periodontitis is an inflammatory disease which is initiated and maintained by the gram - negative bacteria of the subgingival biofilm .
specific pathogen associated molecular patterns ( pamps ) and bacterial virulence factors stimulate an inflammatory host response that finally results in destruction of periodontal tissue and tooth loss .
chronic periodontitis ( cp ) and generalized aggressive forms of periodontitis ( agp ) appear to be associated with certain pathogens , including porphyromonas gingivalis , campylobacter rectus , tannerella forsythia , peptostreptococcus micro , and treponema species [ 3 , 4 ] .
treponema denticola , p. gingivalis , and t. forsythia , characterized as the  red complex ,  were strongly associated with the clinical progression of chronic periodontitis [ 15 ] .
in contrast , agp was more often diagnosed in patients positive for aggregatibacter actinomycetemcomitans , but there were many individuals with agp who did not harbor this microorganism .
in addition to the microbial challenge , other factors , such as genetics , environment , and host factors , play a role in the pathogenesis of these diseases [ 79 ] .
various compounds , such as cytokines , representing an important pathway of connective tissue destruction in periodontitis , have been detected in gingival crevicular fluid ( gcf ) .
il-8 , a member of the cxc chemokine family , was originally described by matsushima and oppenheim .
it is the most important chemoattractant and activator of human neutrophils and an important mediator for granulocyte accumulation .
il-8 is involved in the initiation and amplification of acute inflammatory reactions and chronic inflammatory processes .
functions of il-8 are mediated through two receptors ( cxcr1 and cxcr2 ) ; the expression was detected on numerous cell lineages , including neutrophils and epithelial cells .
gingival epithelial cells ( gec ) are capable of upregulating il8 expression rapidly in response to a. actinomycetemcomitans challenge , facilitating thus the recruitment of neutrophils as a host defense mechanism [ 13 , 14 ] .
il8 expression in gec is induced by p. gingivalis   and t. forsythia ; il-8 production by gingival fibroblast cultures is also affected by lipopolysaccharides of p. gingivalis and p. intermedia .
the il-8 levels in gingival crevicular fluid ( gcf ) are valuable in detecting the inflammation of periodontal tissue [ 1820 ] , and periodontal therapy reduces the il-8 levels in gcf .
il-8 is encoded by the il8 gene located on chromosome 4q13 - 21 ( genbank accession number m28130.1 ) , consisting of four exons , three introns , and the proximal promoter region .
several polymorphisms have been reported in the il8 gene [ 2326 ] and some of them can regulate the il-8 production
. some of snps in the il8 gene such as 845t / c ( rs2227532 ) , 738t / c , 251a / t ( rs4073 , previously referred to as 353a / t ) , + 396t / g ( rs2227307 ) , and + 781c / t ( rs2227306 ) have been studied in patients with agp or cp in the brazilian population [ 2732 ] .
in addition , il8 251 t allele , which was associated with higher production of il-8 , increased the risk of developing acute suppurative form of apical periodontitis ( ap ) , whereas il8 251a  low - producing  allele was associated with chronic nonsuppurative form of ap in the colombian population . in the chinese population , il8 251a allele has been associated with decreased susceptibility to cp .
the cross - sectional study in iran has also focused on the study of polymorphisms in the il8 gene but did not specify whether it was for patients with cp or agp . to date
, no study analyzing allele , genotype , or haplotype frequencies of il8 gene polymorphisms in patients with periodontitis has been performed in caucasians .
the aim of this study was to associate four snps in the il8 gene ( rs4073 , rs2227307 , rs2227306 , and rs2227532 ) and their haplotypes to cp and agp and subgingival bacterial colonization in the czech population .
the study was performed with the approval of the committees for ethics of the medical faculty , masaryk university brno and st .
written informed consent was obtained from all participants before inclusion in the study , in line with the helsinki declaration .
all patients were recruited from the patient pool of the periodontology department , clinic of stomatology , st .
exclusion criteria included history of cardiovascular disorders ( such as coronary artery diseases or hypertension ) , diabetes mellitus , malignant diseases , immunodeficiency , current pregnancy , or lactation .
controls were selected from subjects referring to the clinic of stomatology for reasons other than periodontal disease ( such as dental caries , orthodontic consultations , preventive dental check - ups , etc . ) during the same period as patients and matched for age , gender , and smoking status .
similarly as patients , all controls had at least 20 remaining teeth and were in good general health .
