Is biliary atresia an immune mediated disease?
Authors: Fernando Alvarez
Journal: Journal of Hepatology (2013)

Regulatory T cells inhibit Th1 cell-mediated bile duct injury in murine biliary atresiaJournal of HepatologyVol. 59Issue 4PreviewBiliary atresia (BA) is a pediatric inflammatory disease of the biliary system which leads to cirrhosis and the need for liver transplantation. One theory regarding etiology is that bile duct injury is due to virus-induced autoreactive T cell-mediated inflammation. Regulatory T cell (Treg) abnormalities in BA could result in unchecked bystander inflammation and autoimmunity targeting bile ducts. The aim of this study was to determine if Tregs are dysfunctional in the rotavirus-induced mouse model of BA (murine BA). Full-Text PDF Regional differences in the incidence of biliary atresia (BA) have been reported, with a range of 1:8000 in Taiwan to 1:17,000 in Europe and North America [[1]Jimenez-Rivera C. Jolin-Dahel K.S. Fortinsky K.J. Gozdyra P. Benchimol E.I. International incidence and outcomes of biliary atresia.J Pediatr Gastroenterol Nutr. 2013; 56: 344-354Crossref PubMed Scopus (105) Google Scholar]. Great advances have been made in the treatment of biliary atresia, entitling 80% to 90% of affected children to survive to adulthood [[2]Davenport M. Biliary atresia: clinical aspects.Semin Pediatr Surg. 2012; 21: 175-184Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar]. Around 30% of them survive with their native liver, with better outcomes for those undergoing surgery (Kasai operation) within the first few weeks of life [[1]Jimenez-Rivera C. Jolin-Dahel K.S. Fortinsky K.J. Gozdyra P. Benchimol E.I. International incidence and outcomes of biliary atresia.J Pediatr Gastroenterol Nutr. 2013; 56: 344-354Crossref PubMed Scopus (105) Google Scholar]. Nevertheless, all patients with BA develop progressive cirrhosis, leading to portal hypertension and its complications. BA remains the main indication of orthotopic liver transplantation in Pediatrics. This is probably because no enough progress has been made to allow an earlier diagnosis of the disease. Recently, stool color cards used in Taiwan appeared to increase awareness of BA in parents and physicians [[3]Chiu C.Y. Chen P.H. Chan C.F. Chang M.H. Wu T.C. Biliary Atresia in Preterm Infants in Taiwan: A Nationwide Survey.J Pediatr. 2013; 163: 100-103Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. There is scarce information on the aetiology and pathogenesis of BA. Therefore, it hampers prevention strategies or therapies designed to stop progression of the necro-inflammatory process affecting the bile ducts. Several hypotheses have been explored in the last thirty years: (1) viral infection as a trigger factor; (2) development of an autoimmune response against antigens from the biliary epithelia; and (3) genetic susceptibility to mount such process or defects of genes involved in morphogenesis [[4]Mack C.L. Sokol R.J. Unraveling the pathogenesis and etiology of biliary atresia.Pediatr Res. 2005; 57: 87R-94RCrossref PubMed Scopus (117) Google Scholar]. The latter hypothesis has attracted attention because around 20% of patients with BA also show other malformations: polysplenia, situs inversus abdominis (pre-duodenal portal vein, intestinal malrotation), absence of inferior vena cava, structural cardiac anomalies, and even Kartagener syndrome (situs inversus, ciliary dyskinesia, and bronchiectasis). However, this embryologic form of BA can be the consequence of an early insult during the embryonic period rather than the result of a specific gene defect. There is little evidence for a viral infection as no viral particles could be found in the liver. Such an etiologic factor, known as the hit and run hypothesis, has been considered for other hepatic immune related diseases, autoimmune hepatitis in particular [[5]Beland K. Lapierre P. Djilali-Saiah I. Alvarez F. Liver restores immune homeostasis after local inflammation despite the presence of autoreactive T cells.PLoS One. 2012; 7: e48192Crossref PubMed Scopus (14) Google Scholar]. Viral infections can