9. ANHANG
9.1. A BKÜRZUNGSVERZEICHNIS
9. Anhang
9.1.Abkürzungsverzeichnis
B Belgien
BSA Bovines Serum Albumin
D Deutschland
DMSO Dimethylsulfoxid
dNTP Desoxiribonukleotidtriphosphat
EAE Experimentelle autoimmune Enzephalomyelitis
F Frankreich
FCS Fetales Kälberserum
GPT Glutamat-Pyruvat-Transaminase GOT Glutamat-Oxalacetat-Transaminase
h Stunde/n
ic intrazellulär i.p. intraperitoneal Iono Ionomycin
LDH Lactatdehydrogenase
LN Lymphknoten
PBS phosphatgepufferte Salzlösung PFA Paraformaldehyd
PMA Phorbol 12-Myristate 13-Acetate rpm rounds per minute
s.o. siehe oben (überhaupt verwendet?) s.u. siehe unten
UK Großbritannien
USA Vereinigte Staaten von Amerika
92 9.2.Materialien
9.2.1. Geräte und Pipetten
Acculab Kompaktwaage ATL 4202 Sartorius, Göttingen, D Lichtmikroskop CKX41 Olympus, Hamburg, D
MACS Magnetstand Miltenyi Biotech, Bergisch Gladbach, D Mini und Midi MACS Seperator Miltenyi Biotech, Bergisch Gladbach, D CO2 Inkubator HeraCell 15i Thermo Fischer Scientific, Bonn, D Heraeus Megafuge 40R Centrifuge Thermo Fisher Scientific, Bonn, D Heraeus Tischzentrifuge Fresco 17 Thermo Fisher Scientific, Bonn, D Sterile Werkbank, Typ Safe 2020 Thermo Fisher Scientific, Bonn, D Wasserbad, Typ 1007 GFL GmbH, Burgwedel, D
Vortex Genie2 Scientific Industries, Bohemia, USA Durchflusszytometer (FACS CantoIM II) BD Bioscience, Heidelberg, D GloMax-Multi plate reader Promega, Mannheim, D
Step One Plus Real-Time PCR System Life technologies, Darmstadt, D Thermocycler T3000 combi Biometra, Göttingen, D
Zentrifuge Galaxy ministar VWR, Darmstadt, D Pipettierhelfer ACCU-JET®PRO VWR, Darmstadt, D Mechanische Einkanalpipetten Eppendorf AG, Hamburg, D Mechanische Mehrkanalpipette Eppendorf AG, Hamburg, D
9.2.2. Software
FACSDiva Software BD Biosciences, Massachusetts, USA GraphPad Prism, v5.0 GraphPad Software, La Jolla, USA Kaluza Analysis Software Beckman Coulter, Krefeld, D
Microsoft Office 2013 Microsoft Deutschland GmbH, Berlin, D
9.2.3. Verbrauchsmaterialien 9.2.3.1. Allgemeine Materialien
Mausmodell:
Tuberkulinspritze 1ml 9161406 Braun, Melsungen, D
Insulinkanüle 0,4x12mm, 27G 4665406 Braun, Melsungen, D
Präparierbesteck FST GmbH, Essen, D
93 Zellkultur:
Zellkulturplatte, 96 Well, Rundboden, steril 163320 Greiner Bio-One GmbH, Fri-ckenhausen, D
Zellkulturplatte, 96 Well, Flachboden, steril 167008 Zellgewinnung und -aufarbeitung:
Neubauer Zählkammer T729.1 Carl Roth, Karlsruhe, D
Petrischale 6 cm 82.1194 BD Bioscience, Heidelberg, D
Einweg-Skalpell 200130021 Sterican
Easystrainer Zellsieb 70µm 542070 Greiner Bio-One GmbH, Fri-ckenhausen, D
Zentrifugenröhrchen, 15 ml, 50 ml 861685001/
861689001
Sarstedt, Nürnbrecht, D safe lock tubes, 1,5ml 0030120.094 Sarstedt, Nürnbrecht, D
Glaspasteurpipette 150mm 747715 Brand
Pre-Separation Filter 30µm 130-041-407 Miltenyi Biotec, Bergisch Glad-bach, D
MS Säulen 130-042-201 Miltenyi Biotec, Bergisch
Glad-bach, D
LD Säule 130-042-901 Miltenyi Biotec, Bergisch
Glad-bach, D Durchflusszytometrie:
