Bacterial translocation in liver failure a possible mechanism of impaired innate immune responses and immuneparesis?

Background: Monocyte (Mo) dysfunction is associated with susceptibility to infection in acute-on-chronic liver failure (ACLF). Possible mechanisms include tolerance to persistent microbial stimulation due to increased circulating levels of bacterial products as a consequence of (i) increased translocation of gut-derived bacteria in association with intestinal barrier dysfunction and (ii) impaired hepatic clearance of these microbial ligands. We sought to examine monocyte innate responses to microbial challenges through diverse Toll-like receptor signalling cascades, and the relationship to circulating levels of bacterial DNA (bactDNA). Methods: Patients with ACLF (n=18), cirrhosis (CLD;n=4) and healthy controls (HC;n=9) were studied. Whole blood (WB) was obtained for bactDNA and PBMCs. In a subset of patients undergoing orthotopic liver transplantation (n=8) WB was sampled concurrently from portal (PV), hepatic veins (HV), and peripheral artery for ‘cross-liver’ bactDNA analysis. DNA extraction was performed using QiAMP DNA extraction kit, followed by real-time PCR with a TaqMan probe targeting a 380bp region of bacterial 16s rDNA for bactDNA relative quantification, expressed in pg/mL of WB. In a subset of 7 ACLF, 5 CLD and 9 HC, PBMC production of TNF-α and IL-6 was measured by Toll-like receptor (TLR) 2, 4 and 9 ligand stimulation (5μg/mL, 100ng/mL and 10μg/mL, respectively; InvivoGen™). Results are expressed as median percentage of CD14+ Mo. Results: BactDNA levels in ACLF (2.24 IQR 1.33; p=0.0002) and CLD (1.94 IQR 1.34; p&lt;0.04) were significantly elevated compared to HC (1.08 IQR 0.59) (see figure). Paired cross-liver bactDNA levels (PV vs. HV) demonstrated no significant difference (1.043 vs. 1.291; p=0.11) in ACLF or CLD. BactDNA levels were also similar in peripheral artery and HV (1.162 vs 1.29; p=0.91).TLR stimulation of PBMCs in ACLF patients revealed suppression of TNF-α and IL-6 production. TNF-α production was supressed in response to TLR2 (16.8% IQR 21.6 vs 1.2% IQR 5.5 (p=0.0002)) in HC vs ACLF as was IL-6 ((4.7% IQR 10.3 vs 0.4% IQR 0.39 (p=0.002)). TNF-α production was also significantly reduced in response to TLR4 (52.9% (HC) vs 8.7% (ACLF); p&lt;0.0001) and to TLR9 (8.2% (HC) vs 1.67% (ACLF); p=0.01). Discussion: Our data clearly link attenuated monocyte responses to microbial challenge on diverse signalling cascades with elevated levels of circulating bactDNA in patients with ACLF. Persistent exposure to such bacterial products due to translocation from the gut and other epithelial beds, in combination with a failure of hepatic clearance, might thus explain endotoxin tolerance, immuneparesis and susceptibility to infection in ACLF.<br/