Insulin resistance and necroinflammation drives ductular reaction and epithelial-mesenchymal transition in chronic hepatitis C

Abstract

Objective: To study the mechanism(s) linking insulin resistance (IR) to hepatic fibrosis and the role of the epithelial component in tissue repair and fibrosis in chronic hepatitis C (CHC).

Design: Prospective observational study.

Setting: Tertiary care academic centre.

Patients: 78 consecutive patients with CHC.

Main outcome measures: IR, calculated by the oral glucose insulin sensitivity during oral glucose tolerance test; necroinflammatory activity and fibrosis, defined according to Ishak's score; steatosis, graded as 0 (<5% of hepatocytes), 1 (5-33%), 2 (33-66%) and 3 (>66%). To evaluate the role of the epithelial component in tissue repair and fibrosis, the expansion of the ductular reaction (DR) was calculated by keratin-7 (CK7) morphometry. Nuclear expression of Snail, downregulation of E-cadherin and expression of fibroblast specific protein-1 (FSP1) and vimentin by CK7-positive cells were used as markers of epithelial-mesenchymal transition in DR elements.

Results: IR, the degree of necroinflammation and expansion of the DR (stratified as reactive ductular cells (RDCs), hepatic progenitor cells and intermediate hepatobiliary cells according to morphological criteria) were all associated with the stage of fibrosis. Nuclear Snail expression, E-cadherin downregulation and vimentin upregulation were observed in RDCs. By dual immunofluorescence for CK7 and FSP1, the number of RDCs undergoing epithelial-mesenchymal transition progressively increased together with the necroinflammatory score. By multivariate analysis, total inflammation and insulin resistance were the only factors significantly predicting the presence of advanced fibrosis (Ishak score ≥3) and the expansion of RDCs.

Conclusion: This study indicates that IR is associated with the degree of necroinflammatory injury in CHC and contributes to hepatic fibrosis by stimulating the expansion of RDCs that express epithelial-mesenchymal transition markers.

Figures

Figure 1

Ductular reaction (DR) in patients with chronic hepatitis C (CHC) by immunohistochemistry for CK7 and its topographical association with α-smooth muscle actin (αSMA). (A) Normal liver, with bile ducts of regular morphology without any evidence of DR. (B,C) Evidence of DR in a patient with CHC. Reactive ductular cells appeared as CK7-positive cells with biliary phenotype arranged in irregularly shaped structures (thin arrows). Hepatic progenitor cells appeared as small, oval, or spindle-shaped cells with scant cytoplasm and oval nucleus, alone or in small clamps (arrowheads). Intermediate hepatobiliary cells appeared as cells with morphology and size intermediate between hepatocyte and cholangiocyte with a peculiar pattern of CK7 immunoreactivity, faint on the cytoplasm and reinforced at the plasma membrane (thick arrows); ×50 final magnification. (D) Double immunohistochemistry for CK7 (brown) and αSMA (red) in a patients with CHC, showing a close topographical association between these two elements at the interface between the portal tract and the hepatic parenchyma; ×100 final magnification.

Figure 2

Insulin sensitivity and the presence of intermediate hepatobiliary cells (IHBCs) in patients with chronic hepatitis C. In this ‘box and whiskers’ plot, the boxes identify the median, and the 25th and 75th centiles, whereas the ‘whiskers’ stretch to the 5th and 95th centiles. Note that the presence of IHBCs (grey box) was associated with reduced oral glucose insulin sensitivity (OGIS) levels (ie, higher degree of insulin resistance) (p=0.0012).

Figure 3

Association between the presence of intermediate hepatobiliary cells (IHBCs) and the grading and staging of liver injury in chronic hepatitis C. In this ‘box and whiskers’ plot, the boxes identify the median, and the 25th and 75th centiles, whereas the ‘whiskers’ stretch to the 5th and 95th centiles. Note that the presence of IHBCs (grey box) was associated with a higher grade of necroinflammatory liver injury (p=0.0001) and higher stage of fibrosis (p=0.0033).

Figure 4

Snail expression in ductular reaction cells. (A,B) Immunohistochemical staining for Snail. Snail is strongly expressed by reactive ductular cells in a tissue section from a patient with chronic hepatitis C and severe necroinflammatory damage (B) while it is absent in the normal bile ducts (A). Negative (white arrow) and positive (black arrow) nuclei are indicated; ×400 final magnification.

Figure 5

Vimentin expression in ductular reaction cells. (A–C) Dual immunofluorescence for CK7 (green, A) and vimentin (red, B) in a sample of a patients with chronic HCV-hepatitis, showing that reactive ductular cells express vimentin (merged, C), a late mesenchymal marker, thus indicating their engagement in a phenotypic transitional process; ×200 final magnification.

Figure 6

Downregulation of E-cadherin in ductular reaction cells. Dual immunofluorescence for CK7 (green, A, D) and E-cadherin (red, B, E) (C, F merged) in normal (A–C) and chronic hepatitis C livers (D–F) shows that ductular reaction partially lose E-cadherin expression (arrow, E) with respect to normal bile ducts (B); ×200 final magnification.

Figure 7

Expression of fibroblast specific protein 1 (FSP1) by ductular reaction (DR) cells. Dual immunofluorescence with FSP1 (A, green) and CK7 (B, red) showing co-localisation in DR structures of a chronic hepatitis C (CHC) sample with severe necroinflammatory damage (arrow, C, merged); ×200 final magnification. (D) Morphometric count of DR cells coexpressing FSP-1 significantly increases from mild to severe CHC. *p