Comparison of the expression of cluster of differentiation (CD)39 and CD73 between propofol- and sevoflurane-based anaesthesia during open heart surgery

High expression of cluster of differentiation (CD)39 and CD73 has cardio-protective effects. We hypothesised that the expression of CD39 and CD73 would differ between propofol- and volatile anaesthetic-based anaesthesia in patients undergoing open heart surgery (OHS). The objective of this prospective randomized trial was to compare the changes in CD39 and CD73 levels in CD4+ T cells between propofol- and sevoflurane-based anaesthesia during OHS. The study randomly allocated 156 patients undergoing OHS to a propofol or sevoflurane group. Blood was obtained preoperatively and up to 48 hours after weaning from cardiopulmonary bypass (CPB). The expression levels of CD39 and CD73 in circulating CD4+ T cells, serum cytokines and other laboratory parameters were analysed. The primary outcome was the expression of CD39 and CD73 on CD4+ T cells. Demographic data and perioperative haemodynamic changes did not show significant differences between the two groups. The expression of CD39 and CD73 in the sevoflurane group was significantly lower than in the propofol group (P < 0.001). Other laboratory findings including cardiac enzymes and cytokine levels, did not show significant intergroup differences. Propofol attenuated the decrease in CD39 and CD73 in circulating CD4+ T cells compared to sevoflurane-based anaesthesia during OHS.


Expression of CD39 and CD73 in circulating helper T cells during OHS. The expression of CD39 in
circulating CD4 + T cells was lowest 3 hours after weaning from CPB and increased with time ( Supplementary  Fig. 1). The expression of CD73 in circulating CD4 + T cells was lowest immediately after weaning from CPB and increased with time ( Supplementary Fig. 2).

Expression of CD39 and CD73 in circulating helper T cells between propofol-and sevofluranebased anaesthesia during OHS.
The overall change in CD39 expression in circulating CD4 + T cells was significantly lower in the sevoflurane group than in the propofol group (P < 0.001) (Fig. 2). Especially, the expression of CD39 in circulating CD4 + T cells was significantly lower from 3 hours until 48 hours after weaning from CPB in the sevoflurane group with Bonferroni's correction (Fig. 2). The overall change in CD73 expression in circulating CD4 + T cells was also significantly lower in the sevoflurane group than in the propofol group (P < 0.001) (Fig. 3). Especially, the expression of CD73 in circulating CD4 + T cells from immediately after up to 48 hours after weaning from CPB was significantly lower in the sevoflurane group with Bonferroni's correction (Fig. 3).

Expression of CD39 and CD73 in circulating regulatory T cells during OHS. The pattern of CD39
and CD73 expression in circulating CD4 + CD25 + Foxp3 + T cells was similar to the pattern in CD4 + T cells. The expression of CD39 in circulating CD4 + CD25 + Foxp3 + T cells was lowest 3 hours after weaning from CPB and increased with time ( Supplementary Fig. 3). The expression of CD73 in circulating CD4 + CD25 + Foxp3 + T cells was lowest immediately after weaning from CPB and increased with time ( Supplementary Fig. 4).

Expression of CD39 and CD73 in circulating regulatory T cells in propofol-and sevoflurane-based anaesthesia during OHS.
The expression of CD39 in circulating CD4 + CD25 + Foxp3 + T cells was lower in the sevoflurane group than in the propofol group. The expression of CD73 in circulating CD4 + CD25 + Foxp3 + T cells was also lower in the sevoflurane group. However, the overall change in CD39 and CD73 expression in circulating CD4 + CD25 + Foxp3 + T cells did not differ significantly between the groups (P = 0.161 for CD39 and P = 0.068 for CD73) ( Supplementary Figs 5 and 6).

Mean fluorescence intensity of CD39 and CD73 in circulating helper T cells and regulatory T cells.
The patterns of the mean fluorescence intensity of CD39 and CD73 in circulating helper T cells and regulatory T cells were similar to the results regarding frequency (Supplementary Table 1).

