The Functions of the Avian Receptor Activator of NF-Κb Ligand (RANKL) 2 and Its Receptors, RANK and Osteoprotegerin, Are Evolutionarily 3 Conserved 4 5 Kate M.C

ARTICLE IN PRESS

Developmental and Comparative Immunology ■■ (2015) ■■–■■

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Developmental and Comparative Immunology

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1 The functions of the avian receptor activator of NF-κB ligand (RANKL) 2 and its receptors, RANK and osteoprotegerin, are evolutionarily 3 conserved 4 5 Kate M.C. Sutton a, Tuanjun Hu a, Zhiguang Wu a, Botond Siklodi b, Lonneke Vervelde a,*, 6 Q1 Pete Kaiser a

7 a The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK 8 b CEVA-Phylaxia Veterinary Biologicals Co. Ltd., Szallas u. 5, Budapest H-1107, Hungary 9 10 11 ARTICLE INFO ABSTRACT 12 1413 Article history: A new member of the chicken TNF superfamily has recently been identified, namely receptor activator 1615 Received 11 February 2015 of NF-κB ligand (RANKL), as have its signalling receptor, RANK, and its decoy receptor, osteoprotegerin 1817 Revised 12 March 2015 (OPG). In mammals, RANKL and RANK are transmembrane proteins expressed on the surface of Th1 cells 19 Accepted 13 March 2015 20 and dendritic cells (DC) respectively, whereas OPG is expressed as a soluble protein from osteoblasts and 21 Available online 22 DC. Recombinant soluble chicken RANKL (chRANKL) forms homotrimers whereas chicken OPG (chOPG) 23 24 forms homodimers, characteristic of these molecules in mammals. ChRANKL, chRANK and chOPG are 25 Keywords: 26 expressed at the mRNA level in most tissues and organs. ChRANKL is transcriptionally regulated by Ca2+ 27 Chicken 2928 TNF superfamily mobilisation and enhances the mRNA expression levels of pro-inflammatory cytokines in bone marrow- 3130 Cytokines derived DC (BMDC); this is inhibited by both chOPG-Fc and soluble chRANK-Fc. However, chRANKL does 3332 Pro-inflammatory not enhance the expression of cell surface markers in either BMDC or BM-derived macrophages (BMM). 3534 DC Furthermore, chRANKL enhances the survival of APC similar to its mammalian orthologue. 3736 Macrophages © 2015 Published by Elsevier Ltd.

39 1. Introduction Sprang, 1998). The CRD are typically defined by three intra-chain 65 40 disulphide bridges generated between highly conserved cysteine resi- 66 41 Members of the TNF superfamily are key regulators of the activ- dues that act as a scaffold to produce an elongated structure protruding 67 42 ity of a variety of pathways associated with the regulation and from the cell (Smith et al., 1994). The TNFR superfamily can be further 68 43 modulation of the immune system. The TNF superfamily is made up categorised by the expression of either TNFR-associated factors (TRAF)- 69 44 of TNF ligands and their respective receptor(s). The signals from the binding motifs or death domains (DD). The DD receptors are 70 45 receptor members of the TNF (TNFR) superfamily control survival characterised by the presence of an ~80 amino acids (aa) cytoplas- 71 46 versus death signals but also have a role in regulatory events, such mic sequence necessary for apoptosis (Nagata, 1997). TNF superfamily 72 47 as controlling cytokine and chemokine expression (Locksley et al., ligands are mostly type II transmembrane proteins with an extra- 73 48 2001). Members of the TNFR superfamily are characteristically type cellular site for proteolytic cleavage to release a soluble protein from 74 49 I transmembrane proteins that have low degrees of homology and the membrane. All TNF superfamily ligands share a common struc- 75 50 are grouped due to the presence of conserved cysteine-rich domains tural core, a scaffold of ten hydrogen bonds that assumes a jelly-roll 76 51 (CRD) in their extracellular ligand-binding domains (Naismith and β-sandwich fold (Lam et al., 2001). 77 Analysis of the chicken genome has identified a reduced reper- 78 52 toire for the TNF superfamily compared to those of mammals (Kaiser 79 53 et al., 2005). Other cytokine families, including the type I and type 80 54 Abbreviations: Aa, amino acids; APC, antigen presenting cells; BMDC, bone marrow- III IFN, IL-1, IL-10, IL-12 and chemokine families (Kaiser, 2010, 2012) 81 2+ 55 derived dendritic cells; BMM, BM-macrophages; Ca , calcium ions; CRD, cysteine also have a reduced repertoire compared to their mammalian equiva- 82 56 rich domain; Ct, cycle threshold; DC, dendritic cells; DD, death domain; OPG, 57 osteoprotegerin; PKC, protein kinase C; qRT-PCR, real-time quantitative RT-PCR; RANKL, lents. For the TNF superfamily, 19 ligands and 29 receptors have been 83 58 receptor activator of NF-κB ligand; schRANKL, soluble extracellular domain of identified in mammals, but only 10 ligands and 15 receptors in the 84 59 chRANKL; schRANK, soluble extracellular domain of chRANK; TRAF, TNF receptor- chicken genome. When both a ligand and its respective receptor are 85 60 associated factors. not present in the chicken genome, we hypothesise that these 86 61 * Corresponding author. The Roslin Institute and R(D)SVS, University of Edinburgh, members are really not present rather than not annotated (Kaiser, 87 62 Easter Bush, Midlothian EH25 9RG, UK. Tel.: +44 (0)131 651 9213; fax: +44 131 651 63 9105. 2012). In mammals and chickens, the TNF superfamily members are 88 64 E-mail address: [email protected] (L. Vervelde). mainly found in small sub-families of syntenic genes on different 89

Please cite this article in press as: Kate M.C. Sutton, et al., The functions of the avian receptor activator of NF-κB ligand (RANKL) and its receptors, RANK and osteoprotegerin, are evolutionarily conserved, Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.03.006 ARTICLE IN PRESS

2 K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■

1 chromosomes. Chickens lack certain of these sub-families in their of IL-4 and IL-5 compared to wild-type cells (Kong et al., 1999). These 67 2 genome and this has implications for their biology. For example, the studies strongly suggested that RANKL is a predominant Th1 surface 68 3 lack of an equivalent of the canonical mammalian lymph node in marker. RANKL transcription is upregulated within 2 h of TCR stim- 69 4 chickens may be linked to the absence of the sub-family of genes ulation. Its expression on T cells is regulated by calcium ion (Ca2+) 70 5 encoding the lymphotoxins (LT-α and LT-β) and TNF-α in their mobilisation and is enhanced by protein kinase C (PKC) activation 71 6 genome. LT-α−/− mice have defects in lymph node development and (Bishop et al., 2011; Fionda et al., 2007; Wang et al., 2002). 72 7 splenic architecture (Banks et al., 1995). TNF-α−/− mice develop nor- Various members of the TNF superfamily can upregulate the ex- 73 8 mally but are highly susceptible to infectious agents (Pasparakis et al., pression of costimulatory molecules on the surface of target cells. 74 9 1996). However, two receptors for TNF-α – a partial clone of TNFR1 Mammalian RANKL did not significantly increase the surface ex- 75 10 (Bridgham and Johnson, 2001) and TNFR2 (Abdalla et al., 2004b) – pression of MHC class II, CD80 and CD86 on DC but did upregulate 76 11 have been identified in the chicken genome. Of the ten TNF ligands CD40 expression (Anderson et al., 1997; Wong et al., 1997b). RANKL 77 12 identified in the chicken genome, the biological activities of five have induced partial maturation of monocyte-derived DC and en- 78 13 so far been characterised – CD30 and TNF-related apoptosis induc- hanced the levels of CD83 and CD86 expression (Schiano de Colella 79 14 ing ligand (TRAIL) (Abdalla et al., 2004a), B cell activating factor et al., 2008). Macrophages upregulate MHC class II and CD86 when 80 15 (BAFF) (Schneider et al., 2004), CD40L (Tregaskes et al., 2005) and stimulated with RANKL and can further increase surface expres- 81 16 vascular endothelial growth inhibitor (VEGI) (Takimoto et al., 2005). sion by synergising with LPS and IFN-γ (Park et al., 2005). Mammalian 82 17 Overall, the functions of these chicken TNF family members are evo- RANKL is a survival factor for antigen presenting cells (APC), en- 83 18 lutionarily conserved. couraging cell survival through the upregulation of the anti- 84

19 Mammalian receptor activator of NF-κB ligand (RANKL), a ligand apoptotic molecule, Bcl-XL (Wong et al., 1997a). 85 20 member of the TNF superfamily, is a transmembrane protein of 316 Here we describe the cloning and molecular characterisation of 86 21 aa containing a COOH-terminal receptor-binding domain, a 20 aa hy- chicken RANKL (chRANKL) and its receptors, chRANK and chOPG. 87 22 drophobic transmembrane domain and a relatively long extracellular The main objective of this study was to characterise the role of 88 23 domain that contains a TNF-homologous domain that is the active chRANKL in the function of avian APC, by analysing its ability to 89 24 receptor-binding site (Anderson et al., 1997; Lam et al., 2001). Its ex- enhance pro-inflammatory cytokine mRNA expression levels, alter 90 25 pression in the immune system is limited to the thymus and lymph APC phenotype and enhance cell survival. 91 26 nodes (Anderson et al., 1997; Wong et al., 1997b) a restricted pattern 92 27 of expression not usually seen for TNF superfamily members. RANKL 2. Materials and methods 93 28 has two receptors, a signalling receptor, RANK, and a decoy recep- 94 29 tor, osteoprotegerin (OPG), a secreted TNF-related protein that inhibits 2.1. Identification and molecular cloning of chRANKL, chRANK and 95 30 the RANKL–RANK interaction (Simonet et al., 1997). RANK is a type chOPG 96 31 I transmembrane protein of 616 aa, with the longest extracellular 97 32 domain (residues 234–616) of all TNFR members identified so far, The full-length cDNA of chRANKL and chRANK were cloned using 98 33 containing four CRD (Anderson et al., 1997). It is the only known sig- primers designed to the predicted sequences of the genes in the 99 34 nalling receptor for RANKL (Anderson et al., 1997; Wong et al., 1997b). Ensembl and NCBI databases (see Table 1). ChOPG has previously 100 35 Originally identified in a bone marrow-derived myeloid DC cDNA 36 library, RANK mRNA expression is widely detected in the lungs, spleen, 101 37 skeletal muscle, brain, liver, kidney and surface expression is de- 38 tected in cells of the myelomonocytic lineage ranging from osteoclast Table 1 102 Primers and probes. 103 39 progenitor cells to DC (Anderson et al., 1997; Wong et al., 1997b). 40 OPG was identified through sequence homology with the TNF su- Name Target Sequence (5′–3′) 104 41 perfamily (Simonet et al., 1997). It contains two C-terminal RANKL Full length F: ATGCGGCGCGCCAGCCG 105 42 homologous DD of TNFR1 that are not functional. OPG is a natural- R:TCAGTCTAAATCCCTTACTTTAAATGCCCCA 106 43 ly secreted protein which can form disulphide-linked dimers of RANK Full length F: TTGTCACTACAAAGCACTCTG 107 R: ACAAACTGCATCGGACTTATC 108 44 110 kDa. Transgenic mice overexpressing OPG suffered from osteo- OPG Full length F: ATGAACAAGTTCCTGTGCTGCA 109 45 petrosis and osteoclastogenesis could be inhibited by the addition R: TTAGACACATCTTACTTTCACTGATTTCA 110 46 of OPG to cell cultures (Simonet et al., 1997). RANKL−/− and RANK−/− sRANKL Extracellular F: TGAAGAAGATGGACCCTAGTA 111 47 deficient mice had entirely unexpected phenotypes: early defects in domain R: TCAGTCTAAATCCCTTACTTTA 112 sRANK Extracellular F: ACAAACTGCATCGGACTTATC 113 48 T and B cell development, complete absences of lymph nodes, se- domain R: TCAGTTCAACACCTTCTCA 114 49 verely reduced osteoclastogenesis and failure to develop mammary 28S Taqman F: GGCGAAGCCAGAGGAAACT 115 50 glands (Dougall et al., 1999; Kong et al., 1999). RANKL, RANK and OPG R: GACGACCGATTTGCACGTC 116 51 have been implicated in a number of biological processes, such as P: AGGACCGCTACGGACCTCCACCA 117 52 bone metabolism (Kong et al., 1999; Simonet et al., 1997; Yasuda et al., IL-1β Taqman F: GCTCTACATGTCGTGTGTGATGAG 118 R: TGTCGATGTCCCGCATGA 119 53 1998), lymph node development (Kim et al., 2004), thymocyte de- P: CCACACTGCAGCTGGAGGAAGCC 120 54 velopment (Hikosaka et al., 2008; Rossi et al., 2007), M cell IL-6 Taqman F: GCTCGCCGGCTTCGA 121 55 differentiation and maintenance (Knoop et al., 2009) and immunity R: GGTAGGTCTGAAAGGCGAACAG 122 56 (Anderson et al., 1997). P: AGGAGAAATGCCTGACGAAGCTCTCCA 123 − − − − IL-12α Taqman F: TGGCCGCTGCAAACG 124 57 Although RANKL / or RANK / mice expressed normal DC and R: ACCTCTTCAAGGGTGCACTCA 125 58 macrophages numbers and functions (Darnay et al., 1998; Kong et al., P: CCAGCGTCCTCTGCTTCTGCACCTT 126 59 1999) they were deficient in production of the cytokines that drive RANKL Taqman F: CTGGAACTCGCAAAGTGAACCT 127 60 pro-inflammatory immune responses. RANKL induces pro- R: TTTCCCATCACTGAACGTCATATTT 128 61 inflammatory cytokine expression in monocyte-derived and BM- P: TGGCATCATAACAAAGGCCAGGCAAAC 129 RANK Taqman F: GCCATGTCCC AGAGGATACT 130 62 DC, driving predominantly expression of Th1 effector cytokines R: GCCAATCCCAGAGCTGAACA 131 63 (Anderson et al., 1997; Josien et al., 1999; Schiano de Colella et al., P:TGCTTCATCCACTGATGAGTGTAAATCCTGGACCA 132 64 2008; Wong et al., 1997b). Strong RANKL surface expression is pre- OPG Taqman F: ACAGCCAGGCACTCCTGAGA 133 65 dominantly on activated murine Th1 cell clones rather than Th2 cell R: GCTTTTGACAGACTGCTTTGGA 134 − − P: CCCAGAGGGATTTTTCTCCAATGAAACG 135 66 clones (Josien et al., 1999). RANKL / T cells produce higher levels

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1 been cloned (Hou et al., 2011) and used to designed primers (Table 1). pharmacological inhibitor of Ca2+ mobilisation, 8-(diethylamino)octyl- 67 2 Total RNA was extracted from either ConA (Sigma-Aldrich, Poole, 3,4,5-trimethoxybenzoate (TMB-8) (Sigma-Aldrich) for 18 h. 68 3 UK)-stimulated chicken splenocytes for 4 h or bone marrow cells 69 4 using an RNeasy mini kit (QIAGEN, Crawley, UK) following the ma- 2.4. Protein production 70 5 nufacturer’s instructions. First strand synthesis used Superscript III 71 6 (Invitrogen). After denaturation of the reverse transcriptase at 70 °C COS-7 cells were routinely grown and passaged in complete 72 7 for 15 min, 1 μl of the reaction was used in a 50 μl volume poly- DMEM (10% FCS, 2% L-glutamine, 2% non-essential aa, 1 U/ml pen- 73 8 merase chain reaction (PCR) containing 10 pmol of each primer, 1 μM icillin and 1 μg/ml streptomycin). To produce recombinant protein, 74 9 dNTP and 0.625 U of Taq DNA polymerase (Invitrogen). Each cDNA pCI-IZFLAG-schRANKL, Signal-pKW06-schRANK and pKW06- 75 10 was cloned into pGEM-T Easy and sequences confirmed from three chOPG were transfected into COS-7 cells using DEAE-dextran (Avery 76 11 independent clones on each strand. The cDNA sequences of chRANKL et al., 2004). Cell supernatants (ex-COS-7) were collected 72 h later 77 12 and chRANK were submitted to Ensembl (Accession nos. LM999949 and stored at 4 °C until use. 78 13 and LM999950). Sequences from species containing orthologues of 79 14 RANKL, RANK and OPG were aligned using CLUSTALX v2.1. The sec- 2.5. Western blot analysis 80 15 ondary structure of chRANKL was predicted using PSIPRED v3.0 81 16 Q2 (http://bioinf.cs.ucl.ac.uk/psipred/). The supernatants from COS-7 cells transfected with pCI-IZFLAG- 82 17 schRANKL or pKW06-chOPG-Fc were treated with loading buffer 83 18 2.2. Sub-cloning of chRANKL, chRANK and chOPG into expression with or without β-mercaptoethanol and boiled for 5 min at 95 °C. 84 19 vectors Samples were then loaded on a 4–12% gradient SDS–PAGE gel (Bio- 85 20 Rad) and transferred onto nitrocellulose membrane. Samples were 86 21 To obtain soluble forms of chRANKL (schRANKL) and chRANK blocked with 2% casein for 1 h at room temperature. FLAG tagged 87 22 (schRANK), their extracellular domains were identified using their schRANKL protein was detected using a mouse anti-FLAG mAb 88 23 human orthologues and the SMART prediction program (Sigma-Aldrich, M2) and a goat anti-mouse IgG HRP-conjugated sec- 89 24 (http://smart.embl-heidelberg.de/). Primers containing restriction ondary antibody (SouthernBiotech, 1070-05). ChOPG-Fc was detected 90 25 enzyme sites were designed to flank the extracellular domains of using a goat anti-human HRP-conjugated antibody (SouthernBiotech, 91 26 chRANKL and chRANK (Table 1). To obtain schRANKL, the extracel- 2040-05). Recombinant protein was detected using an ECLTM Western 92 27 lular domain was amplified and sub-cloned into the NhoI and NheI Blotting Detection Reagents system (GE Healthcare Life Sciences, 93 28 sites of a modified pCI-neo vector (Promega, Southampton, UK) ex- Little Chalfont, UK). 94 29 pressing the mouse CD8 signal peptide, an isoleucine zipper and 95

30 an NH2-terminal FLAG-tag, named pCI-IZFLAG-schRANKL (gener- 2.6. BMDC and BMM preparation 96 31 ous gift from John Young, the former Institute for Animal Health, 97 32 UK). The obtained schRANK and chOPG, the extracellular domain Femurs and tibias were collected aseptically from 4 to 6-week- 98 33 of chRANK and the full-length chOPG cDNA were directionally sub- old J-line chickens. Bone marrow-derived DC (BMDC) and 99 34 cloned into two similar plasmids, Signal-pKW06 and pKW06 (Staines macrophages (BMM) were expanded in vitro as described in Wu et al. 100 35 et al., 2013), to generate COOH-human Fc-tagged recombinant pro- (2010) and Garceau et al. (2010), respectively. In brief, bones were 101 36 teins. All sub-cloning was verified by sequencing. flushed with PBS and single cell suspensions were collected using 102 37 Histopaque-1077 (Sigma-Aldrich) followed by two washes with PBS. 103 38 2.3. Chicken tissues and cells BMDC were seeded at 1 × 106 cells/ml in 6-well plates in complete 104 39 RPMI and cultured with appropriate dilutions of exCOS-7 super- 105 40 Tissues were removed from three 6-week-old J-line chickens (The natants of chIL-4 and chCSF-2. For BMM, cells were seeded at 1 × 106 106 41 Roslin Institute, UK) post-mortem. Tissues taken were the heart, kidney, cells/ml in complete BMM RPMI (2% ChS, 3% FCS, 2% L-glutamine, 107 42 liver, muscle, lung, brain, skin, spleen, thymus, bursa of Fabricius, caeca, 1 U/ml penicillin and 1 μg/ml streptomycin) and appropriate dilu- 108 43 bone marrow, crop, gizzard, lower and mid-gut, caecal tonsils, Meckel’s tions of exCOS-7 supernatants of chCSF-1 in 25-well chamber plates 109 44 diverticulum and Harderian gland. Tissues were homogenised using (Thermo Scientific). BMDC and BMM were cultured for 6 days at 110

