Simon T. Schäfer

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA

Toll‐like receptor (TLR) 13 and TLR2 are the major sensors of Gram‐positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA‐derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A‐treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single‐stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence‐derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88‐dependent manner. These ORNs, as well as S. aureus‐, Escherichia coli‐, and mt‐RNA, also activate differentiated human monocytoid THP‐1 cells, provided they express TLR8. Moreover, Unc93b1−/−‐ and Tlr8−/−‐THP‐1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif‐dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria‐driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

The NFKB1 promoter polymorphism (-94ins/delATTG) alters nuclear translocation of NF-κB1 in monocytes after lipopolysaccharide stimulation and is associated with increased mortality in sepsis.

Background:Because the nuclear factor-&kgr;B (NF-&kgr;B) coupled pathway is believed to amplify inflammation prevailing in sepsis, the authors tested the hypotheses that the insertion–deletion polymorphism (−94ins/delATTG) (1) alters nuclear translocation of nuclear factor-&kgr;B and activator protein-1 (NF-&kgr;B1) in monocytes after lipopolysaccharide stimulation; (2) affects lipopolysaccharide-induced NF-&kgr;B1 messenger RNA expression, tumor necrosis factor &agr; concentrations, and tissue factor activity; and (3) may be associated with increased 30-day mortality in patients with sepsis. Methods:Nuclear translocation of NF-&kgr;B1 in monocytes after lipopolysaccharide stimulation from healthy blood donors was performed with immunofluorescence staining (n = 5 each). Lipopolysaccharide-induced NF-&kgr;B1 messenger RNA expression was measured with real-time polymerase chain reaction (PCR; n = 60), tumor necrosis factor &agr; concentrations with a multiplexing system kit (n = 60), and tissue factor activity with thromboelastometry (n = 105). In a prospective study, multivariate proportional hazard analysis tested 30-day mortality in patients with sepsis (n = 143). Methods and Results:The homozygous deletion genotype compared with the homozygous insertion genotype was associated with a nearly twofold increase in nuclear translocation of NF-&kgr;B1 (P = 0.001), a threefold difference in NF-&kgr;B1 messenger RNA expression (P = 0.001), and a twofold increase in tissue factor expression (P = 0.021). The deletion allele in adults with severe sepsis was tested as an independent prognostic factor for 30-day mortality (hazard ratio, 2.3; 95% CI, 1.13–4.8; P = 0.022). Mortality was 25% for homozygous insertion genotypes but 41% for combined heterozygous deletion/homozygous deletion genotypes (P = 0.034). Conclusion:The deletion allele of the NF&kgr;B1 insertion–deletion (−94ins/delATTG) polymorphism is associated with increased 30-day mortality in patients with severe sepsis and increased reaction of the innate immune system.

Intracardiac Transvenous Echocardiography Is Superior to Both Precordial Doppler and Transesophageal Echocardiography Techniques for Detecting Venous Air Embolism and Catheter-Guided Air Aspiration

