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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 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 malities, indicating that the adaptive immune system is critical were intercrossed to produce F2 mice, or they were crossed twice to B6 mice to generate Backcross 2 mice, which were then further intercrossed to to disease pathogenesis (10). Second, CD8 T cells from NOD. generate the backcross 2 intercrossed (BC2i) generation. NOD.Abd3 mice ABD donors, but not NOD donors, transferred disease into backcrossed to B6 mice were genotyped as follows using markers outside NOD.c3c4-scid recipients (10). Finally, NOD-scid recipients the B6 congenic region, which genotype as NOD for NOD.Abd3 mice. The didnotdevelopABDuponadoptivetransfer,evenwhenre- proximal marker is rs13475762 (Fig. 1A) with the forward primer rs13475762_F = 59-TTCCCCCTTTTAATATTTTGCAT-39 and reverse ceiving 20 million splenocytes from NOD.ABD donors that primer rs13475762_R = 59-CAGGGAGGCAGTGATTTAGC-39. The dis- rapidly caused overwhelming ABD in NOD.c3c4-scid recipients tal marker is rs32040516 with forward primer rs32040516_F = 59-

(10). These results showed that the genetic background of the TGAGCCATCTGACAGACCAG-39 and reverse primer rs32040516_R = Downloaded from target tissue was critical to disease pathogenesis; however, the 59-TGGATGGCCATGACAAAAA-39. PCR product was digested meaning of the requirement of B6/B10 genetic components in with restriction enzyme ACC1 (for proximal marker) or MNL1 (for distal marker) (New England Biolabs) at 37˚C for 2 h, and the PCR the adaptive immune system was unclear. An alternate expla- products were run on a 4% agarose gel with 13 Tris/Borate/EDTA nation for our results was that NOD splenocytes did not cause buffer. The size of the digested PCR product of proximal markers is disease, because autoreactive cholangiocyte-directed T cells 87 bp for NOD, and the size of the digested PCR product of distal markers is 182 bp. Comparison of NOD and B6 genomic sequences were not expanded as a result of T cell repertoire develop- http://www.jimmunol.org/ in the congenic portion of the Abd3 region was performed as de- ment in the absence of the cholangiocyte autoantigen(s). This scribed (17). issue is addressed in the current study by constructing bone marrow chimeric mice. Histopathology and disease scoring We used a congenic mapping approach to define the genetic Livers and/or CBDs were scored for visible/clinical liver and CBD pa- origin of ABD in the NOD.c3c4 model (10). Although regions on thology, as previously described (8–10) (see below for methods), isolated 3 and 4 were initially linked to disease, a genome- from mice, immediately fixed in 10% formalin, and embedded in paraffin. wide 5K single nucleotide polymorphism (SNP) chip analysis Samples were stained with H&E, and histology was scored blindly, using showed a small non-NOD region on chromosome 1 in all strains microscopy, for duct epithelial hyperplasia and lymphocytic infiltration. that developed disease. To assess the role of this region, we CBD and liver gross pathological scoring. CBD dilation and the quanti- by guest on September 28, 2021 tation of liver abnormalities were examined and scored separately. The constructed a new chromosome 1–congenic strain (NOD.Abd3). clinical liver score, performed at the University of California at Davis, was The NOD.Abd3 strain develops highly penetrant ABD, similar to assigned based on tissue induration (hardness) and the presence of cysts on the previously described strains, in the absence of the original the surface of liver as follows: 0: soft and no cysts; 1: mild induration, no chromosome 3 and 4 congenic regions present in the NOD.c3c4 or cysts; 2: indurated with cysts on one lobe surface; 3: indurated with cysts on NOD.ABD models. We undertook the present studies to under- two to three lobes; and 4: indurated and cysts on all four lobes. The clinical CBD score was identical to the diameter (in millimeters) of the maximum stand how the chromosome 1 genetic region mediates biliary CBD width; if CBD measured ,1 mm (“normal” CBD), the score was disease and to further characterize the role of genetic background assigned as 0. The clinical biliary duct score performed at Merck (on 7825 3 and the chromosome 1 region in the target tissue (biliary epithe- NOD F2 mice) was assigned as follows: CBD, 0: normal; 1: size just above , ∼ ∼ lium) and hematopoietic system. From these investigations, we normal; 2: CBD clearly enlarged but 2 mm; 3: CBD 2 mm; 4: CBD 2–4 mm; and 5: CBD . 4 mm. The clinical liver score was assigned as discovered that the congenic region on chromosome 1 is itself follows: liver: 0: normal; 1: very few cysts in the liver; 2: cysts in liver are unlikely to cause ABD; rather, the highly likely cause is a mu- easily seen; 3: many cysts easily seen; 4: many cysts in most or all lobes; tation altering the expression of a closely linked (2.5 Mb) to and 5: liver completely full of cysts. the congenic region, polycystic kidney and hepatic disease 1 Histological liver score: duct epithelial hyperplasia. The histological liver (Pkhd1). duct score was calculated as follows: 0: 0–3 ductules per portal triad across the section; 1: at least two times the normal diameter of ductule lumen in 10–25% of the entire section and no fewer than three small ductules Materials and Methods around one triad in 25–50% of the entire section; 2: enlarging ductules Animals and genotyping across 25–50% of the entire section, and the average diameter of a ductule lumen section is ∼2–4 times the normal ductule diameter; 3: larger duct- NOD, NOD.ABD (10), NOD.CD45.2 (14), B10.H2g7, B6.PL-Thy1a/CyJ ules across .50% of the entire section, and the average diameter of (B6.PL), and C57BL/6 (B6) mice and mice derived therefrom during ductule lumen section is ∼4–5.5 times of the normal ductule diameter; and this study were bred and housed under specific pathogen–free condi- 4: dilated, diffuse, torturous ducts (“cysts”) in much of the section (this is tions, and all procedures were conducted according to approved proto- the end stage disease), and the average diameter of ductule lumen section cols of the University of Cincinnati College of Medicine or the Merck is .5.5 times the normal ductule diameter. Research Laboratories Animal Care and Use Committees. We discov- ered during quality control screening of congenic strains in our colony, Histological score: leukocytic infiltration of portal areas. The histological using a 5K mouse single nucleotide polymorphism (SNP) chip (Par- liver leukocyte infiltration score was calculated as follows: 0: none or a few Allele Bioscience, South San Francisco, CA), that the NOD.c3c4 (9) cells in ,25% of entire section; 1: small numbers of cells in multiple areas and NOD.ABD strains included a non-NOD–derived congenic region (25–50%), including some clusters of immune cells; 2: small numbers of (derived from the B6.PL or B10 mouse strains) on chromosome 1 cells (cell infiltrate 2–3 cells deep) diffusely (.50% of entire section) or (Supplemental Table I). This region was isolated by selective back- patchy moderate infiltrates (25–50% of entire section); 3: moderate crossing to NOD as line 7825 (N11) (Fig. 1). Line 7825 was used to numbers of cells diffusely (.50% of entire section) or patchy large in- generate additional chromosome 1 recombinants to isolate smaller filtrates (larger cluster than for “2,” 25–50% of entire section); and 4: congenic segments, resulting in lines NOD.Abd3 (line 8706, N13), 8727 diffuse or significant sections of large cellular infiltrates. The Journal of Immunology 3

