The Role of Neonatal Gr-1+ Myeloid Cells in a Murine Model of Rhesus-Rotavirus–Induced Biliary Atresia

• DOI: 10.1016/j.ajpath.2018.07.024
• Publication Date: 2018-09-08T04:08:24Z
• Authors: Ruizhong Zhang; Zefeng Lin; Ming Fu; Xisi Guan; Jiakang Yu; Wei Zhong; Jixiao Zeng; Vincent C.H. Lui; Paul K.H. Tam; Jonathan R. Lamb; Huimin Xia; Yan Chen
• Keywords: Cholangiocyte

Activation of innate immunity together with cholangiocyte damage occurs in biliary atresia (BA). However, detailed information on the inflammatory cells involved is lacking. This study investigates both pathophysiology CD11b+Gr-1+ a mouse model BA and their presence patients. were targeted by an anti-Ly6G antibody murine induced inoculation rhesus rotavirus. Expression Ly6G homolog CD177+ was examined biopsies from The symptoms ameliorated, survival prolonged those mice receiving 5 to 10 μg per every 3 days for four times compared treated virus alone. later developed chronic persistent low body weight jaundice. Hepatic reduced acute BA. Blockade extrahepatic bile ducts occurred, whereas intrahepatic ductules partially preserved, progressive increase liver fibrosis observed. High levels present these mice. administration again jaundice restored weight. In patients highly expressed liver. Our data suggest that shares key characteristics clinical indicates importance initiation progression essential response microbial infection. newborn infants situation different adults because development adaptive at stage. Maternally inherited as well genetic variation environmental factors all might affect immune reactivity infant bacteria viruses. Biliary (BA) disease affects mainly neonatal infants, resulting high mortality absence transplantation.1Gallo A. Esquivel C.O. Current options management atresia.Pediatr Transplant. 2013; 17: 95-98Crossref PubMed Scopus (21) Google Scholar cause remains unclear immunologic, genetic, maternal suggested potentially contribute development. 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Frokiaer promotes CD11b(+)Gr-1(+) CD4(+) spleen.Immun Inflamm Dis. 3: 309-320Crossref (10) Here, RRV inoculation-induced BA, humans, comparable CD177 demonstrate population, BA; therefore, provides appropriate investigation potential treatments. rat anti-mouse (clone 1A8; Bio X Cell, West Lebanon, NH). antibodies immunohistochemical staining cytokeratin 19 TROMA III; Developmental Studies Hybridoma Bank, Iowa City, IA), (BD Biosciences, San Jose, CA), rabbit polyclonal anti–tumor factor (TNF)-α (Abcam, Francisco, CA). polymorphonuclear leukocytes Upstate Biotechnology (Lake Placid, NY) F4/80 obtained R&D Systems (Minneapolis, MN). flow cytometric analysis, purchased eBioscience Inc. (San Diego, including CD11b-fluorescein isothiocyanate, Gr-1–phycoerythrin/fluorescein Ly6G-phycoerythrin, Ly6C-peridinin chlorophyll protein-Cyanine5.5, CD4-peridinin protein-Cyanine5, CD3e-Alexa Fluor 488, CD8a-allophycoyanin, major histocompatibility complex-II–phycoerythrin, F4/80-allophycoyanin, CD11c-phycoerythrin. real-time PCR quantification, RNeasy Mini Kit Qiagen (Hilden, Germany), reverse transcription reagents Invitrogen (Life Technologies Limited, N.T., Hong Kong), Super Real PreMix Tiangen Biotech Beijing Co., Ltd. (Beijing, China). Day 12.5 pregnant BALB/c (aged weeks; weight, 35 40 g) Guangdong Animal Experimental Center, maintained under pathogen-free conditions, housed 25°C room 12-hour dark–light cycle, fed ad libitum autoclaved chow. (average 1.5 1.6 within first 24 hours birth chosen model. protocol approved Institutional Care Use Committee Sun Yat-Sen University Laboratory Center (number IACUC-DB-16-0602) where experiments performed. strain MMU 18006 ATCC (Manassas, VA). amplified MA104 quantified plaque assay method, described previously.18Arnold Patton J.T. Culturing, storage, quantification rotaviruses.Curr Protoc Microbiol. Chapter 15: Unit 15C.3PubMed separated three groups: group, + group. injected 20 μL × 106 PFU/mL (RRV group) supernatant culture medium (control intraperitoneally birth. pretreated i.p. injection 4 before injection, then (10 μg/mouse) given until day (Figure 1A ). excluded analysis. All recorded daily. icterus skin covered fur acholic stools appeared 6 indicated successful induction anesthetized dissected microscopy (SMZ1000; Nikon, Tokyo, Japan). gross appearance livers photographed. Tissues harvested stored −80°C RNA/protein isolation 10% formalin histologic sample preparation. Mouse samples immunostaining fixed overnight temperature subsequent paraffin-embedding. Paraffin-embedded sections (4 μm thick) dewaxed rehydrated stained hematoxylin eosin immunohistochemistry antigen retrieval performed citrate buffer mmol/L, 0.01% Tween20, pH 6.0) CK19 Tris-EDTA Buffer mmol/L Tris Base, 1 EDTA Solution, 9.0) Gr-1. removal endogenous peroxidase 3% hydrogen peroxide treatment minutes. incubated 30 minutes blocking solution (antibody diluent; Dako Denmark A/S, Glostrup Denmark), followed incubation 4°C contained primary