FACS tubes 2235 BD Bioscience, Heidelberg, D
Polystyrene Tube with Cell-Strainer Cap BD Bioscience, Heidelberg, D ELISA:
MicroWell-Platte 96-well Flachboden 167008 Greiner Bio-One GmbH, Fri-ckenhausen, D
Reagenzien Reservoir 25 ml HAT 65.1 VWR, Darmstadt, D
PCR:
Fast Optical Plates 96well 4346906 Applied Biosystems Abdeckfolie (Optical Adhesive Covers) 4360954 Applied Biosystems
Sonstiges:
Pipettenspitzen(10µl, 100µl, 200µl) 70.760.502 Sarstedt, Nümbrecht, D
Pipettenspitzen1000µl 4665406 Braun, Melsungen, D
Serologische Pipetten 5 ml, 10 ml, 25 ml 861254001 Sarstedt, Nümbrecht, D
94 9.2.3.2. Virus und Zelllinie
Rhesus Rota Virus, MMU18006 VR-1739 ATCC, Manassas, VA, USA
MA-104, Clone 1 CRL-2378.1TM ATCC, Manassas, VA, USA
9.2.3.3. Reagenzien
PBS L 182-50 Biochrom AG, Berlin, D
Isopentan 2001135 Apotheke, MHH
RNAlater 5026072 Qiagen, Hilden, D
PFA
Formalin 4% 41-5313-00 Carl Roth GmbH + Co,
Karls-ruhe, D
RBC-Lysispuffer Apotheke, MHH
Percoll 17-5445-01 GE Healthcare, Chalfont St
Giles, UK
MACS Buffer 130-092-125 Miltenyi Biotec, Bergisch
Glad-bach, D
Trypanblau 0,4% 15250-06 Thermo Fisher Scientific, Bonn,
D
RPMI 1640+ BE12-702F/U1 Lonza, Basel, CHE
FCS (Fetal Calf Serum) Thermo Scientific HyClone,
Er-embodegem, B
L-Glutamin FG0325 Biochrom
Penicillin/ Streptomycin (1000x) A2212 Biochrom
DMSO D5879-100ml Sigma-Aldrich, Steinheim, D
2-Mercapthoethanol Sigma-Aldrich, Steinheim, D
BSA Carl Roth, Karlsruhe, D
EDTA L11-003 PAA
Wash Puffer ELISA WA126 R&D Systems GmbH,
Wiesba-den, D
Reagent Diluent, ELISA DY995 R&D Systems GmbH,
Wiesba-den, D
Substrate Solution DY999 R&D Systems GmbH,
Wiesba-den, D
Stopplösung DY994 R&D Systems GmbH,
Wiesba-den, D 9.2.3.4. Puffer und Medien
cRPMI: RPMI 1640+ + 10% FCS +1% L-Glutamin +1%Penicillin/ Streptomycin
95 FACS Puffer: PBS +0,5 % BSA+ 2mM EDTA
9.2.4. Zytokine
IL-1a Mouse Recombinant Protein 130-094-050 Miltenyi Biotec, Ber-gisch Glad-bach, D IL-2 Mouse Recombinant Protein 130-094-054 Miltenyi Biotec,
Ber-gisch Glad-bach, D IL-4 Mouse Recombinant Protein 130-094-061 Miltenyi Biotec,
Ber-gisch Glad-bach, D IL-6 Mouse Recombinant Protein 130-094-065 Miltenyi Biotec,
Ber-gisch Glad-bach, D IL-21 Mouse Recombinant Protein 594-ML-010 R&D Systems
IL-23 Mouse Recombinant Protein 14-8231 eBioscience GmbH, Frankfurt, D TGF-ß Mouse Recombinant Protein 7666-MB-005 R&D Systems
INF-g Mouse Recombinant Protein 130-094-047 Miltenyi Biotec, Ber-gisch Glad-bach, D
9.2.5. Kits
TCR gamma/delta T Cell Isolation Kit Mouse 130-092-125 Miltenyi Biotec, Bergisch Glad-bach, D
CD4+ T Cell Isolation Kit, mouse 130-104-454 Miltenyi Biotec, Bergisch Glad-bach, D
Mouse IL-17 duo set DY421 R&D Systems GmbH,
Wiesba-den,
Mouse IFN-g duo set DY485 R&D Systems GmbH,
Wiesba-den,
RNeasy Mini Kit 74104 Qiagen
High Capacity RNA to cDNA Kit 4387406 Applied Biosystems
96 9.2.6. Antikörper
Antigen Konjugat Clone Spezifität Artikelnummer Hersteller aCD3 Functional
grade
HIt3a anti mouse 45-0031-82 eBioscience GmbH, Frankfurt, D aCD28 Functional
