Periodontitis association with IL-8 gene polymorphisms

IL-8 and its polymorphisms are involved in multiple acute and chronic inflammatory processes including pathological changes to surrounding structures of the teeth called periodontal diseases or periodontitis. The aim of this manuscript was to systematically review studies from 2006 to 2021 on IL-8 polymorphisms and their association with periodontitis. Literature analysis was done following the PRISMA protocol guidance using articles not older than 15 years (2006-2021). The search was carried out using PubMed (MEDLINE), ScienceDirect and Wiley Online Library databases. For the focus question, the PICO (population (P), intervention (I), control (C), and outcome (O)) study design protocol was used, and the following question was formulated: are IL-8 gene polymorphisms associated with periodontitis? A total of 2422 articles were found at the beginning of the search. After applying the inclusion and exclusion criteria, screening, and full-text article exclusion with reasons, 31 studies were included in the analysis. In conclusion, IL-8 and its gene polymorphisms are associated with an increased risk of periodontal diseases.


INTRODUCTION
Periodontitis is a chronic, multifactorial inflammatory disease of the periodontium, affecting up to 47% of adults over the age of 30 in the United States 1,2 . In Europe, this disease is found in 5-20% of middle-aged adults, and this number increases up to 40% for older individuals 3 . What begins at first as a loss of connective tissue and gingiva, during the course of this disease leads to periodontal ligament loss, resorption of alveolar bone, and the exposure of tooth roots to bacteria 4 . This inflammation can ultimately lead to the tooth loss in the affected region 5 . Tooth loss leads to impaired oral health-related quality of life, and the region from which the teeth are lost can have an impact on the severity of the impairment 6 .
Back in the 1999, periodontitis was classified into 4 different forms: necrotizing, chronic, aggressive, and a manifestation of a systemic disease, but nowadays, a new 2018 classification has been adopted and approved 7 . Periodontitis is classified into three main forms: periodontitis, necrotizing periodontitis and periodontitis as a direct manifestation, each having a localised (<30% of teeth affected) or generalised (>30% of teeth affected) extent 7 . The primary cause of periodontitis are bacteria, which accumulate and colonise mouth tissues. However, multiple factors influence the aetiology of this disease: unhealthy lifestyle habits (such as smoking, drinking), systemic illnesses, calculus accumulation around teeth and restorations, bad oral hygiene and genetic factors 8 .
Various studies have been conducted where the gene polymorphisms were analysed, and some of them have been proven to have an association with periodontitis [9][10][11] . Today at least 65 genes are associated with this mouth disease 7 . Since periodontitis is an infectious disease, interleukin mediates inflammatory and immune responses, and this is one of the main reasons why polymorphisms of this gene may have an impact on the periodontitis pathogenesis 12 . A commonly investigated interleukin-8 gene polymorphism is highly associated with periodontitis susceptibility 13 . Its mechanism is explained by the role of IL-8 in regulating inflammatory reaction to the infection caused by bacteria, as the increased expression of proinflammatory cytokines is highly dependent on the presence of bacterial metabolic products 14 . Various studies analyse different polymorphisms and their association with periodontitis, but mixed results regarding the disease dependency on IL-8 polymorphisms are being apprised 12 .
The aim of this work was to carry out a systematic literature review with no limit to study design of all studies from 2006 to 2021, regarding IL-8 gene polymorphisms and their association with various types of periodontitis.

The aim of the literature review
The aim of this review was to analyse studies from 2006 to 2021 on IL-8 polymorphisms association with periodontitis. The review was done according to the systematic review statement (PRISMA protocol) (ref. 15 ).  Interleukin-8 genotype and allele frequencies in the control group. A2767T, T(1)1722T(2), and C781T polymorphism of IL-8 gene -no statistically significant differences between periodontitis patients and the control group.

None.
A allele had an increased frequency in the disease group. IL-8 mRNA levels were higher in the periodontitis group, presented with TA genotype.
SNP rs4073 associated with the CP group in non-smoker Brazilian subjects.
None. IL-8 / CXCL8 -251 T allele, which is associated with higher production of IL-8/CXCL8, is also associated with a higher risk of developing acute suppurative form of AP. IL-8 / CXCL8 -251 A allele, which is associated with lower production of IL-8/CXCL8, is associated with chronic nonsuppurative form of AP. None. IL-8 polymorphisms had no significant association with risk of developing periodontitis. None.

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PBMCs from the C/C and G/C subjects secreted significantly higher levels of IL-8 in response to LPS than PBMCs from the G/G subjects. This SNP regulates the expression of TLR4 and has some influence on the response to LPS.

