Journal of Natural Science, Biology and Medicine

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 11  |  Issue : 2  |  Page : 145--150

Immunohistochemical evaluation of nucleotide-binding and oligomerization domain 1 and nucleotide-binding and oligomerization domain 2 receptors in periodontal health and disease


CH Neeharika Rao, Lalitha Tanjore Arunachalam, Uma Sudhakar 
 Department of Periodontics, Thai Moogambigai Dental College and Hospital, Chennai, Tamil Nadu, India

Correspondence Address:
Lalitha Tanjore Arunachalam
56/1, Veerabadran Street, Nungambakkam, Chennai, Tamil Nadu
India

Abstract

Introduction: Periodontal disease, infectious in origin and inflammatory in progression ultimately leads to destruction of periodontium. Pattern recognition receptors (PRRs) help in identifying the molecular patterns displayed on the bacteria and mount an immune response. Nucleotide-binding and oligomerization domain receptors (NOD1 and NOD2) are cytosolic PRRs involved in the immunopathogenic process involved in the periodontal diseases. This study was undertaken to evaluate distribution of NOD1 and NOD2 and to compare and correlate the NOD1 and NOD2 expression in gingival samples from healthy, chronic, and aggressive periodontitis participants. Materials and Methods: Sixty participants participated in the study and were divided into three groups of 20 individuals each - Group I (healthy), Group II (chronic periodontitis), and Group III (aggressive periodontitis) based on the inclusion and exclusion criteria. Gingival tissue samples were collected during periodontal flap surgery, crown lengthening procedure in periodontitis individuals and healthy controls, respectively. The levels of NOD1 and NOD2 in the gingival samples were analyzed using immunohistochemistry. Results: The NOD1 and NOD2 levels were higher in Group III (aggressive periodontitis) followed by Group II (chronic periodontitis) and lowest in Group I (healthy). Comparison of mean NOD1 and NOD2 between the three Groups showed statistically significant difference (P < 0.001). Positive correlation was observed on correlating NOD1 and NOD2 with the clinical parameters (gingival index, probing pocket depth, and clinical attachment loss). Conclusion: Epithelial localization of NOD1 and NOD2 was more in periodontitis than in healthy tissue. These findings indicate that NOD1 and NOD2 play an indispensable role at the forefront in innate immunity.



How to cite this article:
Neeharika Rao C H, Arunachalam LT, Sudhakar U. Immunohistochemical evaluation of nucleotide-binding and oligomerization domain 1 and nucleotide-binding and oligomerization domain 2 receptors in periodontal health and disease.J Nat Sc Biol Med 2020;11:145-150


How to cite this URL:
Neeharika Rao C H, Arunachalam LT, Sudhakar U. Immunohistochemical evaluation of nucleotide-binding and oligomerization domain 1 and nucleotide-binding and oligomerization domain 2 receptors in periodontal health and disease. J Nat Sc Biol Med [serial online] 2020 [cited 2020 Nov 26 ];11:145-150
Available from: http://www.jnsbm.org/text.asp?2020/11/2/145/290489


Full Text



 Introduction



Periodontitis begins as an innoxious gingivitis, if left untreated damages the periodontium. The disease process id kick started by dysbiotic microflora, and orchestrated by the host immune response. The war between the host and the biofilm bacteria, will largely determine the destruction that ensues. The pathobionts are well endowed with a plethora of virulence factors that trigger a spew of inflammatory mediators, mediating the collateral damage.

Innate immunity is vital as the initial defense mechanism and identifies the offending microorganisms by a variety of receptors located either on the surface or inside the cytosol. These receptors are expressed by immune cells, ranging from neutrophils, macrophages to dendritic cells, etc., as well as epithelial cells. These specific receptors known as pattern recognition receptors (PRRs) recognize either microbial- or damage-associated molecular patterns (MAMPs or DAMPs, respectively) and help in escalating an immune response. PRRs are classified into five types: toll-like receptors (TLRs), C-type lectin receptors, rig-I-like receptors, AIM2-like receptors, and the nod-like receptors (NLR) proteins. The TLR group of PRR has been explored widely in periodontal health and disease. The NLR group contains a nucleotide-binding and oligomerization domain (NOD) and is thus designated as NLRs. The NLR family has about 22 types identified in human beings and around 30 types in mice.[1] NOD1 and NOD2, belong to this family and are localized intracellularly within the cytosol.