Bone marrow chimeras mice were then sent for next-generation sequencing at Cincinnati Children’s Hospital Medical Center DNA Sequencing and Genotyping Core. Two- to three-month-old NOD (CD45.1) or NOD.Abd3 (CD45.1) mice were irradiated (1200 rad). Fifteen to twenty million bone marrow cells Statistical analysis from 4-mo-old NOD.CD45.2 or 2–3-mo-old NOD.Abd3 (CD45.1) donors were extracted without RBC lysis. Mature CD4, CD8, and CD90 cells GraphPad Prism 6 (GraphPad, San Diego, CA) software package was used were removed using magnetic beads (Miltenyi Biotec), and the bone to perform unpaired two-tailed Student t tests for comparison of the ex- marrow was injected i.v. into the irradiated recipient mice. Recipient mice pression level of genes, as well as the Mann–Whitney U test to estimate were given water treated with antibiotics (neomycin trisulfate salt hydrate) significance levels of histology scores and clinical scores. for 2 wk after transfer. The recipient mice were sacrificed 120 d post–bone marrow transfer or when they developed abdominal swelling indicative of severe hepatobiliary disease (∼2.5 mo for NOD.Abd3 recipients of Results NOD.Abd3 bone marrow). Tissues were analyzed for anatomical and A region on chromosome 1 is necessary for spontaneous histological biliary/liver disease. CD45.1 or CD45.2 quantification was autoimmune cholangitis in NOD congenic mice done for the recipients of different donor cells, and the bone marrow reconstitution rate ranged from 87.6 to 98.9%. We previously reported that NOD.c3c4 and NOD.ABD mice, with B6/ B10 genetic regions on chromosomes 3 and 4 placed on the NOD Quantitative real-time PCR of mRNA background, develop a genetically determined spontaneous autoim- Extrahepatic CBDs were isolated from 2-wk-old NOD.Abd3 and NOD mice mune cholangitis; however, the causative genetic regions were not and stored in RNAlater solution (QIAGEN). For RNA extraction from CBD, clear (8–10). Further analysis of these strains by a 5K SNP chip the tissue was removed from RNAlater solution and disrupted in lysing/binding buffer provided in a mirVana miRNA Isolation Kit (Life Technologies, CA) analysis revealed a region of B6/B10-derived DNA on chromosome 1 using a homogenizer (QIAGEN), and RNA was extracted from the tissues in the NOD.c3c4 and NOD.ABD strains (Supplemental Table I) (10). Downloaded from using a mirVana miRNA Isolation Kit and converted to cDNA by random To assess the significance of this region, we developed, from the primers (High-Capacity cDNA Reverse Transcription Kit; Applied Bio- NOD.ABD strain, a novel congenic mouse that had the chromosome systems). Relative gene expression of Pkhd1 was performed using selective mouse TaqMan gene-expression assays (Mm00467747_m1 for region span- 1 region but lacked the chromosome 3 and 4 regions (line 7825 in ning exons 35 and 36, Mm00467728_m1 for region spanning exons 15 and 16, Fig. 1A). This chromosome 1 congenic mouse has highly penetrant Mm01233737_m1 for regions spanning exon 60 and 61) and an endogenous ABD that is identical in kinetics, penetrance, and phenotype to the

control assay Rn18S (Mm03928990_g1; all from Life Technologies). previously published NOD.c3c4 strain. CBD dilation score, which we http://www.jimmunol.org/ RNA sequencing profiling of CBDs and data analysis previously reported as a sensitive marker of ABD in this strain (8), at 120 d is identical between strain 7825 and NOD.c3c4 (strain 1112) Total CBD RNA was extracted from 2-wk-old NOD or NOD.Abd3 mice (Fig. 1B) as are liver and histology scores (data not shown). There- using a mirVana miRNA Isolation Kit (Life Technologies). Two samples were sequenced for each strain; each of the two NOD.Abd3 samples had fore,wedesignatedthechromosome1regionasAbd3 [chromosome RNA from three individual CBDs, and the two NOD samples had RNA 3 and 4 regions previously described in NOD.ABD mice (10) were from three to five individual CBDs. RNA concentration, purity (28S:18S termed Abd2 and Abd1, respectively]. rRNA ratio), and quality (RNA integrity number) were assessed using a To test whether the Abd3 region is fully recessive and is the only 2100 Bioanalyzer (Agilent, Santa Clara, CA) at the DNA Sequencing and Genotyping Core, Cincinnati Children’s Hospital Medical Center. The segregating genetic region contributing to the ABD phenotype in RNA samples had an RNA integrity score (RNA integrity number) $ 7.9 line 7825, we examined (line 7825 3 NOD) F2 mice. Thirty-four by guest on September 28, 2021 on the Agilent Bioanalyzer (scale of 1–10, with 10 being completely intact). F2 mice were scored at 120 d of age in a blinded manner for CBD The RNA sequencing (RNA-seq) library was generated with an Illumina and liver disease and genotyped at Abd3. All mice genotyped as TruSeq RNA preparation kit and subsequently sequenced on an Illumina homozygous for the NOD allele at Abd3 (n = 6) had clinically HiSeq 2000, using single-end 50-bp read specifications with a read depth $ 10 million (Illumina, San Diego, CA). normal livers and CBDs, whereas all mice genotyped as homozy- RNA-seq analysis was carried out using Bowtie (18) and TopHat2 (19). gous for the B6/B10 allele at Abd3 (n = 10) had macroscopic dis- RNA-seq BAM files generated using the Bowtie–TopHat2 pipeline were ease evident in liver and bile duct (Fig. 1C). All mice genotyped as analyzed for the expression of known and unknown genes/transcripts using heterozygous at Abd3 (n = 18) were also phenotypically normal. the Cufflinks2 pipeline (20) and included removal of reads that did not map uniquely to the mm9 Ensembl genome or ENSEMBL-annotated genes. This confirmed that ABD caused by the B6/B10-derived chromo- Workflows included filtering to remove duplicate reads and those with some 1 region is recessive and that the chromosome 1 region is postaligned read metrics mapping quality , 40. Our samples showed sufficient to cause disease in the context of the NOD genetic normal 3ʹ/59 read distribution ratios compared with hundreds of other background; no other gene influencing ABD to a detectable degree samples run in the Cincinnati Children’s Hospital Medical Center geno- is segregating outside the Abd3 region. The effect of the chromo- mics core. Transcript/isoform and gene-summarized expression tables were filtered to identify entities whose expression was at least five frag- some 1 region on the development of autoimmune diabetes was also ments per kilobase of exon per million fragments mapped (Cufflinks) or examined, and the frequency of diabetes in line 7825 was equivalent five reads per kilobase per million reads mapped (GeneSpring) in at least to that observed in the NOD parental strain, indicating that Abd3 one sample. Differentially expressed gene signatures were identified using had no effect on type 1 diabetes (Supplemental Fig. 1). the Audic–Claverie test (p , 0.05) and the Student t test (false discovery rate , 0.05), followed by a two-fold change requirement. To refine the Abd3 region and aid in the discovery of the causal and other enrichment and biological network analyses were carried out gene, several additional congenic strains were developed by using ToppGene (http://toppgene.cchmc.org) (21) and ToppCluster (http:// screening for recombination events in the congenic segment present toppcluster.cchmc.org) (22). ToppCluster output of xgmml data files were in line 7825 (Fig. 2A). Line 8706 has the smallest congenic interval, network analyzed in Cytoscape, yielding similar and complementary re- a maximal size of 1.0003 Mb (proximal boundary defined by sults compared with the ToppFun functional enrichment analysis. rs13475762 and the distal boundary defined by rs32040516); we PCR and sequencing of Pkhd1 exons and intron 35 designated this strain NOD.Abd3. Like strain 7825, NOD.Abd3 For different exons of Pkhd1, primer pairs were designed using Primer3 mice developed liver histological abnormalities (lymphocytic (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) to amplify rep- infiltrates and biliary ductule epithelial hyperplasia) identical resentative exon sequences, using genomic DNA as template. PCR primer in appearance and kinetics to NOD.c3c4 mice (9) (Fig. 2B). sequences are detailed in Supplemental Table III. (NOD.Abd3 3 NOD) F1 mice lacked clinical disease and had no Primer N7 forward primer and primer 3 reverse primer were used with PrimeSTAR GXL DNA Polymerase (Clontech Laboratories) for long- liver histological abnormalities compared with NOD.Abd3 mice range PCR to identify the inserted DNA fragment in Pkhd1 intron 35 in (Fig. 2C), clearly demonstrating that Abd3 mediates a recessive NOD.Abd3 mice. Long-range PCR products from NOD and NOD.Abd3 trait, even at a microscopic level. 4 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. ABD disease in NOD mice is controlled by a single recessive allele on chromosome 1. (A) The NOD.c3c4 strain (8, 9), the NOD.ABD strain (10), and the newly developed line 7825 all develop ABD and share a non-NOD congenic region (derived from the B6.PL or B10 strain) on chromosome 1 that includes Pkhd1, designated Abd3. The 7825 strain lacks the Abd1 and Abd2 regions on chromosomes 4 and 3, respectively. (B) No difference in NOD. c3c4 versus strain 7825 liver disease kinetics or penetrance. NOD.c3c4 mice (strain 1112; n = 20) and strain 7825 mice (n = 15) were aged to 120 d, and CBD score was assessed (see Materials and Methods). (C) Abd3 region homozygosity required for ABD; mice lacking two copies of 7825-derived Abd3 do not develop any biliary disease. Anatomical liver/biliary scores (CBD score and liver score, see Materials and Methods) of (7825 3 NOD) F2 mice genotyped as B6/B10 Abd3 homozygous (n = 10), NOD Abd3 homozygous (n = 6), and NOD Abd3 3 B6/B10 Abd3 heterozygous (n = 18) using markers for the chromosome 1 congenic region (see Materials and Methods). Error bars show SEM. ***p , 0.001, Mann–Whitney U test.