Focused question of the research (PICO)
For the development of the question focus, PICO (population (P), intervention (I), control (C), and outcome (O)) study design protocol was used, and the following question was formed: Are IL-8 polymorphisms associated with periodontitis pathogenesis?

Synthesis of results
The findings on genes and their polymorphisms associated with periodontitis are shown in Table 1 and Table 2.

Selection of studies for the research
Number of records that were identified through database analysis: 2422.
No additional records were identified through other sources.
Filters that were applied for publications: a) not older than 15 years, b) articles written in English. 703 records were found.
The records were screened for eligibility. During screening, titles and abstracts were analysed, and the inclusion/exclusion criteria were applied.
In total, 37 full-text articles were assessed for eligibility. Six studies were excluded, due to not matching the aim of our research and the criteria: researches not covering IL-8 polymorphisms association with periodontal diseases; researches which did not include patients affected by periodontitis; researches covering taste receptor topics; IL-1 studies; IL-17 studies. 31 articles were included in qualitative synthesis. The search sequence (PRISMA flow diagram) is shown in Fig. 1.

Synthesis of results
This literature analysis revealed that IL-8 polymorphisms and concentrations are associated with periodontal diseases. A study done by Liukkone et al. included 455 participants. A clinical examination was performed by two periodontologists, and 3 groups were formed: BOP (bleeding on probing), pocket depth, alveolar bone loss, and each group had flow cytometry done for the IL-8 concentration measurements 16 . Research results have shown elevated results for patients who had BOP >25%, a higher number of teeth, or deeper gingiva pockets, which concludes that IL-8 concentration has association with periodontal tissues inflammation and tissue destruction 16 .
A study done by Silva et al. analysed 874 Brazilian individuals who were affected by type 2 diabetes mellitus (T2DM) and periodontitis. Three different groups were formed: healthy individuals (n=307), periodontitis group (n=334), and T2DM group (n=233). Genotyping of SNP 251(T>A) rs4073, +396(T>G) rs2227307 and +781(C>T) rs2227306 was done by TaqMan (ref. 17 ). Research conclusions have demonstrated that heterozygous genotypes of the 251 (rs4073), 396 (rs2227307) and 781 (rs2227306) SNPs in the IL-8/CXCL8 gene were related to the diseased phenotypes, and patients who were carrying this haplotype formed by homozygous TTC/TTC were around twice as susceptible to periodontitis and T2DM development, or severe periodontitis development, as non-carriers in Brazilian population 17 .
Additional research covered unrelated individuals. Scientists Linhartova et al. split 153 patients into 4 groups: patients affected with type 1 diabetes mellitus (T1DM) + chronic periodontitis (CP) (n=34), patients affected by T2DM+CP (n=44), healthy individuals (HC) affected with CP (n=32) and non-periodontitis HC (n=41) (ref. 18 ). For the determination of genes, qPCR was used, and for IL-8 plasma levels measurements, ELISA was the first-choice option, whereas the investigators were unaware of the phenotype when genotyping 18 . The research showed that IL-8 plasma levels were scientifically significantly different between non periodontitis HC group and T1DM+CP/T2DM+CP patients (P<0.01), and no significant associations between IL-8 plasma levels and IL-8 and CXCR2 polymorphisms were found (P>0.05) (ref. 18 ).
Another research done by Sajadi et al. included 65 patients from Iran affected by periodontitis (18 with chronic form, and 47 with aggressive form) and 55 healthy controls whose DNA extraction was done for IL-8 +781 C/T and -845 T/C polymorphisms assessment 19 . The results showed a significantly positive association between the IL-8 -845 alleles distribution and the risk of periodontal disease; C allele of IL-8-845 increased the risk of periodontal disease 9.08-fold (9.08 (95% CI 1.14-72.12, P=0.03) (ref. 19 ). As for +781 C/T locus, no statistically significant correlation was found between patients and controls, but there was a statistically significant difference between the TT vs. CC + CT genotypes that had a role of defending against periodontal disease with value of 0.38 (95% CI 0.16-0.90, P=0.02) (ref. 19 ).
Study done by Xiao-Bing et al., in which information was systemically gathered from multiple scientific sources, consisting of over 1938 patients affected by periodontitis, stated that IL-8 C1633T and rs1126580 polymorphisms are associated with this disease 12 . On the other hand, the same review found that IL-8 rs4073, A2767T, T11722T2, rs2234671, rs2230054, rs1126579, rs2227306, rs2227307, rs2227532, and T-738A are not associated with periodontal pathology 12 .