grade
28.2 anti-human 16-0289-81 eBioscience GmbH, Frankfurt, D TCRgd FITC eBioGL3 anti mouse 12-5711-82 eBioscience GmbH,
Frankfurt, D
CD26 PE H194- anti mouse 137803 BioLegend,
Ko-blenz, D
CD3e PerCP-Cy5.5 I45-2C11 anti mouse 45-0031-82 eBioscience GmbH, Frankfurt, D CD8a PE-Cy7 53-6.7 anti mouse 11-0081-82 eBioscience GmbH,
Frankfurt, D
TCRb APC H57-597 anti mouse 17-5961-82 eBioscience GmbH,
Frankfurt, D
CD4 eF780 RM4-5 anti mouse 47-0042-82 eBioscience GmbH,
Frankfurt, D CD45 eF450 30-F11 anti mouse 11-0451-82 eBioscience GmbH,
Frankfurt, D Fixable
Viabil-ity Dye eFlour 506 65-0866-18 eBioscience GmbH,
Frankfurt, D Fc Block
CD16/CD32 Purified 93 anti mouse 14-0161-81 eBioscience GmbH, Frankfurt, D
9.2.6.1. ELISA Antikörper Fänger-Antikörper
Ratten Anti-Maus IFNg R&D, Minneapolis, USA Ratten Anti-Maus IL-17 R&D, Minneapolis, USA
Detektor-Antikörper
Biotinyliertes Ziegen-Anti-Maus IFNg R&D, Minneapolis, USA Biotinyliertes Ziegen-Anti-Maus IL-17 R&D, Minneapolis, USA
97 9.2.7. Primer
Primer Artikelnummer
SABiosciences, Frederick, USA
mGapdh PPM02946E-200
Rorc PPM25095A-200
mIL-17A PPM03023A-200
mIL-17F PPM05398E-200
mIL-22 PPM05481A-200
mIL-23R PPM33761A-200
Primer Artikelnummer Qiagen, Hilden, D
mIL-21 QT01758036
mTbx21 QT00129822
mIL-4 QT00160678
mTNF QT00104006
mCd163l1 QT01077699
mFoxp3 QT00138369
mIL-10 QT00106169
mCcr6 QT02379181
99 10.Abbildungs- und Tabellenverzeichnis
10.1. Abbildungen
Abbildung 1: Makroskopischer und mikroskopischer Vergleich der Gallengänge von ge-sunden Mäusen und Mäusen mit BA
Abbildung 2: Strukturformeln ATRA und AM80
Abbildung 3: Markierung und Gewinnung von gd T-Zellen
Abbildung 4: Reinheit der gd T-Zellen nach MACS Isolation aus Zellsuspension muriner Lymphknoten- und Milz-Leukozyten
Abbildung 5: Ablauf ELISA-Verfahren Abbildung 6: Ablauf RNA Isolierung
Abbildung 7: Etablierung der gd T-Zell-Kultur hinsichtlich Zytokinmilieu, Stimulationszeit und stimulierendem Antikörper
Abbildung 8: Nachweis einer hohen Reinheit der gesorteten gd T-Zellen mittels FACS- Analyse und Darstellung der mangelhaften Reinheitskontrolle mittels Bio-tin-Markierung
Abbildung 9: Dosisabhängige Reduktion der IL-17 Produktion von gd T-Zellen nach Ko-Inkubation mit AM80
Abbildung 10: Il-17A, IL-17F, IFN-g und IL-4 Gehalt in den Kulturüberständen mit und ohne AM80-Koinkubation, analysiert mit Cytometric Bead array (CBA) Abbildung 11: Viabilität und Apoptoserate der mit AM80 koinkubierten Zellen unter-scheiden sich nicht signifikant von denen der unbehandelten bzw. der Kontroll-Zellen
Abbildung 12: IL-17- und RORgt-Expression von gd T-Zellen mit und ohne Behandlung mit AM80
Abbildung 13: Durchflusszytometrische Analyse der gd T-Zell-Kultur hinsichtlich IL-17-und IFNg-Produktion nach 72h Kulturzeit mit und ohne
AM80-Koinkubation (Konzentration 100 nM)
Abbildung 14: Durchflusszytometrische Messung der IL-17 produzierenden gd T-Zellen aus TCRgd reporter Mäusen nach einer Kulturzeit von 24h mit und ohne Zugabe von AM80.