stimulate the immune system to respond to self antigens by several pathways: (1) cross-reactivity (molecular mimicry) between viral and self antigens, (2) release of sequestered proteins, (3) activation of resting auto-reactive T cells, and (4) polyclonal activation of lymphocytes. In addition, inflammation triggered, by a viral infection, could induce the expression of HLA and co-stimulatory molecules on cell surfaces. In the case of BA, these molecules could be expressed on bile ducts epithelial cells. Twenty years ago, bile injury was reported in neonatal Balb/c mice infected with group C rotavirus [[6]Riepenhoff-Talty M. Schaekel K. Clark H.F. Mueller W. Uhnoo I. Rossi T. et al.Group A rotaviruses produce extrahepatic biliary obstruction in orally inoculated newborn mice.Pediatr Res. 1993; 33: 394-399PubMed Google Scholar]. These mice were infected orally at 2 days of life, the virus replicated in bile tract and in the liver, and by the end of the first week post-infection, mice showed inflammation and swelling of bile ducts, half of the affected animals showed complete biliary obstruction (but not biliary atresia). Histology showed bile duct proliferation and liver fibrosis [[6]Riepenhoff-Talty M. Schaekel K. Clark H.F. Mueller W. Uhnoo I. Rossi T. et al.Group A rotaviruses produce extrahepatic biliary obstruction in orally inoculated newborn mice.Pediatr Res. 1993; 33: 394-399PubMed Google Scholar]. When Rhesus rotavirus group A (RRV) was injected intraperitoneally at 24 and 48 hours of life, two third of pups showed cholestasis and delayed growth. Most animals went on to develop biliary obstruction and, few of them spontaneously recovered [[7]Petersen C. Biermanns D. Kuske M. Schakel K. Meyer-Junghanel L. Mildenberger H. New aspects in a murine model for extrahepatic biliary atresia.J Pediatr Surg. 1997; 32: 1190-1195Abstract Full Text PDF PubMed Scopus (124) Google Scholar]. No complete biliary atresia and progressive liver fibrosis as described in humans were recorded, but severe stenosis or short distance atresias was present. RRV showed a strain specific biliary tropism [[8]Wang W. Donnelly B. Bondoc A. Mohanty S.K. McNeal M. Ward R. et al.The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice.J Virol. 2011; 85: 9069-9077Crossref PubMed Scopus (30) Google Scholar], infection of cholangiocytes is determined by RRV gene segment 4, coding for viral protein 4 [[8]Wang W. Donnelly B. Bondoc A. Mohanty S.K. McNeal M. Ward R. et al.The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice.J Virol. 2011; 85: 9069-9077Crossref PubMed Scopus (30) Google Scholar]. A major drawback of this model is the fact that most infected mice died at the third week of life, at that time severe liver fibrosis was not present. It is clear that, histologically, these models do not mimic biliary atresia as observed in humans, to warrant further studies [[9]Petersen C. Biliary atresia: the animal models.Semin Pediatr Surg. 2012; 21: 185-191Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar]. Some other variables are limiting the relevance of this model, as: (1) dependence of a particular mouse strain, (2) importance of the infective viral dosage, and (3) prominence of the time of infection. Balb/c is the most susceptible mouse strain. RRV at high dosage is the only virus known to induce biliary necro-inflammatory changes, and the first 24 h of life at the intra-peritoneal injection is the optimal time to induce significant biliary disease. In most infected pups, the virus is undetectable or cleared by the second week post-infection, but the cellular and immune response persists, is this immune response responsible for the ongoing bile duct injury? In the first week after RRV infection, CD4+ cells producing interferon-gamma (IFN-γ) are found in mouse livers, with an increase in number of macrophages producing tumor necrosis factor-alpha (TNF-α) [[10]Mack C.L. Tucker R.M. Sokol R.J. Kotzin B.L. Armed CD4+ Th1 effector cells and activated macrophages participate in bile duct injury in murine biliary atresia.Clin Immunol. 