Biliary tract instillation of a SMAC mimetic induces TRAIL-dependent acute sclerosing cholangitis-like injury in mice

Abstract

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiopathogenesis characterized by fibrous cholangiopathy of large and small bile ducts. Systemic administration of a murine TNF-related apoptosis-inducing ligand (TRAIL) receptor agonist induces a sclerosing cholangitis injury in C57BL/6 mice, suggesting endogenous TRAIL may contribute to sclerosing cholangitis syndromes. Cellular inhibitor of apoptosis proteins (cIAP-1 and cIAP-2) are negative regulators of inflammation and TRAIL receptor signaling. We hypothesized that if endogenous TRAIL promotes sclerosing cholangitis, then cIAP depletion should also induce this biliary tract injury. Herein, we show that cIAP protein levels are reduced in the interlobular bile ducts of human PSC livers. Downregulation of cIAPs in normal human cholangiocytes in vitro by use of a SMAC mimetic (SM) induces moderate, ripoptosome-mediated apoptosis and RIP1-independent upregulation of proinflammatory cytokines and chemokines. Cytokine and chemokine expression was mediated by the non-canonical activation of NF-κB. To investigate whether downregulation of cIAPs is linked to generation of a PSC-like phenotype, an SM was directly instilled into the mouse biliary tree. Twelve hours after biliary instillation, TUNEL-positive cholangiocytes were identified; 5 days later, PSC-like changes were observed in the SM-treated mice, including a fibrous cholangiopathy of the interlobular bile ducts, portal inflammation, significant elevation of serum markers of cholestasis and cholangiographic evidence of intrahepatic biliary tract injury. In contrast, TRAIL and TRAIL-receptor deficient mice showed no sign of cholangiopathy following SM intrabiliary injection. We conclude that in vivo antagonism of cIAPs in mouse biliary epithelial cells is sufficient to trigger cholangiocytes apoptosis and a proinflammatory response resulting in a fibrous cholangiopathy resembling human sclerosing cholangitis. Therefore, downregulation of cIAPs in PSC cholangiocytes may contribute to the development of the disease. Our results also indicate that inhibition of TRAIL signaling pathways may be beneficial in the treatment of PSC.

Figure 1

cIAP-1 and cIAP-2 are downregulated in cholangiocytes of PSC patients. (a) Representative images of liver sections stained for cIAP-1 (left panel) and cIAP-2 (right panel) in patients with normal, NASH and PSC (stage IV) liver histology. Photomicrographs of small bile ducts (SBD) and large bile ducts (LBD) taken at × 20 magnification. The arrows point to the bile ducts. (b) Histological scoring for cIAP-1 in normal (5, 31), NASH (12, 120) and PSC (16, 131) patients and for cIAP-2 in normal (5, 31), NASH (12, 118) and PSC (16, 123) patients. Numbers in parentheses indicate total number of patients and total number of small and large bile ducts evaluated, respectively. Grade 0=no protein expression; grade 3=high protein expression. **P<0.01, ***P<0.001. (c) Representative images of liver sections stained for TWEAK (left panel) and Fn14 (right panel) from patients with normal or PSC (stage IV) liver histology. Photomicrographs of small bile ducts and large bile ducts taken at × 40 and × 20 magnification, respectively. The arrows point to the bile ducts. (d) Immunoblot analysis showing expression of cIAP-1, cIAP-2 and actin (loading control) in H69 cells treated with human recombinant TWEAK (100 ng/ml) for the indicated times

Figure 2

Normal human cholangiocytes are moderately sensitive to SM-induced, ripoptosome-mediated apoptosis. (a) Apoptosis assessed morphologically after DAPI staining (top panels) and by caspase 3/7 activation (bottom panels) in human cholangiocyte cell lines H69 and NHC, and the human breast cancer cell line MDA-MB-231, incubated for 24 h with or without (cnt) the SMAC mimetic TL32711 (1 μM), in the presence or absence of neutralizing antibodies against TNFα (1 μg/ml) or FasL (1 μg/ml), or recombinant TRAIL-R2:Fc (1 μg/ml). (b) TNFα and TRAIL gene expression analyzed by qPCR in H69, NHC and MDA-MB-231 cells incubated in the presence of TL32711 for the indicated times. Expression normalized to GAPDH RNA. Data are expressed as fold increase over control. Mean±s.e. are depicted from three independent experiments in H69 and NHC (c) H69 cells were treated with TL32711 and subjected to caspase 8 immunoprecipitation at the indicated time points. Affinity-purified proteins and total cell lysates (input) were analyzed by immunoblot for caspase 8, FADD and RIP1. (d) NHC cells were incubated with or without the TL32711 for 24 h, in the presence or absence of the pan-caspase inhibitor Q-VD-OPh (QVD, 10 μM) or the RIP1 kinase inhibitor necrostatin 1 (nec-1, 20 μM). Apoptosis was assessed morphologically after DAPI staining (left panel) and by caspase 3/7 activation (right panel). (e) H69 and RIP1^−/− H69 (clones 1 and 2) were incubated for 24 h in the presence or absence of TL32711. Apoptosis was assessed by DAPI staining (left panel) and by caspase 3/7 activation (right panel). In (a, d and e), mean±S.E. are depicted from three independent experiments performed in triplicate. *P<0.05, **P<0.005

