Clinical relevance of IL-6 gene polymorphism in severely injured patients

In polytrauma, injuries that may be surgically treated under regular circumstances due to a systemic inﬂ ammatory response become life-threatening. Th e inﬂ ammatory response involves a complex pattern of humoral and cellular responses and the expression of related factors is thought to be governed by genetic variations. Th is aim of this paper is to examine the inﬂ uence of interleukin (IL)  single nucleotide polymorphism (SNP) -C/G and -G/A on the treatment outcome in severely injured patients. Forty-seven severely injured patients were included in this study. Patients were assigned an Injury Severity Score. Blood samples were drawn within  h after admission (designated day ) and on subsequent days (, ,  hours and days) of hospitalization. Th e IL- levels were determined through ELISA technique. Polymorphisms were analyzed by a method of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR). Among subjects with diﬀ erent outcomes, no statistically relevant diﬀ erence was found with regards to the gene IL- SNP-G/C polymorphism. More than a half of subjects who died had the SNP-G/C polymorphism, while this polymorphism was represented in a slightly lower number in survivors. Th e incidence of subjects without polymorphism and those with heterozygous and homozygous gene IL- SNP-G/A polymorphism did not present statistically signiﬁ cant variations between survivors and those who died. Th e levels of IL- over the observation period did not present any statistically relevant diﬀ erence among subjects without the IL- SNP- or IL-  SNP - gene polymorphism and those who had either a heterozygous or a homozygous polymorphism.


INTRODUCTION
Trauma is the leading cause of death in the developed societies among the population under  years of age, and it is the th cause of death in general, which therefore makes the understanding of pathophysiological mechanisms exceptionally important [].In polytrauma, injuries that may be surgically treated under regular circumstances -due to system overload -become life-threatening.Direct or indirect consequences of trauma ("trauma load", "antigenic load") overcome the defence mechanisms of an organism.A physiological "host defence response« switches into a self-destructive "host defence failure disease", leading to the immune system shutdown, and subsequently to sepsis and progressive multiple organ dysfunction syndrome (MODS), as the most severe complication of polytrauma that usually involves two or more organ systems with the presence of alerted function in acutely ill patients such that homeostasis cannot be maintained without intervention [].Th e infl ammatory response involves a complex pattern of humoral and cellular responses and the expression of related factors is thought to be governed by genetic variations.Cytokines are integral components of the host immune response to trauma or infection [].Interleukin  (IL-) is a cytokine with both pro-and antiinflammatory properties and its release into peripheral blood seems to be an early marker of injury severity following trauma.Interleukin  is a -kDa glycoprotein that is produced by numerous types of cells, particularly lymphocytes, to which it owes its name, and by fi broblasts and monocytes as well.It exhibits various biological effects, including the activation of B and T lymphocytes, induc- tion of acute-phase protein synthesis in the liver, modulation of hematopoiesis, and it belongs to the groups of both pro-and anti-infl ammatory cytokines.In addition, it acts as a pyrogen, it activates the coagulation system, and in vitro IL- it suppresses the synthesis of TNF-α and IL-β [, ].Th e IL- gene is located on chromosome  and several polymorphisms have been reported.Th e most frequently studied polymorphism is the single nucleotide polymorphism (SNP) -C and -G in the promoter region, which has been associated with transcription rates of IL-.Th e incidence of IL--C alleles is approximately  among the general population, and it decreases in patients suff ering from infl ammatory diseases like juvenile rheumatoid arthritis [].Th e transfection of IL--C alleles into HeLa cells in vitro has resulted in a decrease in IL- production compared to that of IL- -G [].Additional polymorphisms may aff ect the IL- transcription as well, with complex interaction among certain haplotypes [], but the published studies in that regard have been few.As far as the -G/A single-nucleotide polymorphism (SNP) is concerned, relevant literature provides substantially less information on its clinical importance.This aim of this paper is to examine the influence of IL- single nucleotide polymorphism (SNP) -C/G and single nucleotide polymorphism (SNP) -G/A on the treatment outcome in severely injured patients.

