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

THERE is wide variability regarding the outcome in severe sepsis, which in part may be caused by genetic variations.1–3A potential candidate for such variations is the gene encoding nuclear factor-κB and activator protein-1 (NF-κB1), because the ubiquitous transcription factor NF-κB1 binds to recognition elements in the promoter regions of several genes encoding the innate immune and the coagulation system. The nuclear factor-κB family comprises five proteins, NF-κB1, NF-κB2, RelA, RelB, and c-Rel. Nuclear factor-κB family members share structural homology with the retroviral oncoprotein v-Rel, resulting in their classification as NF-κB/Rel proteins. The NFKB1 gene encodes the NF-κB-p105 subunit. NF-κB1 and NF-κB2 proteins are translated as large precursors, p105 and p100, which undergo processing, regulated by the ubiquitin–proteasome pathway, to build the mature nuclear factor-κB subunits, p50 and p52.In most cells, NF-κB1 is found in the cytoplasm in its inactive form, bound to inhibitory proteins. Many extracellular stimuli, including bacterial lipopolysaccharide, viruses, oxidants, inflammatory cytokines, and immune stimuli, can activate NF-κB1.4,5Once activated, it binds to regulatory DNA elements in the promoter regions of inflammatory and immune response genes, such as those encoding proinflammatory cytokines, chemokines, enzymes relevant for inflammation, and adhesion molecules.6,7Furthermore, this pathway induces tissue factor in the blood stream and, thus, activates coagulation.7–10Consequently, disseminated intravascular coagulation is a common finding during severe sepsis, which in turn can evoke ischemia and multiple organ dysfunction.8,9In conclusion, the NF-κB1–coupled pathway is known to amplify and perpetuate inflammatory and coagulatory mechanisms prevailing in sepsis.10Accordingly, genetic variations that alter NFKB gene expression could impact key mechanisms in sepsis and, therefore, influence mortality in severe sepsis.Recently, a functional insertion–deletion polymorphism in the promoter of NFKB1 (−94ins/delATTG in relation to the transcription initiation site or −24.219ins/delATTG in relation to the A as +1 of the initiation codon ATG), which encodes the major isoform of NF-κB1, was reported to be associated with altered NFKB1 gene expression and an increased risk for ulcerative colitis.11Furthermore, we found that the deletion allele influences severity but not mortality of patients suffering from acute respiratory distress syndrome.12In conclusion, there are many reasons to suspect that the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism could impact key mechanisms in sepsis and, therefore, influence mortality in severe sepsis.Accordingly, we prospectively tested the hypotheses that the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism (1) alters nuclear translocation of NF-κB1 in monocytes after lipopolysaccharide stimulation; (2) affects lipopolysaccharide-induced NFκB1 messenger RNA (mRNA) expression, tumor necrosis factor-α (TNFα) concentration, and tissue factor activity; and (3) may be associated with increased 30-day mortality in patients with severe sepsis.13After ethics committee approval (ethics committee of the University Hospital Essen, Essen, Germany) and written informed consent, venous blood (24 ml) was withdrawn from 10 healthy individuals with the homozygous insertion (II) or deletion (DD) genotype, respectively, and centrifuged at 1800g for 20 min using Ficoll density gradient centrifugation tubes (Vacutainer CPT tubes; Becton Dickinson, Franklin Lakes, NJ). Monocytes were resuspended in RPMI 1640 medium (Gibco Products Invitrogen Corporation, Grand Island, NY) containing 5% fetal calf serum (Biochrom AG, Berlin, Germany) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Invitrogen Corporation, Carlsbad, CA). The cell suspension was added to cell culture tubes, and monocytes were allowed to adhere to the surface of the tubes for 2 h. Finally, the supernatant was discarded, fresh RPMI 1640 medium was added, and the cells were allowed to rest for 48 h (37°C; 5% CO2in air) before the experiments. Meanwhile, 12 mm glass plates were coated with 400 μl fibronectin (1 mg/ml; Sigma-Aldrich, St. Louis, MO) inside 24-well plates for 24 h. Afterward, the supernatant was discarded and 500 μl cell suspension (1 × 106monocytes ml-1) was added. Cells were incubated for 60 min either with lipopolysaccharide (10 μg/ml, serotype 0111:B4, Sigma-Aldrich) or without lipopolysaccharide. After incubation, cells were fixed with ice-cold methanol–acetone (1:1) for 10 min at −20°C. Immunofluorescence staining was performed using a primary NF B rabbit antihuman polyclonal p65 antibody (1:200 dilution; Santa Cruz Biotechnologies, Santa Cruz, CA) followed by a Immunoglobulin G - Alexa-flour 568 coupled goat anti-rabbit antibody (1:400 dilution; Molecular Probes, Eugene, OR), as described previously.14An independent investigator, blind for the specific NFKB1 promoter polymorphism and stimulus applied, processed all immunofluorescence slides. Fluorescence microscopy was performed on a Nikon Eclipse E1000 microscope (Nikon GmbH, Düsseldorf, Germany) using the NIS-Elements F.30.0 imaging software (Laboratory Imaging, Prague, Czech Republic). All slides were analyzed in a standardized order, that is, a representative image of each quadrant was captured at 20-fold magnification, and nuclear NFκB-positive cells were counted using an Image J software (ImageJ; National Institute of Health, Bethesda, MD).After approval by the ethics committee of the University Hospital Essen and written informed consent, venous blood was drawn from the antecubital vein of 105 healthy volunteers. After discarding the first 2 ml, blood was collected in one-tenth volume of citrate (Sarstedt Monovette, Nümbrecht, Germany) and samples were immediately used for the experiments. Subsequently, whole blood samples were incubated with lipopolysaccharide at final concentrations of 0, 25, 75, and 225 µg/ml for 4 h at 37°C. Thereafter, samples were subjected to thromboelastometry, and mRNA and DNA were isolated as described in DNA Genotyping. In addition, TNFα concentration was determined in plasma obtained from whole blood samples after incubation with lipopolysaccharide (225 µg/ml; n = 60).To further elucidate the mechanism of the lipopolysaccharide-induced activation of coagulation, lipopolysaccharide- and vehicle (NaCl 0.9%)-treated whole blood samples were incubated with the protein synthesis inhibitor cycloheximide (35 µM) and with an inhibitor of tissue factor effects, active site–inhibited factor seven (50 µg/ml), for 30 min, subsequently challenged with lipopolysaccharide (225 µg/ml) or vehicle (NaCl 0.9%) for 4 h, and then subjected to thromboelastometry as recently described.15,16To assess the tissue factor concentrations induced by lipopolysaccharide, a standard curve was generated by addition of tissue factor standard to whole blood samples and the resulting clotting time was determined.Citrate blood samples were subjected to rotational thromboelastometry (Roteg 5TM, Pentapharm, Munich, Germany), a modification of the original thromboelastography described in 1948 by Hartert,17and coagulation was initiated by recalcification with calcium chloride.18,19Clotting times were determined from the thromboelastogram as described in References 18 and 19. Kinetics of clot formations are measured by the clot formation time and the angle α. The maximum amplitude reflects the strength of the clot and is dependent on both the number and function of platelets and their interaction with fibrin.18,19TNFα plasma levels were determined by using a microsphere-based multiplexing system kit (Invitrogen, Paisley, United Kingdom). As previously shown, the assays were performed according to the manufacturer’s protocol and concentrations measured with a Luminex 100 system (Luminex, Austin, TX).20RNA from whole blood (n = 105) was extracted using the RNeasy kit (Qiagen, Hilden, Germany). First-strand complementary DNA was synthesized from 0.6 µg of total RNA. Quantification of NF-κB1 mRNA was performed by using the intron spanning primer (forward primers) 5′-GTGAAGGCCCATCCCATGGT-3′ and (reverse primer) 5′-TGTGACCAACTGAACAATAACC-3′ resulting in a 122-bp fragment. PCR and primers for the housekeeping gene hβ-actin were used as described.12,21The real-time PCR reaction was performed as described using the Quantitect SYBR Green Kit (Qiagen).21,22A complementary DNA dilution series confirmed a PCR efficiency greater than 95%, which was comparable to the efficiency of hβ-actin. Relative NF-κB1 mRNA expression was measured by two-step real-time PCR with hβ-actin as internal control and calculated as 2−[Ct(NFKB1 )-Ct(β-Actin)].Genomic DNA of patients was extracted from whole blood using standard methods (QIAamp, Qiagen). Genotypes of the insertion–deletion polymorphism were determined by pyrosequencing. A 200-bp PCR fragment was amplified using primer NFKB1 _del/ins_f(5′-ATGGACCGCATGACTCTATCAG-3′) and biotinylated primer NFKB1 _del/ins_BIO_r(5′-GGGGCGCGCGTTAGGCGG-3′). PCR was performed at an annealing temperature of 60°C in a 50-μl reaction mixture applying a commercially available PCR master mix (Eppendorf, Hamburg, Germany). Pyrosequencing was performed on a PSQ96 MA (Pyrosequencing, Uppsala, Sweden) using sequencing primer NFKB1 _del/ins_seq (5′-CGTTCCCCGACCAT-3′). Randomly chosen samples were reanalyzed with a different nucleotide injection order for genotype confirmation.Lipopolysaccharide (Escherichia coli ; serotype 0.111:B4) was obtained from Sigma-Aldrich. Tissue factor standard was obtained from Thrombinoscope BV, Maastricht, Netherlands. Active site–inhibited factor VIIa was a generous gift from Novo Nordisk, Zurich, Switzerland. All other reagents were of analytical grade.This study was reviewed and approved by the ethics committee of the University Hospital Essen. Over a period of 2 yr, 143 patients (93 men and 50 women, mean age: 57 ± 16 yr) admitted to an intensive care unit of the University Hospital of Essen were considered eligible for the study if they fulfilled the criteria for severe sepsis as defined by Bone et al .23Informed consent was obtained for all patients from the guardian of the patient. Exclusion criteria were age more than 18 yr and no affiliation to the Caucasian ethnicity or no informed consent was obtained. All patients were white Germans of Caucasian ethnicity. Clinical and demographic data on study entry, including Simplified Acute Physiology Score II24and the Sequential Organ Failure Assessment score,25were calculated over the first 24 h after the patient met severe sepsis criteria (table 1). All patients were followed up for 30-day mortality. There were no dropouts from the procedures or data lost in any fashion. Patients were treated with a multimodal concept, which included analgosedation, fluid administration, and protective mechanical ventilation, hemodynamic, antibiotic, and diagnostic management. Continuous hemofiltration/dialysis was initiated and technically performed by the Department of Nephrology according to standardized protocols.DNA was isolated and genotyped for the NFKB1 promoter polymorphism (−94ins/delATTG) as described in DNA Genotyping.After incubation, the percentage of nuclear NFκB-positive cells was normalized to the percentage of nuclear NFκB-positive cells in unstimulated controls. The Kolmogorov–Smirnov test and Shapiro–Wilk normality test revealed a normal distribution for nuclear NFκB-positive cells, and the Student t test for unpaired samples was used to analyze differences between groups. Semiquantitative analysis was performed using the Graphpad Prism 5 statistics program (Graph Pad Software, San Diego, CA).To test the effect of the NFKB1 promoter polymorphism (−94ins/delATTG) on NF-κB1 mRNA expression, TNFα concentration, and tissue factor activity after lipopolysaccharide stimulation, heterozygous deletion (ID) and DD genotypes were combined and then tested against the II genotype, because there were only five volunteers with the DD genotype. Potential deviation from the Hardy–Weinberg equilibrium was tested with the statistic program Excel of Microsoft Office 2010 (Microsoft Deutschland, Unterschleißheim, Germany).Statistical analyses were performed using two-way ANOVA followed by post hoc testing with Bonferroni Holm, respectively, using SPSS 13.0 (SPSS, Chicago, IL) or Graphpad Prism 5.0. Data are presented as mean and standard error or SD of the mean, as indicated. For the determination of tissue factor concentrations from clotting time, a four-parameter Hill function was used for the curve fit (SigmaPlot Software, San Jose, CA).The NFKB1 promoter polymorphism (−94ins/delATTG) genotype distributions were tested for deviations from Hardy–Weinberg equilibrium (exact two-sided P value 1.00) in the sepsis cohort. Explorative comparisons by NFKB1 promoter polymorphism (−94ins/delATTG) genotypes (ID/DD vs. II) were performed for several clinical characteristics of the sepsis patients (table 1). ID and DD were combined because of the low frequency of the DD genotype.The clinical endpoint was survival over the first 30 days dependent on NFKB1 promoter polymorphism (−94ins/delATTG) genotypes. Survival probabilities were graphically assessed by the Kaplan–Meier method, and the log-rank test was used to evaluate the univariate relationship between NFKB1 genotypes and clinical outcome. Thereafter, we performed multivariate Cox regression analyses to assess the joint impact of NFKB1 promoter polymorphism (−94ins/delATTG) genotypes, sex, age, Simplified Acute Physiology Score II, Sequential Organ Failure Assessment score, requirement of continuous hemofiltration/dialysis, C-reactive protein, and interleukin 6 concentration as predictors for the clinical outcome (30-day survival). Model diagnostic of the proportional-hazards