Ex Vivo 13C-Metabolic Flux Analysis of Porcine Circulating Immune Cells Reveals Cell Type-Specific Metabolic Patterns and Sex Differences in the Pentose Phosphate Pathway.

In general, females present with stronger immune responses than males, but scarce data are available on sex-specific differences in immunometabolism. In this study, we characterized porcine peripheral blood mononuclear cell (PBMC) and granulocyte energy metabolism using a Bayesian 13C-metabolic flux analysis, which allowed precise determination of the glycolytic, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA) fluxes, together with an assessment of the superoxide anion radical (O2•-) production and mitochondrial O2 consumption. A principal component analysis allowed for identifying the cell type-specific patterns of metabolic plasticity. PBMCs displayed higher TCA cycle activity, especially glutamine-derived aspartate biosynthesis, which was directly related to mitochondrial respiratory activity and inversely related to O2•- production. In contrast, the granulocytes mainly utilized glucose via glycolysis, which was coupled to oxidative PPP utilization and O2•- production rates. The granulocytes of the males had higher oxidative PPP fluxes compared to the females, while the PBMCs of the females displayed higher non-oxidative PPP fluxes compared to the males associated with the T helper cell (CD3+CD4+) subpopulation of PBMCs. The observed sex-specific differences were not directly attributable to sex steroid plasma levels, but we detected an inverse correlation between testosterone and aldosterone plasma levels and showed that aldosterone levels were related with non-oxidative PPP fluxes of both cell types.

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

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 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. 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.

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.

Introduction: Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a 13C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity. Methods: Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (PaO2 = 80 - 120 mmHg) or targeted hyperoxemia (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation, thereafter PaO2 as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48 h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and 13C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-13C2 glucose, U-13C glucose, and U-13C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA). Results: 13C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group. Conclusion: In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O2 concentrations (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian 13C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes' ability to perform phagocytosis or produce superoxide radicals.

Bayesian 13C-Metabolic Flux Analysis of Parallel Tracer Experiments in Granulocytes: A Directional Shift within the Non-Oxidative Pentose Phosphate Pathway Supports Phagocytosis.

The pentose phosphate pathway (PPP) plays a key role in the cellular regulation of immune function; however, little is known about the interplay of metabolic adjustments in granulocytes, especially regarding the non-oxidative PPP. For the determination of metabolic mechanisms within glucose metabolism, we propose a novel set of measures for 13C-metabolic flux analysis based on ex vivo parallel tracer experiments ([1,2-13C]glucose, [U-13C]glucose, [4,5,6-13C]glucose) and gas chromatography-mass spectrometry labeling measurements of intracellular metabolites, such as sugar phosphates and their fragments. A detailed constraint analysis showed that the permission range for net and irreversible fluxes was limited to a three-dimensional space. The overall workflow, including its Bayesian flux estimation, resulted in precise flux distributions and pairwise confidence intervals, some of which could be represented as a line due to the strength of their correlation. The principal component analysis that was enabled by these behaviors comprised three components that explained 99.6% of the data variance. It showed that phagocytic stimulation reversed the direction of non-oxidative PPP net fluxes from ribose-5-phosphate biosynthesis toward glycolytic pathways. This process was closely associated with the up-regulation of the oxidative PPP to promote the oxidative burst.

Cigarette smoke exposure reduces hemorrhagic shock induced circulatory dysfunction in mice with attenuated glucocorticoid receptor function.