BACKGROUND:Venous air embolism (VAE) is a potentially fatal complication during surgical procedures with patients in the sitting position. Since methods for detection of persistent low-volume VAE and targeted air aspiration are limited, we tested the hypotheses that transvenous intracardiac echocardiography (ICE) 1) improves detection of small air emboli in comparison to transesophageal echocardiography (TEE) and precordial Doppler monitoring (PCD) techniques, and that 2) image-guided multiorifice central venous catheter manipulation improves air recovery in moderate and large VAE, when compared with aspiration with the multiorifice central venous catheter in a static position. METHODS AND RESULTS:Adult swine (73 ± 4.6 kg, n = 7) were premedicated, anesthetized with propofol and fentanyl, endotracheally intubated, mechanically ventilated, and placed in a 45° head-up position. First, nine different small volumes of air emboli (0.05–1 mL) were randomly injected via an ear vein, and VAE detection methods were applied in random order. For 378 small volume air injections, ICE had a much higher sensitivity (82.5%, P < 0.0001) on the analysis of VAE detection than TEE (52.8%) or PCD (46.8%), with no difference (P = 0.571) between TEE and PCD. An injected air volume as small as 0.15 mL was detected by ICE in 90% of injections performed, whereas PCD and TEE detected only half of the boluses of 0.25–0.30 mL of air, and required boluses of 0.4–1.0 mL to achieve 100% detection. Air recovery was assessed in a second series of moderate VAE (2, 5, 10 mL); image-guided aspiration-catheter manipulation recovered significantly more (34.1% vs 17.2%, P < 0.0001) intracardiac air than without catheter manipulation. In a third series of injections of large air volumes (25, 50, and 100 mL), air recovery was not significantly different with ultrasound-guided aspiration (41.3% vs 31.8%, P = 0.11). CONCLUSION:Small air emboli are detected by ICE with much greater sensitivity compared with both PCD and TEE techniques. Furthermore, recovery of embolized air is enhanced by image-guided manipulation of a multiorifice central venous catheter. Clinical studies are required to assess this technique during surgery with patients in the sitting position.

Hypoxia-inducible factor and target gene expression are decreased in patients with sepsis: prospective observational clinical and cellular studies.

Background:Hypoxia-inducible factor-1 (HIF-1) is a molecular key player in response to hypoxemic/inflammatory conditions prevailing in sepsis. In a prospective observational study, we tested the hypotheses that sepsis affects HIF-1&agr; messenger ribonucleic acid (mRNA) expression (primary hypothesis) and also (secondary hypotheses) the expression of the HIF-1&agr; target genes adrenomedullin and &bgr;2-integrins. Furthermore, we tested that lipopolysaccharide administration increases HIF-1&agr; mRNA and protein in naive and endotoxin-tolerant monocytes. Methods:In 99 patients with sepsis and 48 healthy volunteers, leukocyte HIF-1&agr; mRNA expression (real-time polymerase chain reaction), cytokine concentrations (enzymelinked immunosorbent assay), and intracellular distribution of HIF-1&agr; protein (immunofluorescence staining) were assessed. In vitro, HIF-1&agr; mRNA expression and protein were measured in naive or endotoxin-tolerant (48h; 0.05ng/ml lipopolysaccharide) monocytes, with/without additional lipopolysaccharide (6h; 1 &mgr;g/ml). Results:In comparison to healthy volunteers, HIF-1&agr; mRNA expression (−67%; P = 0.0001) and HIF-1&agr; protein positive cells (−66.7%; P = 0.01) were decreased in sepsis. mRNA expression of adrenomedullin (−75%), CD11a (−85%), and CD11b (−86%; all P = 0.0001) was also decreased. In contrast, interleukin 6 (P = 0.0001), interleukin 10 (P = 0.0001), and tumor necrosis factor-&agr; (P = 0.0002) concentrations were increased. Of note, HIF-1&agr; mRNA expression was inversely associated with illness severity (Simplified Acute Physiology Score II; r = −0.29; P = 0.0001). In vitro, acute lipopolysaccharide administration of naive monocytic cells increased HIF-1&agr; mRNA expression, whereas HIF-1&agr; mRNA and protein (−60%; P = 0.001) were decreased in endotoxin-tolerant cells, which still up regulated cytokines. Conclusions:In sepsis, HIF-1&agr; mRNA expression was suppressed and inversely associated with illness severity. While acute lipopolysaccharide administration increased HIF-1&agr; mRNA expression, prolonged stimulation suppressed HIF-1&agr; expression. The clinical implications of decreased HIF-1&agr; may include maladaption to tissue hypoxia or depressed immune function.

Mitochondrial DNA: An Endogenous Trigger for Immune Paralysis.

Background:Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. Methods:Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. Results:Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. Conclusions:The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.

Aquaporin 1 and 5 expression evoked by the β2 adrenoreceptor agonist terbutaline and lipopolysaccharide in mice and in the human monocytic cell line THP-1 is differentially regulated.

ABSTRACT Aquaporin 1 (AQP1) and AQP5 expression may impact on key mechanisms in sepsis. However, it is unclear whether these AQPs are expressed to an equal extent or regulated differentially. Accordingly, we investigated the time-dependent expression of AQP1 and AQP5 following stimulation with lipopolysaccharide (LPS) in cultured human THP-1 cells and in the lungs of mice injected with LPS. Furthermore, we tested the hypothesis that the &bgr;2 adrenoreceptor agonist terbutaline or its downstream effector cyclic adenosine monophosphate (cAMP) mitigates LPS-evoked changes of AQP expression. THP-1 cells were stimulated with either LPS (1 &mgr;g/mL; serotype O127:B8), 8-Br-cAMP (1 mM), or both, and RNA and protein were extracted at baseline and after 2, 6, and 24 h. C57BL/6 mice that received LPS (20 mg/kg i.p.), terbutaline (2.5 mg/kg), or both were killed 8 h later, and lungs were excised for RNA extraction and lung wet weight determination. Real-time polymerase chain reaction and Western blot analysis show that LPS increased AQP1 (3 h, P < 0.0001) but not AQP5 mRNA and protein expression in THP-1 cells. cAMP increased AQP1 (6 h, P < 0.0001) but not AQP5 mRNA and protein expression. Incubation with both substances accelerated the increase in AQP1 (2 h, P = 0.001) expression, whereas AQP5 expression decreased after 2 h but increased after 24 h (P = 0.0148). In mice lungs, LPS decreased AQP1 (P = 0.0082) but not AQP5 mRNA expression and increased lung wet weight. Terbutaline increased AQP1 mRNA expression twice (P = 0.0005) but not AQP5 mRNA expression. Terbutaline did neither abolish the LPS-induced decrease in AQP1 and AQP5 expression nor increase lung weight. Thus, AQP1 and AQP5 expression is differentially regulated following exposure to LPS, the &bgr;2 adrenoreceptor agonist terbutaline, and cAMP. Furthermore, neither terbutaline nor cAMP mitigated the LPS-evoked change of AQP1 and AQP5 expression.

DARBEPOETIN α, A LONG-ACTING ERYTHROPOEITIN DERIVATE, DOES NOT ALTER LPS EVOKED MYOCARDIAL DEPRESSION AND GENE EXPRESSION OF BAX, BCL-XS, BCL-XL, BCL-2, AND TNF-α

Darbepoetin &agr; (DA), a long-acting erythropoietin derivative stimulating erythropoiesis, can, by antiapoptotic effects, mitigate myocardial I/R injury. We tested the hypothesis that DA treatment improves left ventricular function (LV) in LPS evoked cardiomyopathy and alters gene expression of apoptosis-regulating proteins (Bcl-XL, Bcl-2, Bax, and Bcl-Xs) and TNF-&agr;. In a prospective, controlled, randomized study in Lewis rats (n = 56; 8 groups), myocardial depression was evoked by LPS administration (serotype O127:B8; 10 mg/kg, i.p.). Darbepoetin &agr; or vehicle was injected either 24 h before (pretreatment) or 2 h after LPS injection (treatment). Hearts were isolated 8 h after LPS injection, perfused (Krebs-Henseleit solution) in a Langendorff apparatus, and LV developed pressure and its derivatives were measured. For gene expression analysis, real-time polymerase chain reaction of LV specimen was performed. LPS decreased LV developed pressure (−64.6 ± 7.9 mmHg) and its derivates by more than 60% in comparison to vehicle (P < 0,01), but this effect was not attenuated by DA pretreatment or DA treatment. LPS administration increased gene expression of Bcl-Xs, Bax, and TNF-&agr;, but this was not altered by DA pretreatment. Furthermore, there was no effect on Bcl-Xl and Bcl-2 expression by DA alone. Whereas proapoptotic genes of the myocardium are up-regulated in LPS-induced cardiomyopathy, neither DA pretreatment nor treatment has significant effects on LV function or gene expression. This may suggest cardiac resistance to darbepoetin in LPS-mediated sepsis.

