10. ABBILDUNGS- UND TABELLENVERZEICHNIS
10.2. T ABELLEN
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,
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Germany) yielding a purity of gdT cells >92 %.
Ex vivo experiments:
Liver leukocytes were isolated as previously described12. Briefly, after homogenization of liver tissue leukocytes were enriched using discontinuous 40%/70% Percoll density gradient centrifugation. To ensure that gdT-cells are stimulated exclusively, a specific anti-TCRgd-antibody was used. T-cells were purified by magnetic activated cell sorting (MACS) using the TCR gd+- Isolation Kit, mouse (Miltenyi Biotech, Bergisch Gladbach, Germany).
gd T-cells (live mononuclear cells (MNC), CD45+, CD3e+, TCRgd+, TCRab-, CD4-) were further
sort-ed using a fluorescence-activatsort-ed cell sorter (FACS) ARIA II (BD Cytometry System, Franklin Lakes, USA).
Cell culture
RPMI 1640 medium (GIBCO, Grand Island, USA) supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, and 100 U/ml penicillin-streptomycin was used for all cell cultures. Cells were activated with either 10 µg/ml immobilized anti-CD3 (2C11, eBioscience) or anti-TCRgd (GL3, eBioscience) and 1µg/ml soluble anti-CD28 monoclonal antibody (mAb, eBioscience). To figure out the best cell culture conditions for gdT-cells, different cytokine-combinations were tested. On the one hand IL-23 (10 ng/ml, eBioscience) was added, on the other hand cells were cultured under Th17 stimulating conditions with 2 ng/ml TGF-b and 20 ng/ml IL-6. Into some cultures AM80 dissolved in dimethyl sulfoxide (Sigma) was added at the required concentrations. Supernatants were harvested at 72 or 96 hours for cytokine measurement and FACS analysis.
Flow cytometry
Murine FACS staining were performed as previously decribed12. A LIVE/DEAD fixable dead cell stain kit (Invitrogen) was used to exclude dead cells. For T-cell phenotyping a surface staining was per-formed using the following antibodys in the presence of anti-FcgRII/III mAb (Biolegend, San Diego, USA): TCRab (Coulter), TCRgd (BioLegend), CD3e (eBiosciences), CD45 (eBiosciences), CD8a (BD), CD4 (eBiosciences) and CD25 (eBiosciences).
To stain cells for production of cytokines (Il-17a and IFN-g), they were re-stimulated with 50 ng/ml
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phorbol 12-myristate 13-acetate (PMA, Merck Millipore) and 500 ng/ml ionomycin (Sigma-Aldrich) for 4 to 6 hours (5% CO2, 37°C). After two hours of incubation brefeldin A was added to inhibit cytokine secretion. For the intracellular staining, cells were diluted in saponin buffer and incubated with IL-17A- and IFN-g antibodies for 30 minutes at 4°C. Samples were acquired on a FACS Canto II flow cytome-ter (BD) and analyzed with FACS Diva software (BD) and KALUZA software (Beckman Coulcytome-ter, Brea, USA).
RNA extraction and quantitative reverse transcription-polymerase chain reaction (qPCR)
Total RNA was extracted from cell populations using RNeasy Micro Kit (Qiagen, Venlo, Neatherlands).
Complementary deoxyribonucleic acid (cDNA) was prepared using a high capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, USA). The transcripts were assessed by QuantiTect Primer Assay (Qiagen) and Maxima SYBR Green/Rox qPCR MasterMix (Thermo Scientific, Waltham, USA) on an Applied Biosystems StepOnePlusTM Real-Time PCR System (Life Technologies, Carlsbad, USA).
Relative gene expression values were normalized with GAPDH as a housekeeping gene using the 2-ΔΔCt method.
Primers used: mGapdh, Rorc, mIL-17A, mIL-17F, mIL-22, mIL-23R, mIL-4, mTNF, mFoxp3, mIL-10, mCcr6
Protein measurements
Murine IL-17 was determined in duplicates using a mouse IL-17 DuoSet Enzyme-linked immuno-sorbent assay (ELISA) (R&D Systems, Minneapolis, USA). All other cytokines were assessed by cy-tometric bead array (CBA) using a mouse inflammation kit (BD).
Histology
Liver paraffin sections were stained with hematoxylin and eosin (H&E), and analyzed by light micros-copy. Liver cryosections (2 µm) were prepared for immunohistochemistry and processed as previously described18 with the following primary antibodies rat mouse GR-1, rat mouse F4/80 and anti-CD3 (Clone: 145-2C11) (AbD Serotec, Oxford, UK).
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Statistics
Statistical analysis was performed using GraphPad Prism software version 6.0 (GraphPad Software, San Diego, USA). Student’s t-test (unpaired, two-tailed, with Welch’s correction)
was used to analyze data between two groups. A one-way analysis of variance (ANOVA) with a Tuk-ey’s multiple comparisons test was used to analyze data contained in three groups. Comparison of survival between groups was calculated with a Log-rank (Mantel-Cox) test and a Gehan-Breslow-Wilcoxon test. Data are displayed as means and standard error of the mean (SEM). P < 0.05 was considered statistically significant.
