Experimental Human Endotoxemia Research Articles

Effect of the antihepcidin Spiegelmer lexaptepid on inflammation-induced decrease in serum iron in humans

AbstractIncreased hepcidin production is key to the development of anemia of inflammation. We investigated whether lexaptepid, an antihepcidin l-oligoribonucleotide, prevents the decrease in serum iron during experimental human endotoxemia. This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy males. At T = 0 hours, 2 ng/kg Escherichia coli lipopolysaccharide was intravenously administered, followed by an intravenous injection of 1.2 mg/kg lexaptepid or placebo at T = 0.5 hours. The lipopolysaccharide-induced inflammatory response was similar in subjects treated with lexaptepid or placebo regarding clinical and biochemical parameters. At T = 9 hours, serum iron had increased by 15.9 ± 9.8 µmol/L from baseline in lexaptepid-treated subjects compared with a decrease of 8.3 ± 9.0 µmol/L in controls (P < .0001). This study delivers proof of concept that lexaptepid achieves clinically relevant hepcidin inhibition enabling investigations in the treatment of anemia of inflammation. This trial was registered at www.clinicaltrial.gov as #NCT01522794.

Experimental human endotoxemia: A model to disentangle the immune-to-brain communication

Experimental human endotoxemia: A model to disentangle the immune-to-brain communication

0730. Plasma endocan levels are associated with endothelial dysfunction during experimental human endotoxemia

The endothelium plays a central role in pathophysiology of sepsis. Systemic inflammation results in endothelial dysfunction, contributing to the development of shock and multiple organ dysfunction. However, an easy to obtain, early, and clinically applicable marker of endothelial dysfunction is currently not available. Such a marker could guide early and targeted therapy such as selective iNOS inhibition. Endocan is a soluble proteoglycan secreted by endothelial cells in response to pro-inflammatory cytokines, bacterial endotoxins, and angiogenic factors, and enhances microvascular permeability. Plasma endocan levels are increased in septic patients, and have been shown to correlate with sepsis severity and mortality. However, the direct relationship between endocan and endothelial function has not yet been investigated in humans in vivo.

Abstract 18773: Discovery and Translation of Novel Long Intergenic Non-Coding RNAs in Inflammatory Cardio-Metabolic Disorders

Objective: The purpose of this study is to identify long intergenic non-coding RNAs (LincRNAs) modulated by inflammation utilizing human experimental endotoxemia and to explore their association with cardiometabolic traits. Methods and results: We performed deep high-throughput RNAseq (~500 million reads per sample) in adipose tissue and whole blood of a healthy subject before and after endotoxin (LPS 1 ng/Kg) administration. Of 8,179 LincRNAs identified by Cabili et al (Gene &amp; Development 2011. 25:1915-1927), we detected 514 present in adipose and 266 in blood, with 69 (13.6%) and 56 (21.2%) LincRNAs respectively modulated by LPS with just six LPS-modulated lincRNAs overlapping in both tissues. In both tissues, LincRNAs had lower expression and more tissue specificity than protein coding RNAs. RNAseq of adipose and blood in 2 additional individuals during endotoxemia was used to confirm findings and qPCR used to validate top findings in an additional 7 study participants. Linc-ARFIP1-4 and Linc-CMC1-2 were consistently reduced by LPS in blood, but not in adipose. Interestingly, Linc-DMRT2 and Linc-TP53I13, the top adipose LPS-modulated LincRNA, was more abundant in human primary adipocytes than monocyte and macrophage cultured in vitro while adipose samples obtained from obese subjects exhibited lower Linc-DMRT2 and Linc-TP63I13 expression (77% and 50% lower respectively, P&lt;0.05) compared to lean individuals (N=10/group). To further investigate biological roles, we generated lentiviral-vectors with short hairpin RNAs (shRNAs) targeting Linc-DMRT2 and Linc-TP53I13 as well as Linc-AQPEP, the top expressed adipose-specific LincRNA. Preliminary studies demonstrated more than 50% knockdown of these lincRNAs in human adipocytes with shRNA application and functional studies are ongoing to characterize their impact on adipocyte differentiation, lipid storage and insulin sensitivity. Conclusion: These novel data from a human endotoxemia experiment suggest that inflammatory modulation of adipose LincRNAs might modulate adipose functions in cardio-metabolic disorders.