assumption for the NFKB1 promoter polymorphism (−94ins/delATTG) genotypes comprised both graphical and formal investigations—none of which indicated strong evidence for a deviation from the proportional-hazards assumption. The multivariate analyses included two steps with a focus on NFKB1 promoter polymorphism (−94ins/delATTG) genotypes and 30-day survival. In the initial model, all main effects were simultaneously investigated (table 2). To avoid overfitting, a restricted model with only four variables was assessed afterward using only those predictors with a P value ⩽0.05 in either the univariate or the initial multivariate (table were calculated with of All reported P are and we a of lipopolysaccharide stimulation, the DD genotype was associated with a in nuclear NFκB-positive cells normalized to with with a in II genotype = mRNA were determined in the and of lipopolysaccharide (225 The mRNA for NF-κB1 increased from ± to ± = on lipopolysaccharide 2). the was associated with genotypes. As in lipopolysaccharide-induced expression of NF-κB1 was times as in the of the deletion allele with the II genotype ± vs. ± P = In the of lipopolysaccharide, NF-κB1 expression was not associated with time was determined in 105 volunteers both in the of lipopolysaccharide and after 4 h of incubation with lipopolysaccharide. the clotting time in a from ± 10 to ± 10 µg/ml ± 10 µg/ml and ± (225 µg/ml of NFKB1 genotypes revealed homozygous for the insertion were and 5 were homozygous for the deletion As in the clotting times of II and combined deletion allele were not different in the of lipopolysaccharide ± vs. ± P = After with lipopolysaccharide, the deletion allele lipopolysaccharide concentrations were associated with a clotting time with the II genotype P = P = and 225 P = maximum clot formation clot formation time was associated with genotypes not the mechanism the lipopolysaccharide-induced of clotting time, the effects of the protein synthesis inhibitor cycloheximide and of the tissue factor active site–inhibited factor were the effect of lipopolysaccharide on clotting time, that lipopolysaccharide its effect protein synthesis of tissue factor the active tissue factor concentration for the lipopolysaccharide-induced of clotting time, a concentration response curve was using tissue factor As in tissue factor the clotting time over a concentration from 20 to 20 by regression analysis in using a four-parameter Hill function was the effects of the genotypes of the NFKB1 insertion–deletion polymorphism on tissue factor concentration in the of lipopolysaccharide as calculated from the standard curve in In to the homozygous II genotype, the lipopolysaccharide-induced tissue factor concentration was to increased in the of the deletion allele P = P = and 225 P = TNFα is by the activity of NF-κB1, we determined the effect of lipopolysaccharide on the concentration of this (225 µg/ml) increased the TNFα concentration more than The effect of lipopolysaccharide was not by the genotypes of the NFKB1 promoter because testing with a two-way ANOVA after post hoc testing with Bonferroni revealed a P value of In addition, in the of lipopolysaccharide, TNFα concentrations were not mortality was associated with NFKB1 insertion–deletion (−94ins/delATTG) polymorphism genotypes = was for II genotypes but for combined genotypes genotypes a risk for than patients with homozygous II genotypes P = including sex, age, Sequential Organ Failure Assessment score, Simplified Acute Physiology Score II, C-reactive protein, or and requirement for continuous hemofiltration/dialysis as revealed the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism as an and independent factor for 30-day mortality. genotypes an greater risk for P = with II genotypes (table requirement of continuous hemofiltration/dialysis was an independent and strong factor for 30-day survival P = and the clinical characteristics of the patients by NFKB1 insertion–deletion (−94ins/delATTG) polymorphism genotypes are in found no of NFKB1 insertion–deletion (−94ins/delATTG) polymorphism genotypes with sex, age, C-reactive protein, or for continuous hemofiltration/dialysis, of or primary at In the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism was associated with a of the Sequential Organ Failure Assessment = (table 1). The deletion allele Sequential Organ Failure Assessment with the II genotype study for the first time that the genotypes of the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism are associated with increased 30-day mortality in patients with severe In addition, study the mechanism by which the allele may amplify and