2005; 115: 200-209Crossref PubMed Scopus (87) Google Scholar]. In addition, the CD4+ cells response has been characterized as Th1 type [[10]Mack C.L. Tucker R.M. Sokol R.J. Kotzin B.L. Armed CD4+ Th1 effector cells and activated macrophages participate in bile duct injury in murine biliary atresia.Clin Immunol. 2005; 115: 200-209Crossref PubMed Scopus (87) Google Scholar]. Initially, natural killer (NK) cells play major roles in virus elimination and probably bile duct injury. NK cells are critical to the innate immune response to virally infected cells [[11]Shivakumar P. Sabla G.E. Whitington P. Chougnet C.A. Bezerra J.A. Neonatal NK cells target the mouse duct epithelium via Nkg2d and drive tissue-specific injury in experimental biliary atresia.J Clin Invest. 2009; 119: 2281-2290Crossref PubMed Scopus (91) Google Scholar]. These cells also have the ability to develop antigen-specific immunological memory and consequently to respond to secondary infections, therefore, NK cells also play a role in the adaptive immune response. Moreover, specific autoantibodies against bile duct antigens and peri-ductular deposits of immunoglobulin were recorded in RRV infected mice [[12]Mack C.L. Tucker R.M. Lu B.R. Sokol R.J. Fontenot A.P. Ueno Y. et al.Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia.Hepatology. 2006; 44: 1231-1239Crossref PubMed Scopus (103) Google Scholar]. CD4+ Th1 help CD8+ cytotoxic T cells become the ultimate aggressor of the biliary epithelium. RRV primed CD8+ cells are known to be cytotoxic for cholangiocytes [[13]Shivakumar P. Sabla G. Mohanty S. McNeal M. Ward R. Stringer K. et al.Effector role of neonatal hepatic CD8+ lymphocytes in epithelial injury and autoimmunity in experimental biliary atresia.Gastroenterology. 2007; 133: 268-277Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar] (Fig. 1). The T-cell immune response persists after virus clearance, and the transfer of CD3+ (helper and cytotoxic) activated cells to syngeneic SCID recipients produces bile duct inflammation [[13]Shivakumar P. Sabla G. Mohanty S. McNeal M. Ward R. Stringer K. et al.Effector role of neonatal hepatic CD8+ lymphocytes in epithelial injury and autoimmunity in experimental biliary atresia.Gastroenterology. 2007; 133: 268-277Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. Therefore, in absence of the virus, it can be considered that such inflammation is the result of the recognition of bile duct autoantigens by the transferred lymphocytes. Considering the above described immune pathogenesis for virus induced BA in Balb/c newborn mice, T regulatory cells (Tregs) become a candidate for the control of bile duct inflammation. Tregs are in few numbers in the first days of life. The behaviour of these cells after RRV infection was reported to show that hepatic Tregs rapidly increase 10-fold when mice were infected at 7 days of life (no bile duct injury), but no such increase was observed when mice were injected at day 1 of life [14Miethke A.G. Saxena V. Shivakumar P. Sabla G.E. Simmons J. Chougnet C.A. Post-natal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia.J Hepatol. 2010; 52: 718-726Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Lages C.S. Simmons J. Chougnet C.A. Miethke A.G. Regulatory T cells control the CD8 adaptive immune response at the time of ductal obstruction in experimental biliary atresia.Hepatology. 2012; 56: 219-227Crossref PubMed Scopus (51) Google Scholar]. Therefore, activation of Tregs their likely proliferation, and homing in the liver play a major role in the control of bile duct injury. Adoptive transfer of Tregs in vivo, followed by RRV inoculation, reduces signs of cholestasis and bile duct inflammation. In addition, a decrease of CD8+ expansion is reported [[15]Lages C.S. Simmons J. Chougnet C.A. Miethke A.G. Regulatory T cells control the CD8 adaptive immune response at the time of ductal obstruction in experimental biliary atresia.Hepatology. 