Introduction: We previously showed that attenuated glucocorticoid receptor (GR) function in mice (GRdim/dim) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GRdim/dim) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS. Methods: After 3 weeks of CS exposure, anesthetized and mechanically ventilated GRdim/dim and GR+/+ mice underwent pressure-controlled HS for 1h via blood withdrawal (mean arterial pressure (MAP) 35mmHg), followed by 4h of resuscitation with re-transfusion of shed blood, colloid fluid infusion and, if necessary, continuous intravenous norepinephrine. Acid-base status and organ function were assessed together with metabolic pathways. Blood and organs were collected at the end of the experiment for analysis of cytokines, corticosterone level, and mitochondrial respiratory capacity. Data is presented as median and interquartile range. Results: Nor CS exposure neither attenuated GR function affected survival. Non-CS GRdim/dim mice had a higher need of norepinephrine to keep target hemodynamics compared to GR+/+ mice. In contrast, after CS exposure norepinephrine need did not differ significantly between GRdim/dim and GR+/+ mice. Non-CS GRdim/dim mice presented with a lower pH and increased blood lactate levels compared to GR+/+ mice, but not CS exposed mice. Also, higher plasma concentrations of some pro-inflammatory cytokines were observed in non-CS GRdim/dim compared to GR+/+ mice, but not in the CS group. With regards to metabolic measurements, CS exposure led to an increased lipolysis in GRdim/dim compared to GR+/+ mice, but not in non-CS exposed animals. Conclusion: Whether less metabolic acidosis or increased lipolysis is the reason or the consequence for the trend towards lower catecholamine need in CS exposed GRdim/dim mice warrants further investigation.

Excretion of oxidated cortisol metabolites is markedly lower than previously assumed: An analysis of urinary cortoic acids in healthy children by GC-MS.

Discovered about 50 years ago, the four C21 steroidal acids (α-)cortolic acid, ß-cortolic acid, (α­)cortolonic acid and ß-cortolonic acid present the oxidative end products of cortisol metabolism. Undergoing renal elimination, these cortoic acids have been assumed to constitute up to 25 % of total urinary cortisol metabolites. However, their analysis has been difficult, only few data has been published in adults, and this class of steroids has become practically forgotten. Since data in children are lacking and nothing is known about their metabolism during human development, we aimed at establishing a more practical analytical method and determined their urinary concentrations in a high number of healthy subjects. In our method, 5-mL-aliquots of 24-hour urine samples were subjected to solid phase extraction (C18 cartridges), followed by strong anion exchange chromatography, and formation of 2-propylester-trimethylsilylether derivatives (2-PR/TMS). The cortoic acids were quantified by targeted gas chromatography-mass spectrometry (GC-MS) using a nonpolar GC column and selected ion monitoring (SIM). Baseline separation of all cortoic acids was achieved. Calibration graphs were linear (R2 > 0.98). Variations in precision and accuracy were less than 15 %, respectively. The detection limit was 100 pg (injected) with a signal-to-noise ratio of 3. 240 specimens from 24-hour urine collections from healthy children (120 boys, 120 girls, aged 3-18 years; DONALD study) were analyzed for cortoic acids and neutral cortisol metabolites to create first reference ranges. The profile of cortoic acids was dominated by α-cortolonic acid with excretion rates up to 70 µg/d. Absolute excretion rates of cortoic acids increased with age, their total excretion rates ranged between 11.0 and 127.3 µg/d (median 45.7 µg/d), but did not show any sexual dimorphism. Since cortoic acids make up only about 1 % of total urinary cortisol metabolites, determination of neutral urinary steroids reliably allows assessment of cortisol production. However, cortoic acids might present potential biomarkers of the body's redox state.

Effects of Sodium Thiosulfate During Resuscitation From Trauma-and-Hemorrhage in Cystathionine-γ-Lyase Knockout Mice With Diabetes Type 1.

