Background
Kidney transplantation is the best option for renal replacement therapy in patients with end-stage renal disease (ESRD), often restoring quality of life in ESRD patients. Allograft rejection, an immune-mediated process, is a common cause of transplant failure [
1‐
4]. Evidence indicates CD4
+CD25
+FoxP3
+ cells, commonly known as regulatory T cells (Tregs), play a critical role in preventing graft rejection by suppression of recipient alloimmune response [
5‐
8]. In healthy subjects, Tregs represent up to 5% of peripheral CD4
+ T cells [
9‐
11]. In kidney transplant patients, high peripheral blood Tregs were associated with stable graft function. Low peripheral blood Tregs was associated with allograft rejection [
12‐
17].
Currently, adoptive transfer of ex vivo expanded Tregs is a promising strategy to induce transplant tolerance and control graft rejection in kidney transplant recipients [
18,
19]. It has been investigated for safety and feasibility in phase I trials, i.e., the ONE (NCT02091232) and TRACT (NCT02145325). Identifying Treg-friendly agents from pharmacologic choices in multiple steps of kidney transplant management may also offer an attractive therapeutic strategy [
19]. Characterization of Tregs under various treatment conditions may help refine current preventive measures or identify novel therapeutic targets.
Volatile anesthetic agents are widely used for general anesthesia during kidney transplantation. A growing body of evidence from ex vivo and clinical studies [
20‐
26] suggest desflurane and sevoflurane (halogenated ether inhaled agents) exhibit immunomodulatory effects (e.g., cell proliferation, activation, migration, cytokine production) on neutrophils, macrophages, natural killer cells, B and T lymphocytes. These effects may be mediated via activation of volatile anesthetic receptors (i.e., γ-aminobutyric acid type A receptor, nicotinic acetylcholine receptor, serotonin receptor and non-canonical β2-integrins) or via binding to surface adhesion molecules such as integrin leukocyte function associated antigen-1, which express differentially on peripheral blood leukocytes [
25,
26]. However, effects of desflurane and sevoflurane on Treg immunomodulation is surprisingly overlooked and has only rarely been investigated. A better understanding of these effects would have translational potential. For example, the early Treg immunomodulation by anesthetic agents may help mitigating the initiation of alloimmune responses during the 24-h perioperative period, and works in conjunction with the standard immunosuppressive regimen to seamlessly maintain the graft survival in LDKT patients.
This interventional trial aims to compare the immunomodulatory effects of desflurane and sevoflurane anesthesia on peripheral blood Treg induction in patients undergoing living donor kidney transplantation (LDKT). Several plasma cytokines were measured as the surrogate outcomes of the volatile anesthetic effects on anti- and pro-inflammatory responses. Evidence from this study would support future investigation of volatile anesthetic agents as part of perioperative management with an aim to improve transplant outcomes.
Methods
Trial design and patient enrollment
This prospective, double-blind, randomized intervention trial was approved by the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects, Faculty of Medicine Ramathibodi Hospital, Mahidol University (protocol ID 045823) and the protocol was registered to
ClinicalTrials.gov (identifier NCT02559297) on September 22, 2015. Patients aged ≥18 years old who received their first living donor kidney transplantation at Ramathibodi Hospital were included in the study. Patients were excluded for hyperacute graft rejection, currently on immunosuppressive drugs due to underlying diseases, receiving blood products during 24-h perioperative period, or patient refusal to participate in the study at any time point. Informed consent was obtained from all subjects. No interim analysis was performed during the trial. This study followed the CONSORT reporting guideline [
27].
Randomization
Randomization was generated in a 1:1 allocation with a block size of 8 and the random number was put in a sealed envelope. Patients were randomly assigned to either desflurane or sevoflurane intervention by drawing a sealed envelope. Randomization took place on the day of surgery just prior to initiation of anesthesia.
Blinding
Subjects and outcome assessors (including laboratory technicians and all investigators except the designated research coordinator) were blinded to group allocation throughout surgery, laboratory investigation and data collection. Blinding was uncovered at the time of data analysis.