There are only a few genes in the 1-Mb Abd3 region, including B3gat2 (Fig. 2D). However, none of these genes showed signifi- two microRNAs (miR-30a and miR-30c-2), Ogfrl1, a predicted cant expression differences in target tissue or CD8 T cells isolated gene with unknown function (Gm6420), and partial sequence of from NOD.Abd3 mice compared with NOD mice (data not The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Congenic mapping defines the Abd3 region as a recessive allele that controls clinical and microscopic ABD. (A) Development of chro- mosome 1 NOD.Abd3 congenic mice. Distal recombination events reduced the size of the 7825 Abd3 region on chromosome 1 to a 1-Mb non-NOD congenic region and the mutated Pkhd1 2.5 Mb proximal to the congenic region in strain 8706 (shown as horizontal black bar). (B) Histological disease (lymphocytic infiltration and biliary duct epithelial hyperplasia) develops in NOD.Abd3 mice with the same kinetics/severity as in NOD.c3c4 mice (9). Representative H&E sections from NOD.Abd3 mice aged 73 d (left panel) and 120 d (right panel). (C) A single copy of Abd3 is insufficient for disease in NOD.Abd3 mice. No significant liver histology abnormalities in NOD.Abd3 mice crossed to NOD mice (NOD n = 10, NOD.Abd3 n = 10, NOD.Abd3 3 NOD n = 22). All mice aged 120 d. Error bars show SEM. (D) Sequence comparison of NOD and B6 in the congenic portion of the Abd3 region present in the NOD.Abd3 strain. The schematic representation of the Abd3 congenic region is shown at the lower part of the panel. The comparison of the B6 and NOD sequences (SNPs per 10 kb) in the 1-Mb congenic portion of Abd3 is shown in the middle portion of the panel. The genes within Abd3 are shown at the top of the figure. Gray bar indicates the B6.PL/B10-derived genome, and the white bar indicates where no NOD/B6 SNPs were genotyped. The thin solid lines indicate the NOD genome. ***p , 0.001, Mann–Whitney U test. 6 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Abnormal Pkhd1 expression pattern in NOD.Abd3 CBD. (A) Abnormal exon expression pattern of Pkhd1. RNA-seq reads aligned to reference B6 genome (mm 9) show that expression of exons 36–67 of Pkhd1 is absent in CBD of 2-wk-old NOD.Abd3 mice (upper two panels) com- pared with NOD (lower two panels). Each panel represents one sample. (B) NOD.Abd3 CBD samples upregulate expression of exons 1–35. Pkhd1 par- tition read density shown for two NOD.Abd3 samples (higher expression) and two NOD samples (lower expression). (C) PCR using genomic DNA shows the presence of representative exons 16, 34, 40, and 61 in NOD, B6, and NOD.Abd3 at the DNA level. (D) Quantitative RT-PCR confirms that NOD.Abd3 CBD upregulates exon 15/16 expression and lacks exons 60/61 and 35/36 expression compared with NOD or B6. n = 3 per group. *p , 0.05, Student t test. shown). A comparison of the NOD and B6/B10 sequence also did Notably, strains developed from proximal recombination events not reveal any functional polymorphisms, such as amino acid– (lines 8727 and 8730, Fig. 2A) did not develop ABD. This led us changing SNPs or SNPs affecting splice sites (data not shown). to consider the possibility that a proximal gene outside, but closely The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. NOD genetic background is necessary for Abd3 homozygosity to mediate ABD; minimal role for the NOD MHC region. Clinical CBD (A)andliver(B) scores (see Materials and Methods)ofNOD.Abd3 (n =24),Abd3 homozygous F2 (n = 17), and Abd3 homozygous BC2i (n = 10) mice. ***p , 0.001, *p , 0.05, Mann–Whitney U test. (C and D) Histology scores (see Materials and Methods)ofNODmice(n =10),NOD.Abd3 mice (n = 10), and F2 and BC2i mice with zero, one, or two copies of Abd3 (F2 with zero copy of Abd3: n = 14; F2 with one copy of Abd3: n = 25; F2 with two copies of Abd3: n = 17; BC2i with zero copy of Abd3: n =9; BC2i with one copy of Abd3: n = 18; BC2i with two copies of Abd3: n = 10). All mice aged 120 d. Error bars show SEM. **p , 0.01, ***p , 0.001, Mann–Whitney U test or Wilcoxon signed-rank test was performed for statistical significance. (E)Two-week-oldNOD.Abd3 mice showed substantial dilation of CBD (left) compared with age-matched NOD control mice (middle) and NOD.c3c4-scid mice (right). Black arrows indicate peribiliary glands; green arrowheads indicate hyperplasia of biliary , consequently merging with peribiliary glands. Histology figures are representative of six CBD samples of NOD.Abd3 mice (left), six CBD samples of NOD mice (middle), and five NOD.c3c4-scid mice (right) at 2 wk of age. The slides were stained with H&E. Original magnification 310; inset, original magnification 340. (F) Minimal role for the NOD MHC region in ABD pathology. Strain 7825 mice were bred to NOD.H2b mice to generate mice expressing Abd3 on the NOD genetic background but with a non-NOD MHC region (strain 8953, see Materials and Methods). Strain 8953 mice (n = 30) and 7925 mice (n = 23) were aged to 120 d and then assessed for clinical liver and CBD scores as above. 8 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE linked to, the congenic interval could be mutated and that the on average, only 12.5% NOD-derived alleles. The pathological congenic interval was carried along with the mutation due to CBD enlargement and anatomically visible liver abnormalities linkage disequilibrium. seen in Abd3 homozygous mice are greatly reduced and absent, respectively, in F2 and BC2i offspring that are homozygous at Bile duct abnormality in NOD.Abd3 mice associated with Abd3 but have with a reduced NOD background (Fig. 4A, 4B). aberrant expression of Pkhd1 Therefore, progressively reducing the NOD genetic background All recombinant strains that did not develop disease had recom- and increasing the B6 genetic background ameliorates clinical bination events removing the proximal portion of the congenic manifestations of ABD in Abd3 homozygous animals, strongly region present in line 7825 (Fig. 2A, lines 8727 and 8730), sug- emphasizing the importance of the NOD genetic background for gesting there could be a genetic mutation proximal to the Abd3 the presence of clinical ABD. Notably, because clinical disease is region that was causing disease (because a recombination event present in a substantial proportion of the F2 mice homozygous for removing the proximal portion of the 7825 congenic region would Abd3, NOD and B6 alleles contributing to disease penetrance also replace a large portion of the DNA upstream of the congenic could be mapped in future studies using a large cohort of such region with NOD DNA derived from the backcross partner). To mice. assess this, we performed RNA-seq analysis of CBD from NOD. We performed histological analysis of the same F2 and BC2i Abd3 and NOD mice. We then compared the RNA-seq reads from crosses. Mice lacking the Abd3 allele, or heterozygous for the NOD and NOD.Abd3 CBDs, focusing on 0–24 Mb (ending at the region in the F2 and BC2i cohorts, had no histological lesions, proximal border of the Abd3 region) on chromosome 1, and found whereas homozygous animals had reduced histology scores for a single major genetic difference between these strains: Pkhd1 was duct proliferation and leukocytic infiltration compared with Downloaded from aberrantly expressed in NOD.Abd3 CBD samples compared with NOD.Abd3 mice (Fig. 4C, 4D). Notably, histological disease was NOD CBD samples (Fig. 3). Pkhd1 is located ∼2.5 Mb upstream not different between F2 and BC2i mice (Fig. 4C, 4D), in contrast from the proximal border of the Abd3 congenic region. In human to the clinical manifestations of disease (Fig. 4A, 4B). The fact and murine models, Pkhd1 mutations mediating biliary-hepatic that histology scores were similar between F2 and BC2i Abd3 disease (in the absence of renal pathology) produce biliary homozygous mice suggests that the B6 immune system does re-