The effect of iron loading and iron chelation on the innate immune response and subclinical organ injury during human endotoxemia: a randomized trial

In this double-blind randomized placebo-controlled trial involving 30 healthy male volunteers we investigated the acute effects of iron loading (single dose of 1.25 mg/kg iron sucrose) and iron chelation therapy (single dose of 30 mg/kg deferasirox) on iron parameters, oxidative stress, the innate immune response, and subclinical organ injury during experimental human endotoxemia. The administration of iron sucrose induced a profound increase in plasma malondialdehyde 1 h after administration (433±37% of baseline; P<0.0001), but did not potentiate the endotoxemia-induced increase in malondialdehyde, as was seen 3 h after endotoxin administration in the placebo group (P=0.34) and the iron chelation group (P=0.008). Endotoxemia resulted in an initial increase in serum iron levels and transferrin saturation that was accompanied by an increase in labile plasma iron, especially when transferrin saturation reached levels above 90%. Thereafter, serum iron decreased to 51.6±9.7% of baseline at T=8 h in the placebo group versus 84±15% and 60.4±8.9% of baseline at 24 h in the groups treated with iron sucrose and deferasirox, respectively. No significant differences in the endotoxemia-induced cytokine response (TNF-α, IL-6, IL-10 and IL-1RA), subclinical vascular injury and kidney injury were observed between groups. However, vascular reactivity to noradrenalin was impaired in the 6 subjects in whom labile plasma iron was elevated during endotoxemia as opposed to those in whom no labile plasma iron was detected (P=0.029). In conclusion, a single dose of iron sucrose does not affect the innate immune response in a model of experimental human endotoxemia, but may impair vascular reactivity when labile plasma iron is formed. (Clinicaltrials.gov identifier:01349699).

Omega‐3 PUFA supplementation and the response to evoked endotoxemia in healthy volunteers

Fish oil-derived n-3 PUFA may improve cardiometabolic health through modulation of innate immunity. However, findings in clinical studies are conflicting. We hypothesized that n-3 PUFA supplementation would dose-dependently reduce the systemic inflammatory response to experimental endotoxemia in healthy humans. The Fenofibrate and omega-3 Fatty Acid Modulation of Endotoxemia (FFAME) study was an 8-wk randomized double-blind trial of placebo or n-3 PUFA supplementation (Lovaza 465 mg eicosapentaenoic acid (EPA) + 375 mg docosahexaenoic acid (DHA)) at "low" (1/day, 900 mg) or "high" (4/day, 3600 mg) dose in healthy individuals (N = 60; age 18-45; BMI 18-30; 43% female; 65% European-, 20% African-, 15% Asian-ancestry) before a low-dose endotoxin challenge (LPS 0.6 ng/kg intravenous bolus). The endotoxemia-induced temperature increase was significantly reduced with high-dose (p = 0.03) but not low-dose EPA + DHA compared to placebo. Although there was no statistically significant impact of EPA + DHA on individual inflammatory responses (tumor necrosis factor-α (TNF-α), IL-6, monocyte chemotactic protein (MCP-1), IL-1 receptor agonist (IL-1RA), IL-10, C-reactive protein (CRP), serum amyloid A (SAA)), there was a pattern of lower responses across all biomarkers with high-dose (nine of nine observed), but not low-dose EPA + DHA. EPA + DHA at 3600 mg/day, but not 900 mg/day, reduced fever and had a pattern of attenuated LPS induction of plasma inflammatory markers during endotoxemia. Clinically and nutritionally relevant long-chain n-3 PUFA regimens may have specific, dose-dependent, anti-inflammatory actions.