Abbildung 15: Hepatische Leukozyten erkrankter Tiere in verschiedenen Kulturansätzen mit und ohne Ko-Inkubation von AM80
Abbildung 16: Überlebenskurven und Gewichtsverläufe von prophylaktisch (ab Lebens-tag 1) mit AM80 behandelten Mäusen im Vergleich zur Kontrollgruppe
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Abbildung 17: Konzentrationen von Bilirubin, GOT, GPT und LDH im Serum von AM80-behandelten Tieren im Vergleich zur unAM80-behandelten Kontrollgruppe Abbildung 18: Genexpression von Il-6, MCP-1 und Collagen 1a1 in den Lebern von
AM80-behandelten Mäuse im Vergleich zur unbehandelten Kontrollgrup-pe
Abbildung 19: Immunhistochemische Färbung von GR-1- und F4/80-positiven Zellen in den Lebern AM80-behandelter Tiere im Vergleich zu den unbehandelten Tieren
Abbildung 19.1.: Anzahl GR-1 (A) und F4/80 (B) positiver Zellen in der murinen Leber, quantifiziert mittels GR-1 bzw. F4/80 cell score
10.2. Tabellen
Tabelle 1: Färbeprotokoll T-Zell-Phänotypisierung 1 Tabelle 2: Färbeprotokoll T-Zell-Phänotypisierung 2 Tabelle 3: Färbeprotokoll Intrazelluläre Zytokinfärbung Tabelle 4: Ablauf und Bedingungen der qRT-PCR
Tabelle 5: Färbeprotokoll T-Zell-Phänotypisierung und Phänotypisierung myeloider Zel-len
101 11.Eingereichtes Manuskript
Synthetic retinoid AM80 inhibits IL-17 production of gamma delta T-cells and ameliorates ex-perimental biliary atresia
Authors:
Nora Möhn1, Arne Schröder1, Stephanie Frömmel1, Faikah Gueler2, Immo Prinz3, Koichi Shudo4, Ta-kashi Yamamura4, Gertrud Vieten1, Joachim F. Kuebler1, Claus Petersen1,Christian Klemann1,5
1Dept. of Pediatric Surgery, Hannover Medical School, Hannover, Germany
2Dept. of Nephrology, Hannover Medical School, Hannover, Germany
3Institute of Immunology, Hannover Medical School, Hannover, Germany
4Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan, Research Foundation ITSUU Laboratory, Tokyo, Japan
5Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hannover, Germany
Corresponding author:
Dr. Christian Klemann, Dept. of Pediatric Surgery, OE 6760, Hannover Medical School, D-30627 Han-nover, Germany, Fax: +49 511 532 8598, Phone: +49 176 1 532 3220
Keywords: IL-17, Retinoids, synthetic retinoid AM80, gd T-cells, biliary atresia, virus induced autoim-munity, neonatal immune system
Word count: 4604 Number of figures: 6 Number of tables: 0
Disclosures: No conflict of interest.
