Decreased activation of NF-κB and expression of related genes in IRAK-1SNP 532 neutrophils from volunteers exposed to endotoxin and in unstimulated neutrophils from septic patients
© BioMed Central Ltd 2005
Published: 9 June 2005
Neutrophils have been involved in sepsis-induced organ damage. Neutrophils could be directly activated by TLR binding ligands including LPS. IRAK-1 is one of many intracellular proteins that are activated upon stimulation of TL receptors. This triggers a series of events that results in the migration of NF-κB into the nucleus and the activation NF-κB-dependent genes.
To identify a single nucleotide polymorphism at position 532 (coding SNP) in volunteers and patients with sepsis. To determine whether IRAK-1SNP532 results in a decrease in neutrophil NF-κB activation in volunteers and patients with sepsis. To evaluate neutrophil gene expression patterns in IRAK-1SNP532 and wildtype patients with sepsis.
Thirty severe sepsis patients and 34 healthy volunteers were enrolled in this study. Peripheral blood was obtained and neutrophils were isolated by plasma–percoll gradients after dextran sedimentation of erythrocytes. Neutrophils from volunteers were resuspended in RPMI and cultured with or without 100 ng/ml LPS for 60 min. The electrophoretic mobility shift assay technique was used to measure the NF-κB activation. Real-time PCR allelic discrimination assays were developed by the assay-by-design service offered by Applied Biosystems (Foster City, CA, USA). Probe and primer combinations were designed at the single nucleotide polymorphism 532. PCR reactions were performed according to the manufacturer's manual using the Applied Biosystems 7500 Real-Time PCR system. Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after LPS stimulus.
IRAK-1SNP532 genotyped neutrophils from volunteers (after LPS ex vivo challenge) and from septic patients are associated with lower NF-κB activation and lower expression of some IRAK1-related genes. These results demonstrate that IRAK1 plays a critical role in the inflammatory response and, potentially, a polymorphism in IRAK1 may alter the immune response impacting clinical outcome.