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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 6  |  Page : 872-876
Protective role of neutrophils in periodontal inflammation


1 Department of Periodontology, Meenakshi Academy of Higher Education and Research, Faculty of Dentistry, Meenakshi Ammal Dental College and Hospital, Dubai, UAE
2 Maktoum Bin Hamdan Dental University College, Dubai, UAE
3 Department of Dentistry, Meenak Academy of Higher Education and Research, Faculty of Dentistry, Meenakshi Ammal Dental College and Hospital, Chennai, Tamil Nadu, India
4 Department of Physiology, Meenakshi Academy of Higher Education and Research, Faculty of Dentistry, Meenakshi Ammal Dental College and Hospital, Chennai, Tamil Nadu, India

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Date of Submission04-Feb-2019
Date of Decision11-Mar-2020
Date of Acceptance13-Jul-2020
Date of Web Publication22-Mar-2021
 

   Abstract 


Aim: The quantification of neutrophils in blood is done to primarily screen the patients with acute infections. On the other hand, the neutrophils have also shown the antimicrobial activity by allowing the host cells to perform the primary function of preventing the wounds from being infected. The aim of this study was to quantify the blood neutrophil count in patients with chronic gingivitis and chronic periodontitis and to compare with the healthy controls in order to assess the protective role of neutrophils in periodontal inflammation. Methods and Material: A total of 30 subjects in the age group of 30–55 years were selected. Based on the clinical examination, the subjects were divided into three groups Group I (healthy), Group II (chronic gingivitis) and Group III (chronic periodontitis). The clinical parameters such as modified gingival index (MGI) and clinical attachment level (CAL) were recorded. Two millilitres of venous blood was collected from each participant in all the groups and the differential neutrophil counts were obtained using the Neubauer's chamber under a light microscope. One way ANOVA test was used to calculate the mean and standard deviation for MGI, CAL and blood neutrophil count among various groups. Results: The mean neutrophil counts in the various study groups were 63.300, 64.100 and 60.800 in Group I, Group II and Group III, respectively. Though there was a decrease in the mean neutrophil count from Group II to Group III, there was no statistically significant difference between the groups. Conclusion: In our study, there was no significant increase in neutrophil count in chronic periodontitis as compared to healthy controls, thereby explaining the protective role of neutrophils in periodontal inflammation.

Keywords: Dysbiotic microbiota, neutrophils, periodontitis

How to cite this article:
Perumalsamy R, Mahendra J, Mahendra L, Sethuraman M, Kavyasri, Rao V M. Protective role of neutrophils in periodontal inflammation. Indian J Dent Res 2020;31:872-6

How to cite this URL:
Perumalsamy R, Mahendra J, Mahendra L, Sethuraman M, Kavyasri, Rao V M. Protective role of neutrophils in periodontal inflammation. Indian J Dent Res [serial online] 2020 [cited 2021 Apr 15];31:872-6. Available from: https://www.ijdr.in/text.asp?2020/31/6/872/311644



   Introduction Top


Periodontitis is a complex disease in which multiple causal factors play a role. Periodontal destruction is not caused by bacterial plaque alone, but also host inflammatory response. Both hyper-responsiveness and hypo-responsiveness are seen in the host. Defence against the tissue irritating effects of the sub-gingival biofilm is mainly through the integrity and activity of epithelial lining, secretion of gingival crevicular fluid (GCF), saliva and local inflammatory reactions.[1]

Periodontitis is initiated by accumulation of biofilms on the tooth and its supporting structures. In susceptible individuals, dysbiosis occurs and an aberrant host-microbe equilibrium can result in the onset of disease, where the microbial biofilm thrives by exploiting the host inflammatory response. The acute inflammatory response is predominantly mediated by neutrophils via activation of innate neutrophil-derived defence mechanism, acquired immune system and humoral immune system.[2]

Neutrophils are the most abundant white blood cells in the gingival crevice and the periodontal pocket, where they play a crucial role in innate immunity response against bacterial infection and are responsible for maintenance of homeostasis. Neutrophils are the most efficient phagocytes and eliminate pathogens by a variety of means.[3] Neutrophils adhere through a cascade of events. They circulate in blood vessels in search of potential targets such as chemokines, produced by endothelial cells, which elaborate signals for pathogen entry and loss of tissue integrity. Host–pathogen interactions are generally initiated via host recognition of molecular structures of microbe known as pathogen-associated molecular patterns.[1]

