Profibrogenic chemokines and viral evolution predict rapid progression of hepatitis C to cirrhosis

Abstract

Chronic hepatitis C may follow a mild and stable disease course or progress rapidly to cirrhosis and liver-related death. The mechanisms underlying the different rates of disease progression are unknown. Using serial, prospectively collected samples from cases of transfusion-associated hepatitis C, we identified outcome-specific features that predict long-term disease severity. Slowly progressing disease correlated with an early alanine aminotransferase peak and antibody seroconversion, transient control of viremia, and significant induction of IFN-γ and MIP-1β, all indicative of an effective, albeit insufficient, adaptive immune response. By contrast, rapidly progressive disease correlated with persistent and significant elevations of alanine aminotransferase and the profibrogenic chemokine MCP-1 (CCL-2), greater viral diversity and divergence, and a higher rate of synonymous substitution. This study suggests that the long-term course of chronic hepatitis C is determined early in infection and that disease severity is predicted by the evolutionary dynamics of hepatitis C virus and the level of MCP-1, a chemokine that appears critical to the induction of progressive fibrogenesis and, ultimately, the ominous complications of cirrhosis.

Fig. 1.

Long-term clinical course and evolution of HCV in two representative patients followed from the time of infection, one with slowly progressive (patient 1) and one with rapidly progressive hepatitis leading to liver-related death (patient 5). Patient numbers are the same as in Table S1. (A and C) Long-term clinical course of slowly and rapidly progressive chronic hepatitis C. The blue areas indicate ALT levels. The red line indicates the titer of serum HCV RNA, as determined by real-time PCR (TaqMan), on a logarithmic scale. The orange horizontal bars indicate antibodies to HCV detected by third-generation enzyme immunoassay (EIA3). “Tx” denotes the time of blood transfusion. The arrows indicate the time and the results of liver biopsies. “CH” denotes chronic hepatitis and “HCC” indicates hepatocellular carcinoma. In patient 5, the diagnosis of HCC was obtained at autopsy. (B and D) Number of viral strains and diversity (genetic distance among variants) of HCV within the 27 amino acids of the HVR1. The vertical bars indicate the number and the proportion of identical clones. The dominant viral variant found in each patient at the first time point is indicated in blue; other variants are indicated by different colors. The same color denotes identity between viral variants detected at different time points within the same patient and not between different patients. The genetic diversity (black line) was calculated by mean p-distance from the predicted amino acid sequences obtained from each sample (Materials and Methods).

Fig. 2.

Differences in serum HCV-RNA titer and ALT and cytokine/chemokine levels in patients with slowly or rapidly progressive chronic hepatitis C, followed from the time of infection. (A–E) Each panel shows the linearly interpolated median curve (Left) and boxplots (Right) of the median, interquartile range, and minimum and maximum values observed during the early acute phase (from the time of infection to the ALT peak), late acute phase (from the ALT peak to 6 mo from infection), and the chronic phase (from 6 mo to 7 y from infection) of HCV infection. *P < 0.05 by Mann–Whitney U test between slow and rapid progressors at different time points.

Fig. 3.

Q-mode Principal Component Analysis showing the relations among the six patients resulting from the median levels of ALT, HCV RNA, and five cytokines/chemokines (IFN-γ, MIP-1β, IL-8, MCP-1, and IP-10) observed during the acute phase (0–6 mo from infection) and the chronic phase (6 mo–7 y from infection) of the disease. A complete separation of low (blue) and rapid (red) progressors is given by the first principal component, which accounts for nearly 50% of the total variance of the 14 variables included in the analysis. The map of the variables points to an increase of IFN-γ and MIP-1β in slow progressors and of MCP-1, IL-8, and IP-10 in rapid progressors in both the acute and the chronic phase. Higher ALT and HCV-RNA levels are also associated with rapid progressors, although in HCV RNA only during the acute phase. Hierarchical clustering (Inset) using the same set of variables confirmed the separation between slow and rapid progressors.

Fig. 4.

Viral evolution in slow and rapid progressors during the acute and chronic phase of hepatitis C. (A) Genetic variation (diversity) of HCV within the HVR1 in slow and rapid progressors during the entire course of the disease. Median values of amino acid sequence diversity, as measured by the mean P-distance among all sequences within each sample. (B) Median values of viral divergence, as measured by the mean P-distance relative to all of the sequences detected in the first PCR-positive sample (founder viral population). Circles denote the mean values for each individual patient. A difference in HVR1 divergence between slow and rapid progressors was seen soon after seroconversion, as well as at 1 y post infection (P = 0.028 by Mann–Whitney U test when the data from these two times points were combined). (C and D) Time-structured Bayesian molecular clock phylogenies of all viral nucleotide sequences of the E1/E2 region, including the HVR1, in two representative patients, one with slowly progressive (C) and one with rapidly progressive chronic hepatitis C (D) leading to end-stage liver disease within 7 y from infection. Patient numbers are the same as in Table S1. In both trees, a red arrow indicates the lineage bottleneck around 1 y post infection. Asterisks along the backbone branches are posterior probabilities >0.95. The shaded areas indicate the acute phase (0–6 mo) of infection. (E) Mean number of synonymous and nonsynonymous substitutions per site in slow and rapid progressors during the first 7 y of infection. The bars indicate the means for all patients for each group, and the results are presented separately for the E1, HVR1, and E2 regions. Circles denote the mean values for each individual patient. (F) Site-by-site analysis of positive selection, as determined by two-parameter fixed-effects likelihood analysis. The blue box denotes the E1 region, the yellow the HVR1, and the pink the E2. Each bar represents an individual patient who showed positive selection at each codon position. The number of codons under positive selection was significantly more concentrated in the HVR1 in slow progressors (9 of 12) than in rapid progressors (3 of 10) (P < 0.01 by Kolmogorov–Smirnov two-sample test). In slow progressors, the analysis at 1 y and during 5–7 y was extended to only two patients because in patient 3 PCR amplification of the E1/E2 region was negative due to undetectable or very low levels of HCV RNA; in rapid progressors, the analysis at 2–4 y and at 5–7 y was also limited to two patients because in patient 6, who died 4 y after transfusion, the amount of serum during the last 2 y was not sufficient for PCR amplification with the set of primers from the E1/E2 region.