Alexander Miethke

Obstruction of extrahepatic bile ducts by lymphocytes is regulated by IFN-γ in experimental biliary atresia

The etiology and pathogenesis of bile duct obstruction in children with biliary atresia are largely unknown. We have previously reported that, despite phenotypic heterogeneity, genomic signatures of livers from patients display a proinflammatory phenotype. Here, we address the hypothesis that production of IFN-gamma is a key pathogenic mechanism of disease using a mouse model of rotavirus-induced biliary atresia. We found that rotavirus infection of neonatal mice has a unique tropism to bile duct cells, and it triggers a hepatobiliary inflammation by IFN-gamma-producing CD4(+) and CD8(+) lymphocytes. The inflammation is tissue specific, resulting in progressive jaundice, growth failure, and greater than 90% mortality due to obstruction of extrahepatic bile ducts. In this model, the genetic loss of IFN-gamma did not alter the onset of jaundice, but it remarkably suppressed the tissue-specific targeting of T lymphocytes and completely prevented the inflammatory and fibrosing obstruction of extrahepatic bile ducts. As a consequence, jaundice resolved, and long-term survival improved to greater than 80%. Notably, administration of recombinant IFN-gamma led to recurrence of bile duct obstruction following rotavirus infection of IFN-gamma-deficient mice. Thus, IFN-gamma-driven obstruction of bile ducts is a key pathogenic mechanism of disease and may constitute a therapeutic target to block disease progression in patients with biliary atresia.

Pharmacological inhibition of apical sodium‐dependent bile acid transporter changes bile composition and blocks progression of sclerosing cholangitis in multidrug resistance 2 knockout mice

Deficiency of multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leads to excretion of low‐phospholipid “toxic” bile causing progressive cholestasis. We hypothesize that pharmacological inhibition of the ileal, apical sodium‐dependent bile acid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2–/– mice. Thirty‐day‐old, female mdr2–/– mice were fed high‐fat chow containing 0.006% SC‐435, a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/day of compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry.

Post-natal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia

BACKGROUND & AIMS Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown. METHODS We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype. RESULTS While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3 days, no increase in Tregs occurred at this time point following infection on day 1. In vitro, Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFalpha and IL-15, following RRV infection. In vivo, adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver. CONCLUSIONS (1) The liver is devoid of Tregs early after perinatal RRV infection; (2) Tregs suppress DC-dependent activation of naive NK cells in vitro, and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo. Thus, the post-natal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury.

Pathogenesis of biliary atresia: defining biology to understand clinical phenotypes

Biliary atresia is a severe cholangiopathy of early infancy that destroys extrahepatic bile ducts and disrupts bile flow. With a poorly defined disease pathogenesis, treatment consists of the surgical removal of duct remnants followed by hepatoportoenterostomy. Although this approach can improve the short-term outcome, the liver disease progresses to end-stage cirrhosis in most children. Further improvement in outcome will require a greater understanding of the mechanisms of biliary injury and fibrosis. Here, we review progress in the field, which has been fuelled by collaborative studies in larger patient cohorts and the development of cell culture and animal model systems to directly test hypotheses. Advances include the identification of phenotypic subgroups and stages of disease based on clinical, pathological and molecular features. Stronger evidence exists for viruses, toxins and gene sequence variations in the aetiology of biliary atresia, triggering a proinflammatory response that injures the duct epithelium and produces a rapidly progressive cholangiopathy. The immune response also activates the expression of type 2 cytokines that promote epithelial cell proliferation and extracellular matrix production by nonparenchymal cells. These advances provide insight into phenotype variability and might be relevant to the design of personalized trials to block progression of liver disease.

Regulatory T cells control the CD8 adaptive immune response at the time of ductal obstruction in experimental biliary atresia.

