Biliary atresia (BA) is a rapidly progressive fibroinflammatory and obliterative disease of the extrahepatic bile ducts that occurs in 1 in 8000 to 15,000 patients (1). The etiology is unknown, although likely involves an environmental insult such as infection or toxic exposure combined with a genetic predisposition (2). Injection of BALB/c mice with rhesus rotavirus (RRV) within 24 hours of birth results in a BA-like syndrome, lending support to an infectious cause (3). Approximately 20% of patients with BA have a variant called syndromic BA characterized by laterality defects (also known as embryonic BA or BA with splenic malformation syndrome) (4–7). There are case reports of patients with syndromic BA having abnormal cilia, suggesting that cilia may be involved in the pathogenesis of the disease (5–7).
Primary cilia are nonmotile organelles that serve as mechanosensors and chemosensors (8–10). They are found on the apical surfaces of most cells, including cholangiocytes, but are notably absent on hepatocytes (11). In addition, they have roles in cell proliferation, cell differentiation, organogenesis, axis orientation during development, and tissue remodeling (8). Ciliary abnormalities have been implicated in many human diseases such as polycystic kidney disease, central nervous system malformations, retinal degeneration, situs defects, polydactyly, and skeletal and gonadal malformations (8,9,12–14). We recently showed that the intrahepatic cholangiocytes of patients with both syndromic and nonsyndromic BA have primary cilia that are decreased in number and are morphologically abnormal (4). In addition, Hartley et al (15) recently found decreased staining for the ciliary protein fibrocystin/polyductin in the livers of children with nonsyndromic BA.
We hypothesized that if cilia abnormalities were part of the pathophysiology of BA, these defects would be prominent in the extrahepatic biliary ducts of human BA livers and would appear early after infection in the mouse RRV model. We therefore examined RRV-infected cholangiocytes in culture, RRV-infected neonatal BALB/c mouse livers at various stages after infection, and human BA extrahepatic biliary remnants for the presence of abnormal cilia.
Formalin-fixed, paraffin-embedded extrahepatic bile duct remnants from hepatoportoenterostomies of patients with BA were obtained with institutional review board approval from The Children's Hospital of Philadelphia. A sample of normal extrahepatic bile duct from an autopsy sample was obtained as a control. For the mouse samples, neonatal mice were injected with RRV and saline within 24 hours of birth, and livers were removed at days 3, 7 to 8, and 12 to 14 (16,17). All of the mice studied at days 7 to 12 after injection were jaundiced. Jaundice in this model is associated with bile duct obstruction and the development of BA in >90% of cases (18). Blocks including the right lobe of the liver, extrahepatic ducts, and a small piece of bowel were dissected, fixed with 10% formalin, arranged in a similar orientation, and paraffin embedded. A liver from a mouse approximately 12 hours after birth was similarly fixed and embedded. Bile duct ligation was carried out in adult mice as described (19); mice were euthanized at 14 days after surgery and livers were removed and processed for immunofluorescence. Animal experiments were carried out with local Institutional Animal Care and Use Committee approval.
Primary Cholangiocytes Isolation and RRV Infection
Neonatal extrahepatic bile ducts were isolated from 0- to 3-day-old pups. Ducts were carefully dissected away from stroma and surrounding cells, then imbedded and cultured in a thick collagen gel (2 mg/mL) for 3 to 4 weeks with cholangiocyte media (20). The cells were then split and cultured on thin (1 mg/mL) collagen-coated dishes. Cells were used for experiments after 2 weeks. Neonatal cholangiocytes were infected with RRV at an multiplicity of infection of 5 for 90 minutes, and then incubated in serum-free media for 12 hours before fixation and staining (21,22).
Tissue samples and cells were stained with antibodies against the cholangiocyte marker K19 (1:10, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), the primary cilia marker acetylated α tubulin (1:5000; Sigma, St Louis, MO), and with the nuclear stain 4,6-diamidino-2-phenylindole. RRV-infected cells in culture were also stained with anti-rotavirus antibody (1:1000; Abcam, Cambridge, MA). Confocal pictures were obtained using a Zeiss LSM 710 microscope and Zen software. Image composites and z-stacks were made using FIJI software.
For all of the samples (tissue and cells in culture), cilia and nuclei were manually counted. For mouse liver analyses, 3 to 5 mice were evaluated for each condition and 2 to 9 high-power fields were assessed per liver, depending on the number of bile ducts visible. Cell data are from 3 independent experiments. Data represent the mean ± standard deviation. Normality tests were performed and the results were subjected to 2-tailed, paired (for cell culture studies) or unpaired (for tissue samples), parametric testing using GraphPad InStat software (GraphPad Software Inc, La Jolla, CA). A P value of <0.05 was considered statistically significant.