Financial Support: This work was supported partly by grants from the Appenrodt-Stiftung (C.P.), Hochschulinterne Leistungsförderung (HiLF) (C.K.) of the Hannover Medical School and a grant from the German Federal Ministry of Education and Research (reference number: 01EO1302) of the "In-tegrierte Forschungs- und Behandlungszentrum Transplantation (IFB-Tx)" (C.K.).
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Author contributions:
N.M. conducted experiments, analyzed data, interpreted the results and wrote the manuscript.
C.K. designed experiments, analyzed data, interpreted the results and wrote the manuscript. F.G.
performed serum analysis and histology. A.S., S.F., G.V. conducted the other experiments. K.S. syn-thesized the AM80. T.Y. critically commented on the manuscript. I.P. helped to design experiments and interpret the results C.P., J.F.K. supervised the project and revised the manuscript. All authors commented on the manuscript.
List of Abbreviations:
Acute promyelocytic leukemia (APL) Alanine transaminase (ALT)
Alpha beta T-cells (ab T-cells) Antigen presenting cells (APC) Aspartate transaminase (AST) Biliary atresia (BA)
C-C chemokine receptor type 6 (CCR6) Cluster of differentiation (CD)
Concanavalin A (ConA)
Competitive enzyme-linked immunosorbent assay (ELISA) Complementary deoxyribonucleic acid (cDNA)
Cytometric bead array (CBA)
Experimental autoimmune encephalomyelitis (EAE) Enzyme-linked immunosorbent assay (ELISA) Fluoresces activated cell sorting (FACS) Focus forming units (FFU)
Gamma delta T-cells (gd T-cells)
Glycerinaldehyd-3-phosphat-Dehydrogenase (GAPDH) Hematoxylin and eosin (H&E)
Interferon gamma (IFN-g) Interleukin- (IL-)
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Ionomycin (INO)
Magnetic activated cell sorting (MACS) Messenger Ribonucleic acid (mRNA) Mononuclear antibody (mAb)
Mononuclear cells (MNC) Natural Killer cell (NK cell)
One-way analysis of variance (ANOVA) Phorbol 12-myristate 13-acetateand (PMA) Phosphate buffered saline (PBS)
Quantitative reverse transcription-polymerase chain reaction (qPCR) Primary biliary cirrhosis (PBC)
Retinoic acid receptor-a (RAR-a) Retinoid-Orphan-Receptor-gt (RORgt)
Reverse transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) Rhesus rota virus (RRV).
Roswell Park Memorial Institute medium (RPMI) Standard error of the mean (SEM)
T-cell receptor (TCR) T helper cells (Th)
Tumor necrosis factor alpha (TNF-a)
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Abstract:
Background & Aims: Recent evidence suggests that Interleukin (IL)-17-producing gamma delta T-cells (gd T-T-cells) are the dominant pathogenic cellular component in selected autoimmune inflammato-ry diseases, including biliainflammato-ry atresia. We have previously demonstrated that retinoids such as all-trans retinoic acid (ATRA) and AM80, a synthetic retinoid with superior biological properties to ATRA, effec-tively suppress T helper cells (Th) 17 differentiation.
Methods: Here, we establish an in vitro system enabling investigations of the effect of AM80 on IL-17 production by gd T-cells. Additionally, we tested the therapeutic effect of AM80 in the Rota-virus in-duced mouse model of biliary atresia.
Results: Co-incubation of gd T-cells with IL-23 proved most effectively in inducing an IL-17 response.
In vitro, AM80 significantly reduced IL-17 production by gd T cells (p<0.001) and expression of the master transcription factor Retinoid-Orphan-Receptor-gt (RORgt) (p<0.001). Moreover, AM80 inhibited IL-17 production by liver infiltrating gd T-cells. In vivo, intraperitoneal (i.p.) administration of AM80 ame-liorated BA associated inflammation in mice. However, AM80 treatment was not sufficient to complete-ly control disease progression in the murine model, despite reduced IL-17 levels in the animals.