2012; 56: 219-227Crossref PubMed Scopus (51) Google Scholar]. By contrast, when Tregs were depleted in vivo, an increase of bile duct injury and its complications occurred, in parallel, hepatic CD8+ cells expansion and enhanced stimulatory capacity of dendritic cells (DCs) were observed. The elegant work published in this issue of the Journal provides new data substantiating the major role of Tregs in the development of bile ducts inflammation and injury in newborn Balb/c mice infected by RRV. Dr Tucker et al. reported lower percentage and large difference in absolute numbers of Foxp3+ (a transcription factor identifying Tregs) cells in the liver from sick mice, but without differences in the percentage of Tregs in the spleen between BA and control mice [[16]Tucker R.M. Feldman A.G. Fenner E.K. Mack C.L. Regulatory T cells inhibit Th1 cell-mediated bile duct injury in murine biliary atresia.J Hepatol. 2013; 59: 790-796Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. Therefore, a defect of Tregs trafficking to the liver or in homing can be most probably suspected. Finding that Tregs from BA mice expressed less molecules characteristic of activation, as CD25 and Foxp3, and adhesion, as CD44 and CD62L, can explain the previously described result [[16]Tucker R.M. Feldman A.G. Fenner E.K. Mack C.L. Regulatory T cells inhibit Th1 cell-mediated bile duct injury in murine biliary atresia.J Hepatol. 2013; 59: 790-796Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. To establish the in vivo role of Tregs in BA mice, adoptive transfer of Tregs was conducted. RRV infected pups receiving Foxp3 positive cells showed more than 85% survival, histological examination confirmed that surviving mice do not show biliary obstruction [[16]Tucker R.M. Feldman A.G. Fenner E.K. Mack C.L. Regulatory T cells inhibit Th1 cell-mediated bile duct injury in murine biliary atresia.J Hepatol. 2013; 59: 790-796Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. In these mice, a decrease of CD8+ and macrophages (that can act as antigen-presenting cells) was recorded. Similarly, minimal production of IFN-γ and TNF-α was found in BA mice receiving Tregs and in control animals. Altogether, these results evidenced the fundamental role of T regulatory cells in the control of bile duct injury in the BA animal model. Tregs would develop BA in Balb/c mice infected after the first 24 h of life, to prove this hypothesis, the authors depleted 4-day old mice of Tregs and infected them at day 7th of life. Almost two thirds of these mice developed biliary disease, as compared with only about 25% of no depleted controls [[16]Tucker R.M. Feldman A.G. Fenner E.K. Mack C.L. Regulatory T cells inhibit Th1 cell-mediated bile duct injury in murine biliary atresia.J Hepatol. 2013; 59: 790-796Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. In conclusion, Balb/c infected by RRV during the first hours of life develop a BA like disease because they have a deficiency in both Tregs number and function. A question motivated by the present work would be: could administration of Tregs stop the necro-inflammatory process destroying bile ducts in patients with BA? The answer may be affirmative in patients diagnosed early, at a time when the Kasai operation shows the higher percentage of success. Again, the key message for clinicians is to raise their level of suspicion in order to obtain an earlier diagnosis. Maternal vaccination against RRV protects newborn pups from the development of bile duct injury [17Bondoc A.J. Jafri M.A. Donnelly B. Mohanty S.K. McNeal M.M. Ward R.L. et al.Prevention of the murine model of biliary atresia after live rotavirus vaccination of dams.J Pediatr Surg. 2009; 44: 1479-1490Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 18Turowski C. Leonhardt J. Teichmann B. Heim A. Baumann U. Kuebler J.F. et al.Preconceptional oral vaccination prevents experimental biliary atresia in newborn mice.Eur J Pediatr Surg. 2010; 20: 158-163Crossref PubMed Scopus (19) Google Scholar]. Most probably, mothers produce neutralizing IgG antibodies thereby to rapidly eliminate the virus. This observation may also have important implications for the prevention of BA, and makes the identification of putative etiological agents possible. The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.