45 matrix tubes containing 1.4 mm ceramic spheres (MD Biomedicals). 41 °C, 5% CO2. 111 46 Primary cell populations were prepared from the spleen, bursa of Fab- 112 47 ricius and thymus of three individual birds. Cells were purified by 2.7. Bioactivity of schRANKL on BMDC and BMM 113 48 passing the tissues through 70 μm mesh cell strainers (Fisher Sci- 114 49 entific) and splenocytes were collected over Histopaque-1077 (Sigma- BMDC and BMM were unstimulated or stimulated with LPS 115 50 Aldrich) followed by two washes with PBS. Cell numbers were adjusted (1–2 ng/ml), schRANKL (1:50 exCOS-7 supernatant) or both for 3 and 116 51 to 5 × 106 cells/ml with pre-warmed complete RPMI (10% chicken 6 h. To determine the antagonistic effects of schRANK-Fc and chOPG- 117 52 serum (ChS), 2% L-glutamine, 1 U/ml penicillin and 1 μg/ml strep- Fc (1:5 exCOS-7 supernatant), either was pre-incubated with schRANKL 118 53 tomycin (Gibco Life Technologies)) and cultured in 25 mm2 flasks for for 3 h prior to cell treatment. The resulting mRNA expression levels 119 54 24 h either unstimulated or stimulated as follows: splenocytes, 1 μg/ml of pro-inflammatory cytokines were analysed by qRT-PCR. 120 55 ConA (Sigma-Aldrich); bursal cells, 500 ng/ml PMA (Sigma-Aldrich) 121 56 and 1 μg/ml ionomycin (Sigma-Aldrich); thymocytes, 25 μg/ml PHA 2.8. Fluorescent activated cell sorting 122 57 (Sigma-Aldrich). Lymphocyte subsets (CD4+, CD8β+, TCRγδ+, TCRαβ1+ 123 58 and TCRαβ2+) were isolated from total splenocytes by positive se- On day 6 of BMDC and BMM cultures, cells were unstimulated 124 59 lection using an AutoMACS Pro Separator (Miltenyi Biotech). Purity or stimulated with LPS (1–2 ng/ml), schRANKL (1:5 exCOS-7 su- 125 60 of the isolated populations was verified by FACS analysis (BD pernatant) or both for 24 h. Cells were removed from culture plates 126 61 FACSCalibur, BD Biosciences). To determine the transcriptional regula- using 0.05 mM EDTA and resuspended in PBS with 0.5% BSA and 127 62 tion of chRANK in avian splenocytes, cells were unstimulated or stimu- 0.001% azide (FACS buffer). Immunofluorescence labelling was carried 128 63 lated with ionomycin (3 μg/ml) or ionomycin and PMA (500 ng/ml) out using mouse anti-chicken MHC class II (IgG1, 2G11), mouse anti- 129 64 for 2, 4 or 18 h. To determine if Ca+2 modulation was linked with chicken CD40 (IgG2a, Serotec, MCA2836) and mouse anti-chicken 130 65 chRANKL transcriptional regulation, cells were either stimulated with KUL01 (IgG1, SouthernBiotech, 8420-01) mAbs on ice for 20 min. 131 66 ionomycin alone or in combination with various amounts of the A FITC-conjugated goat anti-mouse IgG1 antibody (SouthernBiotech, 132

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1 1070-02) was used as a secondary antibody to detect MHC class II are four conserved cysteines between mammalian and chicken 67 2 and KUL01 and a PE-conjugated goat anti-mouse IgG2a antibody RANKL, and three potential N-glycosylation sites in the chRANKL 68 3 (SouthernBiotech, 1080-09S) to detect CD40, incubated on ice for extracellular domain, one of which is conserved with mammalian 69 4 20 min. Negative/isotype controls were used for each sample to RANKL. 70 5 control for non-specific binding. Cells were stained with 7-AAD prior A multiple alignment of the aa sequences of human, mouse and 71 6 to analysis and gated using FSC and SSC using a BD FACSCalibur in- chicken RANK is shown in Fig. 1B. ChRANK has ~40–42% sequence 72 7 strument (BD Biosciences). Data were analysed using FlowJo v7.6.5 identity with mammalian RANK. The TNFR superfamily members 73 8 software (Tree Star, Ashland, USA). have conserved CRD in their extracellular ligand-binding domains 74 9 (Naismith and Sprang, 1998), which are typically defined by three 75 10 2.9. Flow cytometer-based survival assay intra-chain disulphide bridges between highly conserved cysteine 76 11 residues that act as a scaffold to produce an elongated structure 77 12 BMDC and BMM were treated with fresh complete media or protruding from the cell (Smith et al., 1994). All four CRD are present 78 13 schRANKL (1:5 exCOS-7 supernatant) for 24 and 48 h. Cells were har- in chRANK (labelled I–IV) and all of the cysteine residues in the CRD 79 14 vested at indicated time-points and stained with biotin-conjugated are conserved between the three species. There are two potential 80 15 recombinant Annexin-V according to the manufacturer’s instruc- N-glycosylation sites in the extracellular domain of chRANK, both 81 16 tions (eBioscience). Conjugated streptavidin-Alexa 657 (Molecular conserved in the mammalian molecules. All TNFR lack catalytic 82 17 Probes, S-21374) was incubated at RT for 20 min. Propidium iodide domains and require adaptor proteins for downstream signalling, 83 18 (PI) was added to the cells and immediately analysed using a BD called TRAFs, which for mammalian RANK include TRAF1-3, TRAF5-6. 84 19 FACSCalibur instrument. Two binding motifs for TRAF1-3 and TRAF5 in TNFR have been 85 20 identified; a major one, (P/S/A/T)X(Q/E)E, and a minor one, PXQXXD 86 21 2.10. Quantitative real-time RT-PCR analysis (X being any aa) (Devergne et al., 1996; Ishida et al., 1996). The TRAF6 87 22 specific-binding motif is the peptide PXEXX (X being an aromatic or 88 23 RNA from the tissues and cells were extracted using RNeasy Mini acidic residue). Four of the five mammalian RANK TRAF-binding sites 89 24 kit (Qiagen, Crawley, UK) following manufacturer’s instructions. (Cremer et al., 2002; Darnay et al., 1998; Wong et al., 1998)are 90 25 ChRANKL, chRANK and chOPG and pro-inflammatory cytokine mRNA conserved in chRANK (Fig. 1B). The “missing” TRAF-binding motif is 91 26 expression levels were quantified by TaqMan real-time quantita- TRAF6-specific (residues 457–463 in huRANK). ChRANK also contains 92 27 tive RT-PCR (qRT-PCR) using a well described method (e.g. Avery the highly conserved region (HCR; between Pro508 and Gly546 in 93 28 et al., 2004; Eldaghayes et al., 2006). Primers (Sigma) and probes muRANK), which is vital for osteoclastogenesis in mammals (Taguchi 94 29 (Eurogentec, Belgium) (Table 1) were designed using Primer Express et al., 2009). 95 30 (Applied Biosystems). TaqMan assays were performed using the One- To produce soluble protein the extracellular domain of chRANKL 96 31 Step RT-PCR Master Mix reagents and amplification and detection was cloned as a 729 bp cDNA fragment corresponding to 243 aa 97 32 were performed using the FAST Universal PCR Master Mix (Applied (chRANKL75–318) and is henceforth called schRANKL. The extracellular 98 33 Biosystems). Data are expressed in terms of the cycle threshold (Ct) domain of chRANK was cloned as a 498 bp cDNA fragment which cor- 99 34 value, which is normalised using the Ct values of 28S rRNA for each responded to 166 aa (chRANK24–190) and is henceforth called schRANK. 100 35 sample using the formula; Ct + (N′t-C′t)*(S/S′) where N′t is the mean The full-length chOPG cDNA was cloned using primers designed 101 36 Ct value for 28S RNA among all samples, C′t is the mean value for based on the NCBI database sequence. Total RNA was extracted from 102 37 28S RNA in the sample and the S and S′ are the slopes of regres- bone marrow and used to amplify a cDNA fragment of 1.2 kb, corres- 103 38 sion of the standard plots for the cytokine mRNA and the 28S RNA, ponding to 401 aa. A multiple alignment of the aa sequences of 104 39 respectively. Normalised Ct values were corrected against the neg- human, mouse and chicken OPG is shown in Fig. 1C. ChOPG shares 105 40 ative association of the log concentration of mRNA detected using high identity with mammalian OPG (60–64%). It possesses four CRD 106 41 the formula 40 – normalised Ct value of the cytokine of interest. domains (labelled I–IV) with 17 out of 18 cysteine residues conserved 107 42 Final results are shown as corrected 40 − Ct values or as fold dif- and two homologous DD which are conserved between the three 108 43 ference from levels in untreated control cells. species. ChOPG contains a penultimate cysteine residue at position 109 44 401 which is vital for homodimerisation in mammalian OPG (Luan 110 45 2.11. Statistical analysis et al., 2012). 111 46 112 47 Statistical analyses were carried out using the Mann–Whitney U 3.2. Soluble chRANKL and chOPG form homo-trimeric and 113 48 test in Minitab 16.1.0 (State College, USA). Statistical significance was homo-dimeric protein structures 114 49 determined as significant (p < 0.05) or highly significant (p < 0.01). 115 50 The hallmark of TNF ligand and TNFR interactions is their 116 51 3. Results threefold-symmetry. Three subunits of RANKL are required for the 117 52 activation of the receptor, RANK. To enhance the formation of tri- 118 53 3.1. Cloning and analysis of chRANKL, chRANK and chOPG meric schRANKL protein, a modified plasmid was designed to express 119 54 an in-frame isoleucine zipper sequence upstream of the chRANKL 120