The quantification of neutrophils in blood is often used as a primary screen to identify patients with acute infections. Polymorphonuclear leukocytes recruitment to the oral cavity was measured as the migration rate of neutrophils into the oral cavity through GCF. This granulocytic migration rate was found to correlate with the level of periodontal inflammation. There is a sprouting interest in using oral polymorphonuclear leucocyte count as a marker of oral inflammatory load and possibly as a rapid screen for patients with increased oral inflammatory load and periodontal infection. Oral polymorphonuclear leucocyte can be increased in the presence of various oral inflammatory conditions ranging from gingivitis, periodontitis to inflamed mucosal tissues in an implant.[4]

An alteration in the balance, dysbiosis, of oral microbiota is thought to be the initial trigger for periodontitis.[5] The accumulation of bacterial biofilm leads to an increase in the inflammatory infiltrate, composed mainly of neutrophils into the oral tissues. Off late neutrophils have shown to have a defensive role in periodontal inflammation. They form a blockade that prevents bacteria from penetrating into the deeper tissues. Neutrophils have also shown the antimicrobial activity by allowing the host cells to perform a protective role against inflammation and thereby aiding in periodontal wound healing. Thus, lack of neutrophils may also lead to severe periodontitis. Unfortunately, excess of neutrophils is always counted as an inflammatory state which seems to be prejudicially leading to tissue destruction.[6] It is stated that excess presence or absence of neutrophils leads to periodontal tissue damage. Hence, the neutrophil balance becomes important for maintaining the homeostasis. Keeping the above hypothesis in mind, the aim of this study was to quantify the blood neutrophil count in patients with chronic gingivitis and chronic periodontitis and to compare with the healthy controls in order to assess the protective role of neutrophils in periodontal inflammation.


   Methods and Material Top


Patient screening

A total of 50 subjects in the age group of 30–55 years were selected from the outpatient section [Figure 1]. Twenty subjects were screened from the general hospital, who came for a master health check-up. Out of which ten periodontally and systemically healthy subjects volunteered to be a part of the study and were considered as Group I (control group). Thirty subjects were screened for generalized chronic gingivitis and generalized chronic periodontitis from the outpatient section. Of the fifteen subjects screened for generalized chronic gingivitis, five subjects refused to participate in the study. Finally, ten patients were selected as part of Group II (chronic gingivitis). Similarly, of the fifteen subjects screened for generalized chronic periodontitis, three subjects refused to participate in the study and two subjects were excluded due to the presence of type II diabetes mellitus. Finally, ten patients were selected as part of Group III (chronic periodontitis). Finally, the subjects were selected into the above groups based on the inclusion and exclusion criteria. The institutional review board approved the study. Written informed consent was obtained from each patient before enrolment in the study. The study was conducted from July 2018 to September 2018, Chennai, Tamil Nadu, India.
Figure 1: Study flowchart

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The inclusion criteria to participate in this study were as following: Group I (healthy) included subjects with clinically healthy periodontium and with scores of modified gingival index (MGI) <1 and clinical attachment level (CAL) = 0 mm. Group II (chronic gingivitis) included the subjects who showed clinical signs of gingival inflammation with scores of MGI ≤1, CAL = 0 mm. Group III (chronic periodontitis) included subjects who showed clinical signs of inflammation and attachment loss with scores of MGI ≥1, CAL ≥2 mm. The exclusion criteria were: 1) patients with any known systemic diseases or conditions such as diabetes mellitus, cardiovascular diseases, pneumonia, tuberculosis and rheumatoid arthritis; 2) patients who had any course of medication affecting periodontal status within 1 month; 3) patients who had received periodontal therapy within 6 months; 4) subjects with history of smoking, tobacco chewing and alcohol consumption; 5) pregnant and lactating women.

Clinical examination

Clinical periodontal examination was performed by two trained and calibrated investigators (RP and JM) masked to study groups. Overall k value for intra-examiner reliability was 0.67. The clinical parameters recorded were MGI (Lobene et al., 1986)[7] and CAL in the selected subjects. A conventional manual periodontal probe was used for examination. CAL were recorded at six sites of each tooth and measured in millimetres. CAL was measured as the distance from the cementoenamel junction to the base of the periodontal pocket. In the present study, probing pocket depth was used to screen the patients, but CAL was used to assess the severity of periodontitis. CAL shows a better prediction of gingival recession, overgrowth or changes of the marginal gingiva associated with tissue swelling. Hence, CAL was assessed in the selected patients which shows a better reflection of past tissue destruction as compared to probing depth.