Coagulopathy, catecholamines, and biomarkers of endothelial damage in experimental human endotoxemia and in patients with severe sepsis: A prospective study

PurposeThe aim of this study was to investigate associations between circulating catecholamines, endothelial damage, and coagulopathy in experimental human endotoxemia and septic patients. Materials and MethodsNine healthy male volunteers undergoing endotoxemia (4-hour 0.5 ng/kg/hour infusion of E. coli lipopolysaccharide, blood sampling at 0, 4, and 6 hours) and 20 patients with severe sepsis. Analysis of plasma biomarkers (adrenaline, noradrenaline, thrombomodulin, syndecan-1, soluble vascular endothelial cadherin, histone-complexed DNA fragments, soluble CD40 ligand [sCD40L], protein C, tissue-type plasminogen activator, plasminogen activator inhibitor 1) and routine coagulation tests. ResultsEndotoxemia increased heart rate, temperature, white blood cell count, C-reactive protein and procalcitonin, decreased blood pressure and induced a hemostatic response with platelet consumption, reduced protein C and sCD40L levels and enhanced tissue-type plasminogen activator release (all P < .05). Septic patients had increased levels of noradrenaline, syndecan-1, thrombomodulin, histone-complexed DNA and sCD40L but reduced soluble vascular endothelial cadherin and plasminogen activator inhibitor 1 (all P < .05) and plasma catecholamines correlated positively with syndecan-1 (adrenaline and noradrenaline) and sTM (only noradrenaline) (all P < .05), biomarkers reflecting endothelial damage. Furthermore, noradrenaline, syndecan-1 and thrombomodulin levels correlated with INR and disease severity scores (noradrenaline and thrombomodulin) (all P < .05). ConclusionsExperimental endotoxemia induced a discrete hemostatic response without sympathoadrenal activation or endothelial damage. Septic patients had high levels of catecholamines and endothelial damage biomarkers that correlated with each other and with markers of hypocoagulability and disease severity.

Body mass index is not associated with cytokine induction during experimental human endotoxemia

A higher body mass index (BMI) appears to be associated with lower mortality in critically ill patients, possibly explained by an altered innate immune response. However, the precise relationship between BMI and the innate immune response in humans invivo is unknown. We investigated the relationship between BMI and the systemic cytokine response during experimental human endotoxemia. Endotoxemia was induced in 112 healthy male volunteers by intravenous administration of 2 ng/kg Escherichia coli endotoxin. Plasma concentrations of TNF-α, IL-6, IL-10 and IL-1RA were serially determined. The relationship between BMI and the cytokine response, as well as body temperature, was investigated. The BMIs of the participants ranged from 18.3 to 33.6 kg/m(2), (median: 22.7 kg/m(2)). All participants showed a marked increase in plasma cytokine levels [median (interquartile range)] peak levels: TNF-α 509 (353-673) pg/ml; IL-6 757 (522-1098) pg/ml; IL-10 271 (159-401) pg/ml; IL-1RA 4882 (3927-6025) pg/ml; and an increase in body temperature [1.8(1.4-2.2)] during endotoxemia. No significant correlations were found between BMI and levels of any of the cytokines or body temperature. No relationship between BMI and the cytokine response was found in healthy volunteers subjected to experimental endotoxemia. These data question the relationship between BMI and cytokine responses in critical illness.