Discussion
This study showed that the expression of CD39 and CD73 in circulating helper T cells was low immediately after weaning from CPB and recovered over time. In addition, the CD39 and CD73 levels in circulating helper T cells were higher with propofol-based anaesthesia than sevoflurane-based anaesthesia during OHS.    16 . In addition, blocking CD39 and CD73 could induce organ injury by inhibiting adenosine formation after IRI 17,18 . Meanwhile, OHS involves aortic cross clamping for several hours. Organ blood supply depends on CPB during this 'myocardial ischemic' period. After the main surgical procedure, the aortic cross clamp is released and weaning form CPB should follow. Intense IRI related inflammation occurs during this time before and after weaning from CPB 2 . The low expression levels of CD39 and CD73 immediately and 3 hours after weaning from CPB in this study support the notion that this period had the greatest IRI related inflammation during OHS.
Several studies have shown the beneficial effects of volatile anaesthetics relative to propofol during cardiac surgery 19,20 , indexed by lower creatine kinase-MB (CK-MB) levels with volatile anaesthetics compared to propofol-based anaesthesia. However, cardiac enzymes do not guarantee immune status during OHS since they are associated only with myocardial injury related to surgical trauma 21 . In addition, some reports found no differences in cardiac enzyme levels between propofol-and sevoflurane-based anaesthesia during cardiac surgery 22 . Similarly, we found no significant difference in cardiac enzyme and cytokine levels between the two groups. Various perioperative factors, such as the intensity of surgical trauma, haemodynamic changes, transfusion, and drugs, can affect cardiac enzyme and cytokine levels [23][24][25] and could be confounding factors. However, our results     revealed that immune status, which could not be determined based on cytokine expression alone, could be examined more precisely based on the expression of CD39 and CD73 in helper T cells, even with confounding factors. The decrease in CD39 and CD73 immediately and 3 hours after weaning from CPB was greater in the sevoflurane group. Similarly, the recovery of CD39 and CD73 after weaning from CPB was weaker in the sevoflurane group.
In addition, although not significant, IL-6 was higher immediately after weaning from CPB in the sevoflurane group. Because IL-6 can be induced by IRI related inflammation during CPB 3 , we postulated that sevoflurane has a less marked immune regulatory effect against IRI than propofol. Finally, regarding the changes in CD39, CD73, and IL-6, propofol-based anaesthesia might be more beneficial for minimizing IRI related inflammation during OHS. In addition, IRI related inflammation was closely related to postoperative complications after OHS 1 . Therefore, we anticipate potential benefits from propofol use relative to sevoflurane during OHS by reducing IRI related inflammation and postoperative complications.
The clinical outcomes, including the duration of mechanical ventilation and duration of stays in the intensive care unit (ICU) and hospital, did not differ between the groups in our study. Lurati Buse et al. found that propofol and sevoflurane had similar impacts on myocardial ischemia and postoperative complications 26 . In addition, a recent large scale meta-analysis found no difference in survival between propofol and volatile anaesthetics 8 . However, enhanced expression of CD39 and CD73 in animal models was recently considered as a novel therapeutic approach to avoid inflammatory responses against IRI 13,14,[27][28][29][30] . Therefore, regulating the expression of CD39 and CD73 by adjusting the propofol dosage could be a helpful approach for minimizing IRI related inflammation and postoperative complications. Because previous studies of the anti-immunosuppressive effects of CD39 and CD73 used animal models, exploring the effects of CD39 and CD73 according to different anaesthetics may be challenging in the clinical setting.
There could be several considerations in the present study. First, adenosine was not measured directly in the present study. However, the half-life of adenosine is extremely short and it is difficult to measure accurately 31 . Instead, recent studies have focused on blocking the adenosine receptor instead of adenosine itself 10 , or on interventions with several endogenous mediators, such as CD39 and CD73, which are responsible for adenosine formation 13,32 . In this respect, investigating the pattern of CD39 and CD73 expression during OHS may be essential. Second, cytokine production by other leukocyte such as neutrophil and B cells was not measured in the present study. However, CD39 and CD73 are mainly expressed in T cell subpopulation and have main role expressed in T cells. Third, even the patterns of CD39 and CD73 in regulatory T cells were similar in trends of reducing inflammation, there were no statistical significances. Therefore, further clinical investigations about CD39 and CD73 during OHS might help us to understand the exact mechanism of IRI related inflammation during OHS.
In conclusion, the expression of CD39 and CD73 in circulating helper T cells was decreased immediately after weaning from CPB. The decrease in CD39 and CD73 was worse in sevoflurane-based anaesthesia relative to propofol-based anaesthesia. This result may be associated with IRI related inflammation occurring during OHS. Our results suggest that propofol might be better than sevoflurane for reducing IRI related inflammation during OHS.