Analysis of Cilia in the RRV Model
Primary cilia are found on the apical surfaces of cholangiocytes even in newborn mouse livers, as shown by acetylated α tubulin staining of a 12-hour-old mouse liver (Fig. 1A, B). In BALB/c mice injected with RRV within 24 hours of birth, a model of BA, cilia are present and appear normal on intrahepatic cholangiocytes even up to days 12 to 14 (Fig. 1C–J). Minor decreases in the ratio of cilia to nuclei were not statistically significant (Fig. 1E, H, K). Note that all of the day 7 to 12 samples were from jaundiced mice, indicating the development of bile duct obstruction and BA.
In comparison, analysis of the extrahepatic ducts from mice injected with RRV demonstrated no change in primary cilia 3 days after injection with RRV, but did show a marked decrease at days 8 (P ≤ 0.0001) and 12 (P = 0.0084) when compared with saline-injected animals (Fig. 2). Interestingly, there appeared to be a partial recovery of cilia at day 12, with a statistically significant increase compared with day 8 (P = 0.005). To determine whether RRV infection could cause a direct loss of cilia, primary neonatal cholangiocytes were infected with RRV. RRV-infected neonatal cholangiocytes showed a statistically significant reduction in cilia (Fig. 3A–C) when compared with noninfected cells (P = 0.0484) 12 hours after infection.
Loss of cilia may be a nonspecific response to obstruction. We examined the large ducts of adult mice 14 days after bile duct ligation and found no change in cilia (online-only figure, http://links.lww.com/MPG/A225). This suggests that obstruction in and of itself does not lead to cilia damage, although it does not rule out such an effect in the neonatal bile duct.
Analysis of Cilia in Extrahepatic Ducts From Patients With BA
We previously showed that the intrahepatic bile ducts of patients with BA have decreased and morphologically abnormal cilia (4). Given the specific loss of cilia from extrahepatic ducts in the RRV model, we examined the extrahepatic ducts from 4 patients with BA for cholangiocyte ciliation (Fig. 4). Markedly decreased cilia were found in the larger ducts; however, adjacent clusters of peribiliary glands showed numerous cilia (Fig. 4D, I–K). Two of the samples showed ducts with patent lumens (Fig. 4F, H), whereas the other 2 (Fig. 4E, G) showed obliterated lumens. A dense inflammatory infiltrate and expanded fibrotic matrix surrounded all ducts (not shown). For comparison, a normal extrahepatic bile duct from an infant, taken at autopsy, demonstrated abundant cilia (Fig. 4A–C).
We report here that extrahepatic cholangiocytes demonstrate a loss of cilia in both human BA and in the mouse RRV model of the disease. BA is thought to result from an initial insult to the extrahepatic bile ducts. Our present and previous observations are consistent with this hypothesis (4), although it is not clear whether ciliary resorption is a secondary phenomenon (reflecting general cellular disruption) or is a key part of the mechanism of BA. A recent article demonstrating the development of BA in Sox17 haploinsufficient mice found no ciliary loss, suggesting that this is not necessary for the development of BA (23). It is interesting, however, that RRV infection of primary cholangiocytes rapidly alters ciliation. Hartley et al (15) have shown that levels of fibrocystin/polyductin are decreased in BA (15). This ciliary protein is mutated in the disorder autosomal recessive polycystic kidney disease, which is associated with bile duct dilation and peribiliary fibrosis. Our data (in particular the specific loss of cilia from extrahepatic cholangiocytes) in combination with this previous work from Hartley et al raise the intriguing (although speculative) possibility that BA is an acquired ciliopathy.
Testing this hypothesis will require determining the consequences of cilia loss, particularly because it relates to the loss of cholangiocyte polarity and epithelial integrity, and thereby to the potential for bile duct obstruction. It will be particularly critical to make use of 3-dimensional culture systems in which cholangiocytes form ducts, and to determine whether the artificial loss of cilia in these systems leads to obstruction or other damage to the ducts
Regardless of whether cilia damage and loss is a primary or secondary event in BA, it may offer insight into the pathophysiology of the disease. There is compelling evidence in both human BA and the RRV model that duct obstruction is necessary for the disease to develop: the RRV model requires obstruction, even in the setting of autoimmune defects (24); bile duct ligation in neonatal rats (but not adult rats or adult mice) causes a syndrome that appears identical to BA (25); and it has been appreciated since the pre-Kasai era that human BA is also invariably associated with ductal obstruction (26). Although we cannot rule out that ciliary damage is secondary to obstruction of the neonatal bile duct, it is also possible that understanding the cause and functional ramifications of cilia damage may suggest a mechanism for loss of bile duct epithelial integrity and obstruction.
Interestingly, we observed a partial return of cilia on the extrahepatic cholangiocytes of neonatal mice 12 days after RRV infection. This may be related to why icteric RRV mice occasionally recover fully (3) . It is conceivable that cells from the peribiliary glands, which are thought to be a stem cell niche (27,28), participate in this recovery of ciliated cells. Our data suggest that in BA, these glands are relatively resistant (at least in terms of cilia resorption) to damage, raising the possibility that they serve as a reservoir for cholangiocyte renewal. A limitation of our study is that only a small number of human samples were examined. Our data suggest, however, that understanding the relevance of cilia restoration will require a better understanding of the relation between cilia loss and obstruction.