Interventions
Patients were randomly assigned to receive desflurane or sevoflurane for the maintenance phase of anesthesia. In addition to the randomized inhalation agents, patients received the same regimen of 1–2 mg of midazolam for premedication and intravenous anesthetic agents including 1–2 mcg kg− 1 of fentanyl, 1–2 mg kg − 1 of propofol and 0.5–0.6 mg kg − 1 of atracurium for induction of anesthesia and intubation. A balance anesthesia technique was used for maintenance phase. The inhalation agent (sevoflurane or desflurane) was used in conjunction with 50% nitrous oxide in oxygen. Ventilation was adjusted to maintain normocarbia. End-tidal anesthetic gas monitoring was used to ensure 1.0–1.5 minimum alveolar concentration (MAC) of the inhalation agent during maintenance phase in both groups.
During anesthesia, blood pressure, heart rate, oxygen saturation, ETCO2, and temperature were monitored and recorded. Blood pressure was maintained within 20% of baseline values. Hypotension was managed by intravenous fluid and ephedrine IV bolus as needed. Total doses of intravenous medications were recorded. All patients in both interventions were transferred to the kidney transplant unit for postoperative care.
Blood sample collection
Venipuncture was performed at three time-points; pre-exposure (0-h) and post-exposure (2-h, and 24-h) to inhalation agents. Two tubes of 0.5-ml EDTA blood were collected at each time point, one for Treg enumeration and the other for cytokine measurement.
Outcome measures
The primary outcome was the absolute change in number of peripheral blood CD4+CD25+FoxP3+Tregs, which was measured by flow cytometry and expressed as the percentage of the total population of CD4+ T lymphocytes at pre-exposure (0-h) and post-exposure (2-h and 24-h) to anesthetic gas. A secondary outcome was the plasma level of anti-inflammatory cytokine IL-10 (the major cytokine produced by Tregs), TGF-β1 (anti-inflammatory cytokines produced by many types of cells and required for Tregs differentiation), and pro-inflammatory cytokines produced by T helper (Th) 1/Th2, i.e., GM-CSF, IFN-ɣ, IL-2, IL-4, IL-5, IL-12, IL-13 and TNF-α, which measured by multiplex immunoassay. All measurements were performed in triplicate.
Treg enumeration by flow cytometry
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. Approximately 5 × 105 cells were suspended in 20 μL phosphate buffer saline (PBS) in the presence of cell surface marker antibodies (APC-CD4, PE-Cy7-CD25) (#MHCD0404, #25–0259-41; ThermoFisher, Florence, KY), mixed well and incubated at room temperature for 15 min. Thereafter, cells were permeabilized and intracellularly stained using FoxP3-FITC antibody (#11–4776-42; ThermoFisher). Flow cytometry (BD FACSVerse with BD FACSuite software; BD Bioscience, San Jose, CA) was used to measure the number of Tregs, expressed as a percentage of CD4+CD25+FoxP3+ T cells among the CD4+ cell population. The estimated number of CD4 + cells and Treg were calculated by determining the ratio of CD4 + cell count and CD4 + CD25 + FoxP3 + cell count, respectively, to the total count in the flow cytometry, and then multiplied by the number of white blood cells measured from the complete blood count (CBC) which ordered at the pre-operative and post-operative evaluations.
Cytokine measurement by multiplex immunoassay
Multiplex cytokine immunoassay was performed by BioPlex-200 system (Bio-Rad, Hercules, CA). GM-CSF, IFN-ɣ, IL-2, IL-4, IL-5, IL-10, IL-12, IL13, and TNF-α were detected by BioPlex Pro human cytokine Th1/Th2 assay (Bio-Rad), and TGF-β1 was measured by single-plex custom assay (Bio-Rad) as the manufacturer’s instruction.
Sample size estimation and statistical analysis
There was no data related to sevoflurane and desflurane anesthesia on Treg immunomodulation available at the initiation of the study. Nevertheless, Pirbudak Cocelli L et al. [
21], showed that sevoflurane and desflurane anesthesia caused a significant difference in total lymphocyte count at 2-h post-induction in patients undergoing abdominal surgery. Since Treg is a subset of lymphocytes, our study then adopted mean difference and standard deviation to calculate the effect size. The nQuery Advisor program was applied for sample size calculation. Accordingly, at least 40 patients (20 patients per group) were required to determine statistically significant mean difference between groups (the effect size of 0.915, alpha = 0.05 and power = 80%).
Statistical analysis was performed by Excel and R packages. Data were reported in number, percentage, mean ± SD (or SEM) or median [IQR] as appropriate. Parametric and non-parametric tests were used, as appropriate, to determine difference between groups. ANOVA with Tukey post-hoc test was performed for multiple comparison. P-value < 0.05 was considered to be statistically significant.