Experimental human endotoxemia enhances brain activity during social cognition

Acute peripheral inflammation with corresponding increases in peripheral cytokines affects neuropsychological functions and induces depression-like symptoms. However, possible effects of increased immune responses on social cognition remain unknown. Therefore, this study investigated the effects of experimentally induced acute inflammation on performance and neural responses during a social cognition task assessing Theory of Mind (ToM) ability. In this double-blind randomized crossover functional magnetic resonance imaging study, 18 healthy right-handed male volunteers received an injection of bacterial lipopolysaccharide (LPS; 0.4 ng/kg) or saline, respectively. Plasma levels of pro- and anti-inflammatory cytokines as well as mood ratings were analyzed together with brain activation during a validated ToM task (i.e. Reading the Mind in the Eyes Test). LPS administration induced pronounced transient increases in pro- (IL-6, TNF-α) and anti-inflammatory (IL-10, IL-1ra) cytokines as well as decreases in mood. Social cognition performance was not affected by acute inflammation. However, altered neural activity was observed during the ToM task after LPS administration, reflected by increased responses in the fusiform gyrus, temporo-parietal junction, superior temporal gyrus and precuneus. The increased task-related neural responses in the LPS condition may reflect a compensatory strategy or a greater social cognitive processing as a function of sickness.

Randomized double-blind placebo-controlled PK/PD study on the effects of a single intravenous dose of the anti-hepcidin Spiegelmer NOX-H94 on serum iron during experimental human endotoxemia

Anemia is very frequently encountered on the ICU. Increased hepcidin production is one of the cornerstones of the pathophysiology of anemia of inflammation. The first-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of anemia of inflammation. We investigated whether NOX-H94 prevents the inflammation-induced serum iron decrease during experimental human endotoxemia.

Discrepant Fibrinolytic Response in Plasma and Whole Blood during Experimental Endotoxemia in Healthy Volunteers

BackgroundSepsis induces early activation of coagulation and fibrinolysis followed by late fibrinolytic shutdown and progressive endothelial damage. The aim of the present study was to investigate and compare the functional hemostatic response in whole blood and plasma during experimental human endotoxemia by the platelet function analyzer, Multiplate and by standard and modified thrombelastography (TEG).MethodsProspective physiologic study of nine healthy male volunteers undergoing endotoxemia by means of a 4-hour infusion of E. coli lipopolysaccharide (LPS, 0.5 ng/kg/hour), with blood sampled at baseline and at 4 h and 6 h. Physiological and standard biochemical data and coagulation tests, TEG (whole blood: TEG, heparinase-TEG, Functional Fibrinogen; plasma: TEG±tissue-type plasminogen activator (tPA)) and Multiplate (TRAPtest, ADPtest, ASPItest, COLtest) were recorded. Mixed models with Tukey post hoc tests and correlations were applied.ResultsEndotoxemia induced acute SIRS with increased HR, temperature, WBC, CRP and procalcitonin and decreased blood pressure. It also induced a hemostatic response with platelet consumption and reduced APTT while INR increased (all p<0.05). Platelet aggregation decreased (all tests, p<0.05), whereas TEG whole blood clot firmness increased (G, p = 0.05). Furthermore, during endotoxemia (4 h), whole blood fibrinolysis increased (clot lysis time (CLT), p<0.001) and Functional Fibrinogen clot strength decreased (p = 0.049). After endotoxemia (6 h), whole blood fibrinolysis was reduced (CLT, p<0.05). In contrast to findings in whole blood, the plasma fibrin clot became progressively more resistant towards tPA-induced fibrinolysis at both 4 h and 6 h (p<0.001).ConclusionsEndotoxemia induced a hemostatic response with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by downregulation of fibrinolysis, with a discrepant fibrinolytic response in plasma and whole blood. The finding that blood cells are critically involved in the vasculo-fibrinolytic response to acute inflammation is important given that disturbances in the vascular system contribute significantly to morbidity and mortality in critically ill patients.