Figure 3

SM induces upregulation of pro-inflammatory cytokines independent of RIP1. (a) IL6, IL8, MCP1/CCL2, IL1β, RANTES/CCL5 and TNFα gene expression analyzed by qPCR in H69 and RIP1^−/− H69 (clones 1 and 2) incubated in the presence of TL32711 for the indicated times. Expression normalized to 18S rRNA. (b) IL6, IL8 and MCP1/CCL2 gene expression analyzed by qPCR in H69 and NHC incubated in the presence of BV6 (5 μM) at the indicated times. Expression normalized to GAPDH RNA. Data are expressed as fold increase over control. Mean±S.E. are depicted from three independent experiments. *P<0.05, **P<0.005; NS, not significant

Figure 4

SM-induced upregulation of pro-inflammatory cytokines is mediated by activation of the non-canonical NF-κB pathway. (a) Immunoblot analysis showing expression of cIAP-1, cIAP-2, NIK, IκBα, PARP, IL-6 and actin (loading control) in H69 cells treated with TL32711 for the indicated times. (b) Immunoblot analysis showing expression of RIP1, NIK, NF-κB2, actin (loading control), IκBα and GAPDH (loading control) in H69 and RIP1^−/− H69 cells treated with TL32711 for the indicated times. (c) IL6, IL8 and MCP1/CCL2 gene expression analyzed by qPCR in NHC cell line transiently transfected with siRNA against NF-κB2 (p100) or scrambled siRNA for 48 h and incubated in the presence or absence (cnt) of TL32711 for 4 h. Efficiency of the knockdown was evaluated by immunoblot (insert). Data are expressed as fold increase over control. Mean±S.E. are depicted from six independent experiments. *P<0.05, **P<0.005

Figure 5

SM intrabiliary instillation in mice results in a transient PSC-like liver injury. (a) Representative images of liver sections from mice injected with a single dose of BV6 in the biliary tree and killed after 3, 5 or 21 days; and mice injected with saline and killed after 5 days. Hematoxylin–eosin (H&E)-staining, cholangiocyte staining by panCK IHC, collagen staining by picrosirius red-stain and α-smooth muscle actin (αSMA) IHC are depicted. Photomicrographs taken at × 20 magnification. The arrows point to the bile ducts; pv, portal vein. (b) Serum alanine aminotransferase (ALT), alkaline phosphatase (Alk Phos), total bile acids and total bilirubin measured in mice killed 3, 5 or 21 days after a single dose intrabiliary injection of BV6. Day 3: saline (n=4), BV6 (n=6); day 5: saline (n=8), BV6 (n=8); day 21: saline (n=6), BV6 (n=5). *P<0.05, **P<0.005. (c) Representative cholangiographic images of mouse livers 5 days after intrabiliary injection of saline or BV6. Enlargements of the indicated areas are shown next to the original picture. The arrow in the top magnification panel shows a stricture in an intrahepatic duct and adjacent dilatation. The bottom magnification panel shows damage/loss of small bile ducts. The gall bladder was resected to facilitate optimal visualization of the biliary tree during the cholangiograms