Study population. This study was approved by the Institutional Review Board of Konkuk University
Medical Centre, Seoul, Korea (IRB #KUH1160064) and carried out in accordance to the relevant guidelines and regulation of the Declaration of Helsinki. This study was registered before patient enrollment at clinicaltrials.gov (NCT02136979, Principal investigator: Seong-Hyop, Kim, Date of registration: May 13, 2014) and was conducted at a single tertiary medical centre (Konkuk University Medical Centre). All patients signed a written informed consent. The study used a prospective randomized design and was conducted according to the original protocol from May 2014 to December 2016 (full protocol available on request).
Patients undergoing OHS were enrolled, and patients were excluded if any of the following criteria were met: 1) age < 20 years, 2) pre-operative infection, 3) pre-operative use of an immunosuppressive agent, and 4) previous history of cancer. Patients were randomly assigned to groups by opening sequentially numbered envelopes containing the randomization assignment (third party allocation). The allocation sequence was generated by the clinical research coordination centre in our hospital, which was not otherwise involved in the trial, with random-permuted block randomization conducted using an interactive internet-based response system. The propofol and sevoflurane groups were anaesthetised by propofol and sevoflurane, respectively. All involved anaesthesiologists, surgeons and attending physicians were blinded to the study. All data were collected by trained observers who also were blinded and did not participate in patient care.    were titrated at 0.1 μg/ml and 0.1 vol%, respectively, to maintain BIS values between 40 and 50. In both groups, remifentanil was gradually administered using a target-controlled infusion device. A target concentration of 10 ng/ml was achieved after 10 min of administration of remifentanil and maintained throughout the procedure. Following induction of anaesthesia, patients were ventilated with 40% oxygen in air. The tidal volume was 6 ml/kg of lean body mass and the respiratory rate was adjusted to maintain the partial pressure of end-tidal carbon dioxide between 35 and 40 mmHg. Additional rocuronium was administered under the guidance of peripheral monitoring of neuromuscular transmission. A pulmonary artery catheter was inserted, and transoesophageal echocardiography was done, after anaesthesia induction.
Management of haemodynamic changes. Haemodynamic stability was maintained using adequate inotropic and vasoactive agents to ensure that the cardiac index and systemic mean blood pressure were above 2.0 l/min/m and 60 mmHg, respectively. Fluid administration was performed to meet fluid requirements and replace surgically lost blood until the laboratory values met transfusion indications. Perioperative transfusion was performed according to our institutional protocols 33 . After the end of the surgery, the propofol or sevoflurane was stopped and the patient was transferred to the ICU. For the sedation during the transfer, remifentanil 10 ng/ml was maintained and infused continuously for 60 minutes after arrival at the ICU. Decision-making regrading medical treatment was performed in the ICU by the physicians in charge of the unit, who were blinded to the study, based on institutional protocols. 33 Blood samples. In all patients, blood was sampled to analyse CD39, CD73, and cytokine expression in circulating CD4 + T cells and CD4 + CD25 + Foxp3 + T cells. Blood samples were obtained before anaesthesia induction and immediately, and 3, 24, and 48 hours after weaning from cardiopulmonary bypass (CPB). Samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes.  After washing with flow cytometry staining buffer, the PBMCs were incubated with 1 ml freshly prepared Foxp3 fixation/permeabilization buffer for 20 min at 4 °C in the dark. Then, the cells were washed twice with 2 ml freshly prepared 1 × permeabilization buffer. Next, the cells were stained using a PE-conjugated anti-human Foxp3 (eBioscience) antibody for 30 min in the dark at 4 °C. After washing twice, the number of Foxp3-positive cells in the CD4 + CD25 + cell gating was evaluated by flow cytometry, and the frequency of Foxp3 + Treg cells was expressed as a percentage of the total CD4 + CD25 + cells.

Measurements.
The following clinical data were obtained: (1) Patient demographic characteristics, including intraoperative drug dosages, fluids, and transfusion requirements; (2) changes in intraoperative haemodynamics; (3) inflammation-related laboratory tests, including white blood cell, neutrophil and lymphocyte counts, erythrocyte sedimentation rate (ESR), high-sensitivity C-reactive protein (hs-CRP); (4) levels of cardiac enzymes, including CK-MB, high-sensitivity troponin (Tn) I, and N-terminal-pro brain natriuretic peptide (NT-pro BNP); SCIeNTIfIC REPORts | (2018) 8:10197 | DOI:10.1038/s41598-018-28505-8 and (5) arterial blood gas analysis, including the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PF ratio), and haematocrit level, potential of hydrogen (pH), and lactate level. Statistical analysis. The primary outcome was the difference in expression of CD39 and CD73 in circulating CD4 + T cells between the propofol and sevoflurane groups. The data for the sample size calculation were drawn from 10 patients per group in our pilot study using G*power (ver. 3.1.9.2; Universität Kiel, Kiel, Germany). The calculated sample size for the primary outcome was 52 in each group for CD39 and 78 in each group for CD73 from the data of our pilot study with an α of 0.05 and power of 0.8. Therefore, we included 78 patients in each group; a total of 173 patients were enrolled in our study considering a dropout rate of 10%.