Background: Sodium thiosulfate (STS) is a recognized drug with antioxidant and H2S releasing properties. We recently showed that STS attenuated organ dysfunction and injury during resuscitation from trauma-and-hemorrhage in CSE-ko mice, confirming its previously described organ-protective and anti-inflammatory properties. The role of H2S in diabetes mellitus type 1 (DMT1) is controversial: genetic DMT1 impairs H2S biosynthesis, which has been referred to contribute to endothelial dysfunction and cardiomyopathy. In contrast, development and severity of hyperglycemia in streptozotocin(STZ)-induced DMT1 was attenuated in CSE-ko mice. Therefore, we tested the hypothesis whether STS would also exert organ-protective effects in CSE-ko mice with STZ-induced DMT1, similar to our findings in animals without underlying co-morbidity. Methods: Under short-term anesthesia with sevoflurane and analgesia with buprenorphine CSE-ko mice underwent DMT1-induction by single STZ injection (100 µg⋅g-1). Seven days later, animals underwent blast wave-induced blunt chest trauma and surgical instrumentation followed by 1 h of hemorrhagic shock (MAP 35 ± 5 mmHg). Resuscitation comprised re-transfusion of shed blood, lung-protective mechanical ventilation, fluid resuscitation and continuous i.v. norepinephrine together with either i.v. STS (0.45 mg⋅g-1) or vehicle (n = 9 in each group). Lung mechanics, hemodynamics, gas exchange, acid-base status, stable isotope-based metabolism, and visceral organ function were assessed. Blood and organs were collected for analysis of cytokines, chemokines, and immunoblotting. Results: Diabetes mellitus type 1 was associated with more severe circulatory shock when compared to our previous study using the same experimental design in CSE-ko mice without co-morbidity. STS did not exert any beneficial therapeutic effect. Most of the parameters measured of the inflammatory response nor the tissue expression of marker proteins of the stress response were affected either. Conclusion: In contrast to our previous findings in CSE-ko mice without underlying co-morbidity, STS did not exert any beneficial therapeutic effect in mice with STZ-induced DMT1, possibly due to DMT1-related more severe circulatory shock. This result highlights the translational importance of both integrating standard ICU procedures and investigating underlying co-morbidity in animal models of shock research.

Effects of Sodium Thiosulfate During Resuscitation From Trauma-and-Hemorrhage in Cystathionine Gamma Lyase (CSE) Knockout Mice.

BACKGROUND: Sodium thiosulfate (Na2S2O3) is a clinically established drug with antioxidant and sulphide-releasing properties. Na2S2O3 mediated neuro- and cardioprotective effects in ischemia/reperfusion models and anti-inflammatory effects in LPS-induced acute lung injury. Moreover, Na2S2O3 improved lung function during resuscitation from hemorrhagic shock in swine with pre-existing atherosclerosis, characterized by decreased expression of cystathionine γ-lyase (CSE), a major source of hydrogen sulfide (H2S) synthesis in the vasculature. Based on these findings, we investigated the effects of Na2S2O3 administration during resuscitation from trauma-and-hemorrhage in mice under conditions of whole body CSE deficit. METHODS: After blast wave-induced blunt chest trauma and surgical instrumentation, CSE knockout (CSE-/-) mice underwent 1 h of hemorrhagic shock (MAP 35 ±â€Š5 mm Hg). At the beginning of resuscitation comprising retransfusion, norepinephrine support and lung-protective mechanical ventilation, animals received either i.v. Na2S2O3 (0.45 mg g-1, n = 12) or vehicle (saline, n = 13). Hemodynamics, acid-base status, metabolism using stable isotopes, and visceral organ function were assessed. Blood and organs were collected for analysis of cytokines, mitochondrial respiratory capacity, and immunoblotting. RESULTS: Na2S2O3 treatment improved arterial paO2 (P = 0.03) coinciding with higher lung tissue glucocorticoid receptor expression. Norepinephrine requirements were lower in the Na2S2O3 group (P < 0.05), which was associated with lower endogenous glucose production and higher urine output. Na2S2O3 significantly increased renal tissue IκBα and heme oxygenase-1 expression, whereas it lowered kidney IL-6 and MCP-1 levels. CONCLUSION: Na2S2O3 exerted beneficial effects during resuscitation of murine trauma-and-hemorrhage in CSE-/- mice, confirming and extending the previously described organ-protective and anti-inflammatory properties of Na2S2O3. The findings make Na2S2O3 a potentially promising therapeutic option in the context of impaired CSE activity and/or reduced endogenous H2S availability.

Glucocorticoids coordinate macrophage metabolism through the regulation of the tricarboxylic acid cycle.

