Human periodontal ligament cells exhibit no endotoxin tolerance upon stimulation with Porphyromonas gingivalis lipopolysaccharide

Background/Objectives Endotoxin tolerance is characterized by a state of hyporesponsiveness after confrontation with endotoxins such as lipopolysaccharides (LPS) at low concentrations. The aim of this study was to investigate, whether pretreatment with Porphyromonas gingivalis leads to endotoxin tolerance induction and possible alterations in toll‐like receptor (TLR) 2‐ and 4‐induced response in human periodontal ligament cells (hPDLCs). Material and Methods Primary hPDLCs were pretreated with P. gingivalis (0.1 or 0.3 μg/mL) LPS for 24 hours and afterwards treated with one of the following stimuli: P. gingivalis LPS (1 μg/mL); TLR4 agonist Escherichia coli LPS (0.1 μg/mL; 1 μg/mL); TLR2 agonist Pam3CSK4 (0.1 μg/mL; 1 μg/mL). The protein expression of interleukin (IL)‐6, IL‐8 and monocyte chemotactic protein‐1 was analyzed with quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. Gene expression levels of TLR2 and TLR4 were determined by quantitative polymerase chain reaction. Results Pretreatment of cells with low concentrations of P. gingivalis LPS did not result in lower production of IL‐6, IL‐8 and monocyte chemotactic protein‐1 compared to control group. In some cases, pretreated cells exhibited lower gene expression levels of TLR2 and TLR4 compared to non‐pretreated cells. Conclusion The results of this study implicate that hPDLCs do not develop endotoxin tolerance. Furthermore, the amplitude of the inflammatory response shows no significant dependency on TLR2 and TLR4 expression levels.

reasons for adult tooth loss. 6,7 Periodontitis is caused on the one hand by periodontopathogenic bacteria, on the other hand by a dysregulation of the hosts' inflammatory response to bacterial virulence factors. 8,9 One of the most important periodontopathogenic agents is LPS of the strict anaerobic, gram-negative bacterium Porphyromonas gingivalis. 10,11 P. gingivalis LPS induces host cell activation via activation of either toll-like receptor (TLR) 2 and/or TLR4. 12,13 Some studies suggest that TLR2 activation by P. gingivalis LPS might be related in some cases to the impurity of LPS preparations. 14 TLR4 binding occurs by a CD14-dependent mechanism, which can be found either as membrane bound or as a soluble form in the serum. 15 After recognition of LPS, TLRs initiate several signaling pathways resulting in expression of proinflammatory cytokines and activation of hosts' immune response. 16 The main cell population responsible for the inflammatory reaction in periodontal disease are leukocytes such as macrophages, neutrophils and lymphocytes. 8 Even though these cells are known to develop endotoxin tolerance, the inflammatory signs of periodontitis still persist as long as biofilm is present. Considering this fact, Ara et al investigated the inflammatory response of LPS-pretreated human gingival fibroblasts. Their study demonstrated that human gingival fibroblasts do not induce endotoxin tolerance and thus may have a crucial influence on the progression of periodontal disease. 17 Human periodontal ligament cells (hPDLCs) are an important component of the periodontal attachment apparatus and have a morphology similar to those of fibroblasts. 18 These cells are thought to play an important role in the homeostasis of periodontal structures and they share some properties of osteoblasts and cementoblasts. 18 In addition, these cells are able to produce different inflammatory mediators upon stimulation with bacterial stimuli. 19 So far there is only one study concerning endotoxin tolerance in hPDLCs by Wu et al suggesting hyporesponsiveness in these cells after previous stimulation. 20 This finding is rather surprising, because gingival fibroblasts and PDLCs share many common properties. 21 In addition, in this study stimulation with LPS was performed in the absence of soluble CD14 (sCD14). It is known that hPDLCs lack of membrane bound CD14 protein and sCD14 significantly enhances the response of these cells to bacterial LPS. 22 Moreover, sCD14 is present in saliva and gingival crevicular fluid and therefore its application in experiments with LPS reflects physiological conditions more adequately. 23 Therefore, the primary aim of this study was to investigate, whether hPDLCs induce endotoxin tolerance when treated with P. gingivalis LPS in low concentrations combined with physiologically relevant levels of sCD14. As P. gingivalis LPS activates both TLR2 and TLR4, the secondary aim was to evaluate the receptors separately by treatment with TLR4 agonist Escherichia coli LPS and synthetic TLR2 agonist Pam3CSK4.

| Cell culture
Primary hPDLCs were isolated via the outgrowth method from third molars of six healthy donors, which were extracted due to orthodontic indications. Each patient gave written informed consent before the experiment. PDL tissue attached to the middle third of the root surface was scraped off with a scalpel and reduced to small pieces. The tissue fragments were cultured in petri dishes with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, streptomycin (50 μg/mL) and penicillin (100 U/ mL) (Gibco ® , Life Technologies, Carlsbad, CA, USA) and incubated in a humid environment of 5% CO 2 at 37°C. After the outgrowing cells reached confluence, detachment was performed with accutase.
The hPDLCs were seeded in cultural flasks containing DMEM supplemented with fetal bovine serum, streptomycin and penicillin as described above. Cells between passages 3 and 6 were used in the subsequent experiments. Protocol for primary hPDLCs isolation was approved by the Ethics Committee of the Medical University of Vienna.