Vitamin D status is not associated with inflammatory cytokine levels during experimental human endotoxaemia

Vitamin D has been shown to modulate innate immune responses in vitro and ex vivo; however, human in-vivo data are lacking. At high latitudes, seasonal vitamin D deficiency is common due to alternating ultraviolet (UV)-B radiation exposure. In the present study, we investigated whether levels of 25 hydroxyvitamin D(3) [25(OH)D(3) ] and its active metabolite 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] are subject to seasonal variation and whether plasma levels of these vitamin D metabolites correlate with the in-vivo cytokine response during experimental human endotoxaemia [administration of lipopolysaccharide (LPS) in healthy volunteers]. Plasma levels of 25(OH)D(3) and 1,25(OH)(2) D(3) were determined in samples obtained just prior to administration of an intravenous bolus of 2 ng/kg LPS (derived from Escherichia coli O:113) in 112 healthy male volunteers. In the same subjects, plasma levels of the inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 were analysed serially after endotoxin administration. Plasma levels of 1,25(OH)(2) D(3) , but not 25(OH)D(3) , were subject to significant seasonal variation, with lower levels in autumn and winter. 25(OH)D(3) and 1,25(OH)(2) D(3) levels did not correlate with plasma cytokine responses. Furthermore, 25(OH)D(3) deficient subjects (< 50 nmol/l) displayed an identical cytokine response compared with sufficient subjects. In conclusion, plasma levels of vitamin D are not correlated with the LPS-induced TNF, IL-6 and IL-10 cytokine response in humans in vivo. These findings question the direct role of vitamin D in modulation of the innate immune response.

Metformin improves survival in intensive care unit patients, but why?

We read with interest the study by Christiansen and colleagues [1] in the previous issue of Critical Care. In a large cohort study, the authors report that among diabetic patients who are admitted to the ICU, the preadmission use of metformin is associated with a lower mortality rate. Here, we highlight that metformin limits ischemiareperfusion injury (IRI) and modulates inflammation by increased adenosine receptor stimulation and propose that these mechanisms contribute to the reported survival benefit. In prospective studies in patients with diabetes, metformin use is associated with lower cardiovascular mortality compared with alternative glucose-lowering agents, suggesting a direct cardioprotective effect of metformin [2]. In animal studies, metformin potently limits myocardial infarct size [2]. This is caused, at least in part, by increased formation of the endogenous nucleoside adenosine [3]. Adenosine receptor stimulation increases tolerance of various organs against IRI and potently modulates inflammation [4]. Indeed, pharmacological elevation of endogenous adenosine modulates the inflammatory response during experimental human endotoxemia [5]. In the study by Christiansen and colleagues, many patients were admitted to the ICU because of cardiovascular diseases or (non-)cardiac surgery. It is likely that in these patients IRI contributes to organ dysfunction. Therefore, we propose that the survival benefit of metformin is caused by limitation of IRI and inflammation due to increased adenosine receptor signaling. Based on this hypothesis, several randomized controlled trials are currently being performed to investigate whether metformin reduces myocardial injury in patients with a myocardial infarction and patients undergoing cardiac surgery (NCT01438723 and NCT01217307). We await with interest the results of these trials.

Indoleamine-2,3-dioxygenase activity in experimental human endotoxemia

BackgroundExcessive tryptophan metabolism to kynurenine by the rate-limiting enzyme endothelial indoleamine 2,3-dioxygenase 1 (IDO) controls arterial vessel relaxation and causes hypotension in murine endotoxemia. However, its relevance in human endotoxemia has not been investigated so far. We thus aimed to study changes in blood pressure in parallel with tryptophan and kynurenine levels during experimental endotoxemia in humans.FindingsSix healthy male volunteers were given E. coli lipopolysaccharide (LPS; 4 ng/kg) as a 1-min intravenous infusion. They had levels of soluble E-Selectin and soluble vascular cell adhesion molecule-1 as well as IDO activity assessed as the kynurenine-to-tryptophan plasma ratio by liquid chromatography-tandem mass spectrometry at various time points during a 24 h time course. During endotoxemia, IDO activity significantly increased, reaching peak levels at 8 h after LPS infusion (44.0 ± 15.2 vs. 29.4 ± 6.8 at baseline, P<0.0001). IDO activity correlated inversely with the development of hypotension as shown by random effects linear regression models. Finally, IDO activity exhibited a kinetic profile similar to that of soluble endothelial-specific adhesion molecules.ConclusionsLPS is a triggering factor for the induction of IDO in men. Our findings strongly support the concept that the induction of IDO in the vascular endothelium contributes to hypotension in human sepsis.

