Genetic variants of TRAF6 modulate peritoneal immunity and the risk of spontaneous bacterial peritonitis in cirrhosis: A combined prospective-retrospective study.

Alterations of the innate immunity contribute to the development of spontaneous bacterial peritonitis (SBP) in liver cirrhosis. Given its role in immune signaling, antimicrobial function, and macrophage differentiation, we hypothesized that genetic polymorphisms of TRAF6 modulate the risk of SBP. Thus, we determined theTRAF6 haplotype in 432 patients with cirrhosis and ascites using the haplotype-tagging single nucleotide polymorphisms rs331457 and rs5030419. In addition, peritoneal macrophages were immunomagnetically isolated and characterized. Overall, 122 (28%) patients had an episode of SBP. In the combined prospective-retrospective analysis the frequency of SBP differed between the four haplotypes (P = 0.014) and was the highest in 102 patients carrying the rs331457 but not the rs5030419 variant, when compared to other haplotypes (odds ratio 1.95 [1.22-3.12]) or to the wild-type (odds ratio 1.71 [1.04-2.82]). This association was confirmed in multivariate logistic regression (adjusted odds ratio 2.00 [1.24-3.22]) and in prospective sensitivity analysis (hazard ratio 2.09 [1.08-4.07]; P = 0.03). The risk haplotype was associated with lower concentrations of the immune activation marker soluble CD87 in ascitic fluid and with a decreased expression of IL-6 and CXCL8 in isolated peritoneal macrophages. In conclusion, genetic polymorphisms of TRAF6 are associated with decreased peritoneal immune activation and an increased risk of SBP.

Attenuated antigen-specific T cell responses in cirrhosis are accompanied by elevated serum interleukin-10 levels and down-regulation of HLA-DR on monocytes.

BACKGROUND: Advanced liver disease predisposes to bacterial translocation and endotoxaemia which can contribute to elevated circulating levels of IL-10 and down-regulation of MHC class II on antigen-presenting cells. We sought to evaluate antigen-specific T-cell responses toward common viral antigens in order to investigate defects in cellular immunity in cirrhosis. METHODS: Peripheral blood was obtained from 22 cirrhotic patients with systemic inflammation, 13 cirrhotic patients without systemic inflammation and 14 healthy controls. C-reactive protein was used as an indicator for systemic inflammation using a cut-off of 10 mg/l. Intracellular Th1 cytokines were quantified after T cell-stimulation with the viral peptides EBNA1 and BZLF1 or the bacterial superantigen SEB by flow cytometry. Serum levels of lipopolysaccharide-binding protein (LBP) and IL-10 were quantified by ELISA. RESULTS: Compared to healthy controls, patients with cirrhosis had higher circulating levels of LBP and IL-10, an expansion of peripheral blood CD14+ monocytes with low HLA-DR expression and an increased fraction of CD25-positive CD4+ and CD8+ T cells. These findings were most pronounced in cirrhotic patients with systemic inflammation but fell short of reaching statistical significance when comparing against cirrhotic patients without systemic inflammation. In the former group TNF-α production in CD4+ and CD8+ T cells was reduced after stimulation with SEB, whereas there was no significant difference between the total cohort of cirrhotic patients and controls. After stimulation with the overlapping peptide pools for viral antigens EBNA1 and BZLF1, the number of responding T cells and the amount of TNF-α or IFN-γ production did not differ between the three pre-defined groups. However, cirrhotic patients with null-responses to EBV peptides had significantly higher serum IL-10 levels than responders to EBV peptides. Furthermore, TNF-α production in responding T cells was attenuated in patients with a high frequency of CD14+ HLA-DR- monocytes. CONCLUSION: Our data suggest that bacterial translocation, endotoxaemia, inflammation and T cell activation in cirrhosis are accompanied by an increase in circulating anti-inflammatory cytokines, reduced monocytic MHC class II expression and attenuated cytokine production in T cells. These changes are likely to contribute to altered adaptive immune responses during infection or after vaccination.

NOD2 gene variants are a risk factor for culture-positive spontaneous bacterial peritonitis and monomicrobial bacterascites in cirrhosis.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is considered as result of bacterial translocation from the gastrointestinal lumen to the mesenteric lymph nodes and subsequent circulation. Variants of the NOD2 gene contribute to bacterial translocation and were associated with SBP in a recent study. METHODS: We determined common NOD2 variants by TaqMan polymerase chain reaction and analysed the ascitic fluid neutrophil count and bacterial culture results in 175 prospectively characterized hospitalized patients with decompensated cirrhosis who underwent diagnostic paracentesis in two German centres. RESULTS: Ten patients presented with culture-positive SBP, 19 with culture-negative SBP and six had bacterascites. Minor allele frequencies for R702W, G908R and 1007fs in subjects with sterile non-neutrocytic ascites were 3.2, 2.5 and 2.5% respectively. Patients with SBP [odds ratio (OR) 2.7; P=0.036], culture-positive SBP (OR 6.0; P=0.012) and bacterascites (OR 6.0; P=0.050) were more often carriers of NOD2 variants than patients with sterile non-neutrocytic ascites. The mutations 1007fs and G908R were associated with culture-positive SBP (P ≤ 0.005) and R702W with bacterascites (P=0.014). There was no significant association of NOD2 variants with culture-negative SBP (OR 1.6; P=0.493). In logistic regression, previous SBP, a higher model for end-stage liver disease (MELD) score and the presence of a NOD2 variant were independent predictors of ascitic fluid infection. The median survival was insignificantly shorter in patients with NOD2 variants (268 vs. 339 days; P=0.386). In patients without hepatocellular carcinoma at study entry (N=148), NOD2 was a predictor of survival after adjustment for the MELD score and age (hazard ratio 1.89; P=0.045). CONCLUSION: NOD2 variants increase the risk for culture-positive SBP and bacterascites in cirrhosis and may affect survival.

Homozygous carrier of the NOD2 1007fs frame-shift mutation presenting with refractory community-acquired spontaneous bacterial peritonitis and developing fatal pulmonary mucormycosis: A case report.

Genetic variants of the innate immune system contribute to episodes of spontaneous bacterial peritonitis (SBP) in patients with cirrhosis. We herein report the case of a patient with the homozygous nucleotide-binding oligomerization domain containing 2 (NOD2) frame-shift mutation 1007fs presenting with sepsis and community-acquired SBP by Escherichia coli. Secondary peritonitis, pancreatic ascites and malignant causes were excluded by extensive diagnostic work-up. First-line treatment with ceftriaxone was not successful despite in vitro sensitivity of the isolated strain. Despite prolonged second-line treatment with imipenem/cilastatin and intermittent ascites drainage, the ascitic fluid neutrophil count remained markedly elevated in this patient. In the course of the disease the patient developed pneumonia with identification of the typical hyphae of mucormycosis in the bronchoalveolar lavage and died of sepsis with multi-organ failure. On the basis of this observation, variants of the innate immunity have to be considered in therapy-refractory SBP, even when they are community-acquired and caused by cephalosporin-sensitive Enterobacteriaceae.