a total of 492 unrelated caucasian subjects of exclusively czech ethnicity from the region of south moravia were included in this case - control association study .
diagnosis of nonperiodontitis / periodontitis was based on the detailed clinical examination , medical and dental history , tooth mobility , and radiographic assessment .
probing depth ( pd ) and attachment loss ( cal ) were collected with a unc-15 periodontal probe from six sites on every tooth present .
we used the index of mhlemann to evaluate decreases in alveolar bone levels.generalized cp group ( n = 278 ) : all patients with chronic periodontitis ( cp ) fulfilled the diagnostic criteria defined according to cal levels by the international workshop for a classification of periodontal diseases and conditions for chronic periodontitis .
inclusion criteria for patients suffering from generalized chronic periodontitis were as follows : 30% of the teeth were affected , pd was 4  mm , and the amount of cal was consistent with the presence of dental plaque.generalized agp group ( n = 58 ) : patients with aggressive periodontitis with age at disease onset < 35 years , attachment loss of 4  mm or more in at least 30% of the teeth ( at least three of the affected teeth were not first molars and incisors ) , and the severity of attachment loss being inconsistent with the amount of dental plaque were included in this study.control group ( healthy / nonperiodontitis ) ( n = 156 ) : controls were screened using a who probe and the cpitn ( community periodontal index of treatment needs ) was assessed ; values of the cpitn index in controls were less than 3 .
generalized cp group ( n = 278 ) : all patients with chronic periodontitis ( cp ) fulfilled the diagnostic criteria defined according to cal levels by the international workshop for a classification of periodontal diseases and conditions for chronic periodontitis .
inclusion criteria for patients suffering from generalized chronic periodontitis were as follows : 30% of the teeth were affected , pd was 4  mm , and the amount of cal was consistent with the presence of dental plaque . generalized agp group ( n = 58 ) : patients with aggressive periodontitis with age at disease onset < 35 years , attachment loss of 4  mm or more in at least 30% of the teeth ( at least three of the affected teeth were not first molars and incisors ) , and the severity of attachment loss being inconsistent with the amount of dental plaque were included in this study .
control group ( healthy / nonperiodontitis ) ( n = 156 ) : controls were screened using a who probe and the cpitn ( community periodontal index of treatment needs ) was assessed ; values of the cpitn index in controls were less than 3 . in order to adjust for the effect of smoking history on periodontal disease , the subjects ( patients and controls ) were classified into the following groups : subjects who never smoked ( referred to as nonsmokers ) and subjects who were former smokers for 5 pack years or current smokers ( referred to as smokers ) .
the pack years were calculated by multiplying the number of years of smoking by the average number of cigarette packs smoked per day .
dna for genetic analysis was extracted from the peripheral blood leukocytes using standard phenol / chloroform procedures with proteinase k according to sambrook et al . .
isolation and storage of dna ( working samples at concentrations of 50  ng  l at 4c ) as well as the genotyping of samples were conducted in the laboratory of the department of pathophysiology , faculty of medicine , masaryk university , brno , czech republic .
four snps ( 845c / t rs2227532 , 251a / t rs4073 , + 396g / t rs2227307 , and + 781c / t rs2227306 ) in the il8 gene were genotyped using the 5 nuclease taqman assay for allelic discrimination .
individual fluorogenic taqman probes , consisting of an oligonucleotide labelled with both a fluorescent reporter dye , fam , and a quencher dye , vic , were obtained from life technologies ( grand island , ny , usa ) .
each reaction mixture was prepared using taqman genotyping master mix ( 12.5  l ) , taqman snp genotyping assay ( 1.25  l ) , and 50  ng of genomic dna in 17.5  l of dh2o to make a 25.0  l reaction volume .
genotyping was carried out simultaneously with 88 samples on 96-well plate ( + 8 negative controls ) .
the pcr thermocycling protocol consisted of 10  min at 95c , followed by 40 cycles of 15  s at 92c and 1  min at 60c .
each genotyping plate contained eight wells without any dna template ( negative controls ) and randomly selected duplicate samples ( 10% of plate samples ) .
allele genotyping from fluorescence measurements was then obtained using the abi prism 7000 sequence detection system .
sds version 1.2.3 software was used to analyze real - time and endpoint fluorescence data .
genotyping was performed by one investigator ( p. b. l. ) unaware of the phenotype .
subgingival bacterial colonization ( aggregatibacter actinomycetemcomitans , porphyromonas gingivalis , prevotella intermedia , tannerella forsythia , treponema denticola , peptostreptococcus micros , and fusobacterium nucleatum ) in subgingival pockets was investigated by the dna microarray based on a periodontal pathogen detection kit ( protean ltd .