Patients
Forty-seven severely injured patients were included in this study.All patients were admitted in the intensive care unit due to the trauma severity.Patients were assigned an Injury Severity Score (ISS) [] by independent evaluators.Exclusion criteria were (I) age of older than  years or younger than  years, (II) admission more than  h after trauma or secondary admission, (III) penetrating injuries, and (IV) any chronic illnesses.Injuries of the various body regions (head and neck, face, thorax, abdomen, extremities, and skin) were classifi ed by usingthe Abbreviated Injury Scale (AIS) [].Th e clinical course was monitored prospectively in all patients.Patients requiring surgical intervention received standard surgical care and postoperative intensive care.We assumed that blood transfusions that were submitted to patients had no signifi cant eff ect on the results of the study due to the same amount of blood given to the injured patients.Blood samples were drawn within  h after admission (designated day ) and on subsequent days (, ,  hours and  days) of hospitalization.Blood ( ml) was collected in plastic tubes (NH-heparin tube; Sarsted, Nümbrecht, Germany) along with the routine baseline laboratory work-up and was immediately used for stimulation ex vivo.

DNA isolation
Genomic DNA was isolated from peripheral blood with QIAampDNA Mini Kit (Qiagen GmbH, Hilden, Germany), and stored at -°C.

Gene variants detection
All analyzed polymorphisms and primers used for their detection, are shown in Table .Polymorphisms are denoted by both, common nomenclature (used through the whole article), and HGVS nomenclature.

Interleukin- G-C and G-A detection
The genotyping of the polymorphisms in IL- promoter G-C (rs) and G-A (rs) was performed using PCR-RFLP technique as previously described [].A -bp fragment containing both polymorphic sites (- and -) was amplified using one primer set.Th e PCR conditions were: denaturation step at °C for  min,  cycles of amplifi cation (°C for  sec, °C for  sec and °C for  sec) and elongation step of  min at °C.Th e obtained PCR product was digested with Hin II and Fok I (MBI Fermentas, Vilnius, Lithuania).Digestion with Hin II (G-C) gave the following pattern (in base pairs): ++ for GG genotype, ++++ for GC genotype and ++ for CC genotype.Digestion with Fok I (G-A) gave fragments of the following length (in base pairs):  for the GG genotype, ++ for the GA genotype and + for the AA genotype.

Statistical analysis
Statistical analyses were performed using the SPSS software v.. (SPSS Inc., Chicago, IL,USA).Descriptive data for all groups and variables were expressed as mean ± standard deviation (SD), mediana, minimum and maximum for continuous measures, or percent of a group for discrete measures.Categorical data were analyzed using  A statistically relevant change in IL- value over the sevenday observation period was found both in survivors (Friedman test; p=.) and in subjects who died (Freidman test; p=.).In both of these subject groups, a statistically relevant drop in the values was found (Figure ).Among subjects who had a different outcome, no statistically relevant diff erence was noted in the initial  hours, while the cytokine levels on day  with respect to those obtained  h after admission were higher in subjects who died (Table ).Somewhat more than a half of subject did not have the gene IL- SNP-G/A polymorphism, approximately  had heterozygous polymorphism, while about  had homozygous polymorphism.The subjects with homozygous polymorphism were statistically significantly less represented than the patients without polymorphism or those with heterozygous polymorphism (χ  -test; p=.).Among subjects with different outcomes, no statistically relevant difference was found with regards to the gene IL- SNP-G/C polymorphism (Table ).More than a half of subjects who died had the SNP-G/C polymorphism, while this polymorphism was represented in a slightly lower number in survivors (Figure ).Th e incidence of subjects without polymorphism and those with heterozygous and homozygous gene IL- SNP-G/A polymorphism did not present statistically signifi cant variations between survivors and those who died (Figure ).Slightly more than a half of subjects who survived had no polymorphism of this gene, close to a third of subjects had heterozygous polymorphism, while  of subjects had homozygous polymorphism.In subjects who died, somewhat more than a half had the gene IL- SNP-G/A polymorphism, approximately  had heterozygous, while the remaining . had homozygous form (Figure ).The IL- levels over the observed period of measurement did not present a statistically significant difference between the subjects without the gene IL-SNP-G/C polymorphism and those with heterozygous and homozygous polymorphism ( Table , Figure ).No statistically relevant diff erence in IL- levels measured on admission,  h ,  h ,  h and seven days after admission between the subjects without the gene IL- SNP-   G/A polymorphism and with the homozygous and heterozygous polymorphism (Table ).The lowest levels of this interleukin measured during the observation period in the polytrauma group were found in subjects with the homozygous polymorphism of this gene (Figure ).

