Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Receptor Deficiency Promotes the Ductular Reaction, Macrophage Accumulation, and Hepatic Fibrosis in the Abcb4 Mouse
2020; Elsevier BV; Volume: 190; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2020.02.013
ISSN1525-2191
AutoresAnuradha Krishnan, Tomohiro Katsumi, Maria Eugenia Guicciardi, Adiba Azad, Nazlı Begüm Öztürk, Christy E. Trussoni, Gregory J. Gores,
Tópico(s)Liver physiology and pathology
ResumoThe tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr−/−) mice were crossbred with ATP binding cassette subfamily B member 4–deficient (Abcb4−/−, alias Mdr2−/−) mice. Male and female wild-type, Tr−/−, Mdr2−/−, and Tr−/−Mdr2−/− mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2−/− and Tr−/−Mdr2−/− mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2−/− mice, compared with extensive bridging fibrosis in Tr−/−Mdr2−/− mice. DR cell population was greatly expanded in the Tr−/−Mdr2−/− versus Mdr2−/− mice. The expanded DR cell population in Tr−/−Mdr2−/− mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr−/−Mdr2−/− versus Mdr2−/− mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2−/− mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2−/− model of cholestasis. The tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr−/−) mice were crossbred with ATP binding cassette subfamily B member 4–deficient (Abcb4−/−, alias Mdr2−/−) mice. Male and female wild-type, Tr−/−, Mdr2−/−, and Tr−/−Mdr2−/− mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2−/− and Tr−/−Mdr2−/− mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2−/− mice, compared with extensive bridging fibrosis in Tr−/−Mdr2−/− mice. DR cell population was greatly expanded in the Tr−/−Mdr2−/− versus Mdr2−/− mice. The expanded DR cell population in Tr−/−Mdr2−/− mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr−/−Mdr2−/− versus Mdr2−/− mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2−/− mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2−/− model of cholestasis. The bile ducts are lined by cholangiocytes, which modify bile and provide a barrier between biliary constituents in the ductular lumen and the surrounding tissue. Unfortunately, the cholangiocytes are affected by inflammation and fibrosis in a variety of human diseases termed the cholangiopathies.1Lazaridis K.N. Strazzabosco M. Larusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar These cholangiopathies are associated with impaired bile formation, causing cholestasis. Over time, the cholestatic liver injury can result in advanced hepatic fibrosis, cirrhosis, and end-stage liver disease; hence, these diseases are associated with considerable morbidity and mortality. The inflammatory and fibrotic processes mediating the cholangiopathies are incompletely understood, which has limited the development of rational therapies. A histopathologic feature of the cholangiopathies is the development of a ductular reactive (DR) cell population; epithelial cells characterized by a biliary phenotype, organized into irregular shaped structures, often without a lumen, initially localized at the peripheral region of the portal space, from which they often extend into the hepatic parenchyma.2Fabris L. Spirli C. Cadamuro M. Fiorotto R. Strazzabosco M. Emerging concepts in biliary repair and fibrosis.Am J Physiol Gastrointest Liver Physiol. 2017; 313: G102-G116Crossref PubMed Scopus (35) Google Scholar,3Sato K. Marzioni M. Meng F. Francis H. Glaser S. Alpini G. Ductular reaction in liver diseases: pathological mechanisms and translational significances.Hepatology. 2019; 69: 420-430Crossref PubMed Scopus (81) Google Scholar The DR cells exist in a niche intimately associated with myofibroblasts, and accordingly, the magnitude of the DR cell population correlates with the severity of fibrosis in cholestatic liver diseases.3Sato K. Marzioni M. Meng F. Francis H. Glaser S. Alpini G. Ductular reaction in liver diseases: pathological mechanisms and translational significances.Hepatology. 