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

Abstract

Background: Because the nuclear factor-κB (NF-κB) coupled pathway is believed to amplify inflammation prevailing in sepsis, the authors tested the hypotheses that the insertiondeletion polymorphism (−94ins/delATTG) (1) alters nuclear translocation of nuclear factor-κB and activator protein-1 (NF-κB1) in monocytes after lipopolysaccharide stimulation; (2) affects lipopolysaccharide-induced NF-κB1 messenger RNA expression, tumor necrosis factor α concentrations, and tissue factor activity; and (3) may be associated with increased 30-day mortality in patients with sepsis. Methods: Nuclear translocation of NF-κB1 in monocytes after lipopolysaccharide stimulation from healthy blood donors was performed with immunofluorescence staining (n = 5 each). Lipopolysaccharide-induced NF-κB1 messenger RNA expression was measured with real-time polymerase chain reaction (PCR; n = 60), tumor necrosis factor α 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-κB1 (P = 0.001), a threefold difference in NF-κB1 What We Already Know about This Topic• The nuclear factor-κB and activator protein-1-coupled signaling pathway of the innate immune system is known to amplify and perpetuate inflammatory and coagulatory mechanisms in sepsis• Genetic variations that alter nuclear factor-κB gene expression could affect pathophysiologic mechanisms and thereby influence mortality in severe sepsis What This Article Tells Us That Is New• A functional insertion-deletion polymorphism in the promoter of nuclear factor-κB and activator protein-1 affected expression and regulation of mediators of inflammation and coagulation in vitro • Expression of the same polymorphism was associated with increased 30-day mortality in patients with severe sepsis Downloaded from anesthesiology.pubs.asahq.org by guest on 06/07/2019

Hydrocortisone Fails to Abolish NF-κB1 Protein Nuclear Translocation in Deletion Allele Carriers of the NFKB1 Promoter Polymorphism (-94ins/delATTG) and Is Associated with Increased 30-Day Mortality in Septic Shock

“…Blood samples (Vacutainer CPT tubes, Becton Dickinson, Franklin Lakes, NJ) were centrifuged at 1800 g for 20 minutes using Ficoll density gradient centrifugation tubes, as described [12]. Cells were resuspended using RPMI 1640 medium (Gibco Products Invitrogen Corporation, Grand Island, NY) containing 5% fetal calf serum (Biochrom AG, Berlin, Germany) and antibiotics (100 U ml -1 penicillin and 100 µg ml -1 streptomycin; Invitrogen Corporation, Carlsbad, CA).…”

“…Immunofluorescence staining was performed using a primary NF-κB1 rabbit anti-human 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 [12], [18], [19].…”

“…An independent investigator, blind for the treatments and NFKB1 promoter genotypes, processed all immunofluorescence slides in a randomized order [12]. A Nikon Eclipse E1000 fluorescence microscopy (Nikon GmbH, Düsseldorf, Germany) with NIS-Elements F.30.0 imaging software (Laboratory Imaging, Prague, Czech Republic) was used.…”

“…Furthermore, recently we revealed that this polymorphism is associated with increased NFKB1 gene expression as well as nuclear translocation of the NF-κB protein following lipopolysaccharide (LPS) stimulation [12]. In addition, this polymorphism is an independent risk factor for 30-day mortality in patients with severe sepsis [12]. In another study, the deletion allele of this NFKB1 polymorphism was associated with increased illness severity in patients suffering from the acute respiratory distress syndrome [13].…”

“…Furthermore, recently we revealed that this polymorphism is associated with increased NFKB1 gene expression as well as nuclear translocation of the NF-κB protein following lipopolysaccharide (LPS) stimulation [12]. In addition, this polymorphism is an independent risk factor for 30-day mortality in patients with severe sepsis [12].…”