A Novel Pkhd1 Mutation Interacts with the Nonobese Diabetic Genetic Background To Cause Autoimmune Cholangitis This information is current as Wenting Huang, Daniel B. Rainbow, Yuehong Wu, David of September 28, 2021. Adams, Pranavkumar Shivakumar, Leah Kottyan, Rebekah Karns, Bruce Aronow, Jorge Bezerra, M. Eric Gershwin, Laurence B. Peterson, Linda S. Wicker and William M. Ridgway J Immunol published online 20 November 2017 Downloaded from http://www.jimmunol.org/content/early/2017/11/23/jimmun ol.1701087 Supplementary http://www.jimmunol.org/content/suppl/2017/11/20/jimmunol.170108 http://www.jimmunol.org/ Material 7.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published November 27, 2017, doi:10.4049/jimmunol.1701087 The Journal of Immunology ANovelPkhd1 Mutation Interacts with the Nonobese Diabetic Genetic Background To Cause Autoimmune Cholangitis Wenting Huang,*,1 Daniel B. Rainbow,†,1 Yuehong Wu,* David Adams,* Pranavkumar Shivakumar,‡ Leah Kottyan,x Rebekah Karns,{ Bruce Aronow,{ Jorge Bezerra,‡ M. Eric Gershwin,‖ Laurence B. Peterson,# Linda S. Wicker,† and William M. Ridgway* We previously reported that NOD.c3c4 mice develop spontaneous autoimmune biliary disease (ABD) with anti-mitochondrial Abs, histopathological lesions, and autoimmune T lymphocytes similar to human primary biliary cholangitis. In this article, we Downloaded from demonstrate that ABD in NOD.c3c4 and related NOD ABD strains is caused by a chromosome 1 region that includes a novel mutation in polycystic kidney and hepatic disease 1 (Pkhd1). We show that a long terminal repeat element inserted into intron 35 exposes an alternative polyadenylation site, resulting in a truncated Pkhd1 transcript. A novel NOD congenic mouse expressing aberrant Pkhd1, but lacking the c3 and c4 chromosomal regions (NOD.Abd3), reproduces the immunopathological features of NOD ABD. RNA sequencing of NOD.Abd3 common bile duct early in disease demonstrates upregulation of genes involved in cholangiocyte injury/morphology and downregulation of immunoregulatory genes. Consistent with this, bone marrow chimera http://www.jimmunol.org/ studies show that aberrant Pkhd1 must be expressed in the target tissue (cholangiocytes) and the immune system (bone marrow). Mutations of Pkhd1 produce biliary abnormalities in mice but have not been previously associated with autoimmunity. In this study, we eliminate clinical biliary disease by backcrossing this Pkhd1 mutation onto the C57BL/6 genetic background; thus, the NOD genetic background (which promotes autoimmunity) is essential for disease. We propose that loss of functional Pkhd1 on the NOD background produces early bile duct abnormalities, initiating a break in tolerance that leads to autoimmune cholangitis in NOD.Abd3 congenic mice. This model is important for understanding loss of tolerance to cholangiocytes and is relevant to the pathogenesis of several human cholangiopathies. The Journal of Immunology, 2018, 200: 000–000. utoimmune biliary disease (ABD) in humans includes splenomegaly, wasting, abdominal swelling, liver function by guest on September 28, 2021 primary biliary cholangitis (PBC) and primary sclerosing abnormalities, and, eventually, death from obstructive liver dis- A cholangitis in adults (1–3) and biliary atresia in children ease. Histologically, their livers show substantial lymphocytic (4, 5). The biliary epithelial cell (cholangiocyte) is the main auto- infiltration, nonsuppurative destructive cholangitis, and macro- immune target in these diseases (6, 7). Several animal models of phage aggregation in the bile ducts, all features similar to human ABD have been established. NOD.c3c4 (8, 9), NOD.ABD (10), PBC. In addition, NOD.c3c4 mice spontaneously develop anti– and dnTGFbRII mice (11) develop spontaneous ABD similar to pyruvate dehydrogenase E2 autoantibodies (anti-mitochondrial PBC. Infection of neonatal BALB/c mice produces an autoim- Abs), which are highly specific for human PBC. In contrast to mune reaction very similar to biliary atresia (7, 12). The NOD. human PBC, NOD.c3c4 mice also develop common bile duct c3c4 strain arose in a project to refine Idd loci by introgressing B6 (CBD) dilation and inflammation, which more closely resemble and B10 Idd regions onto the NOD genetic background (8). We