antibodies. Immunostaining completed using EnVision system (Dako). analyzed Nikon microscope captured NIS-Elements F4.0 (Nikon Instruments Inc., Melville, NY). detection fibrosis, collagen Picrosirius red. After counterstaining hematoxylin, section red hour temperature; mounted analyzed. Details deposition can be polarized contrast light (Leica DMI8+DFC7000T; Leica Microsystems, Wetzlar, conditions immature green color.19Rich Whittaker Collagen picrosirius staining: assessment fibrillar hue spatial distribution.Braz Morphol Sci. 22: 97-104Google A single-cell suspension isolated spleens gently mincing passing through 70-μm strainer centrifuging (270 g, times). resuspended 30% Percoll (Sigma-Aldrich Corp, St. Louis, MO) centrifuged 400 g 22 minutes, blood lyzed lysis (Tiangen Ltd.) pellet RPMI 1640 (Invitrogen). Specific markers perform cellular phenotyping labeling Flow cytometry BD FACS Canto CA) FlowJo software verison 10.0.7 (FlowJo LLC, Ashland, OR). Cell selected according forward/side scatter, gated isotype controls account background fluorescence, subjected secondary analysis based fluorescence signals sera collected end hospital's laboratory Hitachi Pre-Analytical Process Automation System 7600 Clinical Analyzer (Hitachi, variables follows: alanine aminotransferase; aspartate alkaline phosphatase; total protein; albumin; globulin; bilirubin (TBIL); (DBIL); (IBIL) acids. detection, RNA extracted, SuperScript III First-Strand Synthesis (Thermo Fisher Scientific, Waltham, MA) manufacturer's instructions. PCR-cyber kit Fast SYBR Green Master Mix detect titer determining nonstructural gene NSP3. primer sequence NSP3 forward, 5′-TTGAAGAGAAAATGGAAGTAGATACAA-3′, reverse, 5′-TACTTCTCATTAACCCGATGTTTCA-3′, TNF-α 5′-TAGCTCCCAGAAAAGCAAGC-3′, 5′-TTTTCTGGAGGGAGATGTGG-3′. C1000 Thermal Cycler (Bio-Rad Laboratories Ltd., Hercules, Liver taken wedge biopsy 1– 5–month-old undergoing intraoperative cholangiograms Kasai operations time diagnosis hospital. confirmed histology portal plate obstruction. choledochal cysts control. human protocols conformed ethical guidelines 1975 Declaration Helsinki, Review Board Guangzhou Women Children's Medical China. grading METAVIR score (https://www.uptodate.com/contents/histologic-scoring-systems-for-chronic-liver-disease, subscription required). presented means ± SEM. statistical analyses unpaired two-tailed t-test GraphPad Prism version 6.0 (GraphPad Software La Jolla, P < 0.05 indicating significance. adapted previous well-characterized inoculated birth.6Petersen Compared treatment, result often 14 days, dose (100 totally prevented death, weights same there evidence (Supplemental Figure S1). Mice dosage repeated 1A). RRV-only group median 13 days; contrast, antibody-treated survived minor loss 1B). approximately 25% syndromes lower (8.19 1.14 versus 23.8 1.17 able survive >42 1C Supplemental S2). produced de novo phenotype mouse, termed opposed its counterpart, Time point 21, 28, 42, revealed S3). 42 Other than small size, ear, foot, tail 1D 2). livers. They size pale yellow color enlarged gallbladder filled dark blue 1E). Enlarged 1F) areas normal 1G); moreover, fragment 1H). Analysis showed doses tract inflammation 12, apoptosis observed, unlike RRV-alone still 42-day 2A). slight triad. change expression, greatly 2A When viewed microscope, fibers penetrated nearby tissue. Large deposition, color, 2A) kept ≥49 without augmentation triad, preserved CK19+ 12; mature difficult detect, although could (CK19) 2B). serial dissection area blockade S4). Inflammatory important characteristic (P 0.001 macrophage; n = control, RRV-alone, 2, C). Virus detected quantitative significantly 0.0667) 2D). alone, enzyme levels, aminotransferase (104.40 11.37 U/L 60.75 17.67 U/L; 0.05), (1244.00 135.76 339.50 125.33 0.01), phosphatase (750.00 126.89 439.75 20.10 0.05) (both 3A serum protein changes phase reduction (53.68 0.96 g/L 42.44 1.23 g/L; 0.001), albumin (31.75 0.55 24.26 0.44 globulin (21.93 0.46 17.62 0.93 3B). TBIL, DBIL, IBIL alone TBIL DBIL; 0.01 IBIL) 3C). IBIL, acids (242.0 48.67 μmol/L 1.25 0.17 215.7 37.71 0.5 0.31 μmol/L) bilirubin, respectively acids; 3C), function details biochemistry examination Table S1. cytometry, Gr-1+, CD4+, points 4). CD11b+Gr-1hi sharply week even 12. <5% 0.01) >10% Pretreatment level 1% 3) lower, higher 4, B). CD11b+Gr-1int 5% 2% 50%, fivefold 0.001). Anti-Ly6G earlier points, progressively 0.14% 1.36% Inoculation twofold 3, 7, 0.001, 0.01, respectively). Treatment slightly namely, 0.08% 2.93% 2.24% D E). Similar Enhancement marked threefold increase. Although antibody–treated trend up-regulation statistically significant F). Because administered single 28 (n 5A beneficial general health recovery 5B) 20.0 5C). 5D), rough fully recovered 5E). 5F) 5F), less pronounced (CK19+), tube-like structures 5G), Ly6G+ tissues (28.92 15.77 high-power field 1.22 2.22 field) 5G). pathologic process leading fibrosis. RRV-inoculated phase, inflammation, observed.6Petersen here, phases