In summary, we have demonstrated in an animal model of BA as well as in human tissue that there is significant loss of cilia from extrahepatic cholangiocytes. Future work will need to focus on the functional ramifications of this cilia loss, and on the mechanism by which this occurs after RRV infection and potentially other cholangiocyte insults.
The authors are grateful to the Molecular Pathology and Imaging Core of the UPenn NIDDK Center for Molecular Studies in Digestive and Liver Diseases (P30 DK50306) and to the UPenn Cell and Developmental Biology Microscopy Core for assistance with imaging.
1. Haber BA, Russo P. Biliary atresia. Gastroenterol Clin N Am
2. Bezerra JA. Potential etiologies of biliary atresia. Pediatr Transplant
3. Riepenhoff-Talty M, Schaekel K, Clark HF, et al. Group A rotaviruses produce extrahepatic biliary obstruction in orally inoculated newborn mice. Pediatr Res
4. Chu AS, Russo PA, Wells RG. Cholangiocyte cilia are abnormal in syndromic and non-syndromic biliary atresia. Mod Pathol
5. de Carvalho E, Ivantes CA, Bezerra JA. Extrahepatic biliary atresia: current concepts and future directions. J Pediatr
6. Teichberg S, Markowitz J, Silverberg M, et al. Abnormal cilia in a child with the polysplenia syndrome and extrahepatic biliary atresia. J Pediatr
7. Gershoni-Baruch R, Gottfried E, Pery M, et al. Immotile cilia syndrome including polysplenia, situs inversus, and extrahepatic biliary atresia. Am J Med Genet
8. Irigoin F, Badano JL. Keeping the balance between proliferation and differentiation: the primary cilium. Curr Genomics
9. Oh EC, Katsanis N. Cilia in vertebrate development and disease. Development
10. Berbari NF, O’Connor AK, Haycraft CJ, et al. The primary cilium as a complex signaling center. Curr Biol
11. Huang BQ, Masyuk TV, Muff MA, et al. Isolation and characterization of cholangiocyte primary cilia. Am J Physiol Gastrointest Liver Physiol
12. Masyuk AI, Gradilone SA, Banales JM. Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors. Am J Physiol Gastrointest Liver Physiol
13. Banales JM, Masyuk TV, Gradilone SA, et al. The cAMP effectors epac and protein kinase a (PKA) are involved in the hepatic cystogenesis of an animal model of autosomal recessive polycystic kidney disease (ARPKD). Hepatology
14. Waters AM, Beales PL. Ciliopathies: an expanding disease spectrum. Pediatr Nephrol
15. Hartley JL, O’Callaghan C, Rossetti S, et al. Investigation of primary cilia in the pathogenesis of biliary atresia. J Pediatr Gastroenterol Nutr
16. Allen SR, Jafri M, Donnelly B, et al. Effect of rotavirus strain on the murine model of biliary atresia. J Virol
17. Hand NJ, Horner AM, Master ZR, et al. MicroRNA profiling identifies miR-29 as a regulator of disease-associated pathways in experimental biliary atresia. J Pediatr Gastroenterol Nutr
18. Shivakumar P, Campbell KM, Sabla GE, et al. Obstruction of extrahepatic bile ducts by lymphocytes is regulated by IFN-gamma in experimental biliary atresia. J Clin Invest
19. Olsen AL, Sackey BK, Marcinkiewicz C, et al. Fibronectin extra domain-A promotes hepatic stellate cell motility but not differentiation into myofibroblasts. Gastroenterology
20. Vroman B, LaRusso NF. Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells. Lab Invest
21. Arnold M, Patton JT, McDonald SM. Culturing, storage, and quantification of rotaviruses. Curr Protoc Microbiol
22. Barnes BH, Tucker RM, Wehrmann F, et al. Cholangiocytes as immune modulators in rotavirus-induced murine biliary atresia. Liver Int
23. Uemura M, Ozawa A, Nagata T, et al. Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice. Development
24. Lu BR, Brindley SM, Tucker RM, et al. alpha-enolase autoantibodies cross-reactive to viral proteins in a mouse model of biliary atresia. Gastroenterology
25. Gibelli NE, Tannuri U, de Mello ES, et al. Bile duct ligation in neonatal rats: is it a valid experimental model for biliary atresia studies? Pediatr Transplant
26. Thompson J. On congenital obliteration of the bile ducts. Edinburgh Med J
27. Cardinale V, Wang Y, Carpino G, et al. Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets. Hepatology
28. Nakanuma Y, Hoso M, Sanzen T, et al. Microstructure and development of the normal and pathologic biliary tract in humans, including blood supply. Microsc Res Tech
extrahepatic bile ducts; peribiliary glands; primary cilia; rhesus rotavirus
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