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Results:
Fig. 1. Amount of IL-17 produced by gd T-cells in vitro depends on: I) cytokine milieu II) mode of stimulation III) culture time. (A) 2x105 MACS-sorted gd T-cells isolated from lymph nodes and spleens of healthy B6 mice were stimulated with IL-23 (10 ng/ml) in contrast to stimulation with TGF-b and IL-6. Supernatants were measured after 72 hours for the presence of IL-17 using ELISA. Error bars represent measurements from duplicate wells. Levels of significance: *** p<0.001. (B) Stimulation of 2x105 MACS-sorted gd T-cells via anti-TCRgd antibody GL3 in two doses (2 µg or 10 µg), additional stimulation with soluble antiCD28. Supernatants were measured after 72 hours for the presence of IL-17 assessed by ELISA. Error bars represent measurements from duplicate wells. Levels of signifi-cance: **p<0.01, *p<0.1. (C) Stimulation of 2x105 MACS-sorted gd T-cells with immobilized anti-CD3 antibody 2C11 or anti-TCRgd antibody GL3. Supernatants were measured after 72 hours for the pres-ence of IL-17 using ELISA. Error bars represent measurements from duplicate wells. (D) IL-17 meas-urement in supernatants using ELISA after 24, 48, 72, and 96 hours of stimulation with GL3 (10 µg/ml), antiCD28 (1µg/ml), IL-23 (10 ng/ml) and IL-1b(5 ng/ml). Error bars represent measurements from duplicate wells.
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IL-17 is a cytokine involved in different inflammatory and autoimmune processes. We have previously demonstrated that in livers of mice with BA, IL-17 is exclusively produced by gd T-cells and required for inflammation and destruction of the biliary system. Furthermore, IL17 is up-regulated in liver tis-sues from patients with BA, compared with controls, and thus might serve as a therapeutic target.
In order to establish novel pharmacological strategies for BA we aimed to create a robust system to study IL-17 production of gamma delta T cells in vitro. For this, murine gd T-cells were isolated, stimu-lated under a multitude of different conditions and production of IL-17 was subsequently assessed. We confirmed previous results that IL-23 is indispensable and sufficient alone19 for IL-17 production of gd T-cells (Fig. 1A). In contrast to ab Th17 cells, TGF-b and IL-6 did not yield any production of IL-17 (Fig. 1A). Also, neither addition nor combination of IL-1b or IL-21 did result in any IL-17 production nor did it increase IL-17 production when used together with IL-23 in our system (not shown).
Next, we investigated different kinds of gd T-cell stimulating conditions showing a dose dependent increase of IL-17 with a higher TCR antibody concentration. Of note, addition of a soluble anti-CD28 antibodies lead to maximal IL-17 production of gd T-cells (Fig. 1B). Whether T-cell receptor sig-naling was triggered via an antibody directed against the pan T-cell CD3e epitope (clone 2C11) or specifically the gd T-cell receptor itself (clone GL3), did not impact the amounts of IL-17 produced (Fig.
1C). To find the optimal duration of cell culture we harvested supernatants from gd T-cell stimulation cultures at different time points, with stimulation times of 3 or 4 days showing the highest amounts (Fig. 1D). Collectively, purified gd T-cells stimulated in the presence of IL-23 for 72h hours with immo-bilized anti-CD3 or anti-TCRgd and addition of soluble anti-CD28 lead to the most robust production of IL-17 and was used for all further experiments.
A
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Fig. 2. Synthetic retinoid AM80 suppresses IL-17 production by gd T-cells in vitro dose de-pendently. (A) 2x105 gd T-cells isolated from lymph nodes and spleens of healthy B6 mice were stimu-lated with GL3 (10 µg/ml), antiCD28 (1µg/ml), IL-23 (10 ng/ml) and IL-1b (5 ng/ml) for 72h. Ascending concentrations of AM80 (0.1-100 nM) were added. IL-17 concentration in supernatants was measured using ELISA. Error bars represent measurements from duplicate wells. Levels of significance:
***p<0.001. (B) Quantitative reverse transcription PCR was used to measure the expression of IL17 and RORγt relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping. 2x105 gd T-cells isolated from lymph nodes and spleens of healthy B6 mice were stimulated with GL3 (10 µg/ml), antiCD28 (1µg/ml), IL-23 (10 ng/ml) and IL-1b (5 ng/ml) for 72h. AM80 was added in several concentrations. Error bars represent measurements from triplicate wells. Levels of significance:
***p<0.001. (B) Quantitative reverse transcription PCR was used to measure the expression of IL17 and RORγt relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping. 2x105 gd T-cells isolated from lymph nodes and spleens of healthy B6 mice were stimulated with GL3 (10 µg/ml), antiCD28 (1µg/ml), IL-23 (10 ng/ml) and IL-1b (5 ng/ml) for 72h. AM80 was added in several concentrations. Error bars represent measurements from triplicate wells. Levels of significance: