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

Abstract

Introduction: Sodium 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.

Methods: After 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).

Results: PBMCs showed significantly higher mitochondrial O₂ uptake and lower ${\text{O}}_2^{•-}$ 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.

Conclusion: In 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. ${\text{O}}_2^{•-}$ 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.

Keywords: hemorrhagic shock; hydrogen sulfide; immunometabolism; innate immunity; metabolic flux analysis; metabolic modeling; mitochondrial respiration; reactive oxygen species.