55 A multiple alignment of the aa sequences of human, mouse and sequence with an NH2-terminal FLAG-tag, called pCI-IZFLAG- 121 56 chicken RANKL is shown in Fig. 1A. ChRANKL shares ~59–62% iden- schRANKL. SchRANKL protein was generated in COS-7 cells and 122 57 tity with mammalian RANKL. The extracellular domains of TNF protein structures were analysed by SDS–PAGE under reducing and 123 58 superfamily members express the highly conserved TNF homol- non-reducing conditions and western blot using antibodies against 124 59 ogy domain (residues 163–318 in the chicken molecule) which is the FLAG-tag. In reducing conditions, schRANKL appears as pre- 125 60 required for receptor interaction and trimerisation. This domain is dominantly a monomeric protein at ~37 kDa with a weak band of 126 61 composed of β-strands and loops connected in a “jelly-roll” fold ~75 kDa which is possibly dimeric protein (Fig. 2). Proteins analysed 127 62 (Lam et al., 2001). To investigate the conservation of these β-strands under non-reducing conditions produced bands at ~75, ~ 140 and 128 63 in chRANKL, each was identiﬁed in mammalian RANKL (Fig. 1A) ~200 kDa, suggesting the formation of dimeric, trimeric and pos- 129 64 using PSIPRED. ChRANKL has conserved residues at the positions sibly tetrameric protein structures (Fig. 2A). 130 65 of the β-strands and loops found in mammalian counterparts sug- To analyse the ability of chOPG to form homodimeric protein, 131 66 gesting it has the potential to fold into a trimeric protein. There COS-7 cells were transfected with pKW06-chOPG to produce soluble 132

K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■ 5 A Human MRRASRDYTKYLRGSEEMGGGPGAPHEGPLH.APPPPAPHQPPAASRSMFVALLGLGLGQ 59 Mouse MRRASRDYGKYLRSSEEMGSGPGVPHEGPLHPAPSAPAPAPPPAASRSMFLALLGLGLGQ 60 Chicken MRRASRDYTKYLRGSEEVGSGAAHESAPPPPPAPPLPAHPHPPPASRSLLAALVLLGLGQ 60

Human VVCSVALFFYFRAQMDPNRISEDGTHCIYRILRLHENADFQDTTLESQDTKLIPDSCRRI 119 Mouse VVCSIALFLYFRAQMDPNRISEDSTHCFYRILRLHENADLQDSTLESEDT..LPDSCRRM 118 Chicken VVCSVALFLYFRAQMDPSRISKEDAHCVRMLFRSQESIGLQDTPFENQEVKLMPESCRRM 120 * * *

AA’ loop A Human KQAFQGAVQKELQHIVGSQHIRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINATDIPSGS 179 Mouse KQAFQGAVQKELQHIVGPQRFSGAPAMMEGSWLDVAQRGKPEAQPFAHLTINAASIPSGS 178 Chicken KRALQRAVQKEVQRILGKESPRPEKAAMEAIGMELYRRNKPEKQPFAHLIIDDKNILTGT 180

CD loop A’ B’ B C D Human HKVSLSSWYHDRGWAKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDLATEYLQLM 239 Mouse HKVTLSSWYHDRGWAKISNMTLSNGKLRVNQDGFYYLYANICFRHHETSGSVPTDYLQLM 238 Chicken RKVNLTSWHHNKGQANLSNMTFSDGKLIVNQDGFYYLYANICFRHHETSGNLTKRGLQLM 240 +++ +++ * +++

DE loop EF loop E F G Human VYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNPSLL 299 Mouse VYVVKTSIKIPSSHNLMKGGSTKNWSGNSEFHFYSINVGGFFKLRAGEEISIQVSNPSLL 298 Chicken VYMTKTNLKIRRSDVLMKGGSTKYWSGNSEFHFYSVNIGGFLKLKTGDMISIQVSNPLLL 300

H Human DPDQDATYFGAFKVRDID 317 Mouse DPDQDATYFGAFKVQDID 316 Chicken DSSQEATYFGAFKVRDLD 318

1 Fig. 1. Alignment of the sequences of RANKL, RANK and OPG. Amino acid sequence alignment of (A) chRANKL, (B) chRANK and (C) chOPG with their mammalian orthologues. 2 Shaded areas represent conservation of aa; the darker the shading, the more conserved the residue across species. Dots indicate gaps in the alignment. Asterisks indicate 3 conserved cysteine residues. Predicted N-glycosylation sites in any molecule are indicated by +++. (A) RANKL is a type II membrane protein. The box indicates the trans- 4 membrane domains. The TNF homology domain is underlined. Black lines above the sequences indicate the location of the ten conserved β-strands (labelled A, A′, B′,B,C, 5 D, E, F, G and H) involved in the “β-jelly” roll structure, and the surface loops are shown as grey lines between the β-strands, labelled AA′, CD, DE and EF (based on the 6 mouse RANKL crystal structure (Lam et al., 2001). (B) The four CRD domains are overlined and numbered I–IV, the black box indicates the transmembrane domain and the 7 light grey box the HCD. TRAF-binding motifs are overlined and labelled (6 = TRAF6, 1/2/3/5 = TRAF1, TRAF2, TRAF3, TRAF5 binding motifs). (C) The four CRD domains are 8 overlined and numbered I–IV, the DD are overlined and the triangle indicates the conserved cysteine residue in mammals not conserved in the chicken.

10 chOPG-Fc. Protein structures were analysed under reducing and were detected, suggesting each is ubiquitously expressed in chicken 26 11 non-reducing conditions and visualised by western blot using tissues (Fig. 3). 27 12 antibodies against the Fc tag (Fig. 2). The chOPG-Fc monomer has a In non-lymphoid tissues, chRANKL and chRANK mRNA expres- 28 13 predicted molecular mass of ~72 kDa. In reducing conditions, sion levels are highest in muscle, which had the lowest levels of 29 14 a ~ 75 kDa band was just evident whereas in non-reducing conditions chOPG mRNA expression (Fig. 3A). The lowest levels of chRANKL 30 15 there was a strong band at ~160 kDa, suggesting the formation of and chRANK mRNA expression were in the heart. Levels of mRNA 31 16 homodimers (Fig. 2B). expression of all three cytokine were consistent in the liver, kidney 32 17 and lung and slightly lower in the skin and brain (Fig. 3A). In chicken 33 18 3.3. Tissue distribution and cellular mRNA expression levels of lymphoid tissues, the highest levels of mRNA expression for all 34 19 chRANKL, chRANK and chOPG three cytokines were in the thymus, followed by the upper-gut and 35 20 the bone marrow (Fig. 3B). In the crop, chRANKL mRNA expres- 36 21 The mRNA expression proﬁles of chRANKL, chRANK and chOPG sion levels were higher (~16-fold) than those of chRANK and chOPG. 37 22 were determined in a broad range of non-lymphoid and lymphoid In the Harderian gland, chRANKL was more highly expressed than 38 23 tissues by qRT-PCR. Primers and probes for chRANKL and chRANK chRANK. 39 24 were designed against their extracellular domains. In both non- The mRNA expression levels of chRANKL, chRANK and chOPG 40 25 lymphoid and lymphoid tissues, mRNA transcripts for each gene were also examined in ConA stimulated splenocytes, PMA and 41

6 K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■

B I Human MAPRARRRRPLFA-LLLLCALLAR-LQVALQIAPPCTSEKHYEHLGRCCNKCEPGKYMSS 58 Mouse MAPRARRRRQLPAPLLALCVLLVP-LQVTLQVTPPCTQERHYEHLGRCCSRCEPGKYLSS 59 Chicken -MPGP------PGWLALLCLAAAGRPQLSLQSTLPCESEQHYEYSGRCCKKCEPGKYMSA 53 * * ** * II Human KCTTTSDSVCLPCGPDEYLDSWNEEDKCLLHKVCDTGKALVAVVAGNSTTPRRCACTAGY 118 Mouse KCTPTSDSVCLPCGPDEYLDTWNEEDKCLLHKVCDAGKALVAVDPGNHTAPRRCACTAGY 119 Chicken KCTETSDSVCQPCGPNEYMDVWNEEDKCLLHKICDQGKALKEVNPGNSTFQRQCACTTGY 113 * * * * * +++ * * III IV Human HWSQDCECCRRNTECAPGLGAQHPLQLNKDTVCKPCLAGYFSDAFSSTDKCRPWTNCTFL 178 Mouse HWNSDCECCRRNTECAPGFGAQHPLQLNKDTVCTPCLLGFFSDVFSSTDKCKPWTNCTLL 179 Chicken HWNEDCDCCQRNSICGPGFGIGHPVQQDKDTKCMPCPRGYFSEVASSTDECKSWTNCSAL 173 * ** * * * * * +++ Human GKRVEHHGTEKSDAVCSSSLPARKPPNEPHVYLPGLIILLLFASVALVAAIIFGVCYRKK 238 Mouse GKLEAHQGTTESDVVCSSSMTLRRPPKEAQAYLPSLIVLLLFISVVVVAAIIFGVYYRKG 239 Chicken GLAEIVPGTDKSDAVCSKQKMAEQPEYGTNKILYVFIVILFFV--ALIGIVIFIVYYKNK 231 *

Human GKALTANLWHWINEACGRLSGDKESSGDSCVSTHTANFGQQGACEGVLLLTLEEKTFPED 298 Mouse GKALTANLWNWVNDACSSLSGNKESSGDRCAGSHSATSSQQEVCEGILLMTREEKMVPE- 298 Chicken GKKLTADLQNWANEVCSQIKGTKEPSRDAYVTVNITNTTIPQLSEDVRLLGPAGSPAPG- 290 * ++++++ 6 Human MCYPDQGGVCQGTCVGGGP-YAQGEDARMLSLVSK-TEIEEDSFRQMPTEDEYMDRPSQP 356 Mouse ----DGAGVCGPVCAAGGP-WAEVRDSRTFTLVSE-VETQGDLSRKIPTEDEYTDRPSQP 352 Chicken -----SSPCGHSSCRHGSPGTAPCRAGGNLQEVSVGTGTDDEHFSPVPTEDEYMDKDLNT 345 * * * 6 Human TDQLLFLTEPGSKSTPPFSEPLEVGENDSLSQCFTGTQSTVGSESCNCTEPLCRTDWTPM 416 Mouse STGSLLLIQQGSKSIPPFQEPLEVGENDSLSQCFTGTESTVDSEGCDFTEPPSRTDSMPV 412 Chicken ADYLPLLSQPDNKTLSLFSEPVEVGENDSLNQCFSGIGSAEDVSMPQGSDPSSNADGVHT 405 +++ +++ * 6 Human SSENYLQKEVDSGH-CPHWAASPS------PNWADVCTGC----RNPPGEDCEPLVG-- 462 Mouse SPEKHLTKEIEGDS-CLPWVVSS------NSTDGYTGS----GNTPGEDHEPFPG-- 456 Chicken TMDKYLQKSCQRSHGCPKEIDSKDTDRFAMNRDSEKICVRCGVSYRETPRKGSRPCCAAV 465 * * * * **