Estimation of neutrophil count

Two millilitres of venous blood was collected from each participant in all the groups and the differential neutrophil counts were obtained using the Neubauer's chamber under a light microscope.[8]

Statistical analysis

SPSS software version 16 was used for statistical analysis.

One-way ANOVA test was used to calculate the mean and standard deviation for MGI, CAL and blood neutrophil count among various groups and tukey's test was used for the post-hoc analysis for the same. Spearman's rank order correlation test was used to check the relationship between neutrophil count and various clinical parameters.


   Results Top


The mean MGI in Group I, Group II and Group III was 0.4400, 0.7600 and 1.8500, respectively. The mean MGI in Group III was significantly higher than the mean MGI in Group II followed by Group I (P < 0.05) [Table 1]. On comparing the CAL in various groups, CAL in Group III was significantly higher when compared to Group I and Group II (P < 0.05) [Table 2]. The mean neutrophil count in the various study groups was 63.300, 64.100 and 60.800 in Group I, Group II and Group III, respectively [Table 3]. There was an increase in the neutrophil count in Group II when compared to Group I, followed by a decrease in the mean neutrophil count in Group III when compared to Group II and Group I. Though there was a decrease in the mean neutrophil count from Group II to Group III, there was no statistically significant difference between the groups. There was a negative correlation between the MGI scores and neutrophil count and also between the CAL and neutrophil count, but it was not statistically significant [Table 4].
Table 1: Comparison of mean values of clinical parameters modified gingival index (MGI) in various study groups

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Table 2: Comparison of mean values of clinical parameters clinical attachment level (CAL) in various study groups

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Table 3: Descriptive statistics of neutrophil count in various study groups

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Table 4: Relationship between neutrophil count and various clinical parameters (Spearman's Rank Order Correlation Test)

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   Discussion Top


Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defence during inflammation and infections. Foraying microorganisms evoke an inflammatory response that recruits neutrophils from the circulation into the tissues. There, neutrophils destroy the microorganism by a series of mechanisms, mainly phagocytosis, release of antimicrobial substances and the formation of neutrophil extracellular traps (NETs).[9] Activated neutrophils also release proteinases into the surrounding tissue, causing damage to the host, and can live for much longer than previously thought. It is estimated that neutrophil half-life is days instead of hours.[10] In addition, neutrophils are capable of producing many cytokines and chemokines, which can influence the inflammatory response, as well as the immune response.[11]

Neutrophils are the principal leukocyte (>95%) recruited to the gingival crevice in response to bacteria biofilm deposited on teeth,[12] and their absence or excess leads to periodontal tissue damage, making it evident that neutrophil numbers and distribution play a principal role in maintaining oral health. Neutrophil homeostasis is important to prevent ancillary damage to the host by the potent antimicrobial and pro-inflammatory effects of these cells. Neutrophils homeostasis involves several mechanisms at the level of production, trafficking and clearance.[13] Once neutrophils are in the circulation, they can be quickly mobilized to sites of infection or inflammation through a highly controlled process known as the leukocyte adhesion cascade.[14],[15]

The presence of neutrophils is required for protecting oral health. This is made obvious by the fact that individuals with defects in production and distribution of neutrophils develop severe forms of periodontitis. Some of these defects are rare and congenital and include the Chediak-Higashi syndrome, Papillon-Lefèvre syndrome, neutropenias and leukocyte adhesion deficiency.[16],[17] Thus, inflammation is an important element in periodontitis that is deregulated when neutrophil homeostasis is modified.