In 2014, a meta-analysis was conducted by scientists Chen et al., which included 2233 periodontitis cases, and it concluded that in Brazilian mixed population, IL-8 -251A/T and -845T/C polymorphisms might be associated with the development of periodontitis 9 . Also, -251A/T allele T was also included as a risk factor for the development of periodontitis 9 . On the other hand, Khosropanah et al. composed a research in 2013, which compared 227 chronic periodontitis affected patients with 40 healthy subjects and found that there was no statistically significant correlation between different IL-8 genotypes and the severity of periodontal condition 23 . Kavrikova et al. conducted a study, in which 329 chronic periodontitis affected patients were investigated for SNP in CXCR2 gene, and it concluded that polymorphisms +785C/T (rs2230054), +1208T/C (rs1126579), and +1440A/G (rs1126580)) in the CXCR2 gene are not associated with chronic periodontitis 24 .
Scientists Li et al. examined 122 chronic periodontitis affected Chinese patients, and found, that the MMP-1-1067, MMP-3-1171, MMP-9-1562 and IL-8-251 polymorphisms are related to the susceptibility to chronic periodontitis 25 . Scarel et al., who investigated SNPs rs2227307 (+396) and rs2227306 (+781) and rs4073 (-251) polymorphisms relation with chronic periodontitis, found that +396TT genotype and haplotypes ATC/TTC and AGT/TGC meant a significant risk of this disease's susceptibility in Brazilian patients 26 . Also, Sippert et al. stated that in Brazilian individuals, Erythroid DARC plus IL-8 -353T>A single nucleotide polymorphisms were associated with chronic periodontitis disease 27 . In the same study, a FY*02N.01 with IL-8 -353A nucleotide polymorphism was found to be associated with protection from chronic periodontitis in Afro-Brazilians 27 .
Andia et al. investigation revealed, that in chronic periodontitis affected patients, A allele had an impact of increasing the disease frequency 28 . Furthermore, SNP rs4073 was associated with chronic periodontitis group in Brazilian subjects (non-smokers) (ref. 28 ). In a study done by Li et al., IL-18 plasma levels were found to be higher in periodontitis patients, and C variant of IL-18 -607A>C polymorphism was shown to have an association with increased risk of periodontitis 29 . Zhang et al. reported relation between rs4073, rs2227307 and rs2227306 SNP's and periodontitis in a study done on Han Chinese population 30 . However, in a study done by Kim et al. on 276 periodontitis affected Brazilian patients, it was found that SNP (rs4073) in the IL-8 gene had no relation to susceptibility to periodontitis 31 . In a study by Corbi et al., the results showed that susceptibility to chronic periodontitis had no association with IL-8 cytokine levels or periodontal clinical parameters 32 . In a study by Amaya et al., where individuals with suppurative and non-suppurative forms of periodontitis were analyzed, IL-8 / CXCL8 -251 T allele was associated with higher production of IL-8/CXCL8 and higher risk of aggressive periodontitis acute suppurative form development 33 . Also, Scarrel et al. in their 2011 study done on Brazilian population stated that rs2234671 SNP in the CXCR1 gene was not useful as a genetic risk factor for chronic periodontitis affected patients 39 .
Scientists Da Silva et al. also conducted a meta-analysis with 71531 individuals, in which 25 polymorphisms were analyzed, and a conclusion was approached that IL-8 polymorphism had no significant association with the risk of periodontitis development 34 .
Scientists Rusyanti et al. reported that low levels of IL-8 (below 0.064 pg/μL) showed 34.5 times higher occurrence of aggressive periodontitis 35 . Another factor increasing the prevalence of this disease was polymorphism c576 T>C>G of FPR1 gene, which also correlated significantly with IL-8 (ref. 35  Corbi et al. studied 41 individuals and analyzed their IL-8 levels before and after periodontal therapy and found that nor the outcome of the periodontal therapy (non-surgical), nor the levels of IL-8 were influenced by the ATC/ TTC CP-susceptibility haplotype 37 . In 2017, Cirelli et al. conducted a research, in which they stated that haplotypes in the IL-4 gene, but not in IL-8, influenced levels of A. actinomycetemcomitans in sub-gingival regions, measured before and after non-surgical periodontal treatment 38 . It was also noted that there were significant correlations, when assessing microbiological, genetic and immunological factors, in IL-8 and IL-4 haplotypes 38 . Nevertheless, in Pigossi et al. research, it was concluded that the presence of the ATC/TTC IL-8 haplotype was associated with an enhanced response of neutrophils and leukocytes to Gram-negative bacteria associated with periodontitis 44 . Also, Finoti et al. in their work stated that non-surgical therapy is equally effective in improving clinical parameters and decreasing the levels of periodontopathogens, independent of the genotype groups produced by the IL-8 haplotype 40 .

DISCUSSION
Multiple researchers have analysed association of IL-8 gene polymorphisms with periodontal diseases, although studies regarding this topic are not limited to the aforementioned polymorphisms only. One of the most researched gene families, regarding periodontal pathologies, is the IL-1. In a study done by Papathanasiou et al., IL-1 family members were associated with periodontal inflammation, and therapeutic blockade was assessed, although research in this topic is quite limited 46 . Also, IL-1B was found to influence bone resorption and continuity of bone loss in patients, which was stated in the work done by scientists Cheng et al. They have suggested IL-1B blockage using the antibodies, inhibitors, and even plant-derived substances for reducing IL-1B (ref. 47 ) but stated that more investigations were needed for further analysis of these methods in periodontal treatment. The aforementioned therapeutic blockades were not analysed with IL-8 polymorphisms in our research and could be of interest in later studies.
The same gene IL1B polymorphisms were assessed in a research done by Brodzikowska et al., noting the rs1800587 and rs1143634 association with periodontal inflammation; however, geographical and ethical factors were mentioned as having a role in prevalence of specific polymorphisms in different populations 48 . On the other hand, Lopez et al. research analysed Chilean subjects to determine the prevalence of the IL-1A-889 and IL-1B+3954 polymorphisms and concluded that individuals who carry the positive genotype have a significantly higher risk for periodontitis development 49 .
Another factor found in the research, which increases the prevalence of periodontitis, is diabetes. A study done by Struch et al., in which they genotyped 1515 subjects for IL-1, has shown that diabetes patients had an increased risk of periodontal disease, which was aggravated when combined with IL-1A/1B genotype 50 . Study by Kashiwagi et al. also stated that proinflammatory cytokines are expressed when a patient has high glucose levels, which also explains TLR activation and inflammatory response provoking, although hyperglycaemia did not affect the IL-8 expression 51 . It contradicts the findings of Lihartova et al., explaining that statistically significant levels of IL-8 were found in patients affected by periodontitis and T1DM (ref. 18 ). Nevertheless, more research is needed for additional analysis of IL-8 expression in diabetes affected patients.
Regarding clinical symptoms, a study done by Engrebetson et al. showed a positive gingival crevicular fluid IL-8 correlation with the probing depth of patients affected by periodontitis 52 , although other measurements -clinical attachment level, bleeding on probing or plaque index -did not correlate with the aforementioned levels 52 . IL-8, according to a study done by Lagdive et al., is highly associated with the status of periodontal tissues, and the level of the interleukin-8, found in the gingival crevicular fluid changes according to the severity of the periodontal disease 53 . Some research has been done to engineer the IL-8 gene haplotypes, and to affect the susceptibility of the disease. One such study was done by scientists Benakanakere et al., which provided evidence that ATC/ TTC haplotype may increase neutrophil levels in some cases of inflammatory lesions and could affect the disease susceptibility 54 .
As found in the research, IL-8 polymorphisms are highly associated with periodontal pathologies. In the future, further studies must be done to fully analyse and understand the pathological mechanisms that associate IL-8 with periodontitis.

FUTURE PERSPECTIVES OF CYTOGENETIC MARKERS COMPLEXES DETECTION
In the future, novel molecular marker complexes should be determined in patients with periodontitis. Such markers might suggest additional linking mechanisms in the disease pathogenesis. Considering that the exact etiological agents responsible for the initiation of periodontitis remain unknown, it seems that multifactorial interaction might play a role here. Once significant sets of cytogenetic markers have been identified, we will be able to monitor their changes over time, adjust lifestyle, and initiate early treatment.
Scientific discoveries around the world will allow to develop methods of total analysis of periodontitis development and to select cytogenetic marker complexes required for understanding prognosis and treatment of periodontitis. If these marker complexes could be applied in practice in future, the costs of treatment would be significantly reduced.

CONCLUSIONS
After analysing the articles, the following conclusions can be drawn: Additional scientific literature search was done manually. All of the related and similar articles were reviewed, including the reference lists of the selected articles.
Keywords (included in MESH) "periodontitis, IL-8, gene, polymorphisms" were used along with their variations to ensure the highest number of results possible. The references of the papers were also analysed, in order to identify any potential additional results.
Articles were analysed and investigated by each author independently. Investigators discussed and compared their selections and matched the differences through dis-cussion. The last stage of the research started with the screening of the articles. Exclusion of articles was done after investigating their titles and abstracts. Whether to include the publication or not, was discussed only after analysis of the full text, according to the inclusion and exclusion criteria.