CD8 T‐lymphocytes are effector cells of cholangiocyte injury in human and in rhesus rotavirus (RRV)‐induced experimental biliary atresia (BA). Here we hypothesize that neonatal deficiency in CD25+CD4+ regulatory T cells (Tregs) leads to aberrant activation of hepatic T‐lymphocytes in BA. We found that adoptive transfer of total CD4 cells, but not of CD25‐depleted CD4 cells, prior to RRV inoculation reduced expansion of CD8 cells, plasma bilirubin levels, ductal inflammation, and bile duct epithelial injury at 7 days postinfection (dpi) compared with age‐matched infected controls without adoptive transfer. Searching for mechanisms, we found that in vitro production of interferon‐gamma (IFN‐γ) by naïve CD8 cells upon polyclonal stimulation was enhanced in coculture with hepatic dendritic cells (DCs) from RRV‐infected, but not with DCs from noninfected mice, which was correlated with an increased proportion of CD11b+ myeloid (m)DCs and up‐regulation of the costimulatory molecule CD86 on RRV‐primed DCs. Furthermore, DC‐dependent T‐lymphocyte activation was blocked by anti‐CD86 antibody in dose‐dependent fashion. Importantly, expression of CD86 on mDCs was down‐regulated by Tregs in vitro, and adoptive transfer of Treg‐containing CD4 cells decreased expression of CD86 on hepatic mDCs at 7 dpi. On the contrary, in mice resistant to experimental BA, CD25+ cell depletion aggravated bile duct injury at 12 dpi after RRV inoculation, as plasma bilirubin levels were elevated by >20‐fold compared with nondepleted infected controls. Increased susceptibility to hepatobiliary injury in Treg‐depleted mice was linked to hepatic CD8 expansion and enhanced stimulatory capacity of hepatic DCs. Conclusion: Activation of hepatic T‐lymphocytes driving biliary obstruction in BA is regulated by mDCs by way of CD86‐dependent costimulation and is susceptible to inhibition by Tregs. (HEPATOLOGY 2012;56:219–227)

Portal hypertension in children and young adults with biliary atresia

Objective: Biliary atresia (BA) frequently results in portal hypertension (PHT), complications of which lead to significant morbidity and mortality. The Childhood Liver Disease Research and Education Network was used to perform a cross-sectional multicentered analysis of PHT in children with BA. Methods: Subjects with BA receiving medical management at a Childhood Liver Disease Research and Education Network site were enrolled. A priori, clinically evident PHT was defined as “definite” when there was either history of a complication of PHT or clinical findings consistent with PHT (both splenomegaly and thrombocytopenia). PHT was denoted as “possible” if one of the findings was present in the absence of a complication, whereas PHT was “absent” if none of the criteria were met. Results: A total of 163 subjects were enrolled between May 2006 and December 2009. At baseline, definite PHT was present in 49%, possible in 17%, and absent in 34% of subjects. Demographics, growth, and anthropometrics were similar amongst the 3 PHT categories. Alanine aminotransferase, &ggr;-glutamyl transpeptidase, and sodium levels were similar, whereas there were significant differences in aspartate aminotransferase (AST), AST/alanine aminotransferase, albumin, total bilirubin, prothrombin time, white blood cell count, platelet count, and AST/platelet count between definite and absent PHT. Thirty-four percent of those with definite PHT had either prothrombin time >15 seconds or albumin <3 g/dL. Conclusions: Clinically definable PHT is present in two-thirds of North American long-term BA survivors with their native livers. The presence of PHT is associated with measures of hepatic injury and dysfunction, although in this selected cohort, the degree of hepatic dysfunction is relatively mild and growth is preserved.

Morphologic Findings in Progressive Familial Intrahepatic Cholestasis 2 (PFIC2): Correlation With Genetic and Immunohistochemical Studies