, ceske budejovice , cr ) in a subgroup of randomly selected subjects ( n = 151 for cp , n = 21 for agp , and n = 75 for controls ) before subgingival scaling .
microbial samples were collected from the deepest pocket in periodontitis patients ( and from the deepest sulcus in healthy subjects ) of each quadrant by inserting a sterile paper point into a base of the pocket for 20 seconds .
this test determined the individual pathogens semiquantitatively as follows : (  ) undetected , which corresponds to the number of bacteria less than 10 , ( + ) slightly positive corresponding to the number of bacteria 10 to 10 , ( + + ) positive corresponding to the number of bacteria 10 to 10 , and ( + + + ) strongly positive , with the number of bacteria higher than 10 .
comparisons were made between allelic and genotype frequencies in the patients with chronic or aggressive form of periodontitis and control population .
the significance of differences in the allele frequencies among groups was determined by fisher 's exact test .
 analysis was used to test for deviation of genotype distribution from hardy - weinberg equilibrium and comparison of differences in genotype combinations among groups . to examine the linkage disequilibrium ( ld ) between all snps , pairwise ld coefficients ( d ) and haplotype frequencies
variations in the quantity of subgingival bacteria corresponding to the particular genotypes / alleles were tested by  and fisher 's exact tests .
power analysis was performed with respect to the case - control design of the study taking the incidence rate of markers and estimate of the odds ratio ( or ) as end - point statistical measures .
ors with corresponding 95% confidence intervals ( ci ) were estimated using logistic regression models , adopting age , sex , and smoking as adjusting covariates .
10.0 ( statsoft inc . , tulsa , ok , usa ) and spss software ( spss 20.0.1 , ibm corporation , 2011 )
the mean ages for agp patients ( 37.0  8.2 ; years  sd ) and healthy subjects ( 41.0  12.3 ) did not differ between the two groups .
however , subjects with cp were significantly older ( 47.9  8.7 ) than the patients with agp ( p < 0.05 ) . nearly , 27% of the periodontitis patients ( 28.0% of cp and 26.0% of agp ) and 28.0% of healthy subjects were smokers .
there were no significant differences between the subjects with periodontitis and controls regarding the mean percentage of smokers and ratio of males / females ( 77/79 in controls , 136/142 in patients with cp , and 25/33 in patients with agp ) .
sample size of the study was planned in standard power calculation for case - control design of the study with the null and alternative hypotheses expressed on the basis of or .
the design was prospectively optimized assuming the prevalence of examined attribute among controls to be 0.5 . the recruited sample ( 278 cases , 156 controls ) allowed a statistically significant detection of or out of the range of 0.551.80 ( alpha = 0.05 , power = 0.80 ) . in case of the agp group ( 58 cases , 156 controls ) , the statistically significantly detectable ors estimates were out of the range of 0.392.59 .
allele and genotype frequencies of all investigated il8 polymorphisms were not significantly different between the subjects with cp and/or agp and controls ( p > 0.05 ; table 2 ) . considering that in the czech population , snp il8 845tt genotype occurred in 98.4% of cp patients and even 100% of controls and agp patients , we analyzed this polymorphism only in the subgroup of subjects ( n = 193 ) .
based on the previous study   the haplotype analysis of these selected snps was performed in the il8 gene ( 251t / a rs4073 , + 396t / g rs2227307 , and + 781c / t rs2227306 ) .
all variants in the il8 gene were in tight linkage disequilibrium with each other to various degrees ( d = 0.7931.000 in controls , d = 0.8890.951 in patients with cp , and d = 0.8911.000 in patients with agp ) .
the complex analysis revealed differences in il8 haplotype frequencies . specifically , the a(251)/t(+396)/t(+781 ) and t(251)/g(+396)/c(+781 ) haplotypes were significantly less frequent in patients with cp ( 2.0% versus 5.1% , resp . , 4.5% , p < 0.05 ) ( table 3 ) .
the decreased frequency of the tgc haplotype alleles in patients with cp was confirmed by the observation that tgc / ttc haplotype ( arranged as genotypes ) was less frequent in patients with cp ( 0.4% versus 2.6% , p < 0.05 , or = 0.09 , 95% ci = 0.010.96 ) .
moreover , an uncommon att / att haplotype ( 1.15% of the studied population ) was found more , but nonsignificantly , in non - periodontitis controls ( 2.6% versus 0.4% , p = 0.07 ) .
there was also a nonsignificant trend in the atc / ttc haplotype association with cp ( 2.2% versus 0.0% , p = 0.08 , table 4 ) . 