duct–proliferation abnormalities similar to NOD.Abd3 mice (but spond, albeit less aggressively, to the aberrant regulation of Pkhd1 http://www.jimmunol.org/ largely lacking significant cholangitis/inflammation) (23–26). (i.e., the NOD genetic background does not seem to be necessary RNA-seq reads aligned to the reference genome showed that for initiation of disease [histological lesions] but is necessary for NOD.Abd3 CBD lacked expression of exon 36 to exon 67 of clinical progression of disease). Pkhd1 compared with NOD controls (Fig. 3A). Moreover, the read The development of autoimmune diabetes in NOD mice is density aligned to each exon of Pkhd1 indicated that the expres- dependent on having at least one copy of the NOD MHC, as well as sion level of exons 1–35 of Pkhd1 in NOD.Abd3 CBDs at 2 wk of a large number of other NOD background genes (27). To under- age is higher than that in NOD CBDs; in contrast, the read density stand the interaction between the Abd3 region and the NOD MHC of all 67 Pkhd1 exons in NOD CBDs was relatively constant in the context of the NOD background on the development of (Fig. 3B). ABD, we constructed NOD.H2b Abd3 mice. NOD.H2b Abd3 mice by guest on September 28, 2021 To rule out the possibility that truncated expression of Pkhd1 had a small, but statistically significant, decrease in CBD score, was due to genomic DNA deletion of exons 36–67, we designed but their liver score (Fig. 4F) and histological severity of ABD primers to amplify genomic DNA for proximal and distal exons (data not shown, n = 9 NOD.Abd3 mice and n = 24 NOD.H2b before and after the observed transcript cutoff site at exons 35– Abd3 mice, age 120 d) were not significantly different from 36). PCR showed product bands for all representative exons in NOD.Abd3 mice. This suggests that, unlike type 1 diabetes in the NOD.Abd3 comparable to NOD and B6 (Fig. 3C), clearly dem- NOD model, biliary disease was not dependent on the presence of onstrating that missing expression of Pkhd1 exons 36–67 in the NOD MHC. NOD.Abd3 was not caused by a genomic DNA deletion event. We Early cholangiocyte damage response and abnormal immune then tested Pkhd1 expression level using quantitative RT-PCR activation in NOD.Abd3 CBD (qRT-PCR). Expression of exons 15/16 in CBD from 2-wk-old NOD.Abd3 mice was nearly two times that in NOD and B6 mice, In the NOD mouse, islet inflammation begins as early as 3 wk (27) and the expression of exons 35/36 and 60/61 is dramatically and is associated with developmental processes of the pancreatic diminished (Fig. 3D), consistent with the RNA-seq results (Fig. 3B). islets. We examined the CBD of 2-wk-old mice and found dis- There was no significant difference in the expression of these exons rupted biliary epithelial architecture in NOD.Abd3 CBD compared between NOD and B6 mice (Fig. 3D), and the expression level with the smooth lumen in the CBD from NOD and NOD.c3c4-scid within NOD or B6 mice was relatively consistent (data not shown). mice (Fig. 4E). No significant histological difference was ob- Given these results, we defined Abd3 as including the B6 genetic served in the liver between NOD.Abd3 and NOD control at this region and Pkhd1 (Figs. 1A, 2A). very early time point (data not shown). These findings are con- sistent with our previously published results showing that hepatic Abd3-mediated clinical ABD requires the NOD genetic inflammation was not detectable until ∼8 wk in NOD.ABD mice background but not the NOD MHC region and that NOD.c3c4-scid mice had minimal hepatic disease (8, 10). To examine the influence of non-MHC NOD genes on ABD, we To further understand the striking difference between NOD and crossed NOD.Abd3 and B6 mice and intercrossed the resulting F1 NOD.Abd3 CBD architecture at 2 wk, we performed RNA-seq on mice. This F2 generation allows for the examination of mice CBD from 2-wk-old mice. Two-hundred and fifty-five genes were homozygous for the Abd3 region but, on average, having 50% significantly differentially expressed between the strains (Fig. 5A). NOD and 50% B6 genetic contributions to the remainder of the Cluster enrichment network analysis of these 255 genes, based on genome. To reduce the proportion of NOD-derived genes even biological processes, molecular functions, mouse-knockout phe- further, F1 mice were backcrossed twice to B6 mice and in each notypes, and biological pathways, showed that many of the genes generation breeders were selected to retain the disease-associated upregulated in 2-wk-old NOD.Abd3 CBD were extensively in- Abd3 region and then intercrossed to produce BC2i mice having, volved in cholangiocyte injury (reflecting the tissue target cell in The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 5. RNA-seq of 2-wk-old NOD.Abd3 CBD demonstrates upregulation of cholangiocyte damage response genes and downregulation of immune- related genes. (A) Heat map of 255 genes with $2-fold change and significantly different expression (see Materials and Methods) in NOD.Abd3 CBD compared with NOD CBD. (B) Significantly differentially expressed genes from (A) were used to build a cluster enrichment network. Red hexagons represent genes; squares represents biological process (cyan), molecular function (sky blue), mouse phenotype (light blue), pathways (green), and tran-

scription binding site (purple); and the triangle represents miR-30a. The left cluster represents 2-fold upregulated genes and is highly enriched in chol- by guest on September 28, 2021 angiocyte damage response genes; the right cluster represents 2-fold downregulated genes and is highly enriched in immune-related genes.

ABD), whereas genes involved in the immune response (adaptive antigen (i.e., NOD mice lack abnormal biliary epithelial cells). To and innate) were downregulated in NOD.Abd3 CBD (Fig. 5B). distinguish between these possibilities, we performed bone mar- Biological process–enrichment analysis shows that many of the row chimera studies. NOD.Abd3 irradiated recipients were upregulated genes in NOD.Abd3 mice are involved in epithelial reconstituted with NOD or NOD.Abd3 bone marrow; conversely, cell differentiation, morphology, and morphogenesis (all genes NOD recipients were reconstituted with NOD.Abd3 bone marrow. associated with these processes were upregulated), whereas the The results clearly reproduced the transfer study findings: the downregulated genes are largely involved in abnormal in- recipients must express NOD.Abd3 in biliary tissue to develop flammatory response/inflammation, leukocyte transendothelial disease. NOD.Abd3→NOD recipients had no clinical abnormality migration, hepatobiliary system and gland physiology, and cellular in CBD or liver (Fig. 6A, bars 7 and 8, significantly different from secretion (all genes associated with these processes were down- NOD.Abd3→NOD.Abd3 recipients) and no duct epithelial hy- regulated) (Table I). A few processes involved upregulated and perplasia and minimal lymphocytic infiltrates (Fig. 6B, bars 7 and downregulated genes, including cell adhesion and epithelial cell 8, significantly different from NOD.Abd3→NOD.Abd3 recipi- proliferation (Table I). These results establish that a very early tissue ents). In addition, NOD bone marrow did not cause significant abnormality interacting with an abnormal immune response drives disease in NOD.Abd3 recipients: the clinical gross liver score and the development of NOD.Abd3 biliary disease. histological duct epithelial hyperplasia score at 120 d post–bone mar- row transplant were significantly less than in NOD.Abd3→NOD.Abd3 Bone marrow chimera studies demonstrate that expression of recipients (Fig. 6). NOD.Abd3 recipients already had some dis- Abd3 in target tissue and immune cells is necessary for disease ease at the time of irradiation (Fig. 6, bars 1 and 2). In the We next dissected the level at which the NOD background was NOD.Abd3→NOD.Abd3 transfer, NOD.Abd3 bone marrow acting in the autoimmune process. We had previously shown that repopulated the mouse and caused the disease to progress. In NOD.ABD splenocytes or CD8 T cells could not transfer disease NOD→NOD.Abd3 mice, NOD bone marrow did not accelerate into NOD.scid mice and, conversely, that NOD splenocytes or the disease, and it actually reduced the histological score com- CD8 cells could not transfer disease into NOD.c3c4-scid mice. pared with 10-wk-old NOD.Abd3 mice (Fig. 6B, bars 1 and 2 These results suggested that the Abd3 locus acted on the target compared with bars 5 and 6). Therefore, there is clearly a dif- tissue and the immune system. An alternate explanation, however, ference in the ability of the hematopoietic system to cause dis- was that the NOD CD8 T cell repertoire has the potential to cause ease between the NOD and NOD.Abd3 strains; the Abd3 mutant ABD but that the disease-mediating T cell clones were not ex- Pkhd1 allele must be expressed in the hematopoietic system and panded because they were never exposed to their tissue auto- target tissue for ABD to develop. 10 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE

Table I. Biological processes associated with significantly differentially expressed genes in NOD.Abd3 CBD Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

Biological process enrichment analysis shows the significantly differentially expressed genes in CBD from 2-wk-old mice (from Fig. 5A) associated with biological processes characterized by by upregulated genes (in red) and downregulated genes (in green). The biological processes characterized by all upregulated or downregulated genes are in green and red, respectively. The processes involving some upregulated and some downregulated genes are in black and the individual genes are in green or red depending on whether they are upregulated or downregulated. The Journal of Immunology 11

the splice acceptor site (AG) before the start of exon 36. In con- trast, there were multiple microsatellite repeats in the uniquely transcribed intron region and poly(A) signal (AAUAAA), which suggested that it could be a 39 untranslated region (UTR)-like region (29) in Pkhd1. To evaluate further the unique 3ʹ UTR–like region in intron 35 of NOD.Abd3 Pkhd1 mice, we designed three primer pairs (primers 1–3 in Table II, Supplemental Table III) to perform tiled PCR starting from the end of the 39 UTR–like region in intron 35 to- ward exon 35 (Fig. 8A). The first two pairs of tiled PCR primers at the end of the novel 39 UTR–like region of intron 35 amplified in NOD mice but not in NOD.Abd3 mice (Fig. 8B, Table II). We then designed seven primer pairs (every 5 kb) from exon 36 toward the end of the novel 39 UTR–like region in intron 35 (N1–N7, Table II, Supplemental Table III). Each of these primer pairs amplified in NOD and NOD.Abd3 mice (Fig. 8C). Lastly, using the forward primer of primer pair N7 and the reverse primer of primer pair 3 (Fig. 8A, Table II), we found that a PCR product size

in NOD mice was ∼3 kb (similar to reference target sequence). In Downloaded from striking contrast, the PCR product size in NOD.Abd3 mice was ∼9 kb (Fig. 8D). Therefore, the mutation within Pkhd1 was a DNA insertion of ∼6kb. Using next-generation sequencing technology, we sequenced the PCR products through de novo reads assembly. The assembled

PCR product sequence of NOD mice could be fully aligned with the http://www.jimmunol.org/ reference genome, whereas only part of the three nodes of the assembly sequence of NOD.Abd3 mice matched with the NOD sequence. The node 2–assembled sequence in NOD.Abd3 Pkhd1 intron 35 was analyzed in RepeatMasker and had 99.73% identity with long terminal repeat elements of four class II endogenous retroviruses (ERVs) (Fig. 8E). Given these results of the genetic characterization of Pkhd1 in FIGURE 6. Bone marrow chimera studies show that Abd3 expression is NOD.Abd3 mice, we propose the following potential mechanism: necessary in the recipient (nonhematopoietic tissue) and in the donor the 6-kb long terminal repeat ERV DNA insertion in intron 35 of by guest on September 28, 2021 (hematopoietic tissue) to recapitulate disease. Clinical CBD and liver NOD.Abd3 Pkhd1 mice disrupts normal recognition of splicing scores (A) and histological scores (B) of NOD.Abd3 mice at 10 wk (n = 7), sites in this intron and forces the exposure of an alternative pol- NOD.Abd3 recipients of NOD.Abd3 (n = 3, recipients were 8–10 wk old at yadenylation site within the intron, thus giving rise to a novel the time of bone marrow transplantation [BMT]) or NOD bone marrow shortened transcript that ends after exon 35 (Fig. 8F). Given that (n = 4, recipients were 9–10 wk old at the time of BMT), and NOD recipients qRT-PCR and RNA-seq show an upregulation of the first 35 exons of NOD.Abd3 bone marrow (n = 6, recipients were 8–14 wk old at the time of BMT). Error bars represent SEM. *p , 0.05, Mann–Whitney U test. of Pkhd1 in NOD.Abd3 CBD, this alternative polyadenylation site must predominate over the normal polyadenylation site (after exon 67) in NOD.Abd3 mice. Although the expression of Pkhd1 in immune cells has never In summary, we have shown that ABD in NOD congenic mouse been reported previously, to our knowledge, the Ensembl database strains is associated with a novel Pkhd1 mutation. Uniquely, on the shows that RNA-seq reads from spleen are aligned to Pkhd1 (28). NOD background, this mutation results in an autoimmune disease We hypothesized that a deficient exon expression pattern of Pkhd1 that depends on expression of mutated Pkhd1 in the target tissue could be detected in CBD, the target tissue, as well as in immune and hematopoietic system. Early abnormalities in cholangiocyte cells, explaining why both systems were necessary for ABD. morphology and development are accompanied by dysregulation However, we were unable to detect Pkhd1 expression in unac- of the immune system, resulting in a genetically controlled tivated splenocytes, intrahepatic immune cells, or MACS-sorted autoimmune disease. CD4 or CD8 T cells from spleen and liver using exons 15/16, 35/36, and 60/61 (data not shown). Discussion Details of the Pkhd1 genetic mutation in NOD.Abd3 and In this article, we prove conclusively that NOD ABD is caused by a potential mechanism of truncated transcription recessively acting region on chromosome 1. This region, desig- Pkhd1 is ∼2.5 Mb upstream from the Abd3 allele (Fig. 7A). The nated Abd3, is necessary and sufficient for ABD when on the NOD RNA-seq data, as well as the qRT-PCR results, revealed a novel background. The only genetic difference that we found in the loss of Pkhd1 transcription after exon 35, and we sought to un- Abd3 region of diseased NOD.Abd3 mice compared with NOD derstand how this transcription deregulation occurs in NOD.Abd3 mice was aberrant expression of Pkhd1. Pkhd1 is a large gene with mice. The alignment of the RNA-seq reads from 2-wk-old 67 exons (4059 aa) encoding fibrocystin, a membrane-associated NOD.Abd3 CBD and NOD CBD identified a unique transcribed receptor-like with a single transmembrane domain, a small intronic region immediately after exon 35 (the second longest intracellular domain, and a large extracellular domain containing Pkhd1 intron) in NOD.Abd3 mice but not in NOD mice (Fig. 7B). multiple repeats of Ig-like plexin-transcription factor domains and We found no mutations at the splice donor site (GT) after exon 35 or two G8 domains (predicted to contain 10 b strands and an a helix) 12 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. RNA-seq data reveal early termination of Pkhd1 transcription in NOD.Abd3 CBD. (A) Genomic organization of Pkhd1 and Abd3 alleles. Left and right boxes indicate the Pkhd1 gene and the B6/B10 Abd3 congenic region, respectively. The Pkhd1 gene and the Abd3 region are ∼2.5 Mb apart. (B) Magnified view of read-alignment from CBD RNA-seq data in the intron between exons 35 and 36 of Pkhd1 comparing NOD and NOD.Abd3 samples shows unique expression in NOD.Abd3. Left, middle, and right dashed lines indicate the position of exon 35, 36, and the predicted pseudogene Gm15795, respectively, across all four RNA-seq tracks.