Discussion
Increasing evidence suggests that volatile anesthetic agents exhibit immunomodulatory effects linked to innate and adaptive immunity via induction and suppression of neutrophils, macrophages, NK cells and B and T lymphocytes [
25,
26]. However, their effects on Treg have remained unknown. This study, for the first time, showed that desflurane, but not sevoflurane, increased Treg frequency in peripheral blood of LDKT recipients during 24-h perioperative period. Selection of desflurane anesthesia in kidney transplantation may have additional benefits to kidney graft outcome, particularly preventing allograft rejection.
Studies showed that kidney transplant patients who maintained a high level of peripheral blood Tregs were associated with better outcomes [
14,
15,
28]. San Segundo D, et al. [
14], reported that among 90 kidney transplant recipients, patients who maintained high levels (above 70th percentile) of peripheral blood Tregs at both 6 and 12 months had a better prognosis in the aspect of long-term graft survival after 4 and 5 years follow-up. Liu L, et al. [
15], compared peripheral blood Treg levels between 42 patients with stable kidney graft function and ten patients who suffered from chronic rejection. The results showed that Treg levels were significantly higher in the stable group than the chronic rejection group. Alberu J, et al. [
28], investigated the association between Treg levels and de novo donor-specific HLA-antibody (DSA) production in 53 kidney transplant patients. Although early development of DSA was not associated with Treg numbers, at 12 months after kidney transplant DSA-negative patients had higher number of peripheral blood Treg.
The mechanisms for which higher peripheral blood Tregs help prevention of allograft rejection and maintenance of transplant tolerance meet the same concept of peripheral regulation in autoimmune reaction [
18,
29‐
32]. On a cellular basis, Tregs utilize four modes of action in peripheral regulation including [
29‐
32]; i) secretion or generation of inhibitory cytokines (e.g., IL-10, IL-35, TGF-β and adenosine); ii) direct killing of targets through Granzyme A/B and perforin-dependent cytolysis; iii) IL-2 consumption through high IL-2R expression which leads to cytokine-mediated deprivation and apoptosis of effector cells; and iv) direct interaction with CTLA-4, LAG-3 and PD1 molecules. Although accumulating evidence would favor contact-dependent mechanisms over non-contact/secretory component alone, these different mechanisms should work in concert to control various immune effector cells and regulate different inflammatory settings [
29‐
32]. Given that breadth of regulatory function on autoimmunity and self-tolerance, changes in peripheral blood Tregs in kidney transplant recipients may shift the balance between allograft rejection and transplant tolerance.
According to these lines of evidence, the increment of Tregs and IL-10 (Figs.
2,
3 and
4) after exposure to desflurane anesthesia should be beneficial to graft outcomes in LDKT recipients. In fact, several drugs routinely used in general anesthesia (besides volatile anesthetic agents) have immunomodulatory properties [
25,
26]. It is possible that some of them have positive effects on Tregs. Synergistic effects of multiple Treg-modulated agents may provide a better transplant outcome. Understanding how anesthetic agents exhibit varied effects on the immune system, particularly on Tregs, is important for future development of perioperative medicine in kidney transplantation.
This study was associated with several limitations. First, clinical outcomes, such as short-term and long-term graft survival, that were associated with Treg immunomodulation of desflurane anesthesia were not investigated. Graft survival is influenced by various factors (such as adequacy and toxicity of immunosuppressive drugs, presence of donor-specific antigen, PRA levels, numbers of HLA mismatch). These factors, together with Treg immunomodulation of desflurane, should be taken into account in future studies. Secondly, plasma cytokine levels were measured in 26 out of 40 patients (2/3 of total population in this study) due to the limited budget. A non-significant difference of cytokines between intervention groups may be due to a lack of statistical power, but at least, the upward trend of plasma IL-10 was observed, given the supportive evidence that desflurane anesthesia induced peripheral blood Tregs with potential IL-10 production. Third, the mechanisms of action (MoA) that drive desflurane-Treg immunomodulation were not defined and not the focus of this study. Further studies to characterize receptors and downstream signaling pathways that are responsible for desflurane-Treg effects would give some insight into a new MoA class of volatile anesthetic agent or a new biological process that facilitates transplant tolerance in LDKT recipients.
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