mRNA expression of pattern recognition receptors and their signaling mediators in healthy and diseased gingival tissues.

BACKGROUND: Gingivitis and periodontitis are initiated by inflammation caused by microorganisms. Pathogen-associated molecular patterns (PAMPs) from these microorganisms are recognized through various toll-like receptors (TLRs) and NOD-like receptors (NLRs). In this study, we have chosen five TLRs and two NLRs as representatives taking part in the recognition and inflammation process, along with a few of their signaling mediators including CD14, MYD88, and TRIF to compare their mRNA expression levels between healthy and diseased gingival tissues. This will provide deeper insight into the mechanisms underlying gingivitis and periodontitis. Understanding the mechanisms involved in the onset and progression of the periodontal diseases could greatly help in establishing effective ways for prevention and treatment of these diseases besides decreasing the risk factor for relevant systemic disorders.
MATERIALS AND METHODS: Gingival tissue samples for mRNA extraction and cDNA synthesis were taken from patients with gingivitis and periodontitis and from healthy control subjects. Messenger RNA expression of all genes was assessed using real-time polymerase chain reaction (PCR).
RESULTS: Among the genes studied in different groups, only MYD88 mRNA expression was significantly higher in the periodontitis group compared to that of the controls. The expression level of this molecule was also significantly higher in patients with severe periodontitis compared to other patients and also compared to healthy individuals. In different tissues, positive significant correlations were observed between the mRNA expression levels of some genes.
CONCLUSIONS: Elevated mRNA levels of MYD88 in periodontitis might have a key role in the pathogenesis of this disease. Therefore, MYD88 may be a useful target for the therapy of this disease.

INTRODUCTION

Periodontal diseases including gingivitis and periodontitis are widely prevalent and have been estimated to affect 80% of adults worldwide.[1] Besides the tissue destruction of the periodontium and tooth loss as the final result, studies have shown close associations between these diseases and systemic disorders.[12]

Periodontal diseases result from host response to microorganisms in the subgingival biofilm.[3] The innate immune system recognizes microbial structures known as pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs).[45] There are different families of the PRRs. Two of them are toll-like receptors (TLRs) and NOD-like receptors (NLRs).[5] Upon recognition of PAMPs through PRRs, pro-inflammatory cytokines are produced and host cells set up an inflammatory response in order to eliminate the infection. This response leads to host tissue destruction.[6]

There is limited information on the expression of PRRs and their adaptor molecules in periodontal diseases. Thus, the main purpose of this study is to elucidate the possible expression patterns of some of the TLRs and NLRs, in addition to two of their adaptor molecules including myeloid differentiation primary response protein (MYD88) and toll/interleukin-1 receptor (TIR) domain-containing adaptor including interferon (IFN)-β (TRIF), and also CD14 as a signaling mediator, all involved in the signaling pathways activated through microbial presence in healthy and diseased gingival tissues.

MATERIAL AND METHODS

Subjects

Twenty-five patients participated in this study among whom five (aged 20-58 years) were diagnosed with gingivitis and 20 (aged 23-66 years) with chronic periodontitis. The control group included 15 healthy individuals (aged 16-70 years). The individuals excluded from the study included those with either systemic disorders such as diabetes mellitus and immunological disorders that affect periodontium or the pattern of periodontal diseases, individuals taking certain medications such as phenytoin and nifedipine, smokers, pregnant and lactating women, individuals who had taken antibiotics during the last 2 months before the study, and patients who had undergone periodontal treatment within the preceding 6 months. Sampling for RNA extraction consisted of removing the collar of gingiva including the pocket tissue around the coronal part of the teeth (both the epithelium and the connective tissue). Gingival tissue samples were obtained during periodontal flap surgery in patients with periodontitis. In healthy subjects and those with gingivitis, gingival samples were obtained during surgeries for crown lengthening or orthodontic reasons. Healthy individuals showed no clinical signs of inflammation and periodontitis and no alveolar bone loss in radiography. The modified diagnostic criteria of Kornman et al. were used for the diagnosis of gingivitis and periodontitis and also for the classification of the latter.[7] Subjects with periodontitis were classified into mild, moderate, and severe groups (6, 4, and 10 subjects, respectively) according to their periodontal attachment loss. Written informed consent was obtained from all individuals before getting the samples. Immediately after surgery, samples were snapped frozen and transferred to the laboratory for RNA extraction.

RNA extraction and real-time PCR

Total RNA was extracted from gingival tissues using High Pure RNA Tissue Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Reverse transcription was carried out by random hexamers and oligo-dT on 5 μg of total RNA in a 20 μl reaction mix using First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in 20 μl reaction mixtures in an ABI PRISM 7500 (Applied Biosystems, CA, USA) using SYBR green master mix kit (ABI, Carlsbad, CA, USA) and the appropriate primers (Bioneer, Korea) [Table 1]. The microtubes were incubated at 95ºC for 10 min, followed by 40 cycles consisting of a three-step amplification procedure −95ºC for 15 s, 57ºC for 30 s, and 60ºC for 1 min – and a subsequent dissociation step to obtain the melting curves. The ABI PRISM SDS 2.0 software (Applied Biosystems, USA) was used to analyze the curves. For quantifications, the ratio between the expression amount of each gene and β-actin was determined.