(30, 31). We anticipate that the mutated Pkhd1 protein in sion and proliferation (24). Pkhd1 is extensively alternatively NOD.Abd3 mice, if it is expressed, has lost the G8 domains, as well as spliced and is spliced differently in different cell types, but a role the transmembrane and intracellular domains. Fibrocystin has for alternately spliced products has not been demonstrated domains that are similar to those found in plexin and hepatocyte (32, 33). The human and mouse protein sequences share 73% growth factor families; these domains function in cellular adhe- identity (30, 34). During embryogenesis, fibrocystin is widely The Journal of Immunology 13

Table II. PCR amplification result of different primer pairs

Primer Set or Exon Position Starting Positiona Ending Positiona Amplification of NOD.Abd3 Amplification of NOD Exon 36 20,440,302 20,440,458 NA NA Primer N1 20,440,373 20,440,692 Yes Yes Primer N2 20,445,607 20,445,925 Yes Yes Primer N3 20,450,397 20,450,733 Yes Yes Primer N4 20,455,542 20,455,846 Yes Yes Primer N5 20,460,303 20,460,626 Yes Yes Primer N6 20,465,591 20,465,989 Yes Yes Primer N7 20,470,616 20,470,976 Yes Yes Primer 1 20,472,908 20,473,208 No Yes Primer 2 20,473,134 20,473,447 No Yes Primer 3 20,473,238 20,473,540 Yes Yes Exon 35 20,493,023 20,493,173 NA NA aGenomic coordinates refer to chromosome 1. Genomic assembly: NCBI37/mm9. NA: no PCR was performed; No: no band shown on the gel; Yes: single band with expected PCR product size. expressed in epithelial derivatives, including neural tubules, gut, prevent clinical or histological ABD in the presence of Abd3 pulmonary bronchi, renal cells, and hepatic cells. By embryonic homozygosity (Fig. 4F) (9). This suggests that NOD genes distinct Downloaded from day 15, fibrocystin is strongly expressed in bile duct cholangiocyte from those critical for the development of type 1 diabetes in the cilia, as well as in the epithelial cells in the kidney-collecting NOD model may be essential for the clinical manifestation of tubes (32, 35, 36). mAbs also detect fibrocystin in human pan- ABD. creatic duct, islets, testis, and adrenal gland (37). At this point, we cannot fully explain our findings, because we NOD.Abd3 mice have normal renal structure throughout life. have been unable to verify Pkhd1 expression in the immune cells

Consistent with this, prior mouse models with engineered deletions that we have tested. It is possible that the upregulated Pkhd1 exons http://www.jimmunol.org/ of Pkhd1 have no renal abnormalities. Mice with targeted deletion 1–35 play a role in immune cells; another possibility is that mutant of Pkhd1 exon 3, 4, or 40 had no kidney abnormality, but they Pkhd1 affects alternative splicing in an immune cell subset (e.g., demonstrated intrahepatic bile duct proliferation with progressive macrophages) and that we need to use different primers to detect cyst formation (similar to our model) and associated periportal fi- the immune cell–specific transcript. Pkhd1 expression in the brosis (not seen in NOD.Abd3 mice) (23, 24, 33). Pkhd1ex4del mice thymus could affect the immune repertoire. Finally, we cannot develop splenomegaly, which is also found in our model. exclude that the linked B6-derived congenic region present in the Pkhd1ex4del mice develop a syndrome of congenital hepatic fibrosis, NOD.Abd3 strain plays some role in the pathogenesis of these and Pkhd1ex4del cholangiocytes secrete chemokines, including mice. Although we have not shown any differentially expressed

CXCL1, CXCL2, and CSCL12, which aberrantly recruit macro- genes from this region, we will ultimately only be able to formally by guest on September 28, 2021 phages (38). Macrophage depletion in Pkhd1ex4del mice decreases disprove a role for the 1.0-Mb B6-derived congenic segment fibrosis and cystic disease. The similarity of the biliary epithelial within the Abd3 region in future studies by selecting a recombi- abnormalities and, in some cases, the presence of hematopoietic cell nation event between the congenic region and Pkhd1 and testing infiltrates in these other models of Pkhd1 mutation, leads us to the effect of the mutant Pkhd1 gene in the absence of the congenic conclude that mutated Pkhd1 is the primary cause of ABD in our region (on the NOD background) on disease. model. Our model is unique, however, because none of the previous Bone marrow– and splenocyte-transfer studies show that the models were shown to involve an autoimmune response nor did Abd3 allele/mutated Pkhd1 must be expressed in the immune they require, as far as we know, Pkhd1 expression in hematopoietic system and target tissue to develop cholangitis. The question re- cells. mains how an alteration of a single gene (Pkhd1) involved in Disease expression in other Pkhd1 models was also apparently cilia function and epithelial integrity in cholangiocytes can ulti- unaffected by the genetic background. Thus, the role of the genetic mately lead to an autoimmune disease. Interestingly, the biological background in our model is also unique; we show decreased process–enrichment analysis of RNA-seq data from NOD.Abd3 histological abnormalities (and reduced or no clinical disease CBD reveals a striking pattern, with the downregulated genes ex- depending on the amount of residual NOD genetic material tensively involved in acute immune/inflammatory response (Table I). remaining) when the Pkhd1 mutation is expressed on a genetic Many of these genes can negatively regulate the immune response, background that is predominantly B6. Notably, however, there was thus mediating a protective role against, for example, overexu- no significant difference in histological disease scores between F2 berant immune responses to microbial infection. For example, and BC2i mice (Fig. 4C, 4D), suggesting that disease initiation although there is a large number of reports on its proinflammatory (manifest as histological lesions) does not require the NOD ge- effect, S100A9 (Table I) also has anti-inflammatory effects netic background. In contrast, clinical disease (liver and CBD through inhibition of neutrophil oxidative metabolism (39, 40) and scores in Fig. 4A, 4B) requires the NOD genetic background. neutrophil chemorepulsion. Serpinc1 can induce the anti- Although outside the scope of the current study, mapping the inflammatory cytokine PGI2 from cultured HUVECs (41), Spdef NOD background regions required for clinical disease, in con- negatively regulates TLR signaling in airway epithelial cells junction with Abd3 homozygosity, would be achievable by genetic (42), Vip executes protective humoral responses against patho- analysis of (NOD.Abd3 3 B6) F2 mice having two doses of the gens (43), and CORO1A is linked to inhibition of neutrophil Abd3 region, because disease was observed to be present in a apoptosis in chronic inflammation, which is essential to resolve portion of these mice (Fig. 4A, 4B). We note that introgression of inflammation (44). genetic regions derived from B6 or B10 mice into the NOD strain Downregulation of several of these genes results in increased that prevent type 1 diabetes [i.e., introgression of the MHC region inflammation and autoimmune disease [e.g., ablation of Syt7 in and large segments of chromosomes 3 and 4 (9, 15)] does not mice resulted in inflammatory myopathy (45); C2GnT2-knockout 14 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Mutation characterization of Pkhd1 in NOD.Abd3 and proposed mechanism of deficient NOD.Abd3 Pkhd1 exon expression pattern. (A) Schematic diagram showing strategy of PCR primer design in intron 35 of Pkhd1.(B) Primers 1 and 2 failed to amplify using NOD.Abd3 genomic DNA, whereas primer 3 successfully amplifies using NOD.Abd3 and NOD genomic DNA. (C) Primers N1, N2, N3, N4, N5, N6, and N7 were able to amplify the PCR product with expected product size using NOD.Abd3 and NOD genomic DNA. (D) Long-range PCR using primer N7 forward primer and primer 3 reverse primer showed distinct product sizes on NOD.Abd3 and NOD genomic DNA. (E) Schematic representation of assembled long-range PCR product sequence in NOD and NOD.Abd3 mice with relative position of the primer pair. Three nodes of de novo assembly were generated for NOD.Abd3, with two small gaps present in between node 1/node 2 and node 2/node 3. Parts of node 1 and node 3 can be matched with NOD assembled sequence, and the rest of the NOD.Abd3 assembly matches a class II ERV. Genomic positions of the primers and the sequences are based on B6 NCBI37/mm 9 assembly. (F) Schematic representation of potential mechanism of truncated Pkhd1 transcript in NOD.Abd3 CBD. APA, alternative polyadenylation site. The Journal of Immunology 15 mice have decreased mucosal barrier function in the digestive 4. Mack, C. L. 2007. The pathogenesis of biliary atresia: evidence for a virus- induced autoimmune disease. Semin. Liver Dis. 27: 233–242. tract, reduced levels of circulating IgGs and fecal IgA, and in- 5. Brindley, S. M., A. M. Lanham, F. M. Karrer, R. M. Tucker, A. P. Fontenot, and creased susceptibility to experimental colitis (46); and comple- C. L. Mack. 2012. Cytomegalovirus-specific T-cell reactivity in biliary atresia at ment deficiency is associated with lupus (47)]. We speculate that the time of diagnosis is associated with deficits in regulatory T cells. Hepatology 55: 1130–1138. the downregulation of these anti-inflammatory/regulatory genes in 6. Rong, G., R. Zhong, A. Lleo, P. S. C. Leung, C. L. Bowlus, G.-X. Yang, the innate immune system or in cholangiocytes makes it difficult to C.-Y. Yang, R. L. Coppel, A. A. Ansari, D. A. Cuebas, et al. 2011. Epithelial cell resolve inflammatory responses, which may facilitate the generation specificity and apotope recognition by serum autoantibodies in primary biliary cirrhosis. Hepatology 54: 196–203. of neoantigen in the target tissue. Generation of neoantigens, in the 7. Shivakumar, P., G. Sabla, S. Mohanty, M. McNeal, R. Ward, K. Stringer, presence of altered bile composition and abnormal duct physiology, C. Caldwell, C. Chougnet, and J. A. Bezerra. 2007. Effector role of neonatal hepatic CD8+ lymphocytes in epithelial injury and autoimmunity in experi- could escalate the immune response and progression to an adaptive mental biliary atresia. Gastroenterology 133: 268–277. immune response to self-targets. 8. Irie, J., Y. Wu, L. S. Wicker, D. Rainbow, M. A. Nalesnik, R. Hirsch, Mutation of Pkhd1 likely results in a cholangiociliopathy with L. B. Peterson, P. S. Leung, C. Cheng, I. R. Mackay, et al. 2006. NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and impaired primary cholangiocyte cilia. Defective cilia structure can pathogenetically models human primary biliary cirrhosis. J. Exp. Med. 203: compromise the integrated sensory/transducing functions, resulting 1209–1219. in increased cholangiocyte cAMP and decreased [Ca2+](48), 9. Koarada, S., Y. Wu, N. Fertig, D. A. Sass, M. Nalesnik, J. A. Todd, P. A. Lyons, J. Fenyk-Melody, D. B. Rainbow, L. S. Wicker, et al. 2004. Genetic control of causing cholangiocyte hyperproliferation, changes in metabolic autoimmunity: protection from diabetes, but spontaneous autoimmune biliary processes, abnormal cell–matrix interactions, and altered fluid disease in a nonobese diabetic congenic strain. J. Immunol. 173: 2315–2323. 10. Yang, G. X., Y. Wu, H. Tsukamoto, P. S. Leung, Z. X. Lian, D. B. Rainbow, secretion/absorption (water and bile modification), as reflected by K. M. Hunter, G. A. Morris, P. A. Lyons, L. B. Peterson, et al. 2011. CD8 T cells the gene cluster analysis (Fig. 5B, Table I). Therefore, the entire mediate direct biliary ductule damage in nonobese diabetic autoimmune biliary Downloaded from cholangiocyte environment is changed, and the outcome of the disease. J. Immunol. 186: 1259–1267. 11. Oertelt, S., Z.-X. Lian, C.-M. Cheng, Y.-H. Chuang, K. A. Padgett, X.-S. He, change in one gene (Pkhd1) leads to altered morphogenesis and W. M. Ridgway, A. A. Ansari, R. L. Coppel, M. O. Li, et al. 2006. Anti- altered immune-related responses. mitochondrial antibodies and primary biliary cirrhosis in TGF-b receptor II The results of the bone marrow–transfer studies clearly dem- dominant-negative mice. J. Immunol. 177: 1655–1660. 12. Shivakumar, P., K. M. Campbell, G. E. Sabla, A. Miethke, G. Tiao, onstrate that NOD.Abd3 immune cells are pathogenic, whereas M. M. McNeal, R. L. Ward, and J. A. Bezerra. 2004. Obstruction of extrahepatic

