Tissue Non-specific Alkaline Phosphatase Expression is Needed for the Full Stimulation of T Cells and T Cell-Dependent Colitis

Journal of Crohn's and Colitis, 2017, 857–870 doi:10.1093/ecco-jcc/jjw222 Advance Access publication December 29, 2016 Original Article Original Article Tissue Non-specific Alkaline Phosphatase Expression is Needed for the Full Stimulation of T Cells and T Cell-Dependent Colitis Cristina Hernández-Chirlaque,a,* Reyes Gámez-Belmonte,b,* Borja Ocón,b Patricia Martínez-Moya,a Stefan Wirtz,c Fermín Sánchez de Medina,b Olga Martínez-Augustina Department of Biochemistry and Molecular Biology II, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, Granada, Spain bDepartment of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, Granada, Spain c Department of Medicine 1, University Clinics Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany a Corresponding author: Olga Martínez-Augustin, PhD, Department of Biochemistry and Molecular Biology II, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, Granada, Spain. Tel.: +34 958 241305; fax: +34 958 248960; email: *Both authors contributed equally to this study. Conference presentation: part of this work was presented at the International Congress of Mucosal Immunology [ICMI] in 2015. Abstract Background and Aims: Two alkaline phosphatase isoforms, intestinal [IAP] and tissue non-specific alkaline phosphatase [TNAP], are coexpressed in mouse colon, with the latter predominating in colitis. We aimed to examine the role of TNAP in T lymphocytes, using heterozygous TNAP+/- mice [as TNAP-/- mice are non-viable]. Methods: In vitro primary cultures and in vivo T cell models using TNAP+/- mice were used. Results: Stimulated splenocytes [lipopolysaccharide and concanavalin A] and T lymphocytes [concanavalin A and a-CD3/a-CD28] showed a decreased cytokine production and expression when compared with wild-type [WT] cells. DecreasedT cell activation was reproduced by theTNAP inhibitors levamisole, theophylline, and phenylalanine in WT cells. Intraperitoneal administration of anti-CD3 in vivo resulted in reduced plasma cytokine levels, and decreased activation of splenocytes and T cells ex vivo in TNAP+/- mice. We further tested the hypothesis that TNAP expressed in T lymphocytes is involved in T cell activation and inflammation, using the lymphocyte transfer model of colitis. Rag1-/mice were transferred with T naïve cells [CD4+ CD62L+] from TNAP+/- or WT mice and developed colitis, which was attenuated in the group receiving TNAP+/- cells. Compared with WT, T cells from TNAP+/- mice showed a decreased capacity for proliferation, with no change in differentiation. Conclusions: Our results offer clear evidence that TNAP modulates T lymphocyte function and specifically T cell-dependent colitis. This was associated with distinct changes in the type of TNAP expressed, probably because of changes in glycosylation. Key Words: Alkaline phosphatase; T cells; Rag1-/-; anti-CD3; colitis Abbreviations: AP, alkaline phosphatase; CXCL9, chemokine [C-X-C motif] ligand 9; ConA, concanavalin A; IAP, intestinal alkaline phosphatase; KLF4, Kruppel-like factor 4; LPS, Lipopolysaccharide; MLNC, mesenteric lymph node cells; MPO, myeloperoxidase; noF, not fractionated; OPN, osteopontin; TNAP, tissue non-specific alkaline phosphatase. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: 857 C. Hernández-Chirlaque et al. 858 1. Introduction 2. Material and Methods 2.1. Reagents Except where indicated, all reagents and primers were obtained from Sigma [Barcelona, Spain]. For immediate stabilisation of RNA in tissue, RNAlater was used [Qiagen, Madrid, Spain]. Total RNA was isolated with the RNeasy Mini Kit [Qiagen]. Reverse transcription was achieved with the iScriptTM cDNA Synthesis Kit [Biorad, Alcobendas, Madrid, Spain] and GoTaq® qPCR Supermix for amplification [Promega, Charbonnières-les-Bains, France]. All the mouse cell isolation kits used in the magnetic separation [Pan T Cell and CD4+ CD62L+ T Cell] and MACS columns were provided by Miltenyi Biotec [Cologne, Germany]. Mouse ELISA kits [IL-6, TNF-α, IFN-γ, and IL-17A] were obtained from eBioscience [San Diego, CA, USA] except for IL-10 [R&D Systems, Minneapolis, MN, USA]. Multiplex assay [Procarta plex Mix&Match mouse 17-plex] was provided by eBioscience [San Diego, CA, USA]. Anti-mouse CD3ε antibodies [clone 145-2C11] and hamster IgG1 κ Isotype for in vivo experiments were purchased from BD Pharmingen [San Agustín, Spain]. In vitro experiments were made with anti-mouse CD3ε [clone 145-2C11] and anti-mouse CD28 [clone 37.51] and Armenian hamster IgG iso control [clone Ebio288Arm] of Functional Grade Purified from eBioscience, and LPS from Escherichia coli 055:B5. Cytokines and antibodies for mouse naïve T cell differentiation were purchased in BioLegend [San Diego, CA, USA: IL-4, IL-6, anti-IFN-γ, anti-IL-4, anti-IL-12] and R&D systems [Abingdon, UK: IL-2, IL-12 and TGF-β]. 2.2. Animals All animal procedures in this study were carried out in accordance with existing guidelines and were approved by the Animal Welfare Committee of the University of Granada [registry number: CEEA 2011–354]. We used C57BL/6 [B6.129S7-Akp2tm1Sor/J] heterozygous mice for Alpl [referred to as TNAP+/-], with wild-type [WT] littermates used as controls. Mice were obtained from the Jackson Laboratory. Homozygous Alpl KO mice are not viable.25,26 Genotyping was performed by polymerase chain reaction [PCR] [REDExtract-NAmp™ PCR ReadyMixTM #R4775, Sigma Aldrich] on ear genomic DNA. The Neomycin cassette was used for genotyping heterozygous mice and IL-2 as internal control. Primers were: Neo sense 5’-GGG TGG AGA GGC TAT TCG GCT ATG A-3’, antisense 3’-CCC ATT CGC CGC CAA GCT CTT CAG C-5’; and IL-2 sense 5’-CTA GGC CAC AGA ATT GAA AGA TCT-3’, antisense 3’-GTA GGT GGA AAT TCT AGC ATC ATC C-5’. Mice were maintained at the Unit of Animal Research [Biomedical Research Center, University of Granada, Granada, Spain] in specific pathogen-free conditions with free access to autoclaved tap water and food [HarlanTeklad 2014, Harlan Ibérica, Barcelona, Spain]. Female Rag1−/−mice [T cell receptors in colitis transfer model] were obtained from Jackson Laboratory [Sacramento, CA, USA]. 2.3. Induction of transfer colitis and experimental design C57BL/6J [WT] and TNAP+/- mice [16 weeks] were used as donors. Spleen cells were suspended in Dulbecco’s Modified Alkaline phosphatases are a large family of enzymes distributed from bacteria to man, which cleave phosphate moieties with release of inorganic phosphate at alkaline pH. There are four main alkaline phosphatase [AP] isoforms: the intestinal [IAP], the placental, and the germ cell isoforms, which are tissue-specific, plus the tissue nonspecific alkaline phosphatase [TNAP] which is widely expressed. T (...truncated)