Soluble triggering receptor expressed on myeloid cells 1 ( sTREM-1 ) and polymorphic variants of TREM-1 in the development of multiple organ dysfunction syndrome after coronary artery bypass grafting

Появление тяжелых осложнений в виде полиорганной недостаточности (ПОН) после операций коронарного шунтирования (КШ) является результатом тяжелой стресс-реакции организма. Ключевым этиопатологическим фактором индукционной фазы комплексного патологического процесса, приводящего, в конечном счете, к нарушению функции органов и систем, является нарушение функций иммунного реагирования. Особенности реакций иммунной системы у конкретного индивидуума в определенной степени обусловлены генетически и реализуются через активацию врожденного иммунитета. Цель исследования: определить значение полиморфизма гена TREM-1 в связи с изменением сывороточных концентраций sTREM и их вклад в развитие полиорганной недостаточности у пациентов после операции коронарного шунтирования. Материалы и методы. В исследование включили 132 пациента с атеросклерозом коронарных артерий, перенесших операцию коронарного шунтирования. Ранний послеоперационный период у 30 из них осложнился развитием полиорганной недостаточности. Концентрацию sTREM в сыворотке крови определяли методом иммуноферментного анализа. Генотипирование полиморфных сайтов гена TREM-1 осуществляли методом аллель-специфичной ПЦР в реальном времени по технологии TaqMan. Результаты. В результате исследования обнаружили статистически значимые различия в концентрациях sTREM у пациентов с ПОН и пациентов без осложнений. Установили, что концентрация sTREM зависит от носительства определенных аллельных вариантов в трех полиморфных сайтах гена TREM-1 (rs1817537, rs2234246, rs3804277) Заключение. В данной работе впервые определили взаимосвязь концентраций циркулирующей растворимой формы TREM-1 и полиморфных вариантов кодирующего гена TREM-1 с выраженностью ПОН у пациентов с ишемической болезнью сердца (ИБС) в послеоперационном периоде КШ.


Introduction
The rate of major complications following cardiac surgeries remains high despite recent advances in the treatment of cardiovascular diseases with novel diagnostic and surgical technologies.
Postoperative complications occur in around 30% of cardiac surgery cases.The majority of them are not related to the surgical technique [1].Multiple organ dysfunction syndrome (MODS) is one of the most severe complications commonly associated with poor outcomes.MODS is defined as the presence of cumulative dysfunction of several organs (three and more).This complication requires critical care and remains the main cause of death among patients in the in-hospital period [1,2].
MODS development is associated with systemic inflammatory response syndrome (SIRS).Excessive activation and discoordination between SIRS components (a cascade of pro-and anti-inflammatory cytokines, acute phase proteins, endocrine-metabolic response, etc.) accompanied by alterations of microcirculation, contribute to the development of systemic alterations [3].Despite the fact that SIRS is commonly regarded as a physiological mechanism of adaptation to surgical trauma and reperfusion injury in the early postoperative period, some patients experience its pathological and uncontrollable progress, resulting in MODS and subsequent death [2].Immune reactions are the main pathogenetic links of SIRS and its onset represents the result of both cellular and humoral immunity dysfunction [4].
Triggering receptor expressed on myeloid cells-1 (TREM-1) is a key receptor of the innate immune response, as well as an important participant in the activation of early inflammatory reactions and SIRS [5].
Any sustained increase in sTREM-1 levels may indicate that the overall expression of TREM-1 is constantly increasing with the release of more proinflammatory mediators.Thereafter, any further increase in sTREM-1 suggests a prolonged inflammatory response, commonly associated with poor outcomes.However, the effects of TREM-1 stimulation require further understanding, and many of the studies provide inconsistent data on the clinical significance of TREM-1.It is still unknown whether sTREM-1 levels are genetically regulated or whether gene polymorphism affects expression levels.
Taking into account the participation of this receptor in the regulation of pro-inflammatory activity of cells, it seems to be relevant to assess the potential of its soluble form to be the marker of MODS in the early postoperative period in patients who have undergone cardiac surgery.The understanding of significance of the gene polymorphism encoding it in its individual variable sites can ensure the use of a patient-centered approach in the preoperative period.
Aim: To determine the role of polymorphism of the TREM-1 gene through the changes in sTREM serum levels and evaluate their contribution to the development of MODS in patients after coronary artery bypass grafting.

