Schistosome-induced cholangiocyte proliferation and osteopontin secretion correlate with fibrosis and portal hypertension in human and murine schistosomiasis mansoni

Abstract

Schistosomiasis is a major cause of portal hypertension worldwide. It associates with portal fibrosis that develops during chronic infection. The mechanisms by which the pathogen evokes these host responses remain unclear. We evaluated the hypothesis that schistosome eggs release factors that directly stimulate liver cells to produce osteopontin (OPN), a pro-fibrogenic protein that stimulates hepatic stellate cells to become myofibroblasts. We also investigated the utility of OPN as a biomarker of fibrosis and/or severity of portal hypertension. Cultured cholangiocytes, Kupffer cells and hepatic stellate cells were treated with soluble egg antigen (SEA); OPN production was quantified by quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and ELISA; cell proliferation was assessed by BrdU (5-bromo-2'-deoxyuridine). Mice were infected with Schistosoma mansoni for 6 or 16 weeks to cause early or advanced fibrosis. Liver OPN was evaluated by qRTPCR and immunohistochemistry (IHC) and correlated with liver fibrosis and serum OPN. Livers from patients with schistosomiasis mansoni (early fibrosis n=15; advanced fibrosis n=72) or healthy adults (n=22) were immunostained for OPN and fibrosis markers. Results were correlated with plasma OPN levels and splenic vein pressures. SEA-induced cholangiocyte proliferation and OPN secretion (P<0.001 compared with controls). Cholangiocytes were OPN (+) in Schistosoma-infected mice and humans. Liver and serum OPN levels correlated with fibrosis stage (mice: r=0.861; human r=0.672, P=0.0001) and myofibroblast accumulation (mice: r=0.800; human: r=0.761, P=0.0001). Numbers of OPN (+) bile ductules strongly correlated with splenic vein pressure (r=0.778; P=0.001). S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in liver and blood correlate with fibrosis stage and portal hypertension severity.

Keywords: Schistosomiasis mansoni; Symmers' fibrosis; cholangiocyte; ductular proliferation; osteopontin; portal hypertension.

Figure 1. SEA induces cholangiocyte proliferation and OPN secretion

(A) Cellular proliferation assay (BrdU incorporation). Mouse cholangiocyte cell line 603B and primary human cholangiocytes were treated with 10 μg/ml S. mansoni SEA or 0.0001 μg/ml LPS (control) for 24 h. (B) OPN mRNA expression in mouse cholangiocytes (603B) treated with SEA or control for 2, 6 and 12 h. Results were normalized by mRNA expression at each time point control; means ± S.E.M. are displayed. (C) Quantification of OPN in the conditioned medium of murine cholangiocytes treated with SEA or control (ELISA). Medians are displayed. No difference was observed at 24 h in (B) and (C). (D) OPN mRNA expression in normal human cholangiocyte cell line H69 treated with SEA or control for 12 h. No significant differences were observed at 2, 6 and 24 h. Results were normalized by mRNA expression at each time point control; means ± S.E.M. are displayed. (E) Double immunostaining for OPN (brown) and the cholangiocyte marker K19 (green) in human (left) and murine (right) schistosomiasis. Final magnification ×200. *P<0.05; **P<0.005.

Figure 2. OPN is up-regulated in murine schistosomiasis and correlates with fibrogenesis

(A–C) αSMA immunostaining (A), Picro Sirius Red staining (B) and OPN immunostaining (C) on liver sections from a representative uninfected control mouse (left), from a representative mouse 6 weeks post-infection (low fibrosis; middle) and from a representative mouse 16 weeks post-infection (severe fibrosis; right). Final magnification ×100 (A and B) or ×200 (C). (D) Serum OPN levels in murine schistosomiasis (ELISA; normal (nrl) n=4, 6 weeks n=3, 16 weeks n=6). Medians are displayed; #P<0.05, ##P<0.005 compared with nrl; §P<0.05, §§P<0.005 compared with 6 weeks. (E) Pearson's correlation and linear regression showing strong correlation between liver/blood OPN levels and fibrogenesis.

Figure 3. OPN is up-regulated in human schistosomiasis and correlates with fibrogenesis

(A–C) αSMA immunostaining (A), Picro Sirius Red staining (B) and OPN immunostaining (C) on liver sections from a representative uninfected healthy individual (left), from a representative patient with HI schistosomiasis (middle) and from a representative HS patient (right). Final magnification ×100 (A and B) or ×200 (C). (D) Plasma OPN levels in human schistosomiasis (ELISA). Medians are displayed; #P<0.05, ##P<0.005 compared with uninfected controls (nrl); §§P<0.005 compared with HI. (E) Spearman's correlation (r_s) showing strong correlation between liver/blood OPN levels and fibrogenesis.

Figure 4. OPN correlates with severity of portal hypertension in human schistosomiasis

(A) Spearman's correlation (r_s) showing a strong significant correlation between the number of OPN (+) bile ducts and the severity of portal hypertension in a subgroup of patients with HS schistosomiasis (n=15).

Figure 5. Macrophages and myofibroblasts/HSCs produce and respond to OPN in schistosomiasis

Double immunostaining for OPN (left panels) or one of its receptors (CD44; right panels) and a macrophage marker (F4-80 for mice, CD68 for humans) or myofibroblast/HSC markers (αSMA and desmin) in murine (A) and human (B) schistosomiasis mansoni, showing that those cells produce and respond to OPN (final magnifications: ×400, inset ×1000).