ABSTRACT

INTRODUCTION

Inflammation is clearly a central component of many chronic diseases. Cytokines are regulatory low-weight proteins acting as mediators into genesis and control of immune and inflammatory response.

Interleukin-1 (IL-1) is a proinflammatory cytokine and includes two different but functionally similar molecules: IL-1α e IL-1β, genes which are all located on the long arm of chromosome 2 (region 2q13-14). ^1–3

IL-1 play an important role in immune regulation and inflammatory processes by inducing expression of many effector proteins, e.g. cytokines/chemokines, nitric oxide synthetase (NOS) and matrix metalloproteinases (MMPs) ^1,2.

IL-1α is mainly produced by keratinocytes of junctional epithelium or pocket epithelium, is a mediator of local inflammation and regulates intracellular events. ¹

IL-1β is primarily produced by activated macrophages and fibroblasts that release this extracellular protein. ¹

IL-1 upregulates complement system, Fc-receptor on polymorphonuclear neutrophils and monocytes, and the expression of adhesion molecules upon fibroblast and leukocytes. ^1, 4

It induces the expression of homing receptors into the extracellular matrix for leukocytes and bone resorption by stimulating the proliferation and differentiation of osteoclast progenitors and activating formed osteoclast indirectly. ⁵

It increases the production of MMPs, prostaglandins and IL-1 itself by macrophages, fibroblast and polymorphonuclear neutrophils. ^5,6

It upregulates the expression of major histocompatibility complex molecules (MHC) upon B and T cells facilitating their activation, clonal expansion and immunoglobulin production. ⁶

Excessive and/or dysregulated activity of these mediators is associated with tissue destruction and therefore the synthesis, secretion and biological activity of IL-1 cytokines have been identified as therapeutic targets for common inflammatory disorders such as rheumatoid arthritis (RA), diabetes and periodontal disease ^7–9.

Increase of IL-1 gingival concentration in subjects with periodontal disease and its reduction after periodontal treatment was demonstrated. ^10,11

Periodontal disease (PD) is characterized by loss of connective tissues within the periodontium and destruction of (alveolar) bone support. ^12–15

The most prevalent form of PD is chronic periodontitis, which is an inflammatory disease initiated by pathogenic bacteria in the subgingival plaque biofilm. ¹²

Furthermore, a prominent, localized inflammatory cell infiltrate involving neutrophils, monocytes and both T and B lymphocytes is another characteristic these cells are further sources of MMPs and the cytokines that regulate them.^5,6

Single nucleotide polymorphisms (SNPs) into the IL-1 promoter region could modulate this cytokine function that acts a fundamental role into the pathogenesis of PD, by affecting the susceptibility and severity of the affection. ^16–18

The composite IL-1 genotype, known as periodontitis-associated genotype (PAG), obtained by the combination of the two rare alleles at the above mentioned SNPs, has been extensively investigated with respect to the form of periodontal disease (chronic vs aggressive), smoke, ethnic group, oral microbial pathogens present in the plaque biofilm and other periodontal clinical parameters related to inflammatory status or to the progression of the disease. ^16,17

The aim of this study was to test if polymorphisms of genes of IL-1α⁺⁴⁸⁴⁵ and IL-1β ⁺³⁹⁵⁴ were linked with periodontitis, in a case-control study population, delimited to a specific geographic area, in association with microbiological findings.

PATIENTS, MATERIALS AND METHODS

All subjects were chosen among current and permanent residents of Bari area (Italy).

Apulia (Italian: Puglia) is a Region in Southern Italy. The Region comprises 19,345 square kilometers (7,469 sq mi), and its Population is about 4.1 million Density 210/km2 (540/sq mi). Its capital city is Bari.

Population proportionate technique was employed for sample size estimation. The city is divided in 10 quarters. The average age of Bari residents is 42 compared to the Italian average of 42. The present study includes only resident form 4 generations in the quarter of Old Town that is the one with less heterogeneity of population. According to population census 2011, city itself has a population of about 320.000, over 116 km²; the Old Town in an area of around 0,4 square Kilometers, have a population of around 6000 people, of which the 26-56 old adults constitute 33%. With 95% confidence level, the estimated sample size was 99.

This work was conducted according to the recommendations of the Declaration of Helsinki; all patients received informed consent.

The study included as described in Table ​Table11 and Table ​Table2,2, 195 unrelated patients, recruited only from private dental practice.

All subjects were in good general health conditions.

To identify suitable participants, patient referral records, including a full-mouth series of peri-apical radiographs, were screened.

Complete examinations of soft and hard oral tissues were performed on all patients.

Exclusion criteria from the study were: diseases of the oral hard or soft tissues except caries and periodontal disease; presence/use of orthodontic appliances; need of pre-medication for dental treatment; use of antibiotics for the preceding 6-month period; pregnancy or lactation; inability or unwillingness to sign informed consent; history of diabetes; HIV positive; immunosuppressive chemotherapy; history of any disease known to severely compromise immune function.

No subject with localized periodontitis or acute necrotizing ulcerative gingivitis was included in the study.

All patients were interviewed and smoking habits were recorded. They were subjected to a clinical examination of the periodontal tissues.