Human -----SPKRGPLPQCAYGMGLPPEEEASRTEAR---DQPEDGADGRLPSSARAGAGSGSS 514 Mouse -----SLKCGPLPQCAYSMGFPSEAAASMAEAG---VRPQDRAD------ERGASGSGSS 502 Chicken DGAFTSPETGCYAQCTCGMNFLSAGQSPLASDHGMEDAPSDGTNTKYQNTNRSTSGTNSS 525 * * * +++ +++

1/2/3/5 Human PGGQSPASGNVTGNSNSTFISSGQVMNFKGDIIVVYVSQTSQEG---AAAAAEPMGRPVQ 571 Mouse PSDQPPASGNVTGNSNSTFISSGQVMNFKGDIIVVYVSQTSQEGPGSAEPESEPVGRPVQ 562 Chicken TSDLPPASGNVTGNSNSTFISSGQVMNFKGDIIVVYLSQNSQEGAAASGAAEENVSSPVQ 585 +++ +++ +++

1/2/3/5 Human EETLARRDSFAGNGPRFPDPCG------GPEGLREP------EKASRPVQEQGG 613 Mouse EETLAHRDSFAGTAPRFPDVCA------TGAGLQEQGAPR----QKDGTSRPVQEQGG 610 Chicken EENPSRCETFAGNAQHYKEKCAELHATIPVGSGGTRHHTAPVPSLARHSEASPPVQEEGR 645 * *

Human AKA------616 Mouse AQTSLHTQGSGQCAE 625 Chicken LGHSSEKVLN----- 655

1 Fig. 1. (continued)

3 ionomycin stimulated bursal cells and PHA stimulated thymo- 3.4. ChRANK and chOPG mRNA are differentially expressed in 9 4 cytes (Fig. 4A). After 24 h of stimulation the mRNA expression levels mature BMDC 10 5 of chRANKL, chRANK and chOPG were not statistically signiﬁ- 11 6 cantly altered compared to levels in unstimulated cells (Fig. 4A). At In mammals, RANK surface expression is predominantly found 12 7 a cellular level, the mRNA expression levels of all three molecules on mature DC and osteoclasts (Anderson et al., 1997; Wong et al., 13 8 were detected in CD4+, CD8β+, TCRγδ+ and TCRαβ+ cells (Fig. 4B). 1997b) while OPG expression is in osteoblasts (Yasuda et al., 1998), 14

K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■ 7

C I

Human MNNLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKWKT 60 Mouse MNKWLCCALLVLLDIIEWTTQETLPPKYLHYDPETGHQLLCDKCAPGTYLKQHCTVRRKT 60 Chicken MNKFLCCTLVLLDISVKWSIQDISPPKYLHYDPGTSRQVMCNQCPPGSYVKQHCTAASPT 60 ** * * * II III

Human VCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCLK 120 Mouse LCVPCPDHSYTDSWHTSDECVYCSPVCKELQSVKQECNRTHNRVCECEEGRYLEIEFCLK 120 Chicken VCAPYPDQYYAEDWNSNDECQYCSAVCKELQYIKQECTSTQDRVCECIEGWYLELEFCLK 120 * * * * *+++ * * * IV

Human HRSCPPGFGVVQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQKGNAT 180 Mouse HRSCPPGSGVVQAGTPERNTVCKKCPDGFFSGETSSKAPCIKHTNCSTFGLLLIQKGNAT 180 Chicken HTECPPGFGVAQPGTPESDTVCKSCPEGFFSNETSSKAVCQKHTNCSALGFKTALKGNAV 180 * * * +++ * * +++ +++

Human HDNICSGNSEST.QKCGIDVTLCEEAFFRFAVPTKFTPNWLSVLVDNLPGTKVNAESVER 239 Mouse HDNVCSGNREAT.QKCGIDVTLCEEAFFRFAVPTKIIPNWLSVLVDSLPGTKVNAESVER 239 Chicken RDNICQENTDTSPQKCGIDITLREEAMFRFAVPTRSTPNWLNTLADNLPGTKISTENIER 240 * Δ

Human IKRQHSSQEQTFQLLKLWKHQNKDQDIVKKIIQDIDLCENSVQRHIGHANLTFEQLRSLM 299 Mouse IKRRHSSQEQTFQLLKLWKHQNRDQEMVKKIIQDIDLCESSVQRHLGHSNLTTEQLLALM 299 Chicken IKQRHSSQEQTFQLLKLWKQQNKEQDMVKKIIQDIDHCENSVLKHLGQPSLTFEHLNTLM 300 * +++

Human ESLPGKKVGAEDIEKTIKACKPSDQILKLLSLWRIKNGDQDTLKGLMHALKHSKTYHFPK 359 Mouse ESLPGKKISPEEIERTRKTCKSSEQLLKLLSLWRIKNGDQDTLKGLMYALKHLKTSHFPK 359 Chicken ASLPGKKVGKEDIERTMKLCQHAEQVLKLLNLWRIKNGDQDTIKGLMYGLKHLKTYHFPK 360 *

Human TVTQSLKKTIRFLHSFTMYKLYQKLFLEMIGNQVQSVKISCL 401 Mouse TVTHSLRKTMRFLHSFTMYRLYQKLFLEMIGNQVQSVKISCL 401 Chicken RIIQSLKKVIKFLHGFAMYRLYQKLFLEMVGSQLKSVKVRCV 402 *

1 Fig. 1. (continued)

2 RNRM NR R M 3 suggesting a role for the latter in regulating the interaction between kDa kDa 4 RANKL–RANK in bone metabolism. OPG in human Mo-DC has a 250 5 similar role, where OPG expression levels increase as DC mature 250 6 (Schoppet et al., 2007), suggesting that OPG is also a molecular brake 150 7 for RANKL–RANK interactions in the immune system. Before ex- 150 100 8 amining the bioactivity of schRANKL, the mRNA expression levels 100 9 of both of its receptors were analysed in BMDC. BMDC were 75 10 unstimulated or stimulated with various concentrations of LPS for 75 11 3, 6 and 9 h and mRNA expression levels of the two receptors 50 12 analysed by qRT-PCR. 50 37 13 At 3 h, LPS-stimulated (200 ng/ml) cells had a ~ 2-fold increase 37 14 in chRANK and chOPG mRNA expression levels which dropped to 15 ~1-fold at the lower amounts of LPS used, compared to levels in 25 25 16 unstimulated cells (Fig. 5). At 6 h post-stimulation with high LPS con- 20 20 17 centrations, chOPG mRNA expression levels were increased by nearly 18 3-fold while levels of chRANK mRNA expression were only in- RANKL OPG 19 creased by ~0.5-fold. At lower concentrations of LPS, chRANK mRNA 20 expression levels decreased to basal unstimulated levels or lower Fig. 2. Western blot analyses of schRANKL and chOPG proteins. The extracellular 26 21 (Fig. 5). Cells stimulated with 100 ng/ml of LPS increased chOPG soluble domain of chRANKL (schRANKL) was sub-cloned into a modified pCI-neo 27 22 mRNA expression levels statistically significantly compared to vector, expressing an NH2-terminal FLAG-tag and an in-frame isoleucine zipper se- 28 quence to encourage trimer formation. ChOPG was sub-cloned into a pKW06 vector 29 23 chRANK mRNA expression levels (p < 0.05). At 9 h, chOPG mRNA ex- and expressed as a human Fc-tagged protein. Samples were analysed under reduc- 30 24 pression levels were statistically significantly higher compared to ing (R) and non-reducing (NR) conditions. Marker (M) = protein precision plus 31 25 chRANK mRNA expression levels in all LPS-stimulated cells (p < 0.05). (BioRAD). 32

