The effect of sodium thiosulfate on immune cell metabolism during porcine hemorrhage and resuscitation

Abstract

IntroductionSodium thiosulfate (Na2S2O3), an H2S releasing agent, was shown to be organ-protective in experimental hemorrhage. Systemic inflammation activates immune cells, which in turn show cell type-specific metabolic plasticity with modifications of mitochondrial respiratory activity. Since H2S can dose-dependently stimulate or inhibit mitochondrial respiration, we investigated the effect of Na2S2O3 on immune cell metabolism in a blinded, randomized, controlled, long-term, porcine model of hemorrhage and resuscitation. For this purpose, we developed a Bayesian sampling-based model for 13C isotope metabolic flux analysis (MFA) utilizing 1,2-13C2-labeled glucose, 13C6-labeled glucose, and 13C5-labeled glutamine tracers.MethodsAfter 3 h of hemorrhage, anesthetized and surgically instrumented swine underwent resuscitation up to a maximum of 68 h. At 2 h of shock, animals randomly received vehicle or Na2S2O3 (25 mg/kg/h for 2 h, thereafter 100 mg/kg/h until 24 h after shock). At three time points (prior to shock, 24 h post shock and 64 h post shock) peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from whole blood, and cells were investigated regarding mitochondrial oxygen consumption (high resolution respirometry), reactive oxygen species production (electron spin resonance) and fluxes within the metabolic network (stable isotope-based MFA).ResultsPBMCs showed significantly higher mitochondrial O2 uptake and lower O2•− production in comparison to granulocytes. We found that in response to Na2S2O3 administration, PBMCs but not granulocytes had an increased mitochondrial oxygen consumption combined with a transient reduction of the citrate synthase flux and an increase of acetyl-CoA channeled into other compartments, e.g., for lipid biogenesis.ConclusionIn a porcine model of hemorrhage and resuscitation, Na2S2O3 administration led to increased mitochondrial oxygen consumption combined with stimulation of lipid biogenesis in PBMCs. In contrast, granulocytes remained unaffected. Granulocytes, on the other hand, remained unaffected. O2•− concentration in whole blood remained constant during shock and resuscitation, indicating a sufficient anti-oxidative capacity. Overall, our MFA model seems to be is a promising approach for investigating immunometabolism; especially when combined with complementary methods.

Porcine blood cell and brain tissue energy metabolism: Effects of “early life stress”

“…Isolation of peripheral blood mononuclear cells (PBMC) and granulocytes was performed as described recently ( Wolfschmitt et al, 2023 ). For this purpose, approx.…”

“…Mitochondrial respiration in the isolated immune cells as well as the brain tissue specimens (pre-frontal cortex) was measured by high-resolution respirometry using the Oxygraph-2K ® (Oroboros Instruments, Innsbruck) as described previously ( Datzmann et al, 2023 ; Datzmann et al, 2021 ; Zink et al, 2021 ; Wolfschmitt et al, 2023 ) This device allows for simultaneous recording of the O 2 concentration in two parallel chambers calibrated for 2 mL of respiration medium MiR05 ( Doerrier et al, 2018 ; Zink et al, 2021 ). This medium is composed of 110 mM D-Sucrose (Sigma Aldrich, St. Louis, MO, United States), 60 mM K-Lactobionate (Sigma Aldrich, St. Louis, MO), 0.5 mM ethylene glycol tetra acetic acid (Sigma Aldrich, St. Louis, MO), 1 g/L bovine serum albumin free from essentially fatty acids (Sigma Aldrich, St. Louis, MO), 3 mM MgCl 2 (Scharlau, Hamburg), 20 mM taurine (Sigma Aldrich, St. Louis, MO), 10 mM KH 2 PO 4 (Merck, Darmstadt), 20 mM HEPES (Sigma Aldrich, St. Louis, MO), adjusted to pH = 7.1 with KOH and equilibrated with 21% O 2 at 37°C.…”

“…As a blank sample, KHB added to the respective amount of CMH was measured and subtracted from the sample value. For the determination of immune cell O 2 •− production ( Wolfschmitt et al, 2023 ), 25 µL of a cell suspension containing 2.5 × 106 cells/mL purified granulocytes and mononuclear cells were transferred to FACS tubes and washed in RPMI 1640 medium (Glucose 1.8 mg/mL, Glutamine 0.6 mg/mL, NaHCO 3 100 μg/mL, HEPES 20 mM) while being kept on ice. 50 μL of 1 mg/mL pHrodo ® E. coli BioParticles ® Conjugate (Life technologies TM Carlsbad, CA) was added to the cell suspension before subsequent incubation at 37°C for exactly 20 min.…”

Bayesian13C-metabolic flux analysis of parallel tracer experiments in granulocytes: A directional shift within the non-oxidative pentose phosphate pathway supports phagocytosis

“…Since the model elution curve tends to zero at both ends, a straight line was added to it as a baseline to better track the measured curve. The carbon mass distribution (CMD) was obtained after correction for naturally occurring isotopes like nitrogen, oxygen, hydrogen and silicon with a correction matrix approach (van Winden et al, 2002; Wolfschmitt et al, 2023). The CMDs were corrected for natural abundance of 13 C for model free evaluation, but not for the modeling-based MFA, which had natural 13 C included in its 13 C label definition.…”

13C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock

“…MFA most commonly relies on balancing fluxes within a stoichiometric network and employing nonlinear regressions. We have adapted MFA to a Bayesian approach, which is more computationally intensive than the conservative method, but in turn yields full probability distributions of fluxes, their joint credibility intervals, and their correlations ( 20 ). With a parallel tracer approach of 1,2- 13 C 2 glucose, 13 C 6 glucose, and 13 C 5 glutamine we were able to cover major glycolytic and glutamine-fueled pathways like glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle.…”