The role of osteopontin and tumor necrosis factor alpha receptor-1 in xenobiotic-induced cholangitis and biliary fibrosis in mice

Abstract

Proinflammatory and profibrotic cytokines such as osteopontin (OPN) and tumor necrosis factor-alpha receptor-1 (TNFR(1)) may be critically involved in the pathogenesis of cholangiopathies and biliary fibrosis. We therefore aimed to determine the role of genetic loss of either OPN or TNFR(1) in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice as a model of xenobiotic-induced sclerosing cholangitis with biliary-type liver fibrosis using respective knock-out mice. OPN and TNFR(1) knock-out mice were fed a 0.1% DDC-supplemented diet for 4 weeks and compared with corresponding wild-type (WT) controls. Liver morphology (H&E staining), serum markers of liver injury and cholestasis (ALT, AP, bilirubin), markers of inflammation in liver (CD11b and F4/80 immunostaining, mRNA expression of iNOS, MCP-1, IL-1beta, INF-gamma, TNF-alpha and OPN), degree of ductular reaction (immunohistochemistry with morphometric analysis and western blotting for cholangiocyte-specific marker keratin 19) and degree of liver fibrosis (Sirius-red staining, hepatic hydroxyproline content for quantification) were compared between groups. DDC feeding in OPN and TNFR(1) knock-out mice and respective WT controls resulted in comparable extent of liver injury, inflammatory response, ductular reaction and liver fibrosis. Our data indicate that genetic loss of neither OPN nor TNFR(1) significantly effects on the pathogenesis of DDC-induced sclerosing cholangitis, ductular reaction and resulting biliary fibrosis.

Figure 1. Genetic loss of osteopontin (OPN) and tumor necrosis factor-α receptor-1 (TNFR₁) has no impact on the composition and density of the inflammatory infiltrate in response to DDC feeding

Immunohistochemistry for F4/80 (A–D) and CD11b (E–H) in chow-fed wild type (WT/chow), WT 4 weeks DDC-fed (WT/DDC), OPN knock-out 4 weeks DDC-fed (OPN/DDC), and TNFR₁ knock-out 4 weeks DDC-fed (TNFR/DDC) mice. (B–D) Please note that despite OPN or TNFR₁ loss 4 weeks DDC-fed knock-out mice show a pronounced hepatic inflammation with no differences in regard to density and lobular distribution of the immunoreactivity. (E–H) In contrast to the observed F4/80 staining pattern the CD11b signal is concentrated and accentuated to portal fields and bile ducts again demonstrating no reduction of the inflammatory response in 4 weeks DDC-fed knock-out mice indicating a comparable inflammatory response compared to WT controls. pv, portal vein; bd, bile duct. Original magnification x20.

Figure 2. Genetic loss of osteopontin (OPN) and tumor necrosis factor-α receptor-1 (TNFR1) has no impact on hepatic F4/80 and iNOS mRNA expression

PCR for quantification of hepatic F4/80 and iNOS mRNA levels in chow-fed wild type (WT/chow), WT DDC-fed (WT/DDC), OPN knock-out DDC-fed (OPN/DDC), and TNFR1 knock-out DDC-fed (TNFR/DDC) mice using SYBR Green. Please note that despite OPN and TNFR1 loss, DDC-fed mice show comparable expression levels. n = 5 in each group; *P<0.05 chow-fed vs DDC-fed mice.

Figure 3. Genetic loss of osteopontin (OPN) and tumor necrosis factor-α receptor-1 (TNFR₁) has no impact on hepatic expression of vascular cell adhesion molecule (VCAM) in response to DDC feeding

Immunohistochemistry for VCAM (A–D) and VCAM protein measurement by Western blotting (E) in chow-fed wild type (WT/chow), WT 4 weeks DDC-fed (WT/DDC), OPN knock-out 4 weeks DDC-fed (OPN/DDC), and TNFR₁ knock-out 4 weeks DDC-fed (TNFR/DDC) mice. (B–D) Please note that despite OPN and TNFR₁ loss, DDC-fed mice show a comparable hepatic VCAM induction most pronounced in reactive bile ductules (indicated by asterisks), endothelial cells, and Kupffer cells. (E) Hepatic VCAM protein quantification by Western blotting in 4 weeks DDC-fed mice revealed significant induction in all genotypes. * indicates P<0.05 chow-fed mice vs. DDC-fed mice. pv, portal vein. Original magnification x20.

Figure 4. Genetic loss of osteopontin (OPN) and tumor necrosis factor-α receptor (TNFR₁) has no impact on the degree of ductular reaction in response to DDC-feeding

Immunohistochemistry of keratin 19 (K19) (A–D) and K19 protein measurement by Western blotting (E) in chow-fed wild type (WT/chow), WT 4 weeks DDC-fed (WT/DDC), OPN knock-out 4 weeks DDC-fed (OPN/DDC), and TNFR₁ knock-out 4 weeks DDC-fed (TNFR/DDC) mice. (B–D) Please note that OPN and TNFR₁ loss do not reduce ductular reaction in 4 weeks DDC-fed mice. (E) Hepatic K19 protein quantification by Western blotting in 4 weeks DDC-fed mice revealed significant and comparable induction in all genotypes. * indicates P<0.05 chow-fed mice vs. DDC-fed mice. (pv, portal vein. Original magnification x20.

Figure 5. Effect of osteopontin (OPN) and tumor necrosis factor-α receptor (TNFR₁) deficiency on the degree of α-SMA expression in DDC-fed mice

Immunohistochemistry for α-SMA (A-D) and α-SMA protein measurement by Western blotting (E) in chow-fed wild type (WT/chow), WT 4 weeks DDC-fed (WT/DDC), OPN knock-out 4 weeks DDC-fed (OPN/DDC), and TNFR₁ knock-out 4 weeks DDC-fed (TNFR/DDC) mice. (B–D) Please note that OPN and TNFR₁ loss do not reduce hepatic α-SMA expression in 4 weeks DDC-fed mice. (E) Hepatic α-SMA protein quantification by Western blotting in 4 weeks DDC-fed mice revealed significant induction in all genotypes which was more pronounced in DDC-fed OPN knock-out mice. * indicates P<0.05 chow-fed mice vs. DDC-fed mice. # indicates P<0.05 DDC-fed OPN knock-out mice vs. DDC-fed WT and DDC-fed TNFR₁ knockout mice. Ha, hepatic artery; pv, portal vein. Original magnification x20.

Figure 6. Genetic loss of osteopontin (OPN) and tumor necrosis factor-α receptor-1 (TNFR₁) does not decrease the degree of biliary fibrosis in DDC-fed mice

Sirius-red staining (A–D) and hepatic hydroxyproline measurement (E) in chow-fed wild type (WT/chow), WT 4 weeks DDC-fed (WT/DDC), OPN knock-out 4 weeks DDC-fed (OPN/DDC), and TNFR₁ knock-out 4 weeks DDC-fed (TNFR/DDC) mice. (B–D) Sirius-red staining showed comparable staining pattern in all DDC-fed genotypes. (E) Biochemical quantification of liver fibrosis by hepatic hydroxyproline measurement demonstrating that genetic loss of OPN or TNFR₁ does not reduce the degree of biliary fibrosis in DDC-fed mice. pv, portal vein. * indicates P<0.05 chow-fed mice vs. DDC-fed mice. Original magnification x20.