Host response to translocated microbial products predicts outcomes of patients with HBV or HCV infection

Abstract

Background & aims: Chronic infection with hepatitis B or C virus (HBV or HCV) is a leading cause of cirrhosis by unknown mechanisms of pathogenesis. Translocation of gut microbial products into the systemic circulation might increase because of increased intestinal permeability, bacterial overgrowth, or impaired clearance of microbial products by Kupffer cells. We investigated whether the extent and progression of liver disease in patients with chronic HBV or HCV infection are associated with microbial translocation and subsequent activation of monocytes.

Methods: In a retrospective study, we analyzed data from 16 patients with minimal fibrosis, 68 with cirrhosis, and 67 uninfected volunteers. We analyzed plasma levels of soluble CD14 (sCD14), intestinal fatty acid binding protein, and interleukin-6 by enzyme-linked immunosorbent assay, and lipopolysaccharide (LPS) by the limulus amebocyte lysate assay, at presentation and after antiviral treatment.

Results: Compared with uninfected individuals, HCV- and HBV-infected individuals had higher plasma levels of LPS, intestinal fatty acid binding protein (indicating enterocyte death), sCD14 (produced upon LPS activation of monocytes), and interleukin-6. Portal hypertension, indicated by low platelet counts, was associated with enterocyte death (P=.045 at presentation, P<.0001 after therapy). Levels of sCD14 correlated with markers of hepatic inflammation (P=.02 for aspartate aminotransferase, P=.002 for ferritin) and fibrosis (P<.0001 for γ-glutamyl transpeptidase, P=.01 for alkaline phosphatase, P<.0001 for α-fetoprotein). Compared to subjects with minimal fibrosis, subjects with severe fibrosis at presentation had higher plasma levels of sCD14 (P=.01) and more hepatic CD14+ cells (P=.0002); each increased risk for disease progression (P=.0009 and P=.005, respectively).

Conclusions: LPS-induced local and systemic inflammation is associated with cirrhosis and predicts progression to end-stage liver disease in patients with HBV or HCV infection.

Figure 1

(A,B) Baseline levels of markers of enterocyte death and microbial translocation are elevated in both minimal and severe fibrosis. For I-FABP, n=66 for F5/F6, n=14 for F0/F1, and n=41 for uninfected. For LPS, n=68 for F5/F6, n=16 for F0/F1 and n=67 for uninfected. (C) LPS-induced monocyte activation, reflected by sCD14, is higher in subjects with severe fibrosis (n=68) compared to those without fibrosis (n=16) or uninfected individuals (n=65). (D) IL-6 levels are elevated regardless of fibrosis. n=66 for F5/F6, n=14 for F1/F2, and n=24 for uninfected. Horizontal lines indicate the median value and 5-95% range. P-values were calculated with the Mann-Whitney U test. The number subjects varied based on the volume of plasma available and the amount needed for each assay.

Figure 2

Baseline sCD14 levels correlate with plasma markers of hepatic inflammation (AST, ferritin), fibrosis (GGT, ALP, AFP) and poor synthetic function (direct bilirubin), n=84 for all analyses. Correlations among variables were evaluated using Spearman’s rank correlation.

Figure 3

Enterocyte death (reflected by I-FABP) is associated with portal hypertension (indicated by low platelet counts) and poor synthetic function (indicated by low albumin and high direct bilirubin and INR) at baseline (n=72) and after antiviral treatment (median 4.2 years, n=64). Correlations among variables were evaluated using Spearman’s rank correlation.

Figure 4

I-FABP and IL-6 levels decline with successful treatment of HBV and HCV infections. Subjects with HCV infection with SVR have a significant decrease in I-FABP and IL-6 levels at follow-up (n=17 for both), but subjects who fail to respond to therapy have persistent elevations in IL-6 (n=25) and I-FABP (n=26). I-FABP levels fell in HBV-treated subjects but IL-6 levels did not (n=17 for both). P-values were calculated using a paired t test.

Figure 5

High baseline plasma sCD14 levels predict poor clinical outcome in patients with HBV and HCV infections. For LPS and sCD14, n=43 for non-progressors and n=31 for progressors. n=67 for LPS and n=65 for sCD14 for uninfected individuals. For I-FABP and IL-6, n=40 for non-progressors and n=31 for progressors. n=41 for I-FABP and n=24 for IL-6 for uninfected individuals. Horizontal lines indicate the median value. P-values were calculated with the Mann-Whitney U test.

Figure 6

(A) CD14/CD68 staining of a cirrhotic liver. Brown indicates CD14 and red CD68. (B) CD14/CD68 staining of a non-cirrhotic liver. E. coli staining of cirrhotic (C) and non-cirrhotic (D) liver is indicated by brown. (E,F) Subjects with cirrhosis (n=49) have more CD14⁺ cells than those without (n=14). Those who progressed (n=24) have more CD14+ cells than those who did not (n=32), regardless of fibrosis. Horizontal lines indicate the median value and 5–95% range. P-values for comparisons between groups were calculated with the Mann-Whitney U test.