8 K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■

A RANKL RANKL 20 Corrected RANK A 5 RANK 40-Ct 15 OPG Fold OPG 4 change 10 3 5

2 0 1 234 567 1 B 20 0 Corrected 40-Ct 15 -1 Splenocytes Bursal cells Thymocytes

10 B 20

5 Corrected 40-Ct 15

0 1 2 3 4 586 7 9 10 11 12 10

1 Fig. 3. ChRANKL, chRANK and chOPG mRNA expression in chicken tissues. Expres- 5 2 sion profiles of chRANKL, chRANK and chOPG mRNA in non-lymphoid and lymphoid 3 tissues, as measured by qRT-PCR. (A) Non-lymphoid tissues: (1) brain, (2) muscle, 4 (3) heart, (4) liver, (5) kidney, (6) lung, (7) skin. (B) Lymphoid tissues: (1) thymus, 0 5 (2) spleen, (3) bursa of Fabricius, (4) caecal tonsil, (5) Harderian gland, (6) bone CD4 CD8β TCR1 TCR2/3 6 marrow, (7) Meckel’s diverticulum, (8) caeca, (9) mid-gut, (10) upper-gut, (11) crop, (γδ) (αβ) 7 (12) gizzard. Data are presented as the average corrected 40-Ct values of triplicate 8 assays on each of three individual birds ± SEM. Fig. 4. ChRANKL, chRANK and chOPG mRNA expression in chicken immune cells. 43 9 ChRANKL, chRANK and chOPG mRNA expression in lymphoid cells, as measured by 44 real-time qRT-PCR. (A) Expression levels of chRANKL, chRANK and chOPG in ConA 45 10 3.5. Transcriptional control of chRANKL mRNA expression in avian stimulated splenocytes, PMA and ionomycin stimulated bursal cells and PHA stimu- 46 lated thymocytes for 24 h. Data are presented as fold change in comparison to levels 47 11 splenocytes in unstimulated control cells of triplicate assays on each of three individual birds ± SEM. 48 12 (B) Sorted splenocytes. Data are presented as the average corrected 40-Ct values of 49 13 Our previous data have shown that ConA stimulation has no effect triplicate assays on each of three individual birds ± SEM. 50 14 on chRANKL mRNA expression in chicken splenocytes. Therefore, 51 15 to further understand the regulation of chRANKL transcription, 16 splenocytes were activated with the Ca2+-mobiliser/inducer, chRANKL mRNA expression were analysed in these cells after 18 h 52 17 ionomycin or ionomycin and the PKC activator, PMA, to mimic the of treatment by qRT-PCR (Fig. 6B), and the addition of TMB-8 at all 53 18 signals initiated by TCR activation for 2, 4 and 18 h. Levels of three concentrations reduced chRANKL mRNA expression levels to 54 19 chRANKL mRNA expression were analysed by qRT-PCR (Fig. 6). those in unstimulated cells. 55 20 At 2 h post-stimulation with ionomycin alone, chRANKL mRNA 56 21 expression levels were unaltered compared to those in unstimulated 3.6. The effects of chRANKL on mRNA expression levels of 57 22 cells whereas costimulated cells had a ~ 3-fold, but not significant pro-inflammatory cytokines in BMDC and BMM 58 23 increase in chRANKL mRNA expression levels (Fig. 6A). At 4 h, 59 24 ionomycin-stimulated cells statistically significantly increased To examine the bioactivity of schRANKL on BMDC, recombi- 60 25 chRANKL mRNA expression levels by ~5-fold compared to levels in nant protein (schRANKL fused to FLAG) was produced in COS-7 cells 61 26 unstimulated cells (p < 0.05). The addition of PMA in costimulated and its effect on pro-inflammatory cytokine expression by BMDC 62 27 cells did not increase chRANKL mRNA expression levels any further. was determined. On day 6 of culture, BMDC were unstimulated or 63 28 At 18 h, chRANKL mRNA expression levels in ionomycin-stimulated stimulated with LPS, schRANKL or both, for 3 and 6 h. mRNA ex- 64 29 cells again were not statistically significantly different from levels pression levels of the pro-inflammatory cytokines, IL-1β, IL-6 and 65 30 in unstimulated controls. This could suggest that ionomycin- IL-12α, were analysed by qRT-PCR (Fig. 7A). 66 31 mediated Ca2+-mobilisation declines over time and no longer induces At both time-points, LPS-stimulated cells had upregulated IL- 67 32 chRANKL mRNA expression. At 18 h, chRANKL mRNA expression 12α mRNA expression levels. Stimulation with RANKL on its own 68 33 levels were statistically significantly increased in costimulated cells did not induce IL-12α mRNA expression and costimulation with LPS 69 34 compared to ionomycin-only stimulated cells and unstimulated cells did not affect IL-12α mRNA expression levels compared to those in 70 35 (p < 0.05), indicating that the concomitant activation of Ca2+- cells stimulated only with LPS. IL-12α mRNA expression was not 71 36 mobilisation and the PKC pathway are necessary to drive the detected in unstimulated cells, and therefore these data are pre- 72 37 transcription of chRANKL in chicken splenocytes, after at least 4 h sented as corrected 40-Ct and not as fold change. 73 38 of co-stimulation. At 3 h, IL-1β mRNA expression levels were significantly increased 74 39 To verify that the transcriptional regulation of chRANKL was (26-fold) in LPS-stimulated cells (Fig. 7A) compared to those in 75 40 linked to the activation and mobilisation of Ca2+, splenocytes were unstimulated cells (p < 0.05). Costimulated cells also had a significant 76 41 stimulated with ionomycin or ionomycin with various concentra- increase in IL-1β mRNA expression levels compared to those in 77 42 tions of the Ca2+ channel and PKC inhibitor, TMB-8. The levels of unstimulated cells (p < 0.05) but were not statistically significantly 78

K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■ 9

OPG Fold 5 RANK change 4

3 * ** 2 *

0 200 100 1 200 100 1 200 100 1 LPS (ng/ml) 3 h 6 h 9 h Time post-stimulation

1 Fig. 5. Expression of chOPG and chRANKL in stimulated BMDC. Expression levels of chOPG and chRANK mRNA in BMDC following stimulation with LPS (200, 100 and 1 ng/ml) 2 for 3, 6 and 9 h. Data are presented as fold change in comparison to levels in unstimulated control cells of triplicate assays on each of three individual birds ± SEM. *p < 0.05 3 as indicated (Mann–Whitney U test). 4 5 higher than those in LPS-stimulated cells (Fig. 7A). At 6 h, LPS- unstimulated cells (p < 0.05) (Fig. 7A). At 6 h, IL-6 mRNA expres- 25 6 stimulation led to a significant (~52-fold) increase in IL-1β mRNA sion levels in co-stimulated cells were significantly increased 26 7 expression levels which were further statistically significantly compared to those in LPS-stimulated cells (p < 0.05), suggesting that 27 8 increased to ~79-fold in co-stimulated cells compared to levels in schRANKL enhances IL-6 mRNA expression in BMDC. Cells treated 28 9 unstimulated cells (p < 0.05) and statistically significantly higher with schRANKL alone had a non-significant increase (~5-fold) in IL-6 29 10 than levels in LPS-stimulated cells (p < 0.05) (Fig. 7A). Treatment with mRNA expression levels at 3 h but there was no increase at 6 h 30 11 schRANKL alone increased the mRNA expression levels of IL-1β at (Fig. 7A). 31 12 6 h but this was not statistically significant compared to levels in Antagonistic effects of schRANK-Fc and chOPG-Fc were deter- 32 13 unstimulated cells. mined by their ability to inhibit the schRANKL-mediated 33 14 At 3 and 6 h, IL-6 mRNA expression levels were significantly in- upregulation of pro-inflammatory cytokines in BMDC. SchRANKL 34 15 creased in LPS- and co-stimulated cells compared to levels in was pre-incubated with schRANK-Fc or chOPG-Fc for 3 h prior to 35 stimulation of cells and IL-1β, IL-6 and IL-12α mRNA expressions 36 levels were analysed (Fig. 7A). Incubation of schRANKL with either 37 schRANK-Fc or chOPG-Fc prior to co-culture with LPS resulted in 38 A 10 # * statistically significantly lower pro-inflammatory mRNA expres- 39 Fold 8 sion levels than those induced by co-culture with LPS and schRANKL 40 change (Fig. 7A). Therefore, pro-inflammatory mRNA expression levels 41 6 * * induced by schRANKL in costimulated cells were inhibited by the 42 4 addition of schRANK-Fc and chOPG-Fc. 43 The bioactivity of schRANKL on BMM was also determined by 44 2 examining pro-inflammatory cytokine mRNA expression levels 45 0 (Fig. 7B). Cells were unstimulated or simulated with LPS, schRANKL 46 or both, for 3 and 6 h, similarly to BMDC. IL-12α mRNA expression 47 -2 + +++++ Ionomycin - + -+-+PMA levels were not detected in any sample (data not shown). At 3 h, 48 LPS-stimulated cells increased IL-1β mRNA expression levels sig- 49 16 2418h post-stimulation nificantly by ~50-fold compared to those in unstimulated cells 50 (p < 0.05) (Fig. 7B). SchRANKL treatment alone had no effect. Co- 51 B 10 stimulation with LPS and schRANKL did not increase IL-1β mRNA 52 Fold 8 expression levels further than LPS stimulation alone. At 6 h, IL-1β 53 change mRNA expression levels were still upregulated in LPS-stimulated 54 6 cells but at a lower level than at 3 h. SchRANKL treatment alone again 55 * had no effect. Co-stimulated cells increased IL-1β mRNA expres- 56 4 sion levels but levels were not statistically significantly higher than 57 those in unstimulated or LPS-stimulated cells. 58 2 IL-6 mRNA expression levels were increased in LPS and co- 59 stimulated cells at both 3 and 6 h but levels were not statistically 60 0 ++++Ionomycin significantly compared to unstimulated cells, and co-stimulation did 61 -102050TMB-8 not increase IL-6 mRNA expression levels higher than those in LPS- 62 stimulated cells (Fig. 7B). 63 17 Fig. 6. Transcriptional regulation of chRANKL in chicken splenocytes. ChRANKL mRNA 18 expression levels were analysed by qRT-PCR in (A) splenocytes stimulated with 64 19 ionomycin (3 μg/ml), or ionomycin and PMA (500 ng/ml) for 2, 4 and 18 h and (B) 3.7. Cell surface marker expression of BMDC and BMM treated with 65 20 splenocytes stimulated with ionomycin (3 μg/ml) or ionomycin and TMB-8 (10, 20 schRANKL 66 21 or 50 μM) for 18 h. Data are presented as fold change in comparison to levels in 67 22 unstimulated control cells of triplicate assays on three individual birds ± SEM. *p < 0.05 23 compared to unstimulated control cells and #p < 0.05 as indicated (Mann–Whitney To investigate the ability of schRANKL to enhance the expres- 68 24 U test). sion of cell surface markers on BMDC and BMM, cells were treated 69

10 K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■

IL-12α A 15 Corrected 40-Ct 10

- + - + + + - + - + + + LPS - - + + + + - - + + + + schRANKL - - - - + - - - - - + - chRANK-Fc - - - - - + - - - - - + chOPG-Fc

IL-1β IL-6 ab 100 a Fold ac ac ab a ab ab a a a ac change a a ac ac

+ - + + + + - + + + + - + + + + - + + + LPS - + + + + - + + + + - + + + + - + + + + schRANKL 0.1 - - - + - - - - + - - - - + - - - - + - chRANK-Fc - - - - + - - - - + - - - - + - - - - + chOPG-Fc

β B IL-1 1000 IL-6 Fold 10000 change 1000 100 a 100 a

10 10

1 1

0.1 + - + + - + + - + + - + LPS - + + - + + 0.1 - + + - + + schRANKL

1 Fig. 7. Effect of chRANKL on pro-inflammatory cytokine expression in chicken BMDC and BMM. Pro-inflammatory cytokine mRNA expression levels in (A) BMDC and (B) 2 BMM stimulated with schRANKL, as measured by qRT-PCR. (A) BMDC were unstimulated or stimulated with LPS (2 ng/ml), schRANKL (1:50 ex-COS) or co-stimulated with 3 both for 3 (grey bars) or 6 h (black bars). To determine schRANKL bioactivity, schRANK-Fc or chOPG-Fc (both 1:5 ex-COS) were pre-incubated with schRANKL for 3 h prior 4 to cell stimulation. (B) BMM were unstimulated or stimulated with LPS (1 ng/ml), schRANKL (1:50 ex-COS) or costimulated with both for 3 (grey bars) or 6 h (black bars). 5 IL-12α mRNA expression levels (expression only seen in BMDC) are presented as corrected 40-Ct as expression was undetectable in unstimulated cells, IL-1β and IL-6 mRNA 6 expression levels are presented as fold change in comparison to levels in unstimulated control cells. Data are presented as the average of between three (BMM) and six 7 (BMDC) independent experiments ± SEM. (a) Data are statistically significant (p < 0.05) compared to unstimulated cells; (b) data are statistically significant (p < 0.05) com- 8 pared to LPS-stimulated cells; (c) data are statistically significant (p < 0.05) compared to co-stimulated cells (Mann–Whitney U test).