NOD1 recognizes gamma-D-glutamyl-meso-DAP, found in Gram-negative bacteria[2] and Nod2 recognizes muramyl dipeptide (MDP), which is found in peptidoglycan (PGN) of almost all the bacteria.[3] Once the recognize the offending pathogens, a cascade of signaling events is tipped off and the offenders are eliminated through stimulation of NF-κB.[4] NOD1 and NOD2 receptors are present in many cells within the oral tissues. In gingival health, they show a stronger expression than TLRs.NOD1 and NOD2 are also expressed in oral and periodontal pocket epithelium. NOD1 and NOD2 work synergistically with TLRs to generate cytokines in immune cells as well as in several epithelial cells. In oral epithelial cells, NOD1 and NOD 2 in symphony with TLR upregulate the β-defensin2 secretion.[5]

It is clear that these receptors are expressed in health as well as in inflammation and are thought to play a role in the development of periodontitis. Therefore, this study was undertaken to determine the distribution of NOD 1 and NOD 2 in tissues of healthy, chronic, and aggressive periodontitis individuals using immunohistochemistry and to compare and correlate the NOD 1 and NOD 2 expression in healthy, chronic, and aggressive periodontitis individuals with the gingival and periodontal status.

 Materials and Methods



Patient population

The study population consisted of 60 individuals belonging to both sexes and all individuals were randomly selected from the outpatient clinic of the department of periodontics. Individuals were divided in to three groups of 20 individuals each as - Group I (healthy gingiva), Group II (chronic periodontitis), and Group III (aggressive periodontitis). The study protocol was fully explained to all patients and they signed an informed consent to participate in the study. Institution review board clearance was procured from the university. The inclusion criteria for gingival health were the following - no bleeding on probing (BOP) and gingival index (GI) = 0–<1. Disease groups had the diagnosis of generalized moderate chronic periodontitis and generalized aggressive periodontitis based on the American Academy of Periodontology task force report on the update to the 1999 classification of periodontal diseases and conditions 2015.[6] Individuals with BOP, GI ≥1 with 30% of teeth with one or more sites exhibiting probing pocket depth (PPD) ≥5 mm and ≤7 mm, clinical attachment loss (CAL) 3–4 mm, and radiographic evidence of bone loss were categorized in to Group II as chronic periodontitis. The Group III aggressive periodontitis individuals were included when the following criteria was satisfied namely, BOP, GI ≥1, minimum 4 teeth other than incisors and molars with one or more sites exhibiting PPD ≥5 mm, CAL ≥5 mm at one or more sits in 4 teeth other than incisors and molars with radiographic evidence of bone loss. Individuals were excluded if they were smokers, tobacco users in any form, alcoholics, and systemically or immunologically compromised patients.

Evaluation of gingival and periodontal status

All data were recorded in a standard pro forma by a single examiner to eliminate bias. Oral examination was carried out with proper illumination using mouth mirror and graduated Williams's periodontal probe. The following parameters were evaluated for all the individuals - GI, PPD, CAL, and orthopantomogram (OPG). OPG was taken for each patient to differentiate chronic periodontitis and aggressive periodontitis patients from health. No further delineation was attempted within the chronic periodontitis group based on the extent of alveolar bone loss.

Procurement of gingival tissue samples

Gingival tissue samples with approximate thickness of 2–3 mm and height of 1–2 mm were obtained during periodontal flap surgery and crown lengthening surgery in periodontal patients and healthy group, respectively. Diseased tissue samples were collected from sites with a PPD of 5 mm or more, CAL of at least 3 mm, and presence of BOP. On the other hand, the healthy ones obtained from the sites with minimum pocket depth and the absence of BOP. In all groups, periodontal tissues were excised by internal bevel and sulcular incisions using 15c blade. The tissue samples required for the study were collected after the biopsy procedure from the respective site and directly placed in 10% neutral buffered formalin contained in an uricup for 24 h. The samples were then grossed to record details pertaining to the appearance of the tissue specimen. One section was used for morphological identification by hematoxylin and eosin (H and E) staining, and the other for immunohistochemistry.

Calculation of nucleotide-binding and oligomerization domain 1 and nucleotide-binding and oligomerization domain 2 expression

The staining analysis was assessed by counting the number of positive and negative cells in the samples. Cell counting was done manually by an experienced oral pathologist using a grid from eight different fields and the average was taken. The proportion of stained cells in each field was evaluated by H-SCORE.[7] The membrane staining intensity (0, 1+, 2+, or 3+) was scored for each cell in a fixed field. The H-score was based on the staining intensity, or the sum of individual H-scores were added for each intensity level that was observed. H-score was given using the following formula: (1× [% cells 1+] +2× [percentage cells 2+] +3× [percentage cells 3+]). The final score, ranged from 0 to 300 and the staining intensity was graded from negative staining (0), light staining (1), moderate staining (2), to intense staining (3).

Statistical analysis

To analyze the data SPSS (IBM SPSS Statistics for Windows, Version 23.0, Armonk, NY: IBM Corp. Released 2015) was used. Significance level was fixed at 5% (α = 0.05). To compare the mean values between groups one-way ANOVA is applied followed by Tukey's honest significant difference post hoc tests for multiple pairwise comparisons. Karl Pearson correlation coefficients are calculated to assess the linear relationship between clinical and NOD variable.