2019; 69: 420-430Crossref PubMed Scopus (81) Google Scholar DR cells have also been associated with macrophage-mediated inflammation.2Fabris L. Spirli C. Cadamuro M. Fiorotto R. Strazzabosco M. Emerging concepts in biliary repair and fibrosis.Am J Physiol Gastrointest Liver Physiol. 2017; 313: G102-G116Crossref PubMed Scopus (35) Google Scholar,4Banales J.M. Huebert R.C. Karlsen T. Strazzabosco M. LaRusso N.F. Gores G.J. Cholangiocyte pathobiology.Nat Rev Gastroenterol Hepatol. 2019; 16: 269-281Crossref PubMed Scopus (89) Google Scholar, 5Bird T.G. Lu W.Y. Boulter L. Gordon-Keylock S. Ridgway R.A. Williams M.J. Taube J. Thomas J.A. Wojtacha D. Gambardella A. Sansom O.J. Iredale J.P. Forbes S.J. Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling.Proc Natl Acad Sci U S A. 2013; 110: 6542-6547Crossref PubMed Scopus (100) Google Scholar, 6Hsieh W.C. Mackinnon A.C. Lu W.Y. Jung J. Boulter L. Henderson N.C. Simpson K.J. Schotanus B. Wojtacha D. Bird T.G. Medine C.N. Hay D.C. Sethi T. Iredale J.P. Forbes S.J. Galectin-3 regulates hepatic progenitor cell expansion during liver injury.Gut. 2015; 64: 312-321Crossref PubMed Scopus (32) Google Scholar These observations implicate DR cells as mediators of liver injury in cholestasis. However, few studies have examined the mechanisms regulating the extent of the DR cell population during cholestasis. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), also known as TNF superfamily member 10 (TNFSF10), a death ligand, induces apoptosis in cells expressing its cognate receptors. Two pro-apoptotic TRAIL receptors (TRs) are expressed in humans, TRAIL-receptor 1 or death receptor 4 and TRAIL-receptor 2/death receptor 5. Mice have a single TR.7Martinez F.O. Gordon S. Locati M. Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression.J Immunol. 2006; 177: 7303-7311Crossref PubMed Scopus (1551) Google Scholar Although TRs are ubiquitously expressed, TRAIL is mainly expressed on cells of the immune system, including macrophages.7Martinez F.O. Gordon S. Locati M. Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression.J Immunol. 2006; 177: 7303-7311Crossref PubMed Scopus (1551) Google Scholar Interestingly, TR-mediated signaling has been shown to play a role in inducing acute cholangiocyte injury. Treatment of genetically susceptible mice (C57Bl/6) with agonistic anti-TR antibody induces acute cholestasis with fibrosis.8Takeda K. Kojima Y. Ikejima K. Harada K. Yamashina S. Okumura K. Aoyama T. Frese S. Ikeda H. Haynes N.M. Cretney E. Yagita H. Sueyoshi N. Sato N. Nakanuma Y. Smyth M.J. Okumura K. Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease.Proc Natl Acad Sci U S A. 2008; 105: 10895-10900Crossref PubMed Scopus (97) Google Scholar However, the role of TR signaling in chronic cholestasis remains unclear, especially its role in DR cell biology. As macrophages are a source of TRAIL, participate in cholestatic liver injury,9Guicciardi M.E. Trussoni C.E. Krishnan A. Bronk S.F. Lorenzo Pisarello M.J. O'Hara S.P. Splinter P.L. Gao Y. Vig P. Revzin A. LaRusso N.F. Gores G.J. Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.J Hepatol. 2018; 69: 676-686Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar and are associated with DR cells, their role in TR signaling in chronic cholestasis is likely important. The macrophages associated with DR cell population likely consist of diverse subsets. Macrophages exist as multiple subsets and are characterized using a variety of criteria. For example, liver macrophages are also frequently classified as resident macrophages (Kupffer cells) or recruited macrophages (ie, circulating bone marrow–derived monocytes differentiating into macrophages). Functionally, macrophages also exist as a continuum, with tissue damaging or proinflammatory at one end of the spectrum (M1-like) and restorative macrophages involved in tissue repair and healing at the other end (M2-like). However, the restorative macrophages have been implicated in tissue fibrosis as part of an unrelenting wound healing response.10Krenkel O. Tacke F. Liver macrophages in tissue homeostasis and disease.Nat Rev Immunol. 