10 with LPS (1–2 ng/ml), schRANKL (1:5 exCOS-7) or both, for 24 h expression of KUL01 on the surface of BMDC decreases as the cells 19 11 (Fig. 8). Cells were removed from culture plates and stained for the mature (Wu et al., 2010) and this is evident in the LPS-stimulated 20 12 chicken mononuclear phagocyte marker, KUL01 (Mast et al., 1998), BMDC where there were lower cell surface expression levels of 21 13 and activation markers of chicken APC: MHC class II and CD40 (Wu KUL01 than on the control cells (Fig. 8B). SchRANKL had no effect 22 14 Q3 et al., 2009). on cell surface expression of KUL01 when compared to the control 23 15 Co-stimulation of BMDC with LPS, schRANKL or both did not BMDC. Co-stimulated cells had similar cell surface expression levels 24 16 increase the cell surface expression levels of MHC class II. CD40 cell of KUL01 as the LPS-stimulated cells (Fig. 8A). 25 17 surface expression levels were increased in LPS-stimulated cells but Similarly to BMDC, MHC class II expression levels were not in- 26 18 were not enhanced by addition of schRANKL (Fig. 8A). The creased by stimulation of BMM with LPS (1 ng/ml) or schRANKL, 27

K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■ 11

A MHC class II CD40 KUL01

Control

FL1-H FL2-H FL1-H LPS

FL1-H FL2-H FL1-H schRANKL Counts

FL1-H FL2-H FL1-H

LPS + schRANKL

FL1-H FL2-H FL1-H

B MHC class II CD40 KUL01 Control

FL1-H FL2-H FL1-H LPS

FL1-H FL2-H FL1-H

Counts schRANKL

FL1-H FL2-H FL1-H LPS + schRANKL

FL1-H FL2-H FL1-H

1 Fig. 8. Effect of schRANKL on the phenotype of chicken BMDC and BMM. (A) BMDC were unstimulated or stimulated with LPS (2 ng/ml), schRANKL (1:5 exCOS) or both for 2 24 h. (B) BMM were unstimulated or stimulated with LPS (1 ng/ml), schRANKL (1:5) or both for 24 h. Cell surface expression of MHC class II, CD40 and KUL01 were analysed 3 by FACS. Data are represented as histograms comparing isotype controls (black lines) to surface staining (grey lines) and are representative of between four and six inde- 4 pendent experiments with similar results.

12 K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■

BMDC BMM Control RANKL Control RANKL

24 h FL3-H FL3-H FL3-H FL3-H

PI FL4-H FL4-H FL4-H FL4-H

48 h FL3-H FL3-H FL3-H FL3-H

FL4-H FL4-H FL4-H FL4-H Annexin-V

1 Fig. 9. Survival of BMDC and BMM treated with schRANKL. Cell survival was measured by staining for annexin-V and PI and analysed by FACS. BMDC and BMM were un- 2 treated or treated with schRANKL (1:5 ex-COS) for 24 and 48 h. Data are representative of three independent experiments with similar results.

4 either alone or in combination (Fig. 8B). CD40 cell surface expres- the data suggest that schRANKL acts as a survival factor for chicken 26 5 sion levels were increased in LPS-stimulated BMM but were not BMDC. 27 6 enhanced by the addition of schRANKL (Fig. 8B), which by itself had After 24 h, untreated BMM had a very low number of viable cells 28 7 no effect. KUL01 cell surface expression levels were decreased in (16.1%) whereas for schRANKL-treated BMM viability numbers were 29 8 LPS-stimulated and co-stimulated BMM compared to unstimulated 32.2%. Cells undergoing apoptosis were lower in chRANKL-treated 30 9 or schRANKL-stimulated BMM (Fig. 8B). cells (61.2%) compared to untreated cells (77.9%) (Fig. 9). A further 31 10 24 h of cell culture resulted in similar numbers of viable and 32 11 3.8. Survival of BMDC and BMM treated with schRANKL apoptotic cells between untreated and schRANKL-treated BMM. 33 12 SchRANKL can enhance the survival of BMM at 24 h of stimula- 34 13 To examine whether chRANKL was a survival factor for BMDC tion but this enhanced survival is short-lived, as cells begin to 35 14 and BMM, cells were treated with or without schRANKL (1:5 exCOS- undergo similar rates of apoptosis after 48 h. 36 15 7) for 24 and 48 h (Fig. 9). Cell death and apoptosis were measured 37 16 by annexin-V and PI staining and analysed by FACS. 4. Discussion 38 17 After 24 h, 44.7% of untreated BMDC were viable (annexin-V− PI−) 39 18 and 55.3% positive for annexin-V (annexin-V+ PI+)(Fig. 9). This in- In mammals, the majority of TNF superfamily members are 40 19 dicates that over half of the cell population analysed under normal present as small loci of two or three genes on different chromo- 41 20 conditions was undergoing apoptosis. In contrast, schRANKL- somes – the chicken lacks three of these small sub-families: LT-α, 42 21 treated cells had 57.7% viable cells and 27.8% undergoing apoptosis. TNF-α and LT-β (TNFSF1-3); 4-1BBL, CD27L and LIGHT (TNFSF9, 7 43 22 After 48 h, the number of viable untreated BMDC fell to 29% in com- and 14); TWEAK and APRIL (TNFSF12 and 13) (Kaiser et al., 2005). 44 23 parison to schRANKL-treated cells where 40% were viable (Fig. 9). It has been hypothesised that the lack of lymph nodes in the chicken 45 24 Cells undergoing apoptosis in untreated cells increased to 58.4% but is due to the absence of the lymphotoxin genes. Mice lacking some 46 25 for schRANKL-treated cells apoptotic cells were 49.8% (Fig. 9). Overall of the other missing genes in the chicken have defective T and B 47