f. nucleatum occurred less frequently in nonperiodontitis subjects ( n = 75 ) positive for t allele of il8 + 396g / t variant ( 49.2% versus 77.8% , p < 0.02 ; or = 0.28 , 95% ci = 0.090.89 ) or tt genotype ( 21.2% versus 55.6% , p < 0.05 ; or = 0.22 , 95% ci = 0.050.91 ) . in contrast , il8 251 t allele carriers had an increased or for individual presence of a. actinomycetemcomitans in agp ( n = 21 ) patients ( 91.7% versus 40.0% , p < 0.01 ; or = 16.5 , 95% ci = 1.88145.0 ) and also tt genotype was more often found in a. actinomycetemcomitans presence ( 83.0% versus 13.3% , p < 0.01 ; or = 32.5 , 95% ci = 2.38443.2 ) .
patients with cp ( n = 151 ) carrying cc genotype of il8 + 781t / c variant had less frequent presence of t. forsythia in their subgingival microflora than subjects without this genotype ( 21.6% versus 35.1% , p < 0.05 ; or = 0.51 , 95% ci = 0.251.05 ) .
however , the relationship between periodontal bacteria and il8 gene polymorphisms must be assessed very carefully regarding small numbers of subjects in the respective subgroups .
cytokines involved in the inflammatory process , such as il8 and their genes , are important potential modifiers of individual susceptibility to agp or cp .
although none of the investigated snps in the il8 gene was individually associated with aggressive or chronic periodontitis , the patients with cp showed lower a(251)/t(+396)/t(+781 ) and t(251)/g(+396)/c(+781 ) haplotype frequencies than the controls .
the association of tgc haplotype with cp was confirmed by the relationship between tgc / ttc haplotype ( arranged as genotypes ) and cp .
these results confirm the hypothesis that haplotypes are more powerful for detecting susceptibility alleles than individual polymorphisms .
our results differ from those obtained by scarel - caminaga et al .   who associated atc / ttc and agt / tgc haplotypes with chronic periodontitis in the brazilian population
for example , the frequency of the atc haplotype in the czech population was less than 3.4% , compared to 23.7% in the brazilians . in the brazilian population , some haplotypes of il8 845(t / c)/738(t / a)/353(a / t ) variants showed significant association to , or protection against , cp . of the three il8 snps , only one polymorphism was the same as in our study ( i.e. , snp 251 ( rs4073 ) referred to as 353 in brazilian study ) .
polymorphism 845t / c ( rs2227532 ) was also investigated in this study , but regarding a very low frequency of c allele , it was analyzed only in several individuals and therefore it was not included for any further haplotype assessment . to date , eight studies evaluating the association of il8 snps ( 251 , + 396 , and + 781 ) and cp or agp in different populations ( mostly in brazilian but none in the caucasian population ) have been performed , with contradictory results [ 2732 , 34 , 35 ] . most of these studies analyzed only the individual snp and were focused on examining of cp ; only andia et al
.   studied the relationship between il8 251 variant and agp ; however , no association was found .
similarly , kim et al .   failed to find any association between cp and allele or genotype distribution of il8 251 snp .
in contrast , another study discovered a significant association between il8 251 snp and cp in nonsmokers .
the il8 251ta heterozygote genotype was associated with increased levels of il8 mrna transcripts and a allele had an increased risk for developing periodontitis .
this is consistent with the observation that the a allele of snp il8 251 tended to be associated with higher il-8 production in lipopolysaccharide- ( lps- ) stimulated human whole blood .
very recently , li et al .   found that a allele of il8 251 variant was associated with decreased susceptibility to cp in chinese population .