The present study was aimed to estimate and compare the blood neutrophil count in healthy individuals and patients affected by gingivitis and chronic periodontitis. The clinical parameters recorded were MGI and clinical attachment loss. Our study showed that the mean MGI in Group III was significantly higher than the mean MGI in Group I and Group II which revealed that the signs of gingival inflammation become more evident with the increase in severity of periodontal disease [Table 1]. This was in accordance with the studies done by Huynh et al. and[18] Sharma et al.[19] which showed an increase in MGI scores with increase in the severity of inflammation. This study showed greater attachment loss in chronic periodontitis patients as compared to gingivitis or healthy subjects which revealed increased attachment loss with increase in inflammation [Table 2]. This was in accordance with the study done by Sharma et al.[19]

In the present study, there was an increase in the neutrophil count in Group II when compared to Group I, followed by a decrease in the mean neutrophil count in Group III when compared to Group II [Table 3]. Though there was a decrease in the mean neutrophil count from Group II to Group III, there were no statistically significant differences found between the groups. Gingivitis and periodontitis are inflammatory conditions which are usually seen with increase in the number of neutrophils. However, in our study, the neutrophil counts were found to be more or less the same among the study groups. This could be due to the presence of polymicrobial dysbiotic microbiota having an arsenal of self-defence mechanisms, which can be directed to attack against neutrophils or camouflage the biofilm.[20] Microbiota have the capacity to communicate with each other which is called as quorum sensing, that enables the dysbiotic microbiota to optimize the biofilm conditions and ensure nutrient supply.[21] Quorum sensing molecules control neutrophil ROS response and penetration into P. aeruginosa biofilms.[22] Dysbiotic microbiota, in addition to directly attacking neutrophils in the biofilm, can make themselves resistant to neutrophil-mediated killing by disguising their immunogenicity. P. gingivalis, even in low colonization levels, can cause a shift from symbiotic microbiota to dysbiotic microbiota.[23],[24] These bacteria use varied mechanisms to destabilize neutrophil homeostasis, causing the inhibition of phagocytic killing, resistance to antimicrobial agents and to the oxidative burst, impaired recruitment and chemotaxis of neutrophils, thus delaying the increase in neutrophil count. Also, apoptosis of neutrophils is implicated in the pathogenesis of periodontitis. Reduction in the number of neutrophils could impair the response against pathogenic bacteria such as Porphyromonas gingivalis. Granulocyte monocyte-colony stimulating factor (GM-CSF) present in the gingival crevicular fluid (GCF) and which is secreted during the immune response, reduces the apoptosis of neutrophils, increasing the number of neutrophils.[25]

In the present study, we found a negative correlation between the neutrophil count with MGI and CAL which are both indicative of inflammatory process. Hence, there was no increase in neutrophil count among the various groups with an increase in periodontal inflammation [Table 4]. In contrast to our study, Ana Pejcic et al.[26] in their study reported a significant increase in neutrophil count in subjects with a severe form of periodontitis when compared to its moderate form and healthy controls. This was said to be due to increased response of the host to bacterial presence, manifested through increased inflammatory response in the form of increased leukocyte and neutrophil counts. However, in our study, we could not find a significant difference in the blood neutrophil count between the healthy and gingivitis and chronic periodontitis groups. This may be due to the self-defence mechanisms of polymicrobial dysbiotic microbiota which can attack the neutrophils or camouflage the biofilm, thereby, leading to reduction in the neutrophil count in periodontal inflammation. Thus, oral tissues require constant surveillance by neutrophils in order to maintain homeostasis.

One of the limitations of our study was the lack of comparison of the neutrophil count estimation from various sources such as GCF or saliva. In future, comparative studies can be done based on the comparison among the various sources with periodontal intervention in estimating the protective role of neutrophils.


   Conclusion Top


In our study, there was no significant increase in neutrophil count in chronic periodontitis as compared to healthy controls, thereby explaining the protective role of neutrophils in periodontal inflammation. It is stated that the polymicrobial microorganisms in periodontitis exert different defence mechanisms against neutrophils like inducing cell lysis and degranulation or rapid cell death. Therefore, it can be presumed that the implementation of such survival strategies coexists with the elimination of bacteria by neutrophils. In future, interventional studies are required to elucidate the protective role of neutrophils in inflammatory diseases.

Acknowledgment

The authors would like to thank Dr Shyam, Associate Professor, Department of Public Health Dentistry, Meenakshi Ammal Dental College and Hospital, Chennai, India for the statistical analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Jaideep Mahendra
Department of Periodontics, Meenakshi Academy of Higher Education and Research, Faculty of Dentistry, Meenakshi Ammal Dental College and Hospital, Alapakkam Road, Maduravoyal, Chennai - 600 095, Tamil Nadu
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_106_19

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