2017; 17: 306-321Crossref PubMed Scopus (409) Google Scholar Restorative macrophages often express the scavenger receptors MER proto-oncogene tyrosine kinase (MERTK), CD206, and galectin-3 (alias LGALS3 or Mac-2). The MERTK-expressing macrophages are a cellular source of TRAIL.11Triantafyllou E. Pop O.T. Possamai L.A. Wilhelm A. Liaskou E. Singanayagam A. Bernsmeier C. Khamri W. Petts G. Dargue R. Davies S.P. Tickle J. Yuksel M. Patel V.C. Abeles R.D. Stamataki Z. Curbishley S.M. Ma Y. Wilson I.D. Coen M. Woollard K.J. Quaglia A. Wendon J. Thursz M.R. Adams D.H. Weston C.J. Antoniades C.G. MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.Gut. 2018; 67: 333-347Crossref PubMed Scopus (68) Google Scholar Although recruited macrophages as opposed to resident macrophages participate in the inflammatory periductal milieu in Abcb4−/− mice (a model of chronic biliary tract injury mimicking human cholestatic liver injury; alias Mdr2–/–),9Guicciardi M.E. Trussoni C.E. Krishnan A. Bronk S.F. Lorenzo Pisarello M.J. O'Hara S.P. Splinter P.L. Gao Y. Vig P. Revzin A. LaRusso N.F. Gores G.J. Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.J Hepatol. 2018; 69: 676-686Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar the nature of the macrophage subsets associated with the cholangiocytes and the DR cell population in cholestasis has not been extensively examined. Herein, we report that Mdr2−/− mice genetically lacking the murine TRAIL receptor (TR), the cognate receptor for TRAIL, are characterized by a substantially increased DR population, macrophage accumulation, and fibrosis. All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee of the Mayo Clinic (Rochester, MN). In-house colonies of Trail receptor–deficient (Tnsf10; alias Tr−/−)12Idrissova L. Malhi H. Werneburg N.W. LeBrasseur N.K. Bronk S.F. Fingas C. Tchkonia T. Pirtskhalava T. White T.A. Stout M.B. Hirsova P. Krishnan A. Liedtke C. Trautwein C. Finnberg N. El-Deiry W.S. Kirkland J.L. Gores G.J. TRAIL receptor deletion in mice suppresses the inflammation of nutrient excess.J Hepatol. 2015; 62: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar and Mdr2-deficient (Mdr2−/−)13Tabibian J.H. O'Hara S.P. Trussoni C.E. Tietz P.S. Splinter P.L. Mounajjed T. Hagey L.R. LaRusso N.F. Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis.Hepatology. 2016; 63: 185-196Crossref PubMed Scopus (117) Google Scholar mice on a C57Bl/6 background were used to generate all genotypes employed for these studies. Briefly, Tr−/− and Mdr2−/− were crossbred to generate the heterozygous Tr+/-Mdr2+/- animals that were further crossbred to generate Tr+/+Mdr+/+ [wild type (WT)], Tr−/−Mdr+/+ (Tr−/−), Tr+/+Mdr2−/−(Mdr2−/−), and Tr−/−Mdr2−/−. Mice were reared on a 12-hour light-dark cycle and had ad libitum access to food and water. At 45 ± 5 and 90 ± 5 days of age, male and female mice of the following genotypes (WT, Tr−/−, Mdr2−/−, and Tr−/−Mdr2−/−) were euthanized by exsanguination. Mice were weighed and anesthetized with ketamine (10 mg/kg, intraperitoneally) and xylazine (120 mg/kg, intraperitoneally). Blood was drawn from the inferior vena cava. The liver was isolated, rinsed in phosphate-buffered saline, and weighed. Subsamples of liver tissue were immediately preserved in 4% buffered formalin, embedded in OCT, or snap frozen. In a separate study, 45-day–old male and female (Mdr2−/−) mice were treated with either the drug cenicriviroc, a CCR2/CCR5 inhibitor that prevents the tissue recruitment of macrophages, or placebo control for a period of 2 weeks. Cenicriviroc was administered subcutaneously daily at a dose of 15 mg/kg body weight.9Guicciardi M.E. Trussoni C.E. Krishnan A. Bronk S.F. Lorenzo Pisarello M.J. O'Hara S.P. Splinter P.L. Gao Y. Vig P. Revzin A. LaRusso N.F. Gores G.J. Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.J Hepatol. 2018; 69: 676-686Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Blood samples were centrifuged at 6000 × g for 20 minutes, serum was separated, and 100 μL of serum was analyzed for alanine transferase, bile acids, bilirubin, and albumin in a Vetscan VS2 using the mammalian liver profile rotor (500-7128; Abaxis, Union City, CA). Liver samples were preserved in 4% buffered formalin for 48 hours, paraffin embedded, and divided into section (5 μm thick). Hematoxylin-eosin staining was performed by the Biomaterials and Histomorphometry Core Facility of the Mayo Clinic. To assess fibrosis, deparaffinized and hydrated tissue sections were stained in 1% Sirius red (365548; Sigma, St. Louis, MO) dissolved in saturated picric acid (P6744; Sigma) for 60 minutes using 0.4% fast green (F7252; Sigma) as counterstain. Additional serial sections were used for immunostaining. Briefly, heat-induced epitope retrieval was performed with 10 mmol/L sodium citrate (pH 6.0) on deparaffinized and hydrated serial sections for 20 minutes. Tissue sections were blocked with Rodent Block M (RBM961; Biocare Medicals, Pacheko, CA) and were probed with antibodies against α-smooth muscle actin (ab21027; Abcam, Cambridge, MA) and F4/80 (ab6640; Abcam) at a dilution of 1:100 and sex determining region Y-box 9 (SOX9) (AB5535; EMD Millipore, Burlington, MA) at a dilution of 1:500. Bound primary antibodies were detected with prediluted (K4010, K4065; Dako Cytomation, Glostrup, Denmark), species-specific, horseradish peroxidase–conjugated secondary antibodies using diaminobenzidine tetrahydrochloride as chromogen. Sections were counterstained with hematoxylin. Staining for all antibodies was optimized to ensure minimal background and verified for specificity using secondary antibodies alone. Bright-field digital images were acquired on a Zeiss microscope mounted with an Axiocam camera (Carl Zeiss, GmbH, Jena, Germany). Digital images of Sirius red staining for quantitative analysis were acquired under plane polarized light in an inverted microscope (Nikon Eclipse TE300; Nikon, Tokyo, Japan). Digital image analysis was performed using the software NIS Elements AR 4.60.00 (Nikon). OCT compound embedded frozen tissue was divided into sections (5 μm thick). Air-dried tissue sections were fixed in 4% formalin or acetone and immunostained with antibodies against proliferating cell nuclear antigen (sc-56; Santa Cruz Biotechnology, Dallas, TX) and macrophage inhibitory cytokine 1 (MIC1-1C3) (NBP1-18961; Novus Biologicals, Littleton, CO) or cytokeratin 19 (CK19) (ab52625; Abcam) at a dilution of 1:100. Fluorophore-tagged species-specific secondary antibodies (1:200; A21441, A21070; ThermoFisher, Walthan, MA; T6391; Life Technologies, Carlsbad, CA) were used to detect bound primary antibodies. Sections were stained with 300 nmol/L DAPI solution to visualize nuclei. Images were acquired on a confocal microscope (LSM 780; Zeiss, Jena, Germany). Digital image analysis for MIC1-1C3 was performed using the ImageJ software version 1.52i (NIH, Bethesda, MD; http://imagej.nih.gov), and results are presented as area percentage per field. For proliferation studies, proliferating cell nuclear antigen and CK19 double-positive cells were counted from a minimum of 15 fields per section; and data are presented as a percentage of the total number of CK19-positive cells. Apoptotic cells in frozen liver tissue sections were probed for DNA strand breaks with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) with fluorescein using a commercially available kit following the manufacturer's instructions (In Situ Cell Death Detection Kit; 11684795910; Roche, Indianapolis, IN). Sections were additionally immunostained with CK7 (1:100; sc-53263; Santa Cruz Biotechnology), as described