A TLR9 promoter polymorphism modulates the monocytic immune responses in decompensated cirrhosis

Background: The presence of circulating and peritoneal fragments of bacterial DNA alongside increased pro-inflammatory and anti-inflammatory cytokines is a hallmark finding in advanced cirrhosis and acute-on-chronic liver failure (ACLF). Polymorphisms in the promoter region of toll-like receptor 9 (TLR9), an endosome receptor recognizing unmethylated CpG-rich bacterial DNA, may contribute to hyper-inflammatory immune responses in advanced cirrhosis.

Aims: To investigate the consequences of the single nucleotide polymorphism rs5743836 in the promoter region of TLR9 on inflammatory responses in monocytes and macrophages.

Methods: Whole blood from patients with decompensated cirrhosis was genotyped by endpoint Taqman-PCR. Monocyte and macrophages were isolated from patient blood and ascites using density gradient centrifugation followed by immunomagnetic sorting. Lipopolysaccharides (LPS) and CpG-rich single stranded DNA oligonucleotides (CpG-ODN) were used for in vitro stimulation of CD14-positive cells. mRNA expression was quantified using relative RT-qPCR.

Results: The minor allele frequency of the polymorphism in patients with decompensated cirrhosis was comparable to the general population (0.14). TLR9 expression in monocytes from patients carrying the TLR9 promoter variant tended to be lower than in wild-type patients (p = 0.09) but significantly increased after In vitro stimulation with LPS (p = 0.027). CpG-ODN-induced IL6 (p = 0.003) and IL10 (p = 0.030) mRNA expression in monocytes and IL10 mRNA expression in macrophages (p = 0.05) was increased in patients with the TLR9 promoter polymorphisms as compared to the wild type.

Conclusions: Patients with cirrhosis carrying the frequent TLR9 promoter variant show hyper-inflammatory responses after stimulation with CpG-rich bacterial DNA. Studies are ongoing to investigate whether this association translates into increased complications in clinical practice.