| Study protocol
HPDLCs were seeded in 24-well plates at a density of 5 × 10 4 cells per well and incubated for 24 hours. Subsequently, some hPDLCs were pretreated with P. gingivalis LPS (0.1 or 0.3 μg/mL) in the presence of soluble CD14 (250 ng/mL) in serum-free DMEM supplemented with 1% penicillin/streptomycin. As shown by our recent study and preliminary data, stimulation with such P. gingivalis LPS concentrations result in a submaximal response in hPDLCs. 22 Another group of hPDLCs did not get any treatment and were incubated with serumfree DMEM with 1% penicillin/streptomycin alone. After 24 hours,

| Quantitative polymerase chain reaction
Isolation of mRNA and transcription into cDNA was performed using the TaqMan Gene Expression Cells-to-CT kit (Ambion/Applied Biosystems, Foster City, CA, USA) as described in our previous studies. [24][25][26] The mRNA expression levels of interleukin (IL)-6, IL-8, The reactions were carried out in duplicates with the following thermocycler settings: 95°C for 10 minutes, 40 cycles, each for 15 seconds at 95°C and for 1 minute at 60°C. For each sample, the point at which the PCR product reached a defined threshold (cycle threshold C t ) was determined. ΔΔC t values were calculated according to following formula: ΔΔC t = (C t target -C t GAPDH ) sample -(C t target -C t GAPDH ) control . The difference in the expression of target genes was calculated with the 2 −ΔΔCt method with a control sample. Cells that were not pretreated with P. gingivalis LPS and were not stimulated with any stimuli were used as control.

| Enyzme-linked immunosorbent assay
After the second stimulation and incubation, the supernatants were

| Statistical analysis
The statistical analysis was executed with the statistical program spss 21.0 (IBM Corp., Armonk, NY, USA). A Kolmogorov-Smirnov test was performed to test the normal distribution of all data. Statistical differences between different groups were analyzed by one-way analysis of variance for repeated measures followed by t-test. Data are expressed as mean ± SEM. Differences were considered to be statistically significant at P < .05.

| RE SULTS
3.1 | Effect of human periodontal ligament cell pretreatment with 0.1 μg/mL P. gingivalis lipopolysaccharide on their response to different stimuli    The response of hPDLCs to different stimuli was evaluated based on the production of IL-6, IL-8 and MCP-1. IL-6 is a proinflammatory cytokine that induces bone resorption and plays a major role in acute inflammation. 28 The chemoattractants IL-8 and MCP-1 are of great importance for immunopathogenesis of periodontal disease. 29 Furthermore, gene expression levels of TLR2 and TLR4 were measured. TLRs pertain to a family of transmembrane proteins that are involved in recognition of invading pathogens. 30 The expression of TLR2 and TLR4 is altered in periodontal disease and therefore these receptors are discussed to play an important role in disease progression. 31 TLR4 activation by different bacterial LPS induces the production of inflammatory mediators in hPDLCs. 22 Similarly, TLR2

| D ISCUSS I ON
activation results in cytokine production by PDLs 32 and gingival fibroblasts. 33 Endotoxin tolerance is classically defined as phenomenon when cells pretreated with submaximal LPS stimuli exhibit a lower response to further stimuli with a higher LPS concentration compared to non-pretreated cells. This phenomenon is an important ability of the host to protect itself from damage associated with an excessive immune response. 4 The results of this study did not show any significant decrease of cytokine expression after all applied stimuli in pretreated cells compared to the non-pretreated group. This was observed for cells pretreated with two different submaximal con-  34 However, it seems that the expression level of this receptor had no significant impact on the intensity of inflammatory response in hPDLCs. However, although the changes in the TLR4 expression levels observed in our study were significant, their range was rather small. Therefore, additional studies between TLR4 expression levels and the intensity of the hPDLC responses to bacterial LPS are required.
hPDLC pretreatment with the higher P. gingivalis LPS concentration resulted in lower TLR2 gene expression levels compared to nonpretreated cells. However, decreased TLR2 expression levels did not result in a lower production of proinflammatory mediators. TLR2 expression also was significantly increased upon stimulation with TLR2 agonist Pam3CSK4. This observation is in agreement with our previous study on human gingival fibroblasts. 33 TLR2 seems to play an important role in progression of periodontal disease and particularly in physiology of hPDLCs. Data of the present study show that the response of hPDLCs to TLR2 is significantly higher than that to bacterial LPS, which is in agreement with our previous reports. 22,26 Therefore, detailed examination of TLR2 induced response and its potential modulation might be important for understanding pathogenesis of periodontal disease and development of new treatment modalities.
In conclusion, our data show that hPDLCs exhibit no endotoxin tolerance and therefore might play an important role in sustained inflammation in periodontal disease. These cells exhibit a rather low ability to dampen their own inflammatory response, which might depend on the amplitude and duration of inflammatory stimuli. The results of this in vitro study can only be transferred into an in vivo situation in a limited way. In vivo hPDLCs are exposed to numerous cytokines and growth factors and continuously interact with other cell types. Another limitation of this study was the small number of donors involved in the study. A possibility to extend this study is to expand the number of donors and include hPDLCs isolated from patients with periodontitis.

This work was supported by authors' institution and Austrian
Science Fund (Project P 29440 to O.A.)

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.