DISCUSSION
The term polytrauma involves life-threatening injuries to at least two regions of the body [].The incidence of polytrauma in the under- age group is . and it represents the most frequent cause of death [-].The provision of professional and prompt care directly affects mortality, morbidity and functional recovery.A complicated system of cellular interactions and mediators governs the "generalized inflammatory syndrome" in order to induce recovery and control an invasion by ex-ternal organisms.In some cases of severe trauma, the inflammatory process goes out of control and vital tissue that has not been initially injured sustains damage.As numerous complex mediators and cellular systems are involved, the interpretation of such multiple interactions and their changes over time is highly complicated.IL- production is stimulated by TNF-α and IL-, which was proven in experimental and clinical studies, and it may be found in plasma after these two cytokines [, ].Experimental studies have showed that a high concentration of IL- is in correlation with the poor outcome of trauma and sepsis [].Jastrow et al. [] in their analysis of cytokine concentration in serum every  hours,  hours after trauma, intended to identify a cytokine that could be used as a predictor for the development of MODS.Th is group of authors analyzed a number of cytokines and they came to the conclusion that IP-, MIP-β, IL-, IL-, IL-Ra and eotaxin may be used to predict the development of MODS, as they exhibited higher levels in the period from  to  hours after trauma compared to the patients who did not get MODS.Considering the IL- levels in the period of - hours after trauma, the AU-ROC was ., cut-off value .,sensitivity , while the positive predictive value was at , and in the - hour period after trauma, this value was reduced by -.
Other authors have showed that the increased levels of IL- after trauma may be used as predictors for the development of MODS.It was determined that the increased IL- values were measured on days  and  in correlation with the development of MODS [].Particular authors described a significant increase in IL- on days  and  and the majority agrees that the sensitivity of this cytokine for the prediction of systemic complications development is reduced after  days [].
In our group, the IL- levels were higher in patients who died and exactly on day  of observation, the value exhibited a statistically relevant difference.
Th is SNP G>C at position - in the promoter region of IL- gene is related to the prevalence, incidence and/or prognosis of various diseases like Alzheimer's disease, atherosclerosis, cardiovascular disease, carcinomas, insulin-independent diabetes mellitus, osteoporosis, sepsis and systemic complications of juvenile chronic arthritis [-].Th e G allele of - SNP is coupled with the increase in transcriptional response to stimuli like endotoxins or IL-β in vitro [, ], while the studies that examined in vivo the -G>C promoter polymorphism in correlation with the plasma levels of IL- yielded contrasting results.In the fi rst study, Fishman et al. [] demonstrated that the unstimulated levels of IL- were coupled with the G allele of healthy individuals.Since then, opposing results related to the combination of IL- and SNP-G>C in healthy and ill individuals have been obtained [-].Th us, the plasma levels of IL-, stimulated through coronary artery bypass surgery, correlated both with the G allele [] and with the C allele [], depending on the blood sample drawing after the procedure, while the administration of endotoxins in healthy volunteers did not confi rm the existence of a relation between the IL- concentration and -G>C polymorphism [].Banermo et al. [] studied the role of -SNP in the induction of plasma concentrations of IL- in vivo.Th ey exposed healthy individuals who had the homozygous allele to a standardized infl ammatory stimulus by administering a salmonella typhoid vaccine.Th ey came to the conclusion that the G allele -G>C SNP homozygotes had a stronger IL- infl ammatory response to s. typhoid vaccination than the C allele homozygotes.Th e diff erence was not observed in the serum concentration of TNF-α and IL-β, which was explained by the fact that this vaccine is an infl ammatory stimulus of low potential and a weaker stimulator of cytokine production.Other studies [,] have showed that the G allele - SNP is coupled with the increased transcription upon endotoxin and IL-β stimulation.Further ex vivo studies of full healthy lipopolysaccharide-stimulated blood have showed that the haplotypes of IL- promoters, including the G allele, are related to the higher levels of IL- [].
