specific mechanisms by which these bacteria induce ER stress and to identify potential therapeutic targets within these pathways.