Materials and Methods
Patients with coronary artery disease (CAD) who had undergone coronary artery bypass grafting (CABG) in the period from 2011 to 2012 at the Research Institute of Complex Issues of Cardiovascular Diseases were included in the study.The diagnosis of coronary artery disease was made in accordance with the national guidelines of the Russian Society of Cardiology.The study was performed using CABG Registry (certificate of the state registration of the database No.2012620868 «Electronic archive of patients undergoing coronary artery bypass grafting»).
The inclusion criteria were as follows: clinically and instrumentally confirmed diagnosis of coronary artery disease, angina pectoris, CHF; CABG; Caucasianrace and permanent residency in the Kemerovo region; written informed consent.The exclusion criteria were as follows: a positive history of malignant tumors, autoimmune and mental diseases, acute and exacerbation of chronic infections; surgical complications in the postoperative period.
Материалом для исследования послужила венозная кровь, взятая у пациентов натощак до опера-MODS was confirmed using the objective criteria in the early postoperative period.The serial assessment of the severity of MODS in the postoperative period was performed using the SOFA scale (Sequential Organ Failure Assessment), where the total score consisted of the severity of failure of different organ systems (cardiovascular, respiratory, nervous, excretory, digestive, hemostasis and liver function) [7,8].
30 patients (22.7%) who had MODS in the early postoperative period ( 4 SOFA scores) were included in Group 1 (MODS group) with the 50% mortality rate.102 patients (77.3%) without any major complications in the early postoperative period and without clinically significant signs of MODS ( 4 SOFA scores) were included in Group 2 (non-MODS group).The main clinical and demographic data of the study cohort are presented in table 1.
The data presented in Table 1 indicate that patients with MODS and without it exhibited similar clinical and demographic parameters.However, chronic obstructive pulmonary disease was more frequently detected among patients who had MODS in the postoperative period (P=0.010) as compared to those who did not have MODS.
After overnight fasting (12 h), the venous blood was collected using the standard technique that employs K3EDTA vacuum tubes for subsequent DNA extraction and serum clot activator tubes from all patients before surgery and one day after it.
sTREM-1 serum levels were measured by enzymelinked immunosorbent assay using a commercial kit (Human TREM-1, R&D Systems, USA) according to the manufacturer's protocol.Optical density and concentrations were assessed with the UNIPLAN semi-automatic enzyme immunoassay analyzer (PIKON, Russia).The measurements were performed twice, before and after surgery.
Molecular genetic testing was performed with the inclusion of 8 TREM-1 polymorphic sites (SNP) (rs1817537, rs3804277, rs6910730, rs7768162, rs6917537, rs3804277, rs6910730, rs7768162, rs6917537, rs3804277, rs6910730, rs7768162, rs6917537, rs3804277, rs6910730, rs7768162, rs6917537, rs3804277; rs4711668, rs9471535, rs2234237) to determine the relationships of sTREM-1 levels with TREM-1 gene polymorphism and rare alleles in separate polymorphic sites.The choice of single nucleotide polymorphic sites was due to the sufficient frequency of the alleles, the functional activity of the receptor under study and the absence of studies evaluating the role of specific polymorphic locus in the development of early postoperative complications following CABG.DNA was isolated using phenol-chloroform extraction [9].Genotyping was performed using TaqMan genotyping assays with fluorescence-tagged probes (Applied Biosystems, USA) in a real-time polymerase chain reaction (PCR) on a ViiA TM 7 analyzer (Applied Biosystems, USA) according to the manufacturer's protocol.The characteristics of the TREM-1 gene polymorphic sites are presented in table 2.
The following approach was used to adjust the groups for sex and age [10]: where -is the adjusted parameter score (Y) in Group 1 for the ith person; -mean and standard deviation for Group 1;, -the same for Group 2. The adjustment for mean values,=; the adjustment for dispersions, =.