OBJECTIVES: Glucocorticoids (GCs) are one of the most widely prescribed anti-inflammatory drugs. By acting through their cognate receptor, the glucocorticoid receptor (GR), GCs downregulate the expression of pro-inflammatory genes and upregulate the expression of anti-inflammatory genes. Metabolic pathways have recently been identified as key parts of both the inflammatory activation and anti-inflammatory polarization of macrophages, immune cells responsible for acute inflammation and tissue repair. It is currently unknown whether GCs control macrophage metabolism, and if so, to what extent metabolic regulation by GCs confers anti-inflammatory activity. METHODS: Using transcriptomic and metabolomic profiling of macrophages, we identified GC-controlled pathways involved in metabolism, especially in mitochondrial function. RESULTS: Metabolic analyses revealed that GCs repress glycolysis in inflammatory myeloid cells and promote tricarboxylic acid (TCA) cycle flux, promoting succinate metabolism and preventing intracellular accumulation of succinate. Inhibition of ATP synthase attenuated GC-induced transcriptional changes, likely through stalling of TCA cycle anaplerosis. We further identified a glycolytic regulatory transcription factor, HIF1α, as regulated by GCs, and as a key regulator of GC responsiveness during inflammatory challenge. CONCLUSIONS: Our findings link metabolism to gene regulation by GCs in macrophages.

Temporal-spatial organ response after blast-induced experimental blunt abdominal trauma.

Abdominal trauma (AT) is of major global importance, particularly with the increased potential for civil, terroristic, and military trauma. The injury pattern and systemic consequences of blunt abdominal injuries are highly variable and frequently underestimated or even missed, and the pathomechanisms remain still poorly understood. Therefore, we investigated the temporal-spatial organ and immune response after a standardized blast-induced blunt AT. Anesthetized mice were exposed to a single blast wave centered on the epigastrium. At 2, 6, or 24 h after trauma, abdominal organ damage was assessed macroscopically, microscopically, and biochemically. A higher degree of trauma severity, determined by a reduction of the distance between the epigastrium and blast inductor, was reflected by a reduced survival rate. The hemodynamic monitoring during the first 120 min after AT revealed a decline in the mean arterial pressure within the first 80 min, whereas the heart rate remained quite stable. AT induced a systemic damage and inflammatory response, evidenced by elevated HMGB-1 and IL-6 plasma levels. The macroscopic injury pattern of the abdominal organs (while complex) was consistent, with the following frequency: liver > pancreas > spleen > left kidney > intestine > right kidney > others > lungs and was reflected by microscopic liver and pancreas damages. Plasma levels of organ dysfunction markers increased during the first 6 h after AT and subsequently declined, indicating an early, temporal impairment of the function on a multi-organ level. The established highly reproducible murine blunt AT, with time- and trauma-severity-dependent organ injury patterns, systemic inflammatory response, and impairment of various organ functions, reflects characteristics of human AT. In the future, this model may help to study the complex immuno-pathophysiological consequences and innovative therapeutic approaches after blunt AT.

Evaluation of the gut microbiome in association with biological signatures of inflammation in murine polytrauma and shock.

Severe injuries are frequently accompanied by hemorrhagic shock and harbor an increased risk for complications. Local or systemic inflammation after trauma/hemorrhage may lead to a leaky intestinal epithelial barrier and subsequent translocation of gut microbiota, potentially worsening outcomes. To evaluate the extent with which trauma affects the gut microbiota composition, we performed a post hoc analysis of a murine model of polytrauma and hemorrhage. Four hours after injury, organs and plasma samples were collected, and the diversity and composition of the cecal microbiome were evaluated using 16S rRNA gene sequencing. Although cecal microbial alpha diversity and microbial community composition were not found to be different between experimental groups, norepinephrine support in shock animals resulted in increased alpha diversity, as indicated by higher numbers of distinct microbial features. We observed that the concentrations of proinflammatory mediators in plasma and intestinal tissue were associated with measures of microbial alpha and beta diversity and the presence of specific microbial drivers of inflammation, suggesting that the composition of the gut microbiome at the time of trauma, or shortly after trauma exposure, may play an important role in determining physiological outcomes. In conclusion, we found associations between measures of gut microbial alpha and beta diversity and the severity of systemic and local gut inflammation. Furthermore, our data suggest that four hours following injury is too early for development of global changes in the alpha diversity or community composition of the intestinal microbiome. Future investigations with increased temporal-spatial resolution are needed in order to fully elucidate the effects of trauma and shock on the gut microbiome, biological signatures of inflammation, and proximal and distal outcomes.