 Results



The mean and standard deviation of the clinical parameters is summarized in [Table 1]. The mean value of GI in Group I was 0.23 ± 0.16, Group II was 2.4 ± 0.44, and Group III 1.2 ± 0.40. The difference was statistically significant (P < 0.001). On comparison of PPD in all the three groups - Group I (2.22 ± 0.78), Group II (6.34 ± 0.99), and Group III (8.61 ± 0.80), it was also statistically significant (P < 0.001). Similarly, statistical significance (P < 0.001) was seen for CAL between Group I (2.22 ± 0.78), Group II (5.86 ± 0.83), and Group III (7.51 ± 0.73). [Table 2] shows the comparison of mean NOD 1 and NOD 2 values among Group I, Group II, and Group III. There was a significant difference (P < 0.001) between the three groups for both NOD 1 and NOD 2. [Table 3] shows correlation between NOD 1 and NOD2 and all clinical parameters using Pearson correlation test. In Group II strong positive correlation was noted between NOD 1 and PPD (0.781), which was statistically significant (P = 0.008). In Group I, mild positive correlation was seen between NOD 1 and GI (0.265), PPD (0.327), and CAL (0.327) but no statistically significance was observed. Similarly, in Group III, statistically significant correlation was not seen between NOD 1 and GI (0.121), PPD (0.068), and CAL (0.060). There was strong positive correlation seen between NOD 2 and GI in Group I (0.606) and Group II (0.480). However, there was no statistical significance observed. On comparing NOD 2 and PPD, strong positive correlation noted (0.634) in Group II.{Table 1}{Table 2}{Table 3}

 Discussion



Periodontal disease, albeit infectious in origin is inflammatory in nature. The disease process is kick started by subgingival pathogenic microbiome resulting in localized destruction of both soft and hard tissues supporting the teeth. Although many factors ranging from genetic to environmental contribute to the development and progression, the key player is the dysbiotic microbiota, which starts as well as maintains the inflammation, which after a certain threshold overwhelms the host.[8] The shift from Gram-positive population to the Gram-negative microorganisms is pathognomic, but a single organism cannot be nailed to be the culprit but pathobionts like Porphyromonas gingivalis shift the microbial population from health to dysbiosis. Localized forms of aggressive periodontitis are linked to Aggregatibacter actinomycetemcomitans, while generalized forms of chronic periodontitis are associated with many bacteria including P. gingivalis, T. forsythia, and T. denticola.

Pathogen-associated molecular patterns include lipopolysaccharide, lipoteichoic acid, PGN, and leukotoxin. These are sensed by PRRs and internally localized NOD 1 and NOD 2 are present in immune and nonimmune cells. NOD 1 is specific and recognizes D-γ-glutamyl-meso-DAP dipeptide found in PGN of all Gram-negative and certain Gram-positive bacteria whereas NOD2 identifies the MDP present in almost all bacteria. NOD1 receptors are expressed by cells such as epithelial cells, stromal cells, and endothelial cells,[9] whereas NOD2 expression is not so universal but present in keratinocytes and oral epithelial cells[10] and play a role in triggering immune responses.

This study was undertaken to study the distribution of NOD 1 and NOD 2 in tissues of healthy, chronic periodontitis, and aggressive periodontitis individuals and to compare and correlate the NOD 1 and NOD 2 expression in gingival samples from healthy, chronic, and aggressive periodontitis individuals with and periodontal status namely GI, PPD, and CAL. The expression of NOD 1 and NOD 2 was analyzed using immunohistochemistry and qualitative as well as quantitative analysis was done using H-score.

[Table 1] shows the descriptive statistics of the clinical parameters. GI was lowest in Group I, followed by Group III and highest in Group II. PPD and CAL were lowest in Group I and highest in Group III and NOD 1 and NOD 2 were expressed higher in Group III than Group I and Group II. It is well known that inflammatory component increases from health to disease and this explains for the higher level of GI in periodontitis groups compared to health. One of the features of aggressive periodontitis is the relatively low level of gingival inflammation[11] compared to chronic periodontitis, and this is reflected in the GI scores. However, on comparison of the GI among the three groups, it is statistically significant. Statistical significance was recorded for both PPD and CAL (P < 0.001) among the three groups and our results are consistent with Chandy et al. in 2017,[12] where similar results were noted.

The H and E staining of all the samples were done and increasing number of inflammatory cells was noted in the connective tissue of chronic periodontitis compared to gingival health and aggressive periodontitis. This is akin to the findings of Liljenberg and Lindhe 1980,[13] who observed low number of infiltrated neutrophils in aggressive forms of periodontal disease.