Table 1

Primer sequences used for performing real-time PCR

Statistical analysis

The expression levels of the evaluated genes in patients with gingivitis and different stages of periodontitis were compared with each other and also with those of healthy subjects with SPSS 17 software (SPSS Inc., Chicago, IL, USA) using Mann–Whitney U-test. The correlation between the assessed genes was also investigated using non-parametric Spearman's correlation coefficient. P values less than 0.05 (P < 0.05) were considered statistically significant. GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for drawing the graphs.

RESULTS

Expressions of TLR2, TLR3, TLR4, TLR5, TLR9, NOD2, NALP3 (also called NOD-like receptor (NLR) containing a Pyrin domain), along with CD14, MYD88, and TRIF were detected in gingival tissues of all healthy individuals and also in gingivitis and periodontitis patients. The results are shown in Figures 1 and 2a, b.

Figure 1

Comparison of relative gene expression of TLRs and CD14 among healthy, gingivitis, and periodontitis groups. Graphs are shown as vertical interleaved bars, plotting the median with interquartile range

Figure 2

Comparison of relative gene expression of (a) NLRs and (b) Signaling molecules among healthy, gingivitis, and periodontitis groups. Graphs are shown as vertical interleaved bars, plotting the median with interquartile range. Results were analyzed by Mann–Whitney U-test and P values less than 0.05 are shown as (*)

Comparing gingivitis lesions to the healthy tissues, all assessed genes except TLR9 and NOD2 showed increased but not significant expression levels in gingivitis. A similar non-significant rise was seen in the mRNA levels of all evaluated genes except TLR2 in periodontitis lesions compared to healthy tissues and with the exception of TLR2, TLR5, and CD14 in tissues with gingivitis. In fact, the mRNA expression of the above three genes was non-significantly decreased. The mRNA expression of MYD88 was the only one that increased significantly in the periodontitis group compared to that of the healthy controls (P = 0.044) [Figure 2b].

In other comparisons, the expression level of MYD88 was significantly higher in patients with severe periodontitis compared firstly to that of all other patients with moderate and mild periodontitis and gingivitis and secondly to that of healthy individuals (P = 0.023 and 0.008, respectively) [Figure 3]. No statistically significant difference was observed in the expression levels of the other molecules between different evaluated groups.

Figure 3

Comparison of relative gene expression of MYD88 among healthy subjects, patients with severe periodontitis, and patients with mild and moderate periodontitis and also gingivitis. Graphs are shown as vertical interleaved bars, plotting the median with interquartile range. Results were analyzed by Mann–Whitney U-test. P < 0.05 are shown as (*) and P < 0.01 are shown as (**)

In both healthy and diseased tissues, positive significant correlations were observed between the expression levels of the different genes as shown in Table 2.

Table 2

Significant correlations observed between the mRNA levels of the assessed genes in healthy and diseased tissues

DISCUSSION

The gingival tissue consists of an upper layer of squamous epithelium and an underlying connective tissue. Gingival epithelial cells are the overlying cells of the gingiva and the first cells to be challenged by microbial species populating the periodontal niche, but the microbial virulence factors may diffuse into the connective tissue and stimulate the host cells residing in this area.[68] Studies have shown that these cells express various PRRs such as TLR4 on epithelial cells, neutrophils, monocytes, and fibroblasts[910] and TLR9 on epithelial cells and dendritic cells.[11] Of all other studies evaluating PRRs’ expression in gingival and healthy tissues, most showed increased expression level for these genes in diseased states while one pointed out to a decrease in TLR4 caused by Porphyromonas gingivalis.[8121314151617181920]

Here, we have conducted a more extensive research and assessed mRNA expressions of five TLRs, two NLRs, CD14, MYD88, and TRIF in healthy tissue samples as well as in gingivitis and periodontitis lesions. Most of the evaluated genes were expressed in specific tissue samples to a noticeable extent. In this regard, our findings were consistent with other studies indicated above. The exceptions were TLR5 and NALP3 mRNA expression in healthy tissues which showed negligible expression relative to β-actin. The observed gene expressions in healthy tissues could be due to the fact that microbial components are present in the oral cavity and dental biofilm of healthy individuals and can stimulate immune responses in the healthy gingiva. These responses participate in bacterial clearance and, therefore, maintain equilibrium between the host and normal flora.[621]