Progressive familial intrahepatic cholestasis, type 2 (PFIC2), characterized by cholestasis in infancy that may progress to cirrhosis, is caused by mutation in ABCB11, which encodes bile salt export pump (BSEP). We correlated histopathologic, immunohistochemical, and ultrastructural features in PFIC2 with specific mutations and clinical course. Twelve patients with clinical PFIC2 and ABCB11 mutations were identified, and 22 liver biopsy and explant specimens were assessed. All had hepatocellular cholestasis; most had canalicular bile plugs. At least 1 specimen from every patient had centrizonal/sinusoidal fibrosis, often with periportal fibrosis. Neonatal hepatitis-like features (inflammation, giant cells, necrosis) varied. In 2 of the 5 patients with paired specimens obtained >6 months apart, lobular and portal fibrosis worsened. Transmission electron microscopy (EM) in all 9 patients studied showed canalicular dilatation, microvilli loss, abnormal mitochondrial internal structure, and varying intracanalicular accumulation of finely granular bile. Canalicular staining for BSEP was absent in 10 patients and present in 2 patients, 1 of whom had intermittent symptoms. ABCB11 sequencing of all patients identified 6 novel and 10 previously described mutations, with nonsense, missense, and/or noncoding mutations in the 10 patients without immunohistochemically demonstrable BSEP. Missense and/or noncoding mutations were identified in the 2 patients with demonstrable BSEP, whose clinical course was more indolent. Mutations ending ABCB11 transcription appear linked, through hepatocellular necrosis and fibrosis, to worse outcome. In conclusion, light microscopy and electron microscopy findings in clinical PFIC2 can support diagnosis, but are variable and nonspecific. Therefore, no correlation between specific mutations and histopathology is yet possible.

Analysis of gene mutations in children with cholestasis of undefined etiology

Background: The discovery of genetic mutations in children with inherited syndromes of intrahepatic cholestasis allows for diagnostic specificity despite similar clinical phenotypes. Here, we aimed to determine whether mutation screening of target genes could assign a molecular diagnosis in children with idiopathic cholestasis. Patients and Methods: DNA samples were obtained from 51 subjects with cholestasis of undefined etiology and surveyed for mutations in the genes SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 by a high-throughput gene chip. Then, the sequence readouts for all 5 genes were analyzed for mutations and correlated with clinical phenotypes. Healthy subjects served as controls. Results: Sequence analysis of the genes identified 14 (or 27%) subjects with missense, nonsense, deletion, and splice site variants associated with disease phenotypes based on the type of mutation and/or biallelic involvement in the JAG1, ATP8B1, ABCB11, or ABCB4 genes. These patients had no syndromic features and could not be differentiated by biochemical markers or histopathology. Among the remaining subjects, 10 (or ∼20%) had sequence variants in ATP8B1 or ABCB11 that involved only 1 allele, 8 had variants not likely to be associated with disease phenotypes, and 19 had no variants that changed amino acid composition. Conclusions: Gene sequence analysis assigned a molecular diagnosis in 27% of subjects with idiopathic cholestasis based on the presence of variants likely to cause disease phenotypes.

Evaluation of the child with suspected mitochondrial liver disease

This review was developed by the Mitochondrial Liver Diseases Working Group of the Childhood Liver Disease Research and Education Network (ChiLDREN), supported by the National Institute of Digestive, Diabetes and Kidney Diseases, NIH, to guide evaluation of children with suspected mitochondrial liver disease. Data informing the evaluation guideline was supported by Medline searches of published English language literature and expert opinion from a committee of pediatric hepatologists and a mitochondrial metabolism specialist. Mitochondrial respiratory chain defects can affect any tissue, with the most energy-dependent organs being most vulnerable.(1) In general, clinical manifestations include multisystem involvement such as brain, muscle, heart, or kidney, with acute or chronic liver dysfunction, sometimes in the presence of lactic acidosis, a biomarker of limited sensitivity.(2, 3) Heterogeneous clinical presentations can be explained by the fact that the mitochondrial quantity and function are uniquely influenced by both nuclear and mitochondria DNA (mtDNA) or by the fact that cells in various tissues can contain different mixtures of normal and abnormal mitochondrial genomes (heteroplasmy). Most mitochondrial proteins and enzymes are coded by nuclear genes with Mendelian inheritance, while some respiratory chain subunits, ribosomal and transfer RNAs are encoded by mitochondrial genes that are maternally inherited.(4) Mutations, deletions, or duplications in either of these classes can cause disease, and mutations in nuclear genes that control mitochondrial DNA replication, transcription, and translation may lead to mtDNA depletion syndrome or to a translational disorder.(5-7) The respiratory chain, consisting of 5 multimeric complexes (I-V) in the mitochondrial inner membrane, generates energy as ATP via electron transport and oxidative phosphorylation (Figure 1). Defects in the respiratory chain enzymes or mitochondrial membrane transport proteins result in injury to energy-dependent organs, especially brain, retina, muscle, heart, and liver.(8) In addition, hepatic mitochondria oxidize fatty acids forming ketone bodies, an important source of energy for the brain in the fasting state. Fatty acid oxidation defects, an important group of primary bioenergetic defects, can present similarly with hepatopathy or encephalopathy, often with nonketotic hypoglycemia, acidosis and hyperammonemia, and are thus included in the differential diagnosis and should be simultaneously evaluated.(9) Figure 1 The respiratory chain, consisting of 5 multimeric complexes (I-V) in the mitochondrial inner membrane, generates energy as ATP via electron transport and oxidative phosphorylation. Establishing the diagnosis of primary mitochondrial bioenergetic defects in patients with liver disease requires a high index of suspicion in specific clinical scenarios. A tiered diagnostic evaluation is useful (Table 1). Although mitochondrial hepatopathies are a heterogeneous group of disorders, there are several general laboratory investigations in blood and urine which can reveal an altered redox status suggestive of respiratory-chain defects (lactate:pyruvate molar ratios and ketone body ratios). Specific laboratory tests are considered in those with unique clinical presentations as well, and either tissue analysis or genotyping is used to identify the etiology. Other typically involved organ systems should be evaluated when mitochondrial hepatopathy is suspected (Table 2). Several important management issues should be addressed during this evaluation process. These guidelines outline the evaluation of the infant or child with suspected mitochondrial hepatopathy. Two summary tables (Table 3 and ​and4)4) describing each genetic etiology follow; reference clinical laboratories for the genetic tests can be found on www.genetests.org Table 1 Tiered Approach to Evaluation of Suspected Mitochondrial Disease Table 2 Evaluation for Disease in Other Organ Systems Table 3 Genetic Etiologies of Mitochondrial Hepatopathies Presenting in Neonates or Infants Table 4 Genetic Etiologies of Mitochondrial Hepatopathies Presenting as Chronic Liver Disease or With Later Onset