Immunohistochemical staining of NOD1 and NOD2 was done in all the three groups. This is a first study to quantitatively and qualitatively assess NOD1 and NOD2 in health and periodontitis. Staining was noted in healthy samples as well as periodontitis group. Staining was intense in the gingival epithelium, as shown in [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]. Staining was also noted in the connective tissue in few samples of health and disease, albeit mild. Hence, the focus of the present study was on NOD1 and NOD2 staining in the epithelium. As gingival epithelium is in direct contact with the offending bacteria, they are well equipped with innate receptor system that helps in host defense processes.{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}

The expression pattern of NOD 1 and NOD 2 is shown in [Table 2]. The mean values of NOD1 were higher in aggressive periodontitis group followed by chronic periodontitis and healthy samples. The same pattern was observed for NOD2. Sugawara et al. in 2006[5] was the first to report on the expression of NODs in human oral epithelial cells. Expression of NOD1 and NOD2 on human oral epithelial cells was confirmed by reverse transcription polymerase chain reaction, flow cytometry as well as immunohistochemistry by the authors. They reported that in health, NOD1 and NOD2 showed marked expression and also stronger expression than TLRs. Our results also show the presence of NOD1 and NOD2 in health and periodontitis, similar to the above mentioned study. Our results also agree with Liu et al. in 2014,[14] who demonstrated that NOD1/2 were upregulated in periodontitis. Their immunostaining results showed that NOD1, NOD2, and TLR4 were highly expressed in the gingival tissue of patients with periodontitis, especially in the monocyte infiltrated area. However, in our study, NOD ½ did not show higher expression in the epithelium of periodontitis samples than healthy controls, but in the study by Sugawara et al.,[5] NOD1 and NOD2 were detected in gingival tissue from adult periodontitis patients, similar to that found in healthy gingiva.

NOD1 and NOD2 have also been identified in gingival fibroblasts[15] and that NOD1 and NOD2 signaling also activates NF-kB which markedly up-regulated the production of pro-inflammatory cytokines, interleukin (IL)-6, IL-8, and MCP-1, clearly showing NOD receptors in fibroblasts are involved in recognition of periodontal pathogens.

The expression of NOD2 is more than NOD1 in chronic periodontitis [Table 2]. This is similar to Hasegawa et al. 2010[16] who showed that P. gingivalis possesses low levels of NOD1-stimulatory activity. NOD2 is an inducible protein in most resident cell and the expression level is less but can be increased by specific stimuli.[17] Liu et al. in 2014[18] showed that in periodontal ligament cells, NOD2 are upregulated by P. gingivalis and cause the induction of ICAM-1 production. The role of NOD2 in alveolar bone loss has been studied by Prates et al. in 2014,[19] in an experimental model of periodontitis with P. gingivalis W83. NOD2 deficient mice showed lower bone resorption when compared to wild type. The expression of 2 osteoclast activity markers cathepsin K and matrix metalloproteinase 9 was significantly lower in gingival tissue from NOD2 deficient mice. These results show that the lack of NOD2 receptor impairs the bone resorption, suggesting that NOD2 receptor could contribute to the progression of bone resorption in experimental model of periodontitis. This also substantiates the higher levels of NOD2 observed in Group II of our study.

In aggressive periodontitis (Group III), NOD1 and NOD2 are expressed higher than healthy and NOD2 expression is more than NOD1 [Table 2]. Okugawa et al. in 2010,[20] showed that A. actinomycetemcomitans, Fusobacterium nucleatum strongly stimulated human embryonic kidney (HEK/NOD1) cells in a dose-dependent manner. P. gingivalis also stimulated HEK/NOD1 cells but was much weaker and although A. actinomycetemcomitans showed strongest activity for NOD2 compared to P. gingivalis. Our results are akin to the above study, where NOD1 and NOD2 were expressed more by Group III compared to Group II.

Correlation of NOD1 and NOD2 with clinical parameters in Group I, Group II, and Group III is shown in [Table 3]. In Group II, strong positive correlation was noted between NOD 1 and PPD (0.781), which was statistically significant (P = 0.008). Similarly, strong positive correlation was noted between NOD2 and PPD (0.634), and it was also statistically significant (P ≤ 0.05). This positive linear correlation between PPD and NOD1 and NOD2 in chronic periodontitis group shows that with increasing probing depth, upregulated expression of NOD1 and NOD2 is seen. Since this is the first study to see the association of clinical parameters with NOD expression patterns, we could not find similar studies to relate our findings.

 Conclusion



Detection of NOD 1 and NOD 2 in oral epithelium of health and periodontal disease sheds light on the role of PRRs in periodontal pathogenesis, but is just the tip of the iceberg. In future, advances in epigenetic modulation of these receptors will pave way for therapeutic targets that can help in modulating the disease process.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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