Conclusion: Our in vitro system is feasible to induce IL-17 production in gd T-cells and retinoids are very efficient in down-regulating IL-17 in these cells in vitro, and to a lesser extent also in the BA mouse model. However, retinoids do not suffice in completely controlling disease progression, and thus it is likely that IL-17 is not the only factor responsible for pathogenesis of BA. Moreover, retinoid-signaling seems to not only regulate inflammatory IL17+ gd T-cells, but also other potentially protec-tive players.
Lay summary:
Biliary atresia is a rare disease of infancy with a progressive liver failure in most children and is the most common cause for pediatric liver transplantation. We have previously demonstrated that IL-17, produced by gd T cells, contributes to hepatic inflammation in the murine model of BA and is increased in the livers of infants suffering from BA. In the study at hand, we demonstrate that treatment with AM80, a synthetic retinoid with superior pharmacological properties, effectively inhibits IL-17 produc-tion of gamma delta T cells without generating systemic immuno-suppression. Although all-trans retin-oic acid (ATRA) has been shown to suppress differentiation of IL-17 producing conventional T helper cells (Th17) in vitro, therapeutic application of ATRA in vivo is limited due to its potential side effect
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caused by the instability and the lack of receptor specificity of this compound. Our study is the first to show that AM80 suppresses IL-17 production of gd T cells in a very efficient manner and that mice suffering from BA have an amelioration of the hepatic inflammation. However, AM80 treatment does not suffice to block the disease progression. We conclude that other factors than IL-17 drive the pro-gressive inflammation in BA. However, addition of retinoids in the treatment regime of children suffer-ing from biliary atresia might decrease disease burden, but further research is needed to clarify the pathomechanism and possible therapeutic interventions in humans.
Graphical abstract
Highlights:
• AM80 inhibits liver inflammation in the mouse model of biliary atresia
• Clinical course in the mouse model of biliary atresia is ameliorated by AM80
• AM80 suppresses IL-17 production by gd T cells in vitro dose dependently
• IL-23 and IL-1b-driven IL-17 production by gd T cells in vitro is supported by CD28stimulation
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Introduction:
Biliary atresia (BA) is a rare liver disease of infancy with an incidence of about 1:18000 in Western Europe. Within the first weeks of life a progressive inflammatory destruction of bile ducts eventually results in liver failure in the majority of patients2,3. Thus, biliary atresia is the most common indication for a liver transplant in children1. To date, etiology and pathogenesis of BA remain elusive and a caus-ative treatment is not available. However, an increasing body of evidence suggests an infection with a hepatotropic virus triggering an autoimmune process4,5. This notion is supported by the detection of viral remnants in livers of children, but causality cannot be proven as patients always present in symp-tomatic stages rendering the search for a trigger difficult6. In order to investigate early stages of biliary atresia and possible factors of pathogenesis the rhesus rota virus (RRV) mouse model of BA has been developed7: Newborn mice are infected within 24 hours after birth by intraperitoneal injection of RRV resulting in an periportal (autoimmune-) inflammation response despite timely clearance of the virus resembling many factors of human BA with progressive inflammation of the entire biliary tree and the development of atresia of the extrahepatic bile ducts7. Numerous immunological players have been implicated in disease development8. After an initial response with natural killer cells and antigen pre-senting cells (APC)8 depending on the innate immune system, a T-cell mediated immune response is induced8 and cluster of differentiation (CD)4+ T-cells orchestrate a Th1 cytokine response with secre-tion of Interferon gamma (IFN-g), IL-2, IL-12, and large amounts of tumor necrosis factor alpha (TNF-a).9 Also Th2 cells and B cells have been implicated in disease pathogenesis10,11.In addition, we have recently demonstrated that IL-17, a pro-inflammatory cytokine involved in numerous autoimmune con-ditions, is up-regulated in mice and humans suffering from BA and that antagonization of IL-17 amelio-rated liver inflammation in experimental BA12. Importantly, the source of IL-17 proved to be gd T-cells in this model, while classical Th17 cells were completely absent. Therefore, we aimed to investigate possible therapeutic strategies targeting the IL-17 axis in BA.