Th ree studies examined the role of IL- -G>C SNP in the plasma IL- response in vivo [-].Brull et al. [] have showed that as opposed to in vitro and ex vivo studies, the patients with homozygous C allele have higher plasma levels of IL- up to six hours after CABG stimulation, while a study by Burzotta et al. [] has demonstrated that CABG stimulation in patients with the G allele induces higher concentrations of IL- after  and  h .Th e conclusion of the third study in vivo is that there is no signifi cant diff erence in the plasma levels of IL- in diff erent genotypes after intravenous endotoxin administration [].No adequate explanation for such diverging results has been found, although the diff erent eff ects caused by the infl uence of various stimuli on the IL- promoter cannot be ruled out, while a possible answer may be the lack of a functional relevance of -G>C SNP.Arguments speaking against this are the results of research of relation between the G allele and survival of sepsis [].It has been demonstrated that the G allele is related to the better survival of sepsis, but not to the incidence of sepsis, which is an indicative of the fact that an increased response of IL- in G allele is benefi cial when individuals are exposed to an acute infection.On the other hand, an increased response of IL- combined with the G allele may be harmful when it comes to the development of chronic diseases like the insulin-independent diabetes mellitus and atherosclerosis, considering that the repeated stimulation with minor stimuli increases the infl ammatory stress.For example, it is proven that the insulin resistance is related to the G allele [].However, even the polymorphisms of other genes must be taken into account, as other studies have showed that the C allele, when combined with TNF-α SNP, is also related to the development of insulin-independent diabetes mellitus [].Several studies have concluded that the IL- is of key importance for the development of atherosclerosis, where all but one study [] suggested that the G allele was related to the atherosclerosis [, , ].In addition, several studies pointed to the important role of IL- in bone density regulation in pre-and postmenopausal women, as well as in healthy men [, ].Th e results were that the individuals with C allele had greater bone density when compared to the individuals with G allele.One explanation could be that the prolonged high levels of IL- induced by infections and other chronic conditions may cause the loss of bone mass [, ].It appears that it is not only the -G>C SNP that aff ects the expression of IL- and its production as a response to stimuli.In vitro and in vivo studies have showed that haplotype -G>C, -AnTn, -G>C and -G>A polymorphisms aff ect the IL- concentration [].Haplotype analysis demonstrated that the presence of at least two out of three common haplotypes, including -G, correlated with the increased IL- levels compared to the presence of only one common haplotype, -C included.It is interesting to note that this study showed that individuals with GG haplotype had lower IL- levels, which corresponds to the results obtained by Kelberman et al. [], and it points to the modulating infl uence of AnTn on IL- response within the -G>C SNP.As far as IL- SNP- G>C and IL- SNP- G>A, Terry et al. determined that the IL- transcription positively correlates with these polymorphisms, although the other polymorphism that occurs is in a synergy as far as the eff ect on the promoter activity is concerned.[].Other studies showed that the -A allele constitutes a potent counterbalance of the - C allele, however the limitation to this study was that the information on the origin of IL- were not available [].Due to confl icting results obtained, further investigation both in vitro and in vivo is required for the purpose of learning more and explaining the IL- expression and production.
In an analysis of SNP polymorphism IL- SNP- and SNP- in a group of polytrauma patients in our series, no statistically relevant diff erence in the frequency of a particular outcome, although the - polymorphism was less represented in the group of polytrauma patients who survived.
In the group of polytrauma subjects, the levels of IL-, over the -day period of observation and cytokine measurement, did not produce a statistically relevant diff erence between the subjects who did not have the IL - gene polymorphism and those who had a heterozygous or homozygous polymorphism.Th e lowest levels of this interleukin in the -day observation period were measured in a subject from the group of polytrauma patients, and that subject had a SNP - IL- of this gene in a homozygous form.In the group of critically ill surgical patients, a statistically signifi cant difference, both for the gene IL- SNP- and SNP- gene polymorphism, was not observed among subjects who had diverse outcomes.The levels of IL- over the observation period did not present any statistically relevant diff erence among subjects without the IL- SNP- or IL- SNP - gene polymorphism and those who had either a heterozygous or a homozygous polymorphism (Figures  and ).Furthermore, other polymorphisms may aff ect the IL- transcription with a complex interaction among certain haplotypes [], although few relevant studies have been published thus far.