perpetuate inflammatory mechanism prevailing in of the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism to both an and independent factor for 30-day mortality. The of not only that the allele of the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism effects on NF-κB1 expression but the potential of NF-κB1 expression in severe wide variability regarding the outcome in of this variability may be by the genetic in the promoter of In addition, that the allele is associated with initial because it was associated with an increased Sequential Organ Failure Assessment are in with that the deletion allele of this polymorphism was associated with increased severity of respiratory distress this study mechanism by which the allele of the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism to amplify and perpetuate inflammatory mechanism prevailing in sepsis, which may influence severity and mortality of severe The DD genotype of the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism is associated with increased nuclear translocation of that increased nuclear translocation of alters the expression of genes in the inflammatory response such as or the factor α. factor a is the key inflammatory and of the NF-κB1 gene a in both an inflammatory response in and an of the a of are resulting in or adhesion and in expression in are to be associated with an NF-κB1 binds to recognition elements in the promoter regions of inflammatory genes, it can be that the allele alters the of sepsis the activation of several inflammatory genes of the innate and NF-κB1 influences the coagulation system tissue sepsis, tissue factor is on resulting in disseminated intravascular coagulation, and organ the we for the first time that lipopolysaccharide-induced tissue factor expression was by the allele of the NFKB1 promoter As the of disseminated intravascular coagulation, which is by tissue factor expression, is an independent risk factor in sepsis, it can be that the deletion allele may the of sepsis increased tissue factor the whole blood from volunteers was incubated with lipopolysaccharide in to the effects of the NFKB1 promoter polymorphism on 4 of clotting for tissue factor was by the of active site–inhibited factor because the lipopolysaccharide-induced of clotting time was in of this to clotting time as a of tissue factor using thromboelastometry of protein, because this functional several with other tissue factor of clotting is because lipopolysaccharide-induced tissue factor in whole blood samples the of the more than the functional relevant tissue factor activity on the surface of In in which are used for the tissue and but not a clotting may tissue which is to be of factor concentration was calculated from clotting time using a curve obtained by the addition of tissue factor standard to whole blood In this be by a effect of the NFKB1 genotypes on platelets or coagulation no differences in or coagulation factor activity was as by clot using of the study the the lipopolysaccharide concentration used in in is of clinical To study effects, lipopolysaccharide used in a wide concentration the up to the In most whole blood lipopolysaccharide from µg/ml to most cell culture were performed with lipopolysaccharide concentrations in the is, concentrations in to whole The of different concentration can be by the of the lipopolysaccharide as an in is bound to other such as and is that plasma lipopolysaccharide up to 6 that lipopolysaccharide in plasma before is than lipopolysaccharide in we used 10 µg for the cell culture and 225 µg/ml lipopolysaccharide for the of NFKB1 mRNA expression and TNFα protein concentration in whole In the the lipopolysaccharide-induced activation of coagulation was 18 The lipopolysaccharide in can be by of a of lipopolysaccharide if an intravascular volume of of the is this lipopolysaccharide was used in injection of lipopolysaccharide described in of this be to many genetic be all sepsis patients were treated with a standardized multimodal because of the of this we that the study was and multivariate analysis revealed the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism to be an and strong independent factor for survival. the potential of NF-κB1 expression in severe sepsis, of mechanisms conclusion, after lipopolysaccharide stimulation, the allele of the NFKB1 insertion–deletion (−94ins/delATTG) polymorphism is associated with increased nuclear translocation of NF-κB1, coagulation, and increased 30-day mortality in patients with severe part of the variability in the outcome may be by the genetic in the promoter of NFKB1 and its by altered nuclear translocation of