It has been shown that ATRA, the active metabolite of vitamin A, and synthetic analogues like AM80 suppress the differentiation of alpha beta Th17 cells through ligation to the retinoic acid receptor-a (RAR-receptor-a)14,15, the investigations using ATRA are complicated by instability, poor bioavailability and unspecific retinoid-receptor binding. Therefore, specific RAR agonists have been developed demon-strating superior biological properties. AM80, a synthetic retinoid available in the clinic under the trade name Tamibarotene for treatment of acute promyelocytic leukemia (APL) and psoriasis, has previous-ly been demonstrated to inhibit Th17 cell differentiation and production of IL-17 by effector alpha beta
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T-cells (ab T-cells)16. In the study at hand, we describe a feasible in vitro culture system for gd T-cells enabling pharmacological studies of these cells. Utilizing this system, we show that AM80 suppresses IL-17 production of gd T-cells in a dose dependent manner. Moreover, treatment with AM80 amelio-rates hepatic inflammation in experimental BA. However, continuous AM80 treatment failed to improve chronic hepatic disease despite of apparent suppression of T cell expression of IL-17 and RORgt. We were able to prove that continuous AM80 treatment results in the suppression of IL-10 and IL-22 pro-duction by a possibly regulatory subset of gd T-cells. Eventually, we conclude that treatment with the synthetic retinoid AM80 is an intervention strategy to consider for the acute phase of T-cell receptor (TCR) gd17-mediated autoimmunity.
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Methods:
Animals, Rhesus rotavirus, and experimental biliary atresia
Rotavirus-free Balb/cAnNCrl-mice were obtained from Charles River (Sulzfeld, Germany), kept in spe-cific pathogen-free laminar-flow cages and subjected to a 12-h dark-light cycle with food and water ad libitum. All procedures were approved by the local animal welfare committee (permit number 12/0785 and 4/2012/22). RRV strain MMU18006 was acquired from American Type Culture Collection (Manas-sas, USA), grown in MA104 cells and virus titration was performed immunohistochemically modified according to Lindenbach et al.17. For the induction of BA, mice were intraperitoneally infected within the first 24h of life with 50μL phosphate buffered saline (PBS) solution containing 230 focus forming units RRV. For AM80-treatment experiments mice received i.p. injections with 50 µl AM80 (0,18-1,0 mg/kg) in PBS every other day. Controls received intraperitoneal injections with 50 µl PBS every two days. Mice were monitored daily and their survival and weight was assessed. In some experiments, mice were sacrificed by decapitation, blood was collected, and neonatal livers were explanted. Speci-mens were freshly prepared for leukocyte extraction, paraffin embedded after formalin fixation, or snap frozen and stored at -80°C for protein and RNA analysis.
All in vitro experiments were performed with C57BL/6J mice that were kept in the central animal labor-atory of Hannover Medical School in barrier-maintained mouse colonies and fed with sterilised animal feed. So-called TCRgd reporter mice obtained from AG Prinz, Hannover Medical School were utilized for distinct flow cytometry experiments as they exhibit an intrinsic green fluorescence signal. Mice were kept according to the standards of AG Prinz and T-cells were isolated as previously described12.
Cell isolation and purification In vitro experiments:
Pooled gd T-cells from lymph nodes and spleens of B6 mice were isolated as previously described12. Briefly, leukocytes of spleen and lymph nodes were isolated by cutting the tissue with a scalpel into small pieces. The tissue was then homogenized by passing it through a 70 µm cell strainer. After ex-tensive flushing with PBS, several centrifugation steps and lysis of erythrocytes cell numbers were counted with an improved Neubauer counting chamber. The number of cells per experimental animal was between 0,43 x 108 and 3,07 x 108 cells. gdT cells were purified by MACS using the TCR gd+- Isolation Kit mouse according to the manufacturer’s instructions (Miltenyi Biotech,Bergisch Gladbach,
109
Germany) yielding a purity of gdT cells >92 %.
Ex vivo experiments:
Liver leukocytes were isolated as previously described12. Briefly, after homogenization of liver tissue
Liver leukocytes were isolated as previously described12. Briefly, after homogenization of liver tissue