The parameters investigated were: plaque accumulation (0/1/2), overt gingivitis (0/1), bleeding on probing (0/1), and suppuration (0/1).

A manual periodontal probe was used to assess the attachment loss which was measured at six sites per tooth at all teeth excluding third molars.

Regarding these parameters, the cases were divided into four groups:

(A) Healthy controls patients;

(B) With mild periodontitis (plaque accumulation, overt gingivitis, attachment loss ≤ 4 mm with or without bleeding);

(C) Moderate periodontitis (bleeding and/or attachment loss between 4-6 mm);

(D) Severe periodontitis (bleeding or suppuration and attachment loss > 6 mm).

Periodontally intact subjects were qualified as controls if they displayed: no sites with probing depth exceeding 3 mm and no more than 4 sites with bleeding on probing.

Our study recruited only Italian Caucasian subjects, and none of them had a history of diabetes mellitus or current manifestation of systemic diseases, which correlates with destructive periodontal disease.

Radiographic evaluation included Orthopantomography and 21 intraoral radiographs.

All data from clinical and radiographic evaluation were collected into a dedicated periodontal folder.

The periodontal probe (PCP15 -HuFriedy, Chicago, IL, USA) was inserted parallel to the vertical axis of the tooth and ‘walked’ circumferentially clockwise around each surface of the tooth, to detect the area of deepest penetration ¹⁹.

GENOMIC ANALYSIS

RESULTS

The present study determined the frequencies of the two IL-1 polymorphisms, IL-1α⁺⁴⁸⁴⁵ and IL-1β⁺³⁹⁵⁴in a sample group of 195 individuals.

Sixty-four subjects were positive for IL-1α⁺⁴⁸⁴⁵ genotype (33 %), with a major frequency of positive subjects observed among the group B subjects and the least frequency observed among the healthy subjects. (Table 4)

This difference achieved statistical significance because p < 0.05 at d.f.=1.

Similar trend was noticed for the SNPs of IL-1β⁺³⁹⁵⁴ gene, with the frequency progressively increasing from group A through group C, B, to group D, which was significant at p<0.05, d.f.=1. (Table ​(Table55)

The combined presence of both the IL-1α and IL-1β composite genotype, dived for severity of disease was studied. (Table 6)

This difference brought out a significant correlation between the severe form of periodontitis and the presence of composite genotype (p < 0.05, d.f.=1, calculated among healthy vs. severe).

Furthermore, microbiological data were available for all the subgingival plaque samples collected from all cases and controls. (Table ​7)

As expected a statistically significant association between the presence of bacteria and periodontitis was detected (p<0.05, d.f.=1).

The most frequent pathogens found in the subgingival plaque of patients positives for the presence of bacteria were Fusobacterium nucleatum (27%), Campylobacter rectus (20%), Eikenella corrodens (20%), Tannerella forsythia (2%), Treponema denticola (6%), Parvimonas micra (7%),, Porphyromonas gingivalis (10%), and Prevotella intermedia (8%). (Figure 1)

When the allele distribution was compared between cases and healthy controls, statistically insignificant values were obtained. (Table 8)

The heterozygous genotypes (GT) and (CT) are more frequent respectively in, IL-1α⁺⁴⁸⁴⁵ and IL-1β⁺³⁹⁵⁴, for both cases and controls.

In the current investigation findings were concordant with literature observations. ^21–22.

However, contradictory results were obtained in a recently published data ²⁸ where they have found that for IL-1β⁺³⁹⁵⁴ CC genotype was the more frequently genotype identified in cases than in controls.

DISCUSSION

In the present work, we have directed our effort primarily on the study of a homogeneous population from both ethnic and clinical points of examination.

This approach, given the small extent of our region, reduces the possibilities for establishing large sample sizes, but the homogeneous population has fewer problems with population stratification.

A second limitation of the present study is represented by the limited size of case samples.

Current evidence from complex diseases (with large heritable components) indicates that the predicted odds ratios for many genes may be 1.5 or less and, to identify such associations, as a general rule at least 100 cases and controls should be genetically characterized. ^29, 30

The subjects may not be representative of the true disease distribution because of recruitment from a clinical practice.

This phenomenon is known as the “clinicians’ illusion,” where the identified cases among the subjects are likely to be more numerous and severe than cases identified from a general representative population sample. ³¹

The sample is very small as to provide for any generalized conclusions based on the results that apply to the whole population.

Due to the phenomenon of genetic heterogeneity, the periodontal phenotype may be due to several different genotypes. ^{21–22, 32–33}

The clinical categorization of PD has been different in different studies due to changes in classification and nomenclature.

Since many risk factors contribute to PD, the ideal method of studying genetic relationship would be to start before the disease sets in and following it up through the natural history, i.e., to establish the genotypes required first and then follow it up as the subject is exposed to various predisposing and risk factors.

Also, as recommended, further researches are needed to validate the biologic basis for genetic susceptibility testing, to evaluate the ability of IL-1 genotypes to predict disease initiation and to evaluate the effectiveness of IL-1 genotyping in making diagnostic or treatment intervention strategies, especially in dental “new age” ³⁴ tissue engineering approach ^35–40.

ACKNOWLEDGMENTS

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REFERENCES