Lipopolysaccharide evokes resistance to erythropoiesis induced by the long-acting erythropoietin analogue darbepoetin alfa in rats.

BACKGROUND:Anemia is common in patients with sepsis but its mechanism is unknown. We tested the hypothesis that effects on erythropoiesis evoked by darbepoetin alfa (DA), a long-acting erythropoietin analog, are diminished by lipopolysaccharide (LPS). METHODS:We performed a prospective, controlled, randomized animal study (male Lewis rats n = 44). The interventions we used were intraperitoneal injection of Escherichia coli LPS (10 mg/kg) or vehicle followed by either DA (25 &mgr;g/kg) or vehicle (four experimental groups). Blood and reticulocyte counts and variables of iron metabolism were measured at baseline and 3 and 14 days after interventions. RESULTS:Animals treated with DA alone showed an eightfold increase in reticulocyte count from baseline on Day 3, whereas no increase was seen in animals administered LPS or LPS/DA. On Day 14, the red blood cell count and hemoglobin concentration had increased by approximately 10% from baseline (P < 0.001) in the DA group but had decreased after LPS on Days 3 and 14 (P < 0.05) and in animals administered LPS/DA. Consumption of iron was seen on Day 3 in the DA group but not after LPS or LPS/DA combined. Values of ferritin and transferrin did not change between groups. CONCLUSION:LPS abolishes erythropoiesis and iron use evoked by DA and this is accompanied by a decrease in hemoglobin concentration and red blood cell concentration. Accordingly, endotoxin suppresses DAs ability to increase erythropoiesis.

Increased circulating microRNA-122 is a biomarker for discrimination and risk stratification in patients defined by sepsis-3 criteria

Background Sepsis is now operationally defined as life-threatening organ dysfunction caused by an infection, identified by an acute change in SOFA-Score of at least two points, including clinical chemistry such as creatinine or bilirubin concentrations. However, little knowledge exists about organ-specific microRNAs as potentially new biomarkers. Accordingly, we tested the hypotheses that micro-RNA-122, the foremost liver-related micro-RNA (miR), 1) discriminates between sepsis and infection, 2) is an early predictor for mortality, and 3) improves the prognostic value of the SOFA-score. Methods We analyzed 108 patients with sepsis (infection + increase SOFA-Score ≥2) within the first 24h of ICU admission and as controls 20 patients with infections without sepsis (infection + SOFA-Score ≤1). Total circulating miR was isolated from serum and relative miR-122 expression was measured (using spiked-in cel-miR-54) and associated with 30-day survival. Results 30-day survival of the sepsis patients was 63%. miR-122 expression was 40-fold higher in non-survivors (p = 0.001) and increased almost 6-fold in survivors (p = 0.013) compared to controls. miR-122 serum-expression discriminated both between sepsis vs. infection (AUC 0.760, sensitivity 58.3%, specificity 95%) and survivors vs. non-survivors (AUC 0.728, sensitivity 42.5%, specificity 94%). Multivariate Cox-regression analysis revealed miR-122 (HR 4.3; 95%-CI 2.0–8.9, p<0.001) as independent prognostic factor for 30-day mortality. Furthermore, the predictive value for 30-day mortality of the SOFA-Score (AUC 0.668) was improved by adding miR-122 (AUC 0.743; net reclassification improvement 0.37, p<0.001; integrated discrimination improvement 0.07, p = 0.007). Conclusions Increased miR-122 serum concentration supports the discrimination between infection and sepsis, is an early and independent risk factor for 30-day mortality, and improves the prognostic value of the SOFA-Score, suggesting a potential role for miR-122 in sepsis-related prediction models.