NOD immune cells are not. NOD T cells are unable to cause bile ducts by lymphocytes is regulated by IFN-g in experimental biliary atresia. http://www.jimmunol.org/ + J. Clin. Invest. 114: 322–329. disease, even when they develop in an Abd3 host with abnormal 13. Torres, V. E. 1995. Polycystic liver disease. Contrib. Nephrol. 115: 44–52. cholangiocytes. Taken together with a similar result in our transfer 14. Hamilton-Williams, E. E., X. Martinez, J. Clark, S. Howlett, K. M. Hunter, studies (10), this suggests that the Pkhd1 mutation must be D. B. Rainbow, L. Wen, M. J. Shlomchik, J. D. Katz, G. F. Beilhack, et al. 2009. Expression of diabetes-associated genes by dendritic cells and CD4 T cells expressed in the target tissue and in the hematopoietic tissue. In drives the loss of tolerance in nonobese diabetic mice. J. Immunol. 183: 1533– contrast, requirement of the NOD genetic background (which is 1541. highly disposed to autoimmunity) for disease manifestation sup- 15. Wicker, L. S., M. C. Appel, F. Dotta, A. Pressey, B. J. Miller, N. H. DeLarato, P. A. Fischer, R. C. Boltz, Jr., and L. B. Peterson. 1992. Autoimmune syndromes ports the idea that the immune system perpetuates and enhances in major histocompatibility complex (MHC) congenic strains of nonobese dia- cholangitis in NOD.Abd3 mice. Future studies will be directed to betic (NOD) mice. The NOD MHC is dominant for insulitis and

cyclophosphamide-induced diabetes. J. Exp. Med. 176: 67–77. by guest on September 28, 2021 discover the critical immune subsets and the mechanism by which 16. Bednar, K. J., H. Tsukamoto, K. Kachapati, S. Ohta, Y. Wu, J. D. Katz, the Pkhd1 mutation affects the immune system. D. P. Ascherman, and W. M. Ridgway. 2015. Reversal of new-onset type In summary, we have identified a novel mutation in Pkhd1 as a 1 diabetes with an agonistic TLR4/MD-2 monoclonal antibody. Diabetes 64: 3614–3626. key genetic factor causing the biliary tissue abnormality and ac- 17. Fraser, H. I., S. Howlett, J. Clark, D. B. Rainbow, S. M. Stanford, D. J. Wu, tivating the adaptive immune system in the context of the NOD Y. W. Hsieh, C. J. Maine, M. Christensen, V. Kuchroo, et al. 2015. Ptpn22 and genetic background. Although we have clearly demonstrated that Cd2 variations are associated with altered protein expression and susceptibility to type 1 diabetes in nonobese diabetic mice. J. Immunol. 195: 4841–4852. the target tissue (cholangiocytes) and the immune system must 18. Langmead, B., C. Trapnell, M. Pop, and S. L. Salzberg. 2009. Ultrafast and express the Pkhd1 mutation, we do not yet understand the role of memory-efficient alignment of short DNA sequences to the . Genome Biol. 10: R25. the mutation in each system or how the systems interact. Abnor- 19. Kim, D., G. Pertea, C. Trapnell, H. Pimentel, R. Kelley, and S. L. Salzberg. 2013. malities involving hundreds of tissue-specific (cholangiocyte TopHat2: accurate alignment of transcriptomes in the presence of insertions, specific) and immune-origin genes are affected by 2 wk of age, deletions and gene fusions. Genome Biol. 14: R36. 20. Trapnell, C., A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel, indicating a complex orchestrated event producing this organ-specific S. L. Salzberg, J. L. Rinn, and L. Pachter. 2012. Differential gene and transcript autoimmune disease. Thus, our mouse model of spontaneously expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. mutated Pkhd1 is valuable for exploring several cholangiopathies, Protoc. 7: 562–578. 21. Chen, J., H. Xu, B. J. Aronow, and A. G. Jegga. 2007. Improved human disease including primary biliary cholangitis, primary sclerosing chol- candidate gene prioritization using mouse phenotype. BMC Bioinformatics 8: angitis, and polycystic hepatobiliary disease. 392. 22. Kaimal, V., E. E. Bardes, S. C. Tabar, A. G. Jegga, and B. J. Aronow. 2010. ToppCluster: a multiple gene list feature analyzer for comparative enrichment Acknowledgments clustering and network-based dissection of biological systems. Nucleic Acids We thank Mehdi Keddache and Satwica Yerneni for assistance with RNA-seq. Res. 38(Web Server): W96–102. 23. Gallagher, A.-R., E. L. Esquivel, T. S. Briere, X. Tian, M. Mitobe, L. F. Menezes, G. S. Markowitz, D. Jain, L. F. Onuchic, and S. Somlo. 2008. Biliary and Disclosures pancreatic dysgenesis in mice harboring a mutation in Pkhd1. Am. J. Pathol. 172: 417–429. The authors have no financial conflicts of interest. 24. Moser, M., S. Matthiesen, J. Kirfel, H. Schorle, C. Bergmann, J. Senderek, S. Rudnik-Scho¨neborn, K. Zerres, and R. Buettner. 2005. A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease References (ARPKD). Hepatology 41: 1113–1121. 1. Kaplan, M. M., and M. E. Gershwin. 2005. Primary biliary cirrhosis. N. Engl. J. 25. Woollard, J. R., R. Punyashtiti, S. Richardson, T. V. Masyuk, S. Whelan, Med. 353: 1261–1273. B. Q. Huang, D. J. Lager, J. vanDeursen, V. E. Torres, V. H. Gattone, et al. 2007. 2. Invernizzi, P., C. Selmi, and M. E. Gershwin. 2010. Update on primary biliary A mouse model of autosomal recessive polycystic kidney disease with biliary cirrhosis. Dig. Liver Dis. 42: 401–408. duct and proximal tubule dilatation. Kidney Int. 72: 328–336. 3. Eaton, J. E., J. A. Talwalkar, K. N. Lazaridis, G. J. Gores, and K. D. Lindor. 26. Williams, S. S., P. Cobo-Stark, L. R. James, S. Somlo, and P. Igarashi. 2008. 2013. Pathogenesis of primary sclerosing cholangitis and advances in diagnosis Kidney cysts, pancreatic cysts, and biliary disease in a mouse model of auto- and management. Gastroenterology 145: 521–536. somal recessive polycystic kidney disease. Pediatr. Nephrol. 23: 733–741. 16 GENETIC CAUSE OF NOD AUTOIMMUNE BILIARY DISEASE