Analysis of surgical interruption of the enterohepatic circulation as a treatment for pediatric cholestasis

To evaluate the efficacy of nontransplant surgery for pediatric cholestasis, 58 clinically diagnosed children, including 20 with Alagille syndrome (ALGS), 16 with familial intrahepatic cholestasis‐1 (FIC1), 18 with bile salt export pump (BSEP) disease, and 4 others with low γ‐glutamyl transpeptidase disease (levels <100 U/L), were identified across 14 Childhood Liver Disease Research Network (ChiLDReN) centers. Data were collected retrospectively from individuals who collectively had 39 partial external biliary diversions (PEBDs), 11 ileal exclusions (IEs), and seven gallbladder‐to‐colon (GBC) diversions. Serum total bilirubin decreased after PEBD in FIC1 (8.1 ± 4.0 vs. 2.9 ± 4.1 mg/dL, preoperatively vs. 12‐24 months postoperatively, respectively; P = 0.02), but not in ALGS or BSEP. Total serum cholesterol decreased after PEBD in ALGS patients (695 ± 465 vs. 457 ± 319 mg/dL, preoperatively vs. 12‐24 months postoperatively, respectively; P = 0.0001). Alanine aminotransferase levels increased in ALGS after PEBD (182 ± 70 vs. 260 ± 73 IU/L, preoperatively vs. 24 months; P = 0.03), but not in FIC1 or BSEP. ALGS, FIC1, and BSEP patients experienced less severely scored pruritus after PEBD (ALGS, 100% vs. 9% severe; FIC1, 64% vs. 10%; BSEP, 50% vs. 20%, preoperatively vs. >24 months postoperatively, respectively; P < 0.001). ALGS patients experienced a trend toward greater freedom from xanthomata after PEBD. There was a trend toward decreased pruritus in FIC1 after IE and GBC. Vitamin K supplementation increased in ALGS after PEBD (33% vs. 77%; P = 0.03). Overall, there were 15 major complications after surgery. Twelve patients (3 ALGS, 3 FIC1, and 6 BSEP) subsequently underwent liver transplantation. Conclusion: This was a multicenter analysis of nontransplant surgical approaches to intrahepatic cholestasis. Approaches vary, are well tolerated, and generally, although not uniformly, result in improvement of pruritus and cholestasis. (Hepatology 2017;65:1645‐1654).