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Matthias Unterberg, Maximiliane Kreuzer, Simon Thomas Schäfer, Zainab Bazzi, Michael Adamzik, Katharina Rump

• Sepsis, with a persistently high 90-day mortality of about 46%, is the third most frequent cause of death in intensive care units worldwide. Further understanding of the inflammatory signaling pathways occurring in sepsis is important for new efficient treatment options. Key regulator of the inflammatory response is the transcription factor NF\(\scriptsize k\)B. As we have recently shown, the -94 Ins/Del \(\textit {NFKB1}\) promoter polymorphism influences sepsis mortality. However, a molecular explanation is still missing. Thus, promoter activity might be varying depending on the \(\textit{NFKB1}\) genotype, explaining the genotype dependent mortality from sepsis, and one likely mechanism is the degree of promoter methylation. Therefore, we tested the hypothesis that NF\(\scriptsize κ\)B mRNA expression is regulated by promoter methylation in human cell lines and primary immune cell cultures. First, we examined the methylation of the \(\textit {NFKB1}\) promoter in U937, REH and HL-60 cells. In the promoter region of nt+99/+229 methylation in all analyzed cell lines was below 1%. Following incubation with bacterial cell wall components, no significant changes in the frequency of promoter methylation in U937 and REH cells were measured and the methylation frequency was under 1%. However, NF\(\scriptsize κ\)B1 mRNA expression was two-fold increased in U937 cells after 24 h incubation with LPS. By contrast, demethylation by 5-Aza-2’-deoxycytidine incubation enhanced \(\textit {NF\(\scriptsize K\)B1}\) expression significantly. In addition, we analyzed \(\textit {NFKB1}\) promoter methylation in primary cells from healthy volunteers depending on the \(\textit {NFKB1}\) –94 Ins/Del genotype. Methylation in the promoter region from nt+402 to nt+99 was below 1%. Genotype dependent differences occurred in neutrophil cells, where DD-genotype was significantly more methylated compared to II genotype at nt+284/+402. Besides in the promoter region from nt-227/-8 in ID-genotypes methylation of neutrophils was significantly decreased compared to lymphocytes and in II-genotypes methylation in neutrophils was significantly decreased compared to lymphocytes and monocytes. In addition, CHART-PCR showed that the hypomethylated promoter regions are highly accessible. Therefore we assume that the demethylated regions are very important for \(\textit {NFKB1}\) promoter activity.