Metabolic monitoring via on-line analysis of 13C-enriched carbon dioxide in exhaled mouse breath using substrate-integrated hollow waveguide infrared spectroscopy and luminescence sensing combined with Bayesian sampling.

In studies that target specific functions or organs, the response is often overlaid by indirect effects of the intervention on global metabolism. The metabolic side of these interactions can be assessed based on total energy expenditure (TEE) and the contributions of the principal energy sources, carbohydrates, proteins and fat to whole body CO2 production. These parameters can be identified from indirect calorimetry using respiratory oxygen intake and CO2 dioxide production data that are combined with the response of the 13CO2 release in the expired air and the glucose tracer enrichment in plasma following a 13C glucose stable isotope infusion. This concept is applied to a mouse protocol involving anesthesia, mechanical respiration, a disease model, like hemorrhage and therapeutic intervention. It faces challenges caused by a small sample size for both breath and plasma as well as changes in metabolic parameters caused by disease and intervention. Key parameters are derived from multiple measurements, all afflicted with errors that may accumulate leading to unrealistic values. To cope with these challenges, a sensitive on-line breath analysis system based on substrate-integrated hollow waveguide infrared spectroscopy and luminescence (iHWG-IR-LS) was used to monitor gas exchange values. A Bayesian statistical model is developed that uses established equations for indirect calorimetry to predict values for respiratory gas exchange and tracer data that are consistent with the corresponding measurements and also provides statistical error bands for these parameters. With this new methodology, it was possible to estimate important metabolic parameters (respiratory quotient (RQ), relative contribution of carbohydrate, protein and fat oxidation fcarb, ffat and fprot , total energy expenditure TEE) in a resolution never available before for a minimal invasive protocol of mice under anesthesia.

Effects of Acute Subdural Hematoma-Induced Brain Injury On Energy Metabolism in Peripheral Blood Mononuclear Cells.

ABSTRACT: In activated immune cells, differentiation and function are determined by cell type-specific modifications of metabolic patterns. After traumatic brain injury both immune cell activation and suppression were reported. Therefore, we sought to explore immune cell energy metabolism in a long-term, resuscitated porcine model of acute subdural hematoma (ASDH)-induced acute brain injury devoid of impaired systemic hemodynamics and oxygen transport.Before and up to 50 h after induction of ASDH, peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation, and cell metabolism was analyzed using high-resolution respirometry for mitochondrial respiration and electron spin resonance for reactive oxygen species production. After incubation with stable isotope-labeled 1,2-13C2-glucose or 13C5-glutamine, distinct labeling patterns of intermediates of glycolysis or tricarboxylic acid (TCA) cycle and 13CO2 production were measured by gas chromatography-mass spectroscopy. Principal component analysis was followed by a varimax rotation on the covariance across all measured variables and all measured time points.After ASDH induction, average PBMC metabolic activity remained unaffected, possibly because strict adherence to intensive care unit guidelines limited trauma to ASDH induction without any change in parameters of systemic hemodynamics, oxygen transport, and whole-body metabolism. Despite decreased glycolytic activity fueling the TCA cycle, the principal component analysis indicated a cell type-specific activation pattern with biosynthetic and proliferative characteristics.

Metabolic substrate utilization in stress-induced immune cells.

Immune cell activation leads to the acquisition of new functions, such as proliferation, chemotaxis, and cytokine production. These functional changes require continuous metabolic adaption in order to sustain ATP homeostasis for sufficient host defense. The bioenergetic demands are usually met by the interconnected metabolic pathways glycolysis, TCA cycle, and oxidative phosphorylation. Apart from glucose, other sources, such as fatty acids and glutamine, are able to fuel the TCA cycle.Rising evidence has shown that cellular metabolism has a direct effect on the regulation of immune cell functions. Thus, quiescent immune cells maintain a basal metabolic state, which shifts to an accelerated metabolic level upon immune cell activation in order to promote key effector functions.This review article summarizes distinct metabolic signatures of key immune cell subsets from quiescence to activation and demonstrates a methodical concept of how to assess cellular metabolic pathways. It further discusses why metabolic functions are of rising interest for translational research and how they can be affected by the underlying pathophysiological condition and/or therapeutic interventions.

