Monocyte Metabolism and Function in Patients Undergoing Cardiac Surgery

Abstract

Objective: Cardiopulmonary bypass (CPB) can lead to systemic inflammation, which is associated with higher morbidity. Therefore, we investigated the metabolism of isolated blood monocytes before and after CPB compared to healthy controls.

Methods: In this prospective, monocentric, observational study, we included 30 patients undergoing CPB and 20 controls. We isolated monocytes from heparinized blood and investigated their metabolism by using Seahorse technology before (t0), 4 h (t4), and 24 h (t24) after the start of the CPB. We also examined programmed cell death 1 ligand (PD-L1), PD-L2, V-domain Ig suppressor of T cell activation (VISTA), and human leukocyte antigen-DR isotype (HLA-DR) using fluorescence-activated cell sorting analysis. Additionally, we investigated plasma cytokine levels in patients without and after ex vivo stimulation.

Results: CPB-induced inflammatory responses are shown by significantly elevated plasma interleukin-6 levels in the CPB group compared to baseline and controls [t0: 0 ng/ml (95%CI 0-0 ng/ml); t4: 0.16 ng/ml (95%CI 0.1-0.197 ng/ml), p < 0.0001; t24: 0.11 ng/ml (95% CI 0.1-0.16 ng/ml), p < 0.0001, and controls: 0 ng/ml (95% CI 0-0 ng/ml)]. The cytokine release in the ex vivo stimulation is reduced for lipopolysaccharide stimulation at t4 [t0: 35.68 ng/ml (95% CI 22.17-46.57 ng/ml) vs. t4: 15.02 (95% CI 10.25-24.78 ng/ml), p < 0.0001]. Intracellular metabolism of monocytes after CPB showed a protracted shift to aerobic glycolysis [t0: 179.2 pmol/min (95% CI 138.0-205.1 pmol/min) vs. t24: 250.1 pmol/min (95% CI 94.8-300.2 pmol/min), p < 0.0001]. Additionally, we observed an altered metabolism in monocytes in patients undergoing cardiac surgery compared to controls even before any surgical procedure [t0: 179.2 pmol/min (95% CI 138.0-205.1) vs. controls 97.4 (95% CI 59.13-144.6 pmol/min), p = 0.0031].

Conclusion: After CPB, patients' monocytes show a shift in metabolism from oxidative phosphorylation to aerobic glycolysis, which is associated with energy-demanding and proinflammatory processes. This is the first study to show changes in monocyte immunometabolism in cardiac surgery. Monocytes of patients undergoing cardiac surgery were leaning toward aerobic glycolysis even before any surgical procedure was conducted. Leaving the question of the pathophysiological mechanisms for future studies to be investigated and paving the way for potential therapy approaches preventing inflammatory effects of CPB.

Keywords: Warburg effect; cardiac surgery; immune reaction; immunometabolism; inflammation; monocytes.

Figure 1

Representative gating strategy for monocytes and antibody staining of surface markers. (A) Chronological from left to right: debris were excluded from all cellular events. Afterward, single cells were identified via FSC-A and FSC-H. Using CD14 FITC antibodies, monocytes were finally detected as CD14⁺. (B–D) Antibody staining of PD-L1, PD-L2, and VISTA in CD14⁺ monocytes. The gray line represents FMO, and the red line represents stained monocytes. APC, allophycocyanin; FMO, fluorescence minus one; FSC-A, forward scatter area; FSC-H, forward scatter height; PE, phycoerythrin; SSC-A, side scatter area.

Figure 2

Leukocytes, CD14⁺ monocytes, and surface markers HLA-DR, PD-L1, PD-L2, and VISTA. Leukocyte count of patients (A) and the percentage of CD14⁺ monocytes (B) in full blood; FACS analyses of surface marker: (C) expression of HLA-DR on isolated blood monocytes; (D) expression of PD-L1 on isolated blood monocytes; (E) expression of PD-L2 on isolated blood monocytes; (F) expression of VISTA on isolated blood monocytes; all measurements in (C–F) were taken from isolated blood monocytes before surgery (baseline), 4 h after CPB (4 h CPB), 24 h after CPB (24 h CPB), and compared to the healthy control group; data shown as median and the whiskers of the 5th and 95th percentile; MWU test was used to test for significance; CPB, cardiopulmonary bypass; FACS, fluorescence-activated cell sorting; HLA, human leukocyte antigen system; ΔMFI, delta mean fluorescence intensity; PD-L, programmed cell death ligand; VISTA, V-domain Ig suppressor of T-cell activation.

Figure 3

Analyses of metabolic flux. Basal glycolysis (A), compensatory glycolysis (B), basal respiration (C), maximum respiration (D), spare respiratory capacity (E), non-mitochondrial respiration (F), proton leak (G), and ATP production (H) were taken from isolated blood monocytes before surgery (baseline), 4 h after CPB (4 h CPB), 24 h after CPB (24 h CPB), and compared to monocytes of the healthy control group; data shown as median and the whiskers of the 5th and 95th percentile; MWU test was used to test for significance; ATP, adenosine triphosphate; CPB, cardiopulmonary bypass; OCR, oxygen consumption rate; PER, proton efflux rate.

Figure 4

Cytokine levels in plasma and after ex vivo stimulation. ELISA analyses of cytokine levels in plasma (A–C), after ex vivo stimulation with LPS (D–F) and zymosan (G–I); all measurements were taken from blood samples before surgery (BL), 4 h after CPB (4 h), 24 h after CPB (24 h), and compared among each other and to the healthy control group; the y-axis in (A–C) is presented in a log scale and zero data has been set to 0.001; cytokine levels after stimulation were only compared to controls (D–I); data shown as median and the whiskers of the 5th and 95th percentile; MWU test was used to test for significance; CPB, cardiopulmonary bypass; IL, interleukins.