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1 cell responses (Locksley et al., 2001). The absence of the other TNF then unstimulated or stimulated with various concentrations of LPS 67 2 superfamily members has not yet been linked to any abnormal de- and the levels of chRANK and chOPG mRNA expression analysed 68 3 velopment or obvious difference in the chicken’s ability to mount by qRT-PCR (Fig. 5). The data suggest that the mRNA expression levels 69 4 an immune response. However, it is therefore important to study of chRANK and chOPG in BMDC are both dose- and time-dependent. 70 5 the biological function of those chicken TNF superfamily members The more the BMDC matured, the lower the levels of chRANK mRNA 71 6 that are present in more detail. expression and the higher the levels of chOPG mRNA expression ob- 72 7 Mammalian RANKL is a type II transmembrane protein that can served. Mammalian OPG expression is dependent on activation of 73 8 function as either a membrane-bound or secreted form (Ikeda et al., the NF-κB pathway and increases as DC mature (Schoppet et al., 74 9 2001; Walsh et al., 2013), with no known functional difference 2007). It is therefore possible that mature chicken BMDC increase 75 10 between the two (Nakashima et al., 2000). An aa alignment of full- levels of chOPG expression to regulate the magnitude of chRANKL+ 76 11 length chRANKL with those of human and mouse showed high T cells interacting with chRANK+ DC and chOPG is therefore a po- 77 12 conservation across all species. The RANKL monomer consists of two tential marker for mature chBMDC. 78 13 anti-parallel β-sheets which are formed by various β-strands (Lam The expression of RANKL by mammalian T cells is induced by 79 14 et al., 2001). Using the PSIPRED prediction program, the locations their activation and is dependent on Ca2+ mobilisation (Wang et al., 80 15 of the residues involved in β-sheet formation in chRANKL were found 2002). To understand whether the mechanism of chRANKL expres- 81 16 to be conserved. SchRANKL contains ten β-strands and the four loops sion was conserved between mammals and birds, chicken 82 17 required for assembly of a homotrimer (Fig. 1A). To investigate the splenocytes were stimulated with the intracellular calcium mod- 83 18 ability of schRANKL to form homotrimers, the extracellular domain ulator, ionomycin, and the PKC activator, PMA, which mimic TCR 84 19 of chRANKL was sub-cloned into a modified vector expressing an activation, for 2, 4 and 18 h and levels of chRANKL mRNA expres- 85 20 in-frame isoleucine zipper previously shown to enhance the bio- sion were analysed by qRT-PCR (Fig. 6A). In co-stimulated cells the 86 21 activity of members of the TNF superfamily by encouraging and levels of chRANKL mRNA expression were significantly increased 87 22 stabilising trimeric protein structures (Kim et al., 2004; Morris et al., after 4 and 18 h indicating that ionomycin and PMA work in synergy 88 23 1999). SchRANKL protein structures were analysed under both re- to induce chRANKL mRNA expression. In murine T cells, ionomycin 89 24 ducing and non-reducing conditions (Fig. 2) and the data suggest alone was sufficient to induce RANKL expression and no synergy 90 25 that schRANKL forms homotrimeric protein structures, a signa- was found when cells were co-stimulated with ionomycin and PMA 91 26 ture of TNF superfamily members. (Wang et al., 2002). In similar studies, murine T cell hybridomas 92 27 Human and mouse OPG cDNAs encode a 401 aa protein that is weakly induced RANKL after PMA stimulation but a combination 93 28 further processed to form a 380 aa mature protein. OPG is of PMA and ionomycin synergised and induced higher levels of 94 29 synthesised as a glycosylated 55 kDa protein which self-associates RANKL expression (Bishop et al., 2011; Fionda et al., 2007). The tran- 95 30 to form disulphide-linked dimers prior to secretion from cells. scriptional control of chRANKL and mammalian RANKL by Ca2+ 96 31 ChOPG was previously cloned by Hou et al. (2011).However,no modulation and PKC activation, as verified by inhibition studies 97 32 sequence alignment or analysis was presented in that study to (Fig. 6B), is conserved between mammals and chickens. 98 33 investigate the conservation of the CRD, DD and penultimate The ability of schRANKL to induce or enhance pro-inflammatory 99 34 cysteine residue required for chOPG dimer assembly. An aa cytokine expression in immature or mature chicken APC was 100 35 alignment of chOPG with those of human and mouse showed it to analysed by qRT-PCR (Fig. 7). APC were either unstimulated or stimu- 101 36 be highly conserved (64–65% aa identity). ChOPG, similar to lated with LPS, schRANKL or both for 3 and 6 h. Costimulation of 102 37 mammalian OPG, naturally forms soluble homodimeric proteins BMDC with schRANKL and LPS led to a significant increase in pro- 103 38 (Fig. 2). inflammatory cytokine mRNA expression levels compared to those 104 39 ChRANKL, chRANK and chOPG mRNA expression levels were in unstimulated BMDC and, in some cases, LPS-stimulated BMDC. 105 40 ubiquitous across a range of lymphoid and non-lymphoid tissues SchRANKL-treatment alone did not significantly alter pro- 106 41 (Fig. 3) and immune cells (Fig. 4) in the chicken. In the bone marrow, inflammatory cytokines mRNA expression levels. BMM costimulated 107 42 all three molecules were highly expressed at the mRNA level, which with LPS and schRANKL significantly increased IL-1β mRNA ex- 108 43 was not surprising as all three have vital roles in bone metabolism pression levels in comparison to unstimulated cells but not LPS- 109 44 in mammalian species (Simonet et al., 1997; Yasuda et al., 1998). stimulated cells. Pre-incubation with both schRANK-Fc and chOPG- 110 45 In non-lymphoid organs, chRANKL and chRANK mRNA expression Fc decreased the schRANKL-mediated increase in pro-inflammatory 111 46 levels were highest in muscle, similarly to mammalian RANKL and cytokine mRNA expression levels (Fig. 7). This also indicated that 112 47 RANK (Anderson et al., 1997; Wong et al., 1997a). The heart had the soluble receptors of chRANKL were capable of interacting with 113 48 the lowest mRNA expression levels of all three molecules amongst their ligand. 114 49 the non-lymphoid tissues examined. SchRANKL treatment did not affect the expression levels of MHC 115 50 In the upper gut, levels of chRANKL mRNA expression were high. class II either alone or in combination with LPS (Fig. 8). CD40 ex- 116 51 In the mammalian gut-associated lymphoid tissues (GALT), sam- pression levels were increased in LPS-stimulated cells but were not 117 52 pling of lumenal pathogens is achieved by M cells (Mabbott et al., enhanced by the addition of schRANKL. The expression pattern of 118 53 2013). M cells are highly efficient phagocytic cells that uptake bac- KUL01 was also analysed; although it is not an activation marker 119 54 terial antigen from the lumen into the Payer’s patches, priming CD4+ like CD40 and MHC class II, KUL01 expression decreases as chicken 120 55 T cells and IgA+-committed B cells (VanCott et al., 1996). In mammals, APC mature (Wu et al., 2010). This was therefore a good marker to 121 56 RANKL is necessary for the differentiation of RANK-expressing identify the ability of schRANKL to induce cell maturation. As ex- 122 57 enterocytes into M cells (Knoop et al., 2009). Recently the gener- pected, at 24 h LPS-treated cells had decreased KUL01 expression 123 58 ation of CSF1 transgenic chickens identified the large number of but expression levels thereof were unaltered by schRANKL treat- 124 59 macrophages in the chicken gut (Balic et al., 2014) therefore it would ment. Overall schRANKL does not mediate the upregulation of cell 125 60 be interesting to determine if the high levels of chRANKL mRNA ex- surface activation markers in chicken BMDC, similarly to mamma- 126 61 pressed in the upper gut correlated with the presence of large lian RANKL (Anderson et al., 1997; Wong et al., 1997a). 127 62 numbers of M cells in this tissue. Studies on the biological effect of mammalian RANKL on DC have 128 63 The expression of both RANK and OPG has been observed on produced contrasting evidence as to its effect on their phenotype. 129 64 mature DC in mammals (Anderson et al., 1997; Wong et al., 1997a; One of the first studies examining the biological role of mamma- 130 65 Yasuda et al., 1998). To investigate the expression patterns of the lian RANKL proposed that RANKL-treated murine BMDC did not 131 66 chRANKL receptors on chicken BMDC, BMDC were generated and significantly increase expression levels of MHC class II, CD80 or CD86 132

14 K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■

1 but did slightly (not significantly) upregulate the expression of CD40 for IL-5 that appears to be a pseudogene, and a gene encoding another 69 2 (Anderson et al., 1997; Wong et al., 1997a). Monocyte-derived DC cytokinelike transcript, KK34. J. Interferon Cytokine Res. 24, 600–610. 70 Balic, A., Garcia-Morales, C., Vervelde, L., Gilhooley, H., Sherman, A., Garceau, V., et al., 71 3 treated with RANKL increased surface expression of MHC class II, 2014. Visualisation of chicken macrophages using transgenic reporter genes: 72 4 CD80 and CD86 (Seshasayee et al., 2004), suggesting that DC derived insights into the development of the avian macrophage lineage. Development 73 5 from different sources react differently to RANKL exposure. One study 141, 3255–3265. 74 Banks, T.A., Rouse, B.T., Kerley, M.K., Blair, P.J., Godfrey, V.L., Kuklin, N.A., et al., 75 6 (Park et al., 2005) on the bioactivity of mammalian RANKL on mac- 1995. Lymphotoxin-alpha-deficient mice. Effects on secondary lymphoid organ 76 7 rophages suggested that RANKL-treated cells upregulated MHC class development and humoral immune responsiveness. J. Immunol. 155, 1685– 77 8 II expression which was enhanced when cells were co-stimulated 1693. 78 Bishop, K.A., Coy, H.M., Nerenz, R.D., Meyer, M.B., Pike, J.W., 2011. Mouse Rankl 79 9 with LPS but not with IFN-γ. It is important to note that DC may expression is regulated in T cells by c-Fos through a cluster of distal regulatory 80 10 express RANK but react differently in their response to RANKL de- enhancers designated the T cell control region. J. Biol. Chem. 286, 20880– 81 11 pending on their tissue of origin. Our data indicate that chRANKL 20891. 82 Bridgham, J.T., Johnson, A.L., 2001. Expression and regulation of Fas antigen and tumor 83 12 has no effect on the phenotype of BMDC or BMM alone or in com- necrosis factor receptor type I in hen granulosa cells. Biol. Reprod. 65, 733–739. 84 13 bination with LPS (Fig. 8). Cremer, I., Dieu-Nosjean, M.C., Marechal, S., Dezutter-Dambuyant, C., Goddard, S., 85 14 The lifespan of DC can influence the duration of lymphocyte ac- Adams, D., et al., 2002. Long-lived immature dendritic cells mediated by 86 TRANCE-RANK interaction. Blood 100, 3646–3655. 87 15 tivation, thereby affecting the outcome of immune responses. Darnay, B.G., Haridas, V., Ni, J., Moore, P.A., Aggarwal, B.B., 1998. Characterization 88 16 Mammalian RANKL is a survival factor for DC and macrophages of the intracellular domain of receptor activator of NF-kappaB (RANK). Interaction 89 17 (Anderson et al., 1997; Cremer et al., 2002; Park et al., 2005; Wong with tumor necrosis factor receptor-associated factors and activation of NF- 90 18 et al., 1997a). To determine the ability of schRANKL to enhance the kappab and c-Jun N-terminal kinase. J. Biol. Chem. 273, 20551–20555. 91 Devergne, O., Hatzivassiliou, E., Izumi, K.M., Kaye, K.M., Kleijnen, M.F., Kieff, E., et al., 92 19 survival rates of BMDC and BMM, cells were untreated or treated 1996. Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 93 20 with schRANKL for 24 and 48 h (Fig. 9). BMDC survival rates were domain important for B-lymphocyte transformation: role in NF-kappaB activation. 94 21 increased in schRANKL-treated cells compared to untreated cells Mol. Cell. Biol. 16, 7098–7108. 95 Dougall, W.C., Glaccum, M., Charrier, K., Rohrbach, K., Brasel, K., De Smedt, T., et al., 96 22 after 24 h and 48 h (Fig. 9). After 24 h, the number of viable cells 1999. RANK is essential for osteoclast and lymph node development. Genes Dev. 97 23 was increased in schRANKL-treated BMM compared to untreated 13, 2412–2424. 98 24 cells. However, after 48 h schRANKL-treated cells had similar Eldaghayes, I., Rothwell, L., Williams, A., Withers, D., Balu, S., Davison, F., et al., 2006. 99 Infectious bursal disease virus: strains that differ in virulence differentially 100 25 numbers of viable cells as untreated cells. It seems that the ability modulate the innate immune response to infection in the chicken bursa. Viral 101 26 of schRANKL to enhance the survival of BMM is short-lived. Various Immunol. 19, 83–91. 102 27 mammalian studies have shown that the upregulation of the Bcl-2 Ellis, R.E., Yuan, J.Y., Horvitz, H.R., 1991. Mechanisms and functions of cell death. Annu. 103 Rev. Cell Biol. 7, 663–698. 104 28 family members, Bcl-X and Bcl-2, are linked to RANKL-mediated L Fionda, C., Nappi, F., Piccoli, M., Frati, L., Santoni, A., Cippitelli, M., 2007. 15-deoxy- 105 29 cell survival (