.   found a significant difference in the genotype frequencies of il8 251a / t and + 396g / t snps between subjects with periodontitis and a control group in hamedan , iran , but did not specify whether it was for patients with cp or agp .
conversely , corbi et al .   showed that the genetic susceptibility to cp in the il8 gene was not associated with worse periodontal clinical parameters and increased il-8 concentration . with the exception of scarel - caminaga et al
, no association between snp at position + 396 or + 781 and cp in the brazilian population was discovered .
several studies have examined polymorphisms in interleukins in connection with a subgingival bacterial colonization in patients with periodontitis . in this study ,
snps in the gene encoding il-8 were associated with the presence of pathogenic bacteria in subgingival dental plaque .
the results showed that il8 251 t allele carriers had an increased or for the individual presence of a. actinomycetemcomitans in agp patients ( p < 0.01 ) and an increased or was also found for the presence of t. forsythia for t allele of il8 + 781 in cp patients ( p < 0.05 ) .
these data are in agreement with the notion that individual genetic susceptibility may influence the host response to infection .
nibali et al .   found association between il6 snps and a. actinomycetemcomitans , which they confirmed by the haplotype analysis .
specifically , il6 174gg genotype was associated with high ( above median ) counts of a. actinomycetemcomitans ( both in all subjects and periodontally healthy subjects only ) in indians . but nibali et al .   also suggested that only a detection of known periodontopathogenic bacteria could not discriminate different forms of periodontitis .
in contrast , schulz et al .   found no evidence that snps in il1 gene cluster could be associated with subgingival colonization with a. actinomycetemcomitans and could thus be an independent risk indicator of agp .
in addition , finoti et al .   observed that periodontal destruction may occur in patients who are considered to be genetically susceptible to cp with a lower microbial challenge because of the presence of the il8 atc / ttc haplotype than in patients without this haplotype .
first , the major complicating factor in the study of isolated locus ( such as il8 ) is the nature of periodontitis as a multifactorial disease in which interaction between multiple genes plays a role and each genetic polymorphism has generally only a small effect . in addition , the interaction of gene variants with environmental factors ( such as bacterial pathogens ) , socioeconomic factors , bmi , and others not analyzed in this study , potentially affect the observed phenotype .
second , the case - control approach used is generally quite vulnerable to the population stratification , for example , due to different ethnic origin
. the present sample , however , is exclusively of the czech caucasian origin , restricted to the limited geographical area populated by quite homogeneous population with low admixture .
finally , we did not measure rna expression or protein levels of il-8 , therefore , we do not know the functional consequences of these polymorphisms in our subjects . in conclusion ,
although none of the investigated snps in the il8 gene were individually associated with periodontitis , some haplotypes can be protective against cp in the czech population .
clinical significance of these findings is low due to a very low frequency of the  protective  haplotypes .
the individual il8 variants were associated with subgingival colonization with a. actinomycetemcomitans in agp and with t. forsythia in cp in the czech population . however
, these relationships must be assessed very carefully regarding small numbers of subjects in the respective subgroups .
further studies are needed to clarify the association of these polymorphisms with periodontal diseases in other populations .
 
 objectives . periodontitis is an inflammatory disease characterized by connective tissue loss and alveolar bone destruction . 
 interleukin-8 ( il8 ) is important in the regulation of the immune response . 
 the aim of this study was to analyze four polymorphisms in the il8 gene in relation to chronic ( cp ) and aggressive ( agp ) periodontitis . 
 methods . 
 a total of 492 unrelated subjects were included in this case - control association study . 
 genomic dna of 278 patients with cp , 58 patients with agp , and 156 controls were genotyped , using the 5 nuclease taqman assay , for il8 ( rs4073 , rs2227307 , rs2227306 , and rs2227532 ) gene polymorphisms . 
 subgingival bacterial colonization was investigated by the dna - microarray detection kit in a subgroup of subjects ( n = 247 ) . 
 results . 
 allele and genotype frequencies of all investigated il8 polymorphisms were not significantly different between the subjects with cp and/or agp and controls ( p > 0.05 ) . nevertheless , the a(251)/t(+396)/t(+781 ) and t(251)/g(+396)/c(+781 ) haplotypes were significantly less frequent in patients with cp ( 2.0% versus 5.1% , p < 0.02 , or  =  0.34 , 95% ci : 0.150.78 , resp . , 2.0% versus 4.5% , p < 0.05 , or  =  0.41 , 95% ci : 0.180.97 ) than in controls . conclusions . 
 although none of the investigated snps in the il8 gene was individually associated with periodontitis , some haplotypes can be protective against cp in the czech population . 