primary sclerosing cholangitis or biliary atresia. Finally, they showed that NOD.c3c4 mice are completely protected from develop extensive proliferation of intrahepatic bile ductules, far diabetes but develop ABD that is characterized by hepato- exceeding the ductule proliferation seen in stage II human PBC *Division of Immunology, Allergy and Rheumatology, University of Cincinnati Col- Veterans Affairs Merit Grant BX000827-01A1 (to W.M.R.). L.S.W. and D.B.R. lege of Medicine, Cincinnati, OH 45267; †JDRF/Wellcome Trust Diabetes and In- were supported by Wellcome Trust Grant 107212/Z/15/Z and Juvenile Diabetes flammation Laboratory, Wellcome Trust Center for Human Genetics, Nuffield Research Foundation Grant 4-SRA-2017-473-A-N. The Wellcome Trust Center for Department of Medicine, National Institute for Health Research Oxford Biomedical Human Genetics is supported by a core award from the Wellcome Trust (203141/Z/ Research Centre, University of Oxford, Oxford OX3 7BN, United Kingdom; 16/Z). W.M.R., L.S.W., and M.E.G. were supported by National Institutes of ‡Division of Pediatric Gastroenterology, Hepatology and Nutrition, Cincinnati Child- Health Grant R01DK074768. ren’s Hospital Medical Center, Cincinnati, OH 45229; xDepartment of Pediatrics, { Address correspondence and reprint requests to Dr. William M. Ridgway, University Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; Division of Cincinnati College of Medicine, 231 Albert Sabin Way, 7510 MSB, Cincinnati, of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincin- ‖ OH 45267. E-mail address: [email protected] nati, OH 45229; Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, CA 95616; and #Department of Pharmacol- The online version of this article contains supplemental material. ogy, Merck Research Laboratories, Rahway, NJ 07065 Abbreviations used in this article: ABD, autoimmune biliary disease; B6, C57BL/6; 1W.H. and D.B.R. contributed equally to this work. BC2i, backcross 2 intercrossed; B6.PL, B6.PL-Thy1a/CyJ; CBD, common bile duct; ERV, endogenous retrovirus; NOD.H2b, NOD.B10-H2b; PBC, primary biliary ORCIDs: 0000-0002-2095-0623 (P.S.); 0000-0003-3979-2220 (L.K.); 0000-0001- cholangitis; Pkhd1, polycystic kidney and hepatic disease 1; qRT-PCR, quantitative 9667-7742 (J.B.); 0000-0001-7771-0324 (L.S.W.). RT-PCR; RNA-seq, RNA sequencing; SNP, single nucleotide polymorphism; UTR, Received for publication July 28, 2017. Accepted for publication October 19, 2017. untranslated region. This work was supported by the Biomedical Laboratory Research and Devel- Ó opment Service of the Veterans Affairs Office of Research and Development, Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701087 2 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE and more resembling polycystic liver disease (8, 9, 13). However, (N11), 8728 (N11), and 8730 (N11) (Fig. 2A). Novel primers developed transfer of splenocytes from NOD.ABD donors with severe dis- to screen for recombination events and to define recombination points in detail are listed in Supplemental Table II. To assess whether the NOD ease resulted in overwhelming inflammation, nonsuppurative de- b MHC contributed to ABD, line 7825 mice were bred to NOD.B10-H2 structive cholangitis, high titer anti–pyruvate dehydrogenase E2 (NOD.H2b) mice (15) and then backcrossed to line 7825 mice. Mice Abs, and severe illness in NOD.c3c4-scid recipients, in the homozygous for the chromosome 1 congenic region and heterozygous absence of any additional significant ductular proliferation (10). for the H2b MHC were intercrossed to fix both regions as homozygous We concluded that the NOD.c3c4 mouse strain was a useful model for the non-NOD haplotype. Line 7825 mice were crossed to NOD, and the F1 mice were intercrossed to assess whether the Abd3 region was for understanding the mechanisms of ABD. recessive and sufficient to cause ABD on the NOD background in We have previously identified several immune mechanisms of (7825 3 NOD) F2 mice. A cohort of line 7825 mice was compared with NOD ABD. First, NOD.c3c4-scid mice do not develop clinical a cohort of NOD mice for diabetes development, as described (16). disease and have much diminished hepatic histological abnor- NOD.Abd3 mice were crossed once with B6 mice and then the F1 offspring