Randomized Double Blind Placebo Controlled PK/PD Study On the Effects of a Single Intravenous Dose of the Anti-Hepcidin Spiegelmer Nox-H94 On Serum Iron During Experimental Human Endotoxemia

AbstractAbstract 3452 ObjectiveAnemia of chronic disease (ACD) is generally attributed to increased hepcidin production. The first-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of ACD. We investigated whether NOX-H94 prevents the inflammation-induced serum iron decrease during experimental human endotoxemia. Effects on the innate immune response were studied as secondary endpoints. Materials and MethodsThe study protocol (ClinicalTrials.gov identifier NCT01522794) was approved by the local ethics committee of the Radboud University Nijmegen Medical Centre and was conducted in accordance with the Declaration of Helsinki (2000) of the World Medical Association.This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy young men. At T=0 hours (h), 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T=0.5h. Blood was drawn serially for 24h and at day 3, 8 and 15 after endotoxin administration for measurements of inflammatory parameters, cytokines, NOX-H94 pharmacokinetics, total hepcidin-25 (free and NOX-H94-bound), and iron parameters. The difference of serum iron change from baseline at T=9h was defined as primary endpoint. Data are expressed as mean±SD. ResultsEndotoxin administration led to flu-like symptoms peaking at T=1.5h, irrespective of the treatment group. Body temperature rose by 1.9±0.5 °C in both groups. Peak CRP at T=24h was also similar in NOX-H94 and placebo treated groups (34.3±18.0 vs. 36.8±8.7 mg/L) as was the rise in leucocytes at T=6 h (12.1±2.2 vs. 12.1±2.3 ×109/L). Plasma levels of TNF-α, IL-6, IL-10, and IL-1RA peaked markedly and similarly in both treatment groups.NOX-H94 was well tolerated. Plasma concentrations peaked at 0.7±0.4 h after the start of administration, after which they declined according to a two-compartment model, with rapid initial elimination followed by a slower elimination phase with a plasma half-life of 22.5 ± 4.28h. Hepcidin-25 rose to a peak of 23.0±5.2 nM at T=5.5±0.05 h in control subjects and normalized at about T=24 h. In the NOX-H94 treated group, total hepcidin-25 concentrations, which represent the sum of free hepcidin-25 and hepcidin-25 bound to NOX-H94, rose to 559±112 nM at T=9.5±1.5 h and normalized slowly until day 15.The observed serum iron concentrations are shown in the table. In the placebo group, serum iron increased from baseline to a peak at T=3h, returned close to baseline at T=6h and decreased under the baseline concentration at T=9h reaching its lowest point at T=12h. In the NOX-H94 group, serum iron concentrations rose until T=6h and then slowly declined until T=24h. From 6 to 12 h post LPS, the serum iron concentrations in NOX-H94 treated subjects were significantly higher than in placebo treated subjects (P<0.0001, ANCOVA). At T=24 h and 48 h, serum iron was lower in NOX-H94 treated subjects than in placebo controls.TableMean serum iron concentrations (μmol/L, ± SD) in subjects treated with LPS at T= 0h and with NOX-H94 or Placebo at T=0.5hDayTime (h)Placebo (n=12)1.2 mg/kg NOX-H94 (n=12)10 (baseline)19.0 ± 7.6418.0 ± 6.821332.6 ± 6.7133.1 ± 6.291622.8 ± 6.3138.3 ± 8.09 *1910.7 ± 5.6033.9 ± 9.04 *1128.0 ± 2.9528.7 ± 9.62 *22413.7 ± 5.969.3 ± 5.8334817.4 ± 3.9610.1 ± 4.17821.2 ± 4.3221.6 ± 5.761520.8 ± 7.8523.1 ± 8.03*p<0.0001, ANCOVA. ConclusionExperimental human endotoxemia induced a robust inflammatory response with hepcidin release and a subsequent decrease in serum iron. Treatment with NOX-H94 had no effect on innate immunity, but effectively prevented the inflammation-induced drop in serum iron concentrations. These findings demonstrate the clinical potential of the anti-hepcidin drug NOX-H94 for further development to treat patients with ACD. Disclosures:van Eijk:NOXXON Pharma AG: Research Funding. Swinkels:NOXXON Pharma AG: Research Funding. Schwoebel:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment. Summo:NOXXON Pharma AG: Employment. Stéphanie:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Pikkers:NOXXON Pharma AG: Research Funding.