27. Bouma, G., J. M. C. Coppens, S. Mourits, T. Nikolic, S. Sozzani, Cellular and subcellular localization of the ARPKD protein; fibrocystin is H. A. Drexhage, and M. A. Versnel. 2005. Evidence for an enhanced adhesion of expressed on primary cilia. Hum. Mol. Genet. 12: 2703–2710. DC to fibronectin and a role of CCL19 and CCL21 in the accumulation of DC 38. Locatelli, L., M. Cadamuro, C. Spirlı`, R. Fiorotto, S. Lecchi, C. M. Morell, around the pre-diabetic islets in NOD mice. Eur. J. Immunol. 35: 2386–2396. Y. Popov, R. Scirpo, M. De Matteis, M. Amenduni, et al. 2016. Macrophage 28. Flicek, P., M. R. Amode, D. Barrell, K. Beal, K. Billis, S. Brent, D. Carvalho- recruitment by fibrocystin-defective biliary epithelial cells promotes portal fi- Silva, P. Clapham, G. Coates, S. Fitzgerald, et al. 2014. Ensembl 2014. Nucleic brosis in congenital hepatic fibrosis. Hepatology 63: 965–982. Acids Res. 42(D1): D749–D755. 39. Sroussi, H. Y., Y. Lu, Q. L. Zhang, D. Villines, and P. T. Marucha. 2010. S100A8 29. Mignone, F., C. Gissi, S. Liuni, and G. Pesole. 2002. Untranslated regions of and S100A9 inhibit neutrophil oxidative metabolism in-vitro: involvement of mRNAs. Genome Biol. 3: REVIEWS0004. adenosine metabolites. Free Radic. Res. 44: 389–396. 30. Onuchic, L. F., L. Furu, Y. Nagasawa, X. Hou, T. Eggermann, Z. Ren, 40. Sroussi, H. Y., Y. Lu, D. Villines, and Y. Sun. 2012. The down regulation of C. Bergmann, J. Senderek, E. Esquivel, R. Zeltner, et al. 2002. PKHD1, the neutrophil oxidative metabolism by S100A8 and S100A9: implication of the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein protease-activated receptor-2. Mol. Immunol. 50: 42–48. containing multiple immunoglobulin-like plexin-transcription-factor domains 41. Horie, S., H. Ishii, and M. Kazama. 1990. Heparin-like glycosaminoglycan is a and parallel beta-helix 1 repeats. Am. J. Hum. Genet. 70: 1305–1317. receptor for antithrombin III–dependent but not for thrombin-dependent pros- 31. Xiong, H., Y. Chen, Y. Yi, K. Tsuchiya, G. Moeckel, J. Cheung, D. Liang, tacyclin production in human endothelial cells. Thromb. Res. 59: 895–904. K. Tham, X. Xu, X.-Z. Chen, et al. 2002. A novel gene encoding a TIG multiple 42. Korfhagen, T. R., J. Kitzmiller, G. Chen, A. Sridharan, H.-M. Haitchi, domain protein is a positional candidate for autosomal recessive polycystic R. S. Hegde, S. Divanovic, C. L. Karp, and J. A. Whitsett. 2012. SAM-pointed kidney disease. Genomics 80: 96–104. domain ETS factor mediates epithelial cell-intrinsic innate immune signaling 32. Nagasawa, Y., S. Matthiesen, L. F. Onuchic, X. Hou, C. Bergmann, E. Esquivel, during airway mucous metaplasia. Proc. Natl. Acad. Sci. USA 109: 16630– J. Senderek, Z. Ren, R. Zeltner, L. Furu, et al. 2002. Identification and characterization 16635. of Pkhd1, the mouse orthologue of the human ARPKD gene. J. Am. Soc. Nephrol. 43. El Karim, I. A., G. J. Linden, D. F. Orr, and F. T. Lundy. 2008. Antimicrobial 13: 2246–2258. activity of neuropeptides against a range of micro-organisms from skin, oral, 33. Bakeberg, J. L., R. Tammachote, J. R. Woollard, M. C. Hogan, H. F. Tuan, M. Li, respiratory and gastrointestinal tract sites. J. Neuroimmunol. 200: 11–16. J. M. van Deursen, Y. Wu, B. Q. Huang, V. E. Torres, et al. 2011. Epitope-tagged 44. Moriceau, S., C. Kantari, J. Mocek, N. Davezac, J. Gabillet, I. C. Guerrera, Pkhd1 tracks the processing, secretion, and localization of fibrocystin. J. Am. F. Brouillard, D. Tondelier, I. Sermet-Gaudelus, C. Danel, et al. 2009. Coronin-1 Soc. Nephrol. 22: 2266–2277. is associated with neutrophil survival and is cleaved during apoptosis: potential Downloaded from 34. Ward, C. J., M. C. Hogan, S. Rossetti, D. Walker, T. Sneddon, X. Wang, implication in neutrophils from cystic fibrosis patients. J. Immunol. 182: 7254– V. Kubly, J. M. Cunningham, R. Bacallao, M. Ishibashi, et al. 2002. The gene 7263. mutated in autosomal recessive polycystic kidney disease encodes a large, 45. Chakrabarti, S., K. S. Kobayashi, R. A. Flavell, C. B. Marks, K. Miyake, receptor-like protein. Nat. Genet. 30: 259–269. D. R. Liston, K. T. Fowler, F. S. Gorelick, and N. W. Andrews. 2003. Impaired 35. Masyuk, T. V., B. Q. Huang, C. J. Ward, A. I. Masyuk, D. Yuan, P. L. Splinter, membrane resealing and autoimmune myositis in synaptotagmin VII-deficient R. Punyashthiti, E. L. Ritman, V. E. Torres, P. C. Harris, and N. F. LaRusso. mice. J. Cell Biol. 162: 543–549. 2003. Defects in cholangiocyte fibrocystin expression and ciliary structure in the 46. Stone, E. L., S. H. Lee, M. N. Ismail, and M. Fukuda. 2010. Characterization of

PCK rat. Gastroenterology 125: 1303–1310. mice with targeted deletion of the gene encoding core 2 beta1,6-N-acetylglucos- http://www.jimmunol.org/ 36. Zhang, M.-Z., W. Mai, C. Li, S. Y. Cho, C. Hao, G. Moeckel, R. Zhao, I. Kim, aminyltransferase-2. Methods Enzymol. 479: 155–172. J. Wang, H. Xiong, et al. 2004. PKHD1 protein encoded by the gene for autosomal 47. Manderson, A. P., M. Botto, and M. J. Walport. 2004. The role of complement in recessive polycystic kidney disease associates with basal bodies and primary cilia the development of systemic lupus erythematosus. Annu. Rev. Immunol. 22: 431– in renal epithelial cells. Proc. Natl. Acad. Sci. USA 101: 2311–2316. 456. 37. Ward, C. J., D. Yuan, T. V. Masyuk, X. Wang, R. Punyashthiti, S. Whelan, 48. Masyuk, A. I., T. V. Masyuk, and N. F. LaRusso. 2008. Cholangiocyte primary R. Bacallao, R. Torra, N. F. LaRusso, V. E. Torres, and P. C. Harris. 2003. cilia in liver health and disease. Dev. Dyn. 237: 2007–2012. by guest on September 28, 2021