above, to identify cholangiocytes/ductular reactive cells and counterstained with DAPI for visualization of nuclei. Digital images were acquired on a confocal microscope (Axiovert; Carl Zeiss Microimaging GmbH, Jena, Germany) at a magnification of ×25. TUNEL-positive nuclei were individually verified for DAPI costaining. TUNEL and CK7 double-positive cells were quantified visually on a confocal microscope from 10 to 12 fields per section. Data are expressed as number of double-positive cells per field. Hepatic hydroxyproline content was determined from approximately 200 mg tissue, adopting previously published methods.14Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Briefly, hepatic collagen was extracted from tissue by homogenizing in 0.5 mol/L acetic acid containing 2 mg/mL pepsin (P7012; Sigma) at 50 mg/mL and incubating overnight at 4°C. Digested lysates were hydrolyzed with 4 volumes of 6N HCL. Hydroxyproline content in hydrolysates was determined using a standard biochemical protocol.15Reddy G.K. Enwemeka C.S. A simplified method for the analysis of hydroxyproline in biological tissues.Clin Biochem. 1996; 29: 225-229Crossref PubMed Scopus (979) Google Scholar Intrahepatic leukocytes were isolated from whole livers of WT, Tr−/−, Mdr2−/−, and Tr−/− Mdr2−/− female mice. Briefly, liver tissue was isolated from euthanized mice, weighed, minced, and digested with a liver dissociation enzyme mix (130-105-807; Miltenyi Biotec, San Diego, CA) in a gentleMACS Octo-tissue dissociator using the manufacturer's provided protocol. Dissociated cells were centrifuged at 300 × g for 5 minutes at 4°C. Red blood cells were lysed (155 mmol/L ammonium chloride, 12 mmol/L sodium bicarbonate, and 0.1 mmol/L EDTA) over ice for 5 minutes. The remaining cells were filtered through a 100-μm cell strainer. Kupffer cells and recruited monocyte and macrophage fractions were collected from the interphase of a 25% to 50% Percoll density gradient. The cell pellet containing the polymorphonuclear and lymphocyte fraction was also collected. Both fractions were combined, washed twice, and resuspended in buffer (phosphate-buffered saline with 10 mmol/L EDTA and 0.5% bovine serum albumin). A minimum of three million freshly isolated intrahepatic leukocytes per animal were used for analysis. The mixed immune cell population was spun down at 300 × g for 5 minutes, resuspended in ultrapure water, and stained with an antibody cocktail (Table 1) conjugated to lanthanide-based metal isotopes, according to manufacturer guidelines (Fluidigm, San Francisco, CA). Preconjugated antibodies were purchased directly from Fluidigm. Custom conjugations were performed by the Mayo Clinic Hybridoma Core (Rochester, MN). Mass cytometry was performed at the Mayo Clinic Immune Monitoring Core (Rochester, MN) on a Helios mass cytometry system (Fluidigm). EQ four element calibration beads were spiked into each sample to allow for signal normalization. Normalization was performed using time-of-flight mass cytometry software version 6.7.1014 (Fluidigm). High-dimensional data analysis was performed after identifying cell singlets (191Ir+ and 193Ir+) and viable (195 Pt−) events using the Cytobank software version 7.2 (Santa Clara, CA). All live singlet events were exported to new fcs files before analysis. Visualization for t-distributed stochastic neighbor embedding clustering analysis was performed on 10,000 equivalent events from each sample with selection of all parameters. Cellular phenotypes were assigned to the visualization for t-distributed stochastic neighbor embedding plot on the basis of distribution and expression characteristics of all markers after clustering. Phenographs were generated in the R language–based Cytofkit software version 1.11.3.16Becher B. Schlitzer A. Chen J. Mair F. Sumatoh H.R. Teng K.W. Low D. Ruedl C. Riccardi-Castagnoli P. Poidinger M. Greter M. Ginhoux F. Newell E.W. High-dimensional analysis of the murine myeloid cell system.Nat Immunol. 