Results and Discussion
Preoperative sTREM-1 levels differed among patients who had and did not have MODS following CABG.sTREM-1 serum levels were significantly higher in patients who had MODS in the early postoperative period in comparison to those patients who did not have MODS (P 0.0001).
sTREM-1 serum levels increased in both groups in the early postoperative period compared to the baselines (P 0.0001 for the MODS group and P 0.0001 for the non-MODS group) (one day after the surgery).Thus, patients with MODS demon-
However, the observed relationships of high sTREM-1 serum levels with the carriage of rare alleles in individual polymorphic sites of the TREM-1 gene were not confirmed statistically (P 0.05) for the preoperative and early postoperative periods after all the patients were assigned into the study groups according to the presence of MODS.The obtained results are related to the fact that sTREM-1 levels have not been changed significantly within the groups.The relationship between the genotype of the TREM-1 gene polymorphic variants and high sTREM-1 serum levels in the total cohort of patients at different time points (before surgery and one day after surgery) has not been found either.The obtained findings confirmed the hypothesis that none of the genotypes of the eight polymorphic loci of the TREM-1 gene control reduction of the increased sTREM-1 levels following CABG.
Previous studies have shown that sTREM-1 can be a valuable diagnostic biomarker for various infectious diseases [11][12][13][14][15][16].It is generally assumed that receptor expression is modulated on the cell surface during active infectious invasion, then TREM-1 is released from the cell membrane by shading and the formation of a freely circulating soluble form of sTREM-1.
Moreover, there are numerous studies comparing sTREM-1 with CRP (C-reactive protein) and PCT (procalcitonin) to determine a biomarker that better predicts the onset of sepsis [20,21].
To date, sTREM-1 is actively involved in autoimmune diseases and inflammatory reactions.The studies demonstrating the involvement of TREM-1 in pathological conditions without underlying infectious process are of particular importance.Dai et al. focused on the estimation of the relationships between sTREM-1 serum levels and coronary artery disease.sTREM-1 serum levels were significantly lower in patients with coronary artery disease than in the control group (P 0.001) [22].The authors suggested that TREM-1 may act as a vascular protective factor.
Patients (n=280) undergoing coronary artery bypass grafting were included in the study by Wang et al.One year after CAG, 130 patients were diagnosed with restenosis.After sTREM-1 serum levels measurements, they found that patients with restenosis had a 1.35-fold increase in sTREM-1 levels in comparison with patients without restenosis (P=0.001)[23].
Ранее было проведено исследование о возможности использования sTREM-1 как маркера выраженности СВО и его осложнений в периоперационном периоде прямой реваску-and its complications in the perioperative period after direct myocardial revascularization under extracorporeal circulation [4].They found increased sTREM-1 serum levels in the early postoperative period.The obtained data on the levels increase have proved its importance in the onset of non-infectious SIRS and some other complications in patients who underwent on-pump CABG.However, an increase in sTREM-1 levels was found on day 1 and day 7 post CABG (74.98 pg/mL (58.99-107.9) vs. 101.20 pg/mL (68.45-162.55),respectively), as compared with the preoperative values (58.06 pg/ml (46.53-109.20) in patients enrolled in the group with an uncomplicated postoperative period (n=57).Patients with a complicated postoperative period (n=5) had lower preoperative sTREM-1 levels as compared to the levels measured on days one and seven after CABG (67.46 pg/ml (54.71-77.90)vs. 131.10pg/ml (130.50-135.10)and 157.50 pg/ml (134.00-249.30),respectively).Despite a progressive increase of sTREM-1 serum levels in patients with complications, there were no significant differences found between its values on days 1 and 7 within the groups.
Nevertheless, we found that there were obvious differences in sTREM-1 levels in patients with coronary atherosclerosis in the preoperative period.The higher are sTREM-1 levels before the surgery, the greater is the likelihood of complications in the postoperative period.Moreover, we found an active increase in sTREM-1 levels in the early postoperative period, regardless of its initial levels.However, peak sTREM-1 levels were observed in patients with complicated postoperative period.
Unfortunately, we could not estimate serial changes in sTREM-1 serum levels on day 7 post surgery.The restrictions on statistical analysis were caused by high mortality rate in the MODS group accounting for 50%.
Several studies compared sTREM-1 serum levels between patients admitted to the intensive care unit with non-infectious SIRS and infection or sepsis.sTREM-1 serum levels in septic patients were significantly higher than those in patients with SIRS (P 0.05) [24].Another study reported that patients with infection also had significantly higher sTREM-1 levels than patients with SIRS (P 0.0001) [25].Thus, the authors conclude that sTREM-1 may be useful for early differentiation of the systemic inflammatory response syndrome from infection, assessment of the severity of the disease and prediction of outcomes in ICU patients with sepsis and SIRS.
The first study [27] on the relationship of the polymorphic locus rs2234246 of the TREM-1 gene with sTREM-1 levels has been recently published.Among 10 SNPs of the TREM-1 gene studied by the authors, the minor allele T rs2234246 was associated with an increase in sTREM-1 levels in the cohort of healthy European subjects (P=0.003).Thus, the authors suggest that the carrying the T rs2234246 minor allele of the TREM-1 gene can be considered as a risk factor, while carrying the C allele can be considered as a protective factor.

Conclusion
This study is the first one to determine the relationship between the level of the circulating soluble TREM-1 and the polymorphic variants of the coding TREM-1 gene with the severity of MODS in patients with CAD following CABG.
of TREM-1 on the w w w .r e a n i m a t o l o g y .c o m G E N E R A L R E A N I M AT O L O G Y, 2 0 1 9 , 1 5 ; 3 DOI:10.15360/1813-9779-2019-3-48-60Clinical Practice & Studies
The study design was approved by the Local Ethics Committee of the Research Institute of Complex Issues of Cardiovascular Diseases.All patients were enrolled retrospectively in two groups either with MODS or without it.The presence of