Cholangiocyte secretion of chemokines in experimental biliary atresia

Abstract

Biliary atresia (BA) is a disease of the newborn that results in obstruction of the biliary tree. The cause of BA remains unknown; however, recent studies using the murine model of biliary atresia have found that rotavirus infection of the biliary epithelial cell (cholangiocyte) triggers an inflammatory response. We hypothesized that rotavirus infection of cholangiocytes results in the release of chemokines, important mediators of the host immune response.

Methods: In vivo, Balb/c pups were injected with rhesus rotavirus (RRV) or saline, and, their extrahepatic bile ducts were microdissected 2, 5, 7, and 14 days after injection. Next, an immortalized cholangiocyte cell line (mCl) was incubated with RRV or serum-free media. Qualitative and quantitative chemokine assessment was performed using enzyme-linked immunosorbent assay, polymerase chain reaction, and immunohistochemistry.

Results: In vivo, increased levels of the chemokines macrophage inflammatory protein 2, monocyte chemotactic protein 1, KC and Regulated upon Activation, Normal T Expressed and Secreted were found in RRV-infected murine bile ducts. In vitro, infected mCl cells produced increasing amounts of these same chemokines in relation to dose and time.

Conclusion: These novel results suggest that chemokine expression by RRV-infected cholangiocytes may trigger a host inflammatory process that causes bile duct obstruction. Understanding how viral infection initiates this response may shed light on the pathogenesis of biliary atresia.

Figure 1. Chemokine levels (panels A–E) in extra-hepatic bile ducts following infection with RRV

Bile ducts were harvested from new born mice 2, 5, 7 and 14 days post infection with RRV and homogenized (n=3–7). Chemokine levels were determined by ELISA and expressed as means with standard error. Significantly higher levels of RANTES, KC, and MCP-1 were observed starting at day 5 and continuing through day 14 (p<0.05). RANTES and MCP-1 peaked at day 7 while KC peaked at day 5. MIP-2 was significantly higher at all time points tested (p<0.05), peaking at day 7.

Figure 2. Chemokine levels in the supernatant (panels A–E) of in vitro cholangiocytes infected with increasing amounts of RRV

In vitro, cholangiocyte cells were infected with increase amounts of RRV (n=3–5 tubes). Chemokine expression in the supernatants was determined by ELISA and expressed as means with standard error. Some chemokine expression was seen in the absence of infection in controls, though virus at MOIs of 50 and 100 resulted in significant increases in the quantity of MIP-2, RANTES and KC found in supernatant fluid from cholangiocytes (p<0.05) 24 hours after infection. MCP-1 was found to be significantly elevated over background expression at an MOI of 100 (p<0.05). Experiment conditions repeated 3 times.

Figure 3. Chemokine levels over time in the supernatant (panels A–D) of in vitro cholangiocytes infected with RRV

Cholangiocytes were exposed to RRV at an MOI of 100 for 1 hour, washed and let incubated for 6, 12, 24 and 48 hour periods (n=3–7). Chemokine expression in the supernatants was determined by ELISA and expressed as means with standard error. There was no difference in chemokine levels expressed by the cholangiocytes at 6 and 12 hours post infection. Conversely at 24 and 48 hours post infection significantly more of all four chemokines were observed (p<0.05).

Figure 4. A. MIP-2 mRNA in the extra-hepatic biliary tract harvested from mice infected with RRV

RT-PCR on mRNA extracted from samples harvested at day 2, 5 and 7 days post RRV infection of new born mice for MIP-2 and Beta-actin. mRNA for MIP-2 was only detectable on the infected samples of day 5 and 7 post infection. B. In vitro expression of MIP-2 mRNA in cholangiocytes after RRV infection. mRNA from cholangiocytes infected with RRV at an MOI of 100 for 1 hour. After 24 hours of incubation MIP-2 was unregulated in the infected cells.

Figure 5. Immunohistochemistry for MIP-2 in the hepatobiliary system following RRV infection

Hepatobiliary tissue was harvested from mice on day 2, 7 and 14 post infection with RRV and stained with an anti-mouse MIP-2 anti-body. Positive staining, small arrows () were observed in liver samples harvested from mice on post infection day 2 and day 7. Large arrow () indicates portal vein.