2014; 15: 1181-1189Crossref PubMed Scopus (242) Google Scholar,17Chen H. Lau M.C. Wong M.T. Newell E.W. Poidinger M. Chen J. Cytofkit: a bioconductor package for an integrated mass cytometry data analysis pipeline.PLoS Comput Biol. 2016; 12: e1005112Crossref PubMed Scopus (136) Google ScholarTable 1List of Markers and Antibody Clones Used for Mass Cytometry AnalysisMarkerCloneMetal tagCD4530-F1189 YLGALS3202213141 PrCD11CN418142 NdTcRbH57-597143 NdMHCI28-14-8144 NdTIM4370901149 SmMHCIIM5/114.15.2150 NdCD3e145-2C11152 SmCD206C06802153 EuTER-119TER-119154 SmMERTK108928155 GdCCR2475301158 GdF4/80BM8159 TbCD64290322160 GdLy6G1A8161 DyCx3CR1SA011F11164 DyCD14Sa14-2165 HoCD196D5166 ErCD8a53-6.7168 ErNK1.1PK136170 ErCD11bM1/70172 YbCD115AFS98174 YbLy6CHK1.4175 LuB220RA3-6B2176 YbTRAILN2B2169TmB220, CD45 isoform expressed in B cells; CCR2, C-C motif chemokine receptor 2; CD11b, cluster of differentiation 11b, integrin alpha-M; CD11C, cluster of differentiation 11c, integrin alpha-X; CD115, cluster of differentiation 115, macrophage colony stimulating; CD14, cluster of differentiation 14, monocyte differentiation antigen; CD19, cluster of differentiation 19, B lymphocyte antigen CD19; CD206, cluster of differentiation 206, macrophage mannose receptor 1; CD3e, cluster of differentiation 3 e, T cell surface glycoprotein epsilon chain; CD45, cluster of differentiation 45, receptor-type tyrosine protein phosphatase C; CD64, cluster of differentiation 64, high affinity immunoglobulin gamma Fc receptor 1; CD8a, cluster of differentiation 8a, T cell surface glycoprotein CD8 alpha chain; Cx3CR1, CX3C chemokine receptor 1; F4/80, adhesion G protein-coupled receptor E1; LGALS3, galectin-3; Ly6C, lymphocyte antigen 6 complex c; Ly6G, lymphocyte antigen 6G; MERTK, tyrosine-protein kinase mer; MHCI, major histocompatibility complex I; MHCII, major histocompatibility complex II; mus, musculus; NK1.1, killer cell lectin-like receptor subfamily B, member 1c factor 1 receptor; TcRb, T-cell receptor beta chain; TER-119, erythroid specific antigen; TIM4, T-cell immunoglobulin and mucin domain-containing protein 4; TRAIL, TNF-related apoptosis inducing ligand. Open table in a new tab B220, CD45 isoform expressed in B cells; CCR2, C-C motif chemokine receptor 2; CD11b, cluster of differentiation 11b, integrin alpha-M; CD11C, cluster of differentiation 11c, integrin alpha-X; CD115, cluster of differentiation 115, macrophage colony stimulating; CD14, cluster of differentiation 14, monocyte differentiation antigen; CD19, cluster of differentiation 19, B lymphocyte antigen CD19; CD206, cluster of differentiation 206, macrophage mannose receptor 1; CD3e, cluster of differentiation 3 e, T cell surface glycoprotein epsilon chain; CD45, cluster of differentiation 45, receptor-type tyrosine protein phosphatase C; CD64, cluster of differentiation 64, high affinity immunoglobulin gamma Fc receptor 1; CD8a, cluster of differentiation 8a, T cell surface glycoprotein CD8 alpha chain; Cx3CR1, CX3C chemokine receptor 1; F4/80, adhesion G protein-coupled receptor E1; LGALS3, galectin-3; Ly6C, lymphocyte antigen 6 complex c; Ly6G, lymphocyte antigen 6G; MERTK, tyrosine-protein kinase mer; MHCI, major histocompatibility complex I; MHCII, major histocompatibility complex II; mus, musculus; NK1.1, killer cell lectin-like receptor subfamily B, member 1c factor 1 receptor; TcRb, T-cell receptor beta chain; TER-119, erythroid specific antigen; TIM4, T-cell immunoglobulin and mucin domain-containing protein 4; TRAIL, TNF-related apoptosis inducing ligand. Intrahepatic macrophages collected from mice, as described above, were resuspended in serum-free RPMI 1640 medium and Primocin (100 μg/mL), seeded in 6-well plates, and incubated with protein transport inhibitors brefeldin A at 5 ng/mL (420601; BioLegend, San Diego, CA) and monensin at 2 nmol/L (420701; BioLegend). At 4 hours, cells were detached using 2 