Deficient Candida-Specific T-Helper 17 Response During Sepsis

Patients with sepsis in the intensive care unit (ICU) are prone to develop Candida infections. Here, we investigated Candida-induced T-helper 17 (Th17) responses during experimental human endotoxemia and in patients with sepsis admitted to the ICU. Peripheral blood mononuclear cells were stimulated with Candida albicans. The Th17 response was significantly lower during endotoxemia, compared with baseline. Patients with gram-negative sepsis had a significantly lower Th17 response as compared to healthy controls. These data demonstrate that the Th17 response is deficient during endotoxin-related systemic inflammation, which likely represents an important risk factor for increased susceptibility to develop Candida infection in patients with sepsis.

How systemic inflammation modulates adenosine metabolism and adenosine receptor expression in humans in vivo

Adenosine modulates inflammation and prevents associated organ injury by activation of its receptors. During sepsis, the extracellular adenosine concentration increases rapidly, but the underlying mechanism in humans is unknown. We aimed to determine the changes in adenosine metabolism and signaling both in vivo during experimental human endotoxemia and in vitro. We studied subjects participating in three different randomized double-blind placebo-controlled trials. In order to prevent confounding by the different pharmacological interventions in these trials, analyses were performed on data of placebo-treated subjects only. Intensive care research unit at the Radboud University Nijmegen Medical Center. In total, we used material of 24 healthy male subjects. Subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide) intravenously. Following experimental endotoxemia, endogenous adenosine concentrations increased. Expression of 5'ectonucleotidase messenger RNA was upregulated (p = .01), whereas adenosine deaminase messenger RNA was downregulated (p = .02). Furthermore, both adenosine deaminase and adenosine kinase activity was significantly diminished (both p ≤ .0001). A2a and A2b receptor messenger RNA expression was elevated (p = .02 and p = .04, respectively), whereas messenger RNA expression of A1 and A3 receptors was reduced (both, p = .03). In vitro, lipopolysaccharide dose-dependently attenuated the activity of both adenosine deaminase and adenosine kinase (both p ≤ .0001). Adenosine metabolism and signaling undergo adaptive changes during human experimental endotoxemia promoting higher levels of adenosine thereby facilitating its inflammatory signaling.

Adipose Genes Down-Regulated During Experimental Endotoxemia Are Also Suppressed in Obesity

Adipose inflammation is a crucial link between obesity and its metabolic complications. Human experimental endotoxemia is a controlled model for the study of inflammatory cardiometabolic responses in vivo. We hypothesized that adipose genes down-regulated during endotoxemia would approximate changes observed with obesity-related inflammation and reveal novel candidates in cardiometabolic disease. Healthy volunteers (n = 14) underwent a 3 ng/kg endotoxin challenge; adipose biopsies were taken at 0, 4, 12, and 24 h for mRNA microarray. A priority list of highly down-regulated and biologically relevant genes was validated by RT-PCR in an independent sample of adipose from healthy subjects (n = 7) undergoing a subclinical 0.6 ng/kg endotoxemia protocol. Expression of validated genes was screened in adipose of lean and severely obese individuals (n = 11 per group), and cellular source was probed in cultured adipocytes and macrophages. Endotoxemia (3 ng/kg) suppressed expression of 353 genes (to <67% of baseline; P < 1 × 10(-5)) of which 68 candidates were prioritized for validation. In low-dose (0.6 ng/kg) endotoxin validation, 22 (32%) of these 68 genes were confirmed. Functional classification revealed that many of these genes are involved in cell development and differentiation. Of validated genes, 59% (13 of 22) were down-regulated more than 1.5-fold in primary human adipocytes after treatment with endotoxin. In human macrophages, 59% (13 of 22) were up-regulated during differentiation to inflammatory M1 macrophages whereas 64% (14 of 22) were down-regulated during transition to homeostatic M2 macrophages. Finally, in obese vs. lean adipose, 91% (20 of 22) tended to have reduced expression (χ(2) = 10.72, P < 0.01) with 50% (11 of 22) reaching P < 0.05 (χ(2) = 9.28, P < 0.01). Exploration of down-regulated mRNA in adipose during human endotoxemia revealed suppression of genes involved in cell development and differentiation. A majority of candidates were also suppressed in endogenous human obesity, suggesting a potential pathophysiological role in human obesity-related adipose inflammation.

Transcriptome Kinetics of Circulating Neutrophils during Human Experimental Endotoxemia

Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1β. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.

Abstract 482: Peroxisome Proliferator-Activated Receptor α Modulation with Fenofibrate Does Not Alter the Clinical or Inflammatory Responses to Evoked Endotoxemia in Humans

Introduction: Inflammation plays a crucial role in the pathophysiology of cardiometabolic disease. Some experimental data and clinical trials suggest that PPAR-α activation and fibrate therapy suppress inflammation in humans, potentially promoting anti-atherogenic actions. Human experimental endotoxemia provides a controlled model for the study of atherogenic inflammatory responses and their modulation in vivo . We hypothesized that fenofibrate would suppress the inflammatory responses to endotoxin (LPS) in healthy humans. Methods: In the Fenofibrate and Omega-3 Fatty Acid Modulation of Endotoxemia study, 36 healthy volunteers (mean age 26 ± 7 years; mean BMI 24 ± 3 kg/m 2 ; 56% male; 72% Caucasian, 19% African, 8% Asian ancestry) were randomized to fenofibrate 145 mg or placebo daily. After 8 weeks of treatment, subjects underwent a low-dose LPS challenge (0.6 ng/kg intravenous bolus). Anthropometric, clinical, and blood measures were collected at baseline, pre-LPS, and serially for 24 hours after LPS exposure. Results: Compared to placebo, fenofibrate reduced total cholesterol (170 ± 35 to 137 ± 31 mg/dL, p=0.009) and tended to decrease triglycerides (78 ± 40 to 57 ± 23 mg/dL, p=0.611) prior to LPS. LPS induced a modest clinical response with increased temperature, heart rate, and blood pressure ( p &lt;0.001 for all). Cytokines and chemokines increased significantly, peaking 2 to 4 hours post-LPS (TNFα 12-fold, IL-6 13-fold, IL-10 9-fold, MCP-1 10-fold; p &lt;0.001 for all). Acute phase reactants also increased, peaking at 24 hours (serum amyloid A (SAA) 14-fold, C-reactive protein (CRP) 9-fold; both p &lt;0.001). Compared to placebo, fenofibrate had no effect on LPS-induced clinical responses, cytokine or chemokine release as measured by peak response or area under the curve (AUC). Fenofibrate also had no effect on plasma CRP or lipid parameters following LPS. Although peak SAA and SAA AUC tended to be lower with fenofibrate vs. placebo, the differences were not statistically significant ( p =0.08 for both). Conclusions: PPAR-α modulation with fenofibrate did not attenuate clinical or inflammatory responses during low-grade human endotoxemia. These data suggest the anti-inflammatory properties of fenofibrate at clinically-relevant dosing are limited.