CONCLUSION
Based on the results of our study, we concluded that presensce of IL- polymorphism (SNP- or -) have no influence to production of IL- and due to this inflammatory response to severe trauma, but future studies with more subjects included are necessary to clarify the mechanism of injury and patophysiological response to severe trauma.

DECLARATION OF INTEREST
Authors declare no confl ict of interest for this study.

FIGURE 3 .
FIGURE 3. Gene IL-6 SNP-174G/C polymorphism in groups with diff erent treatment outcomes

TABLE 1 .
Analyzed gene polymorhismsthe Pearson chi-square test.A normal distribution was tested using the Koglomorov-Smirnov test.If the data were normally distributed, the t-test was used.Non-parametric data were analyzed using the Mann Whitney U test, and Kruskal Wallis test.Diff erences were considered significant when the p value was less than . (p<.).Th is study received an approval from the local ethics committee.RESULTSA total of forty-seven severely injured patients were included in the fi nal study.Demographic features of patients involved in this research were analyzed.The mean age of patients was .±;  patients were male and  females.Patients were stratifi ed acording to outcome to survivors (mean age .±.male patients and  female) and non survivors (mean age . ±. male and  female patients).Th ere was no signifi cant statistical diff erence between these two groups acording to gender and age (p>.)Th e main mechanism of injury was traffi c, followed by fall and assault.Th e non survivors were the most frequent in the group of patients which were injuried as motorcycle drivers.Using χ  test no statistically signifi cant diff erence was recorded between survivors and non-survivors (p=.).Th e Injury Serverity score (ISS) provided an over all score for patients with multiple injuries.Each injury was assigned an Abberviated Inury Scale score (AIS) allocated to one of six body regions (Head, Face, Chest, Abdomen, Extremities, External).Analysing the AIS and ISS score we observed statistically significant higher values of ISS and AIS for head and neck in patients with lethal outcome usin Mann Whitnez U-test (p=. and ., respectively).Th e IL- value analysis during the observation period yielded a statistically relevant difference (Friedman test; p=.).Th e highest values with statistical relevance were measured on admission (Wilcoxon test; p=.).Th e IL- level measurement after  h resulted in statistically relevant lower values compared to those on admission, but these values were higher in turn from the values obtained after  h (Wilcoxon test; p=.),  h (Wilcoxon test; p=.) and after  days (Wilcoxon test; p=.).For values of IL- measured  h and  h after admission (Wilcoxon test; p=.),  h and  days (Wilcoxon test; p=.) and  h and  days after admission, no diff erence was found between them (Figure ).

TABLE 2 .
IL-6 levels measurements according to outcome JEREMIĆ ET AL.: CLINICAL RELEVANCE OF IL6 GENE POLYMORPHISM IN SEVERELY INJURED PATIENTS Subjects without the IL- SNP-G/C gene polymorphism (.) and those with heterozygous polymorphism (.) were represented in closely equal number, while trauma patients with homozygous polymorphism were represented in a statistically signifi cantly lower number (χ  -test; p=.).
*statistically signifi cant; a t-test; b Mann Whitney U test FIGURE 1.The IL-6 level measurements FIGURE 2. IL-6 levels measurements according to outcome VASILIJE

TABLE 3 .
Gene polymorphism incidence according to outcome *statistically signifi cant; a χ 2 -test b p=0.369

TABLE 4 .
Cytokine IL-6 levels and genetic polymorphism * statistically relevant; b Kruskal Wallis test