Compared to the healthy controls, some of the evaluated genes showed higher expression levels in disease circumstances while others showed lower levels of expression (TLR9 and NOD2 in gingivitis and TLR2 in periodontitis), though not significant. In gingivitis and periodontitis, the difference in the expression level of the assessed PRRs and other related molecules, though not significant in this survey, as stated in other articles depends on the microbial pattern of the disease and thus the organism's PAMPs[19] and also the host's immuno-inflammatory response.[20] Higher expression of TLRs in inflamed gingiva might be a result of stimulation by periodontopathic bacteria and their products, which results in the release of inflammatory cytokines.[8] This could also be the case for NLRs. Down-regulation of some TLRs compared to the healthy state may indicate an effect of possible intracellular negative regulators, apoptosis, or other controlling mechanisms that result in the inhibition of non-specific and harmful inflammatory host responses. It has been suggested that complete down-regulation of all TLRs is very unlikely to happen because new cells migrate to the inflammation area, and therefore, the cells form a heterogeneous population of responding cells including the cells from the late and early phases of response.[13]

CD14 is one of the two proteins essential for mediating TLR4 inflammatory activity. Wang et al. confirmed the enhanced expression of CD14 (and TLR4) on inflammatory human gingival fibroblasts (HGFs).[12] It has been reported that anti-CD14 Ab inhibits the development of periodontitis in mice.[22] Some studies have shown that healthy tissues express higher levels of membrane CD14 (mCD14) compared to diseased tissues or clinically healthy tissues from patients with chronic periodontitis. These studies indicated that elevated levels of mCD14 expression in healthy tissues contribute to maintain periodontal homeostasis.[23] Our results showed no significant alteration in CD14 mRNA levels between healthy and diseased states, which is not consistent with these studies. We observed a significant correlation between CD14 and TLR2, which complies with the fact that CD14 is a co-receptor of TLR2.[24]

Two NLRs were evaluated in our study, NOD2 and NALP3. The changes in expression levels of these two proteins which were assessed between healthy and diseased tissues and also between gingivitis and periodontitis groups were not significant. There are limited studies investigating NLR expression in gingival tissues. These studies have demonstrated that oral epithelial cells express NOD1 and NOD2 and have marked elevated levels of these molecules in inflamed tissues resulting in higher immune responses to PAMPs in sites of inflammation.[816] Studies have shown a synergistic cooperation between TLRs and NOD1 and 2 proteins in oral epithelial cells and also in human CD14+ monocytes and dendritic cells.[2526] NOD1 and NOD2 mRNA expression is induced by TLR activation and cytokines such as tumor necrosis factor-alpha (TNF-α) and IFN-γ.[25] Consistently, we observed significant correlations between NOD2 and TLR2 in patients and between NOD2 and TLR9 in patients and healthy individuals. NOD proteins have also been shown to stimulate the NALP3/cryopyrin inflammosome.[27] We also observed a positive significant correlation between NALP3 and TLR3 in healthy tissues and between NALP3 and TLR5 in healthy and diseased tissues, which could be attributable to their synergistic function.

Our results showed a significant correlation between NOD2 and MYD88. Studies have indicated that activation of both TLRs and NLRs might be important for the fine-tuning of innate immune inflammatory response. It has been shown that NOD2 ligand enhances MYD88 expression, which could be a possible explanation of the high sensitivity of TLR responses after peptidoglycan priming indicating the cross-talk between TLRs and NLRs.[25] The result we obtained here is consistent with this study. NALP3 and NOD2 showed a significant correlation with MYD88 in patients and TRIF in both patients and healthy subjects, respectively. This could possibly be explained with regards to NOD protein and TLR family synergistic function, considering MYD88 and TRIF as a connection link between these two families.

This study showed increased non-significant levels of TRIF mRNA expression in both gingivitis and periodontitis. This may be a possible indication that the TRIF signaling pathway does not have an important role in gingivitis and periodontitis events, but may also be due to the relatively small number of cases in each group. In contrast, MYD88 expression levels showed significant differences between healthy state and periodontitis and also between the healthy controls and severe periodontitis patients. MYD88 activation results in igniting intracellular signaling cascades that stimulates nuclear factor-kappa B (NF-κB) and pro-inflammatory cytokine secretion.[5] The significant difference of MYD88 expression in different tissues can indicate a possible role for this molecule in the development of periodontitis. Our results were consistent with other studies investigating the role of MYD88 in periodontal disease. Wang et al. showed MYD88 expression in HGFs by western blot analysis, which is used in the signal transduction induced by P. gingivalis lipopolysaccharide (LPS).[28] Papadopoulos et al. showed that MYD88 is a required downstream adaptor in the TLR signaling pathway for pro-inflammatory cytokine production in periodontal disease.[29] Burns et al. showed in their results that MYD88 signaling is critical for bacterial clearance in infection with P. gingivalis.[30]

Obtaining definite conclusions and confirming the present results require further investigation with larger sample sizes and more extensive research because mere changes in mRNA expression cannot indicate the changes in the protein expression of the relevant genes. Thus, alterations in protein expressions should also be investigated because there might be regulating mechanisms at the translational level that could affect the transcribed genes inversely as what has been shown in some studies.[14] For clarifying the pathogenic mechanism of periodontal diseases, the relation between the cellular expression of PRRs and different periodontal conditions should be unraveled. This would greatly help in the prevention and, especially, therapy of these diseases, knowing the fact that PRRs are considered as therapeutic targets in inflammatory disorders.