The Pro-Inflammatory Deletion Allele of the NF-κB1 Polymorphism Is Characterized by a Depletion of Subunit p50 in Sepsis

Abstract

The functionally important NF-κB1 promoter polymorphism (-94ins/delATTG) significantly shapes inflammation and impacts the outcome of sepsis. However, exploratory studies elucidating the molecular link of this genotype-dependent pattern are lacking. Accordingly, we analyzed lipopolysaccharide-stimulated peripheral blood mononuclear cells from both healthy volunteers (n = 20) and septic patients (n = 10). All individuals were genotyped for the -94ins/delATTG NF-κB1 promoter polymorphism. We found a diminished nuclear activity of the NF-κB subunit p50 in ID/DD genotypes after 48 h of lipopolysaccharide stimulation compared to II genotypes (p = 0.025). This was associated with higher TNF-α (p = 0.005) and interleukin 6 concentrations (p = 0.014) and an increased production of mitochondrial radical oxygen species in ID/DD genotypes (p = 0.001). Although ID/DD genotypes showed enhanced activation of mitochondrial biogenesis, they still had a significantly diminished cellular ATP content (p = 0.046) and lower mtDNA copy numbers (p = 0.010) compared to II genotypes. Strikingly, these findings were mirrored in peripheral blood mononuclear cells taken from septic patients. Our results emphasize the crucial aspect of considering NF-κB subunits in sepsis. We showed here that the deletion allele of the NF-κB1 (-94ins/delATTG) polymorphism was associated with the lower nuclear activity of subunit p50, which, in turn, was associated with aggravated inflammation and mitochondrial dysfunction.

Keywords: NF-κB; NF-κB1 promoter polymorphism; mitochondrial dysfunction; nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells; sepsis; subunit p50.

Figure 1

Lipopolysaccharide (LPS) decreases nuclear DNA-binding activity of NF-κB subunit p50 in deletion allele carriers compared to II genotypes. LPS stimulation of PBMCs from healthy volunteers before and after 0.5, 4, 24, and 48 h, respectively. Upper panel: mRNA expression (quantitative polymerase chain reaction) normalized to beta actin of (a) NF-κB1 and (b) RelA in LPS-stimulated PBMCs of healthy volunteers. (c–g) Nuclear binding activity of NF-κB subunits of PBMCs from healthy volunteers. Columns represent means with error bars as standard deviation. p values relate to post hoc Šídák’s test adjusted for multiple comparisons. No labeling of p-values designates no statistically significant difference at respective time points. There were no missing data.

Figure 2

The deletion allele of the NF-κB1 promoter polymorphism is associated with an aggravated inflammation in lipopolysaccharide-stimulated PBMCs. LPS stimulation of PBMCs from healthy volunteers before and after 0.5, 4, 24, and 48 h, respectively. Concentrations of selected cytokines in PBMC cell culture supernatants. (a) TNF-α; (b) interleukin 6; (c) interleukin 10. Columns represent means with error bars as standard deviation. p values relate to post hoc Šídák’s test adjusted for multiple comparisons. No labeling of p-values designates no statistically significant difference at respective time points. Cytokine concentrations were derived from a calibration curve. There were no missing data.

Figure 3

Deterioration of mitochondrial function in LPS-stimulated PBMCs from healthy volunteers is aggravated in deletion allele carriers. Upper panel: (a) PGC-1α (ELISA of nuclear protein extracts) in PBMCs of healthy volunteers. (b) TFAM protein expression in LPS-stimulated PBMCs from healthy volunteers. Middle Panel: (c) Mitochondrial ROS production in LPS-stimulated PBMCs from healthy volunteers. Lower panel: Time course of mitochondrial function indicators (d) mitochondrial DNA copy number and (e) cellular ATP content in PBMCs from healthy volunteers before and 0.5, 4, 24, and 48 after LPS stimulation. Cellular ATP was determined using a luciferase-based assay and expressed as relative fluorescent units normalized to 2.25 × 10⁵ cells per well. Columns represent means with error bars as standard deviation. p values relate to post hoc Šídák’s test adjusted for multiple comparisons. No labeling of p-values designates no statistically significant difference at respective time points. There were no missing data.

Figure 4

Sepsis is associated with a diminished DNA binding activity of NF-κB subunit p50 in deletion allele carriers. Results representing PBMCs from septic patients (n = 10) sampled within 24 h after the diagnosis and stratified according to II-genotypes and ID/DD genotypes of the NF-κB1 promoter polymorphism. Upper panel: mRNA expression (quantitative polymerase chain reaction) normalized to beta actin of (a) NF-κB1 and (b) RelA in PBMCs of septic patients. (c–g) Nuclear binding activity of NF-κB subunits of sepsis patients. Each dot represents an individual patient; line represents mean. There were no missing data. p-values were determined using the Mann–Whitney test; ns designates no statistically significant difference. Cytokine concentrations pg/mL were derived from a calibration curve.

Figure 5

Sepsis is associated with an aggravated inflammation and intensified mitochondrial dysfunction in deletion allele carriers. Results representing PBMCs from septic patients (n = 10) sampled within 24 h after the diagnosis and stratified according to II-genotypes and ID/DD genotypes of the NF-κB1 promoter polymorphism. Upper panel: Concentrations of selected cytokines in PBMC cell culture supernatants. (a) TNF-α; (b) Interleukin 6; (c) Interleukin 10. Middle a lower Panel: (d) Mitochondrial ROS production in PBMCs from sepsis patients. (e) PGC-1α (ELISA of nuclear protein extracts) in PBMCs of healthy volunteers. (f) TFAM protein expression in PBMCs from sepsis patients. Mitochondrial function indicators (g) cellular ATP content and (h) mitochondrial DNA copy number in PBMCs from sepsis patients. Cellular ATP (g) was determined using a luciferase-based assay and expressed as relative fluorescent units normalized to 2.25 × 10⁵ cells per well. Each dot represents an individual patient; line represents mean. There were no missing data. p-values were determined using the Mann–Whitney test; ns designates no statistically significant difference. Cytokine concentrations pg/mL were derived from a calibration curve.