Figure 6

SM intrabiliary instillation in mice causes early cholangiocyte apoptosis and triggers periductal inflammation. (a) Representative images of TUNEL staining on fixed mouse liver specimens 12 h after intrabiliary injection of saline or BV6, 5 days after intrabiliary injection of BV6, or 4 days after systemic injection of MD5-1. The latter mice received two consecutive injections of MD5-1 (300 μg/mouse) on day 1 and day 4 and were killed at day 8. This experimental paradigm induces cholangiocyte apoptosis and was used as positive control. The black arrows point to the bile ducts; the white arrows point to TUNEL-positive/apoptotic cholangiocytes. Photomicrographs taken at × 40 magnification. (b) Serum ALT, alkaline phosphatase, total bile acids and total bilirubin measured in mice killed 5 days after intrabiliary instillation of BV6 or BV6+IDN-7314. BV6 (n=7); BV6+IDN-7314 (n=5). **P<0.005. (c) Representative images of H&E staining, collagen staining by picrosirius red-stain and immunohistochemical staining for α-smooth muscle actin and Mac-2/galectin3 (marker of phagocytically active macrophages) on liver sections of mice 5 days after intrabiliary instillation of BV6 or BV6+IDN-7314. The arrows point to the bile ducts. Photomicrographs taken at × 20 magnification; pv, portal vein. (d) Representative images of immunohistochemical staining for Mac-2/Galectin 3 (macrophages, top panel), mouse neutrophil differentiation antigen (neutrophils, middle panel) and CD3 (lymphocytes, bottom panel) on fixed mouse liver specimens 5 days after intrabiliary instillation of saline or BV6. Photomicrographs taken at × 20 magnification. Enlargements of the indicated areas are shown next to the original pictures; pv, portal vein. (e) Representative transmission electron microscopy (TEM) images of mouse liver specimens 5 days after intrabiliary injection of saline or BV6. The arrow points to a macrophage adjacent to the bile duct

Figure 7

Dr5^−/− and Trail^−/− mice are resistant to biliary injury following SM intrabiliary instillation. (a) In situ hybridization using mRNA detection probes for Trail and Dr5 performed on liver tissue sections of saline- or BV6-treated mice at day 5. The white arrows point to cholangiocytes. The orange arrows point to surrounding infiltrating cells (inflammatory cells, fibroblasts); BD, bile duct. (b) Representative images of liver sections from Dr5^/- (top) and Trail^−/− (bottom) mice injected with a single dose of BV6 or saline in the biliary tree and killed after 5 days. H&E staining, panCK IHC, picrosirius red-stain (collagen), α-smooth muscle actin IHC and Mac2 staining are depicted. Photomicrographs taken at × 20 magnification. The arrows point to the bile ducts; pv, portal vein. (c) Serum ALT, alkaline phosphatase, total bile acids and total bilirubin. Dr5^−/−: saline (n=5), BV6 (n=5); Trail^−/−: saline (n=4), BV6 (n=7). *P<0.05. (d) Il6, Il8, Il1β, Tnfα, Mcp-1/Ccl2, Cd68 and Coll-1α1 gene expression analyzed by qPCR in total liver lysates of wild-type, Dr5^−/− and Trail^−/− mice. Expression normalized to 18S rRNA. Data are expressed as fold increase over control. Mean±S.E. are depicted. *P<0.05, **P<0.005; NS, not significant

Figure 7

Dr5^−/− and Trail^−/− mice are resistant to biliary injury following SM intrabiliary instillation. (a) In situ hybridization using mRNA detection probes for Trail and Dr5 performed on liver tissue sections of saline- or BV6-treated mice at day 5. The white arrows point to cholangiocytes. The orange arrows point to surrounding infiltrating cells (inflammatory cells, fibroblasts); BD, bile duct. (b) Representative images of liver sections from Dr5^/- (top) and Trail^−/− (bottom) mice injected with a single dose of BV6 or saline in the biliary tree and killed after 5 days. H&E staining, panCK IHC, picrosirius red-stain (collagen), α-smooth muscle actin IHC and Mac2 staining are depicted. Photomicrographs taken at × 20 magnification. The arrows point to the bile ducts; pv, portal vein. (c) Serum ALT, alkaline phosphatase, total bile acids and total bilirubin. Dr5^−/−: saline (n=5), BV6 (n=5); Trail^−/−: saline (n=4), BV6 (n=7). *P<0.05. (d) Il6, Il8, Il1β, Tnfα, Mcp-1/Ccl2, Cd68 and Coll-1α1 gene expression analyzed by qPCR in total liver lysates of wild-type, Dr5^−/− and Trail^−/− mice. Expression normalized to 18S rRNA. Data are expressed as fold increase over control. Mean±S.E. are depicted. *P<0.05, **P<0.005; NS, not significant