mmol/L EDTA for 15 minutes. Cells were resuspended in PEB buffer (phosphate-buffered saline, 2 mmol/L EDTA, and 0.5% bovine serum albumin) and blocked with FcR blocking reagent (130-092-575; Miltenyi Biotec). Macrophages were stained with fluorochrome-conjugated surface markers against CD45 (130-102-469; Miltenyi Biotec), CD11b (130-097-336; Miltenyi Biotec), and intracellular antigen TRAIL/CD253 (130-102-562; Miltenyi Biotec). Flow cytometry was performed on a MACSQUANT X (Miltenyi Biotec) using appropriate fluorescence minus one controls. The Viobility fixable dye (Mitenyi Biotec) was used to discriminate between live and dead cells. Data were analyzed on FlowJo software version 10.6.1 (FlowJo, LLC, BD Life Sciences, Ashland, OR). Total RNA was isolated from 50 to 100 mg of frozen liver tissue using standard TRIZOL protocol. Total RNA (1000 ng) was transcribed to cDNA using iScript cDNA synthesis kit (BioRad, Carlsbad, CA). Quantitative real-time PCR was performed on a Roche LC480 using SYBR Green technology. Details of primer pairs are provided in Table 2. Fold change in expression of genes of interest was calculated using the ΔΔCt method, normalizing to the geometric mean of two housekeeping genes (18S and glyceraldehyde-3-phosphate dehydrogenase).Table 2List of Primers Used in the Study (Mus musculus)GeneReverse primerForward primer18S5′-CGCTCCACCAACTAAGAACG-3′5′-TCAACACGGGAAACCTCAC-3′Gapdh5′-CCTGTTGCTGTAGCCGTATT-3′5′-TTGTCTCCTGCGACTTCA-3′Hprt15′-CCTGGTTCATCATCGCTAATC-3′5′-TCCTCCTCAGACCGCTTTT-3′TWEAK (Tnfsf12)5′-CCCAGACACCTGGCACAAA-3′5′-TCAGGGCTGGGCTCTACTAC-3′Fn14 (Tnfrsf12a)5′-GCCAAAACCAGACCAGACT-3′5′-GGACCTCGACAAGTGCATGG-3′Krt235′-TCCAAGGTCTTTCGGAGGCCC-3′5′-CGCCAGGATGGCAGTGGATGA-3′Trail (Tnsf10)5′-GCAAGCAGGGTTGTTCAAGA-3′5′-ATGGTGATTTGCATAGTGCTCC-3′ Open table in a new tab Data are expressed as means ± SEM, representing mouse numbers within an experiment. Statistical significance between multiple genotypes was determined by two-tailed analysis of variance, whereas statistical difference between two groups was defined by paired t-test. P < 0.05 was considered statistically significant. There was no significant difference in body weights of male and female mice among the different genotypes at 45 days (Figure 1A). However, the Tr−/−Mdr2−/− male mice and all of the female Mdr2−/− and Tr−/−Mdr2−/− mice were significantly smaller than their WT counterparts by the later time point of 90 days (Figure 1B). The liver/body weight ratio was significantly higher in Mdr2−/− and Tr−/−Mdr2−/− mice for males and females at both time points. In male mice, serum alanine transferase levels were significantly elevated in the Tr−/−Mdr2−/− mice at 45 days and in both Mdr2−/− and Tr−/−Mdr2−/− mice by 90 days. In female mice, serum alanine transferase levels were comparatively higher and significantly elevated in Mdr2−/− and Tr−/−Mdr2−/− over that of the WT and Tr−/− mice (Figure 1). Increased bile acids were also observed in both male and female Mdr2−/− and Tr−/−Mdr2−/− mice for both age groups (Supplemental Figure S1). Interestingly, serum albumin levels were significantly decreased in male and female Tr−/−Mdr2−/− and in Mdr2−/− in the later age groups (Supplemental Figure S1), suggesting increased hepatic dysfunction. Histologic evaluation of hematoxylin-eosin stained liver sections displayed the characteristic cholestatic pattern of injury in the male Mdr2−/− mice with expanded portal tracts due to portal and periportal inflammation and increased number of bile ductules (Figure 2). In female Mdr2−/− and Tr−/−Mdr2−/− mice, inflammation extended into the hepatic lobule with injury more pronounced in the Tr−/−Mdr2−/− mice. No injury was observed in the Tr−/− and the WT mice (Figure 2). Hepatic fibrosis was evaluated by Sirius red staining and indicated accumulation of collagen around the bile ducts under both plane polarized light (Figure 3A) and br
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