Thirty-Eight-Negative Kinase 1 Is a Mediator of Acute Kidney Injury in Experimental and Clinical Traumatic Hemorrhagic Shock.

Trauma represents a major socioeconomic burden worldwide. After a severe injury, hemorrhagic shock (HS) as a frequent concomitant aspect is a central driver of systemic inflammation and organ damage. The kidney is often strongly affected by traumatic-HS, and acute kidney injury (AKI) poses the patient at great risk for adverse outcome. Recently, thirty-eight-negative kinase 1 (TNK1) was proposed to play a detrimental role in organ damage after trauma/HS. Therefore, we aimed to assess the role of TNK1 in HS-induced kidney injury in a murine and a post hoc analysis of a non-human primate model of HS comparable to the clinical situation. Mice and non-human primates underwent resuscitated HS at 30 mmHg for 60 min. 5 h after the induction of shock, animals were assessed for systemic inflammation and TNK1 expression in the kidney. In vitro, murine distal convoluted tubule cells were stimulated with inflammatory mediators to gain mechanistic insights into the role of TNK1 in kidney dysfunction. In a translational approach, we investigated blood drawn from either healthy volunteers or severely injured patients at different time points after trauma (from arrival at the emergency room and at fixed time intervals until 10 days post injury; identifier: NCT02682550, https://clinicaltrials.gov/ct2/show/NCT02682550). A pronounced inflammatory response, as seen by increased IL-6 plasma levels as well as early signs of AKI, were observed in mice, non-human primates, and humans after trauma/HS. TNK1 was found in the plasma early after trauma-HS in trauma patients. Renal TNK1 expression was significantly increased in mice and non-human primates after HS, and these effects with concomitant induction of apoptosis were blocked by therapeutic inhibition of complement C3 activation in non-human primates. Mechanistically, in vitro data suggested that IL-6 rather than C3 cleavage products induced upregulation of TNK1 and impaired barrier function in renal epithelial cells. In conclusion, these data indicate that C3 inhibition in vivo may inhibit an excessive inflammatory response and mediator release, thereby indirectly neutralizing TNK1 as a potent driver of organ damage. In future studies, we will address the therapeutic potential of direct TNK1 inhibition in the context of severe tissue trauma with different degrees of additional HS.

Effects of sodium thiosulfate (Na2S2O3) during resuscitation from hemorrhagic shock in swine with preexisting atherosclerosis.

Controversial data are available on hydrogen sulfide (H2S) during hemorrhage and resuscitation, depending on timing, dosing, mode of application, and the H2S donor used. Sodium thiosulfate (Na2S2O3) is a recognized drug devoid of major side effects, which attenuated murine acute lung injury and cerebral ischemia/reperfusion injury. Therefore, we tested the hypothesis whether Na2S2O3 would mitigate organ dysfunction in porcine hemorrhage-and-resuscitation. We studied animals with pre-existing coronary artery disease because of the reduced coronary arterial expression of the H2S producing enzyme cystathionine-γ-lyase (CSE) in this prospective, randomized, controlled, blinded experimental study. 20 anesthetized and instrumented pigs underwent 3 h of hemorrhage (removal of 30 % of the blood volume and subsequent titration of mean arterial pressure to 40 mmHg). Resuscitation (72 h) comprised re-transfusion of shed blood, crystalloids, and continuous i.v. norepinephrine. Animals randomly received vehicle or Na2S2O3 (0.1 g·kg-1 h-1) for 24 h. Before, at the end of and every 24 h after shock, hemodynamics, metabolism, blood gases, lung, heart, kidney, and liver function and injury were evaluated together with cytokines and parameters of oxidative and nitrosative stress. Immediate post mortem lung, kidney, heart, and liver specimen were analyzed for marker proteins of inflammation and oxidative and nitrosative stress and mitochondrial respiratory activity in the heart, kidney, and liver. Immuno-histochemical analysis comprised lung extra-vascular albumin accumulation, nitrotyrosine formation, and CSE and glucocorticoid receptor (GCR) expression. Na2S2O3 significantly attenuated shock-induced impairment of lung mechanics and gas exchange (plateau and positive end-expiratory pressure at 72 h p = 0.0006/p = 0.0264; Horovitz index at 48 h p = 0.0261), which coincided with a higher tissue GCR expression (p = 0.0415). During resuscitation from hemorrhagic shock Na2S2O3 attenuated shock-induced acute lung injury in co-morbid swine, most likely due to a GCR expression related mechanism.

Strategies for 13C enrichment calculation in Fourier-transform infrared CO2 spectra containing spectral overlapping and nonlinear abundance-amount relations utilizing response surface fits.

The metabolism can be explored via 13C labeling of biological active substances and subsequent quantification of 13C enrichment in the exhaled carbon dioxide in breath. The resulting tracer enrichment values can be determined by Fourier-transform Infrared Spectroscopy (FTIR), since different CO2 isotopologues result in distinct absorption lines in the spectrum.The corresponding determination poses two challenges: first, FTIR absorbance can contain a nonlinear relationship between analyte amount and spectral signal and second, the spectral peaks for the different isotopologues overlap. The overlap precludes a separate calibration to asses the isotopologue concentration values and with it a determination of enrichments from concentration values. We propose here, first, a data reduction step like Principal Component Analysis (PCA) to convert the spectral information into one score pertaining to the 13CO2 enrichment. In a second step, a calibration function between score and enrichment values was established. The enrichment score can be derived by normalizing a subset of the spectrum by some measure for the 12CO2 sample content. Alternatively, the overlapping spectra were decomposed into two isotopologue spectra and the intensity of the separated spectra was used to form an enrichment score. For spectral separation, either Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) was used or a novel decomposition strategy developed for this paper called Rotation and Angle-Bending Bayesian induced Transformation - Multivariate Curve Resolution (RABBIT - MCR) that operates in a Principal Component Analysis (PCA) subspace and is derived from MCR. We compared 13C enrichment estimates from FTIR CO2 spectra using different normalization variants with the two spectral separation models. In conclusion, the two spectral separation variants performed nearly equal, but better than any normalization variant.

In-Depth Characterization of the Effects of Cigarette Smoke Exposure on the Acute Trauma Response and Hemorrhage in Mice.

INTRODUCTION: Hemorrhagic shock accounts for a large amount of trauma-related mortality. The severity of trauma can be further aggravated by an additional blunt chest trauma (TxT), which independently contributes to mortality upon the development of an acute lung injury (ALI). Besides, cigarette smoke (CS) exposure before TxT enhanced posttraumatic inflammation, thereby aggravating ALI. We therefore aimed to characterize the impact of an acute and/or chronic lung injury on organ dysfunction in a murine model of traumatic hemorrhagic shock (HS). METHODS: After 3 weeks of CS exposure, anesthetized mice underwent HS with/without TxT. Hemorrhagic shock was implemented for 1 h followed by retransfusion of shed blood and intensive care therapy for 4 h including lung-protective mechanical ventilation, fluid resuscitation, and noradrenaline titrated to maintain mean arterial pressure ≥50 mmHg. Lung mechanics and gas exchange were assessed together with systemic hemodynamics, metabolism, and acid-base status. Postmortem blood and tissue samples were analyzed for cytokine and chemokine levels, protein expression, mitochondrial respiration, and histological changes. RESULTS: CS exposure and HS alone coincided with increased inflammation, decreased whole blood sulfide concentrations, and decreased diaphragmatic mitochondrial respiration. CS-exposed mice, which were subjected to TxT and subsequent HS, showed hemodynamic instability, acute kidney injury, and high mortality. CONCLUSIONS: Chronic CS exposure per se had the strongest impact on inflammatory responses. The degree of inflammation was similar upon an additional TxT, however, mice presented with organ dysfunction and increased mortality rates. Hence, in mice the degree of inflammation may be dissociated from the severity of organ dysfunction or injury.