Third International Symposium on Intensive Care and Emergency Medicine for Latin America

Introduction 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. Objectives 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. Methods 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. Results The median AUC for NF-κB activation was higher in wildtype genotyped neutrophils as compared with IRAK-1SNP532 genotyped neutrophils (85.2 vs 100.5, P = 0.05) (Fig. 1). In terms of kinetics pattern, we found some differences on nuclear levels of NF-κB in neutrophils from volunteers cultured with LPS. At 30 min after LPS, the culture nuclear translocation of NK-κB was significantly greater in wildtype genotyped neutrophils than in IRAK-1SNP532 genotyped neutrophils. Even after 60 min, the NF-κB translocation remained high in wildtype genotyped neutrophils, while in IRAK-1SNP532 genotyped neutrophils the NF-κB translocation was similar to baseline (Fig. 2). In unstimulated neutrophils from septic patients, the NF-κB translocation was significantly lower in IRAK-1SNP532 genotyped neutrophils than in wildtype genotyped neutrophils (1.20 vs 2.10, P = 0.05) (Fig. 3). Finally, the expression of some inflammatory related genes (IL-8, IL1β, MIP-2, COX-2, and SOD2) was decreased in IRAK-1SNP532 genotyped neutrophils. Conclusion 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 Figure 2