Elsevier

Journal of Hepatology

Volume 55, Issue 5, November 2011, Pages 1010-1016
Journal of Hepatology

Research Article
Toll-like receptor (TLR) 2 promoter and intron 2 polymorphisms are associated with increased risk for spontaneous bacterial peritonitis in liver cirrhosis

https://doi.org/10.1016/j.jhep.2011.02.022Get rights and content

Background & Aims

Toll-like receptor (TLR) 2 and nucleotide-binding oligomerisation domain (NOD) 2 recognize distinct pathogen-associated molecular patterns (PAMS) on the cell surface and in the cytoplasm, respectively. Since they may contribute to susceptibility to spontaneous bacterial peritonitis (SBP), we studied the effects of TLR2 gene variants on susceptibility for SBP in relation to the previously reported NOD2 alleles.

Methods

Overall, 150 patients with liver cirrhosis and ascites were genotyped for TLR2 gene variants −16934 (rs4696480), Arg753Gln (rs5743708), Pro631His (rs5743704) and the TLR2 GT microsatellite polymorphism in intron 2. Patients were monitored for SBP over two years. TLR2 SNPs were identified by hybridization probe assays on a LightCycler system. Numbers of GT repeats were determined with an ABI310 sequencer and Genescan Analysis 2.1 software.

Results

Fifty two patients (35%) had SBP. Unlike the TLR2 Arg753Gln and Pro631His mutations, SBP was significantly more frequent in patients with the TLR2 −16934 TT genotype (38.5% vs. 15.3%; p = 0.002) and in carriers with two long tandem GT repeat alleles (>20) (53.8% vs. 25.5%; p = 0.001). A multivariate analysis confirmed TLR2 GT microsatellite polymorphism (OR = 3.8, p = 0.002) and NOD2 variants (OR = 3.3, p = 0.011) as independent predictors of SBP, and the simultaneous presence of both risk factors indicated a particularly high risk for SBP (OR = 11.3, p = 0.00002).

Conclusions

Analogous to NOD2 risk variants, TLR2 polymorphisms indicate increased susceptibility toward SBP in cirrhotic patients with ascites, and the combination of the TLR2 GT microsatellite polymorphism with at least one NOD2 risk variant enables improved identification of patients with a high risk for SBP.

Introduction

Spontaneous bacterial peritonitis (SBP) is one of the most frequent bacterial infections in patients with decompensated cirrhosis and ascites [1]. SBP is associated with in-hospital mortality rates up to 15%, and patients who recover from a first episode of SBP still have significantly reduced survival as compared to cirrhotic patients without prior SBP [2].

The pathomechanisms underlying SBP are only poorly understood. It is generally assumed that bacteria migrate from the intestinal lumen to the mesenteric lymph nodes because of delayed bowel transit and increased intestinal permeability, a process termed bacterial translocation [3], [4]. In addition to increased bacterial translocation, immunodeficiency associated with liver cirrhosis facilitates survival of bacteria in the mesenteric lymph nodes. For instance, functions of peritoneal macrophages, which contribute to the antimicrobial defense in ascites by producing proinflammatory cytokines, are impaired in SBP [5], [6].

Bacteria trigger pattern recognition receptors, which comprise various members of the NOD (nucleotide-binding oligomerisation domain) protein family and Toll-like receptor (TLR) proteins. NODs and TLRs 1, 2, 4 and 6 are expressed in macrophages, monocytes, and neutrophils and recognize a broad range of microbial components, e.g. the muramyl dipeptide (MDP) component of peptidoglycan, lipopolysaccaride (LPS) and lipoproteins. Activation of macrophages via NOD and TLR molecules plays a pivotal role in the initiation of an efficient antimicrobial defense.

Genetic variation in the genes coding for pattern recognition receptors is probably an important factor to determine individual susceptibility to infection. For example, the TLR4 D299G polymorphism has been found to predispose to infections in cirrhotic patients [7]. Impaired mucosal barrier function in Crohn’s disease has been linked to genetic variants of the NOD2 gene [8], and recently, we have reported variants in the NOD2 gene to increase the risk to develop SBP [9].

NOD2 and Toll-like receptors (TLR) interact with each other in a synergistic fashion to trigger release of proinflammatory cytokines [10], [11], [12]. Polymorphisms in the TLR2 gene may be particularly important in SBP, because Gram-positive organisms, the cognate ligands for TLR2, have nowadays become a major cause of peritoneal fluid infection [13], [14], which in our hospital accounted for 38% of peritoneal fluid infections between 2004 and 2010. Furthermore, NOD2 co-activation modulates TLR 2-mediated cytokine responses which thus may be affected by the NOD2 genotype [15]. A great variety of polymorphisms exist in the TLR2 gene, which may be linked to differential disease susceptibility: a single nucleotide polymorphism (SNPs) each at position −16934 A>T (rs4696480) and at positions +2258 G>A (rs5743708, Arg753Gln) and +1892 C>A (rs5743704, Pro631His) as well as a short tandem GT repeat polymorphism in intron 2 of the TLR2 gene. For instance, Veltkamp et al. suspected an association between the number of GT repeats in intron 2 and susceptibility to sarcoidosis, probably reflecting individual variation in the production of inflammatory cytokines such as TNF-alpha, IL-6 and IL-12 [16]. Finally, TLR2 gene variants have been reported to predispose individuals to infection with Gram-positive agents and tuberculosis [17], [18]. In this study, we tested the hypothesis that polymorphisms in the TLR2 gene might be linked to individual susceptibility for SBP in patients with liver cirrhosis and ascites.

Section snippets

Patient cohort

Between May 2006 and October 2007, we had prospectively recruited 150 consecutive patients with liver cirrhosis and ascites who were admitted for hospitalization to the Department of Internal Medicine I, University Hospital Bonn, Germany. The individuals in this study were identical to the cohort previously studied to identify NOD2 variants as a genetic risk factor for SBP in cirrhosis [9]. These patients had been monitored for the presence of SBP over a median observation period of 155 days

Patients‘characteristics and SBP frequency

Liver cirrhosis was caused by alcohol abuse in 94 patients (63%), viral hepatitis in 26 patients (17%), autoimmune liver disease in 13 (9%), and other etiologies in 17 patients (11.3%). All patients were Caucasians of German descent. They had a median age of 57 years, and 108 were males (72%). 141 patients were classified as Child-Pugh class B or C, the median MELD score was 16. Further characteristics of the patients are summarized in Table 1.

In 52 patients SBP was diagnosed. However, a

Discussion

In this study, we identified genetic polymorphisms in the TLR2 gene as novel susceptibility factors for spontaneous bacterial peritonitis in cirrhotic patients with ascites. In detail, we observed an increased incidence of SBP in patients with the TLR2 gene variants −16934 TT and/or two long alleles of the TLR2 GT microsatellite polymorphism. If compared as single factors, the TLR2 GT microsatellite polymorphism correctly predicted patients developing SBP more frequently than either the TLR2

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgments

This study was funded by the Deutsche Krebshilfe (107865) to HDN and US, the SFB TR57 TP01 & TP12 of the Deutsche Forschungsgemeinschaft to FL, JN and US, HOMFOR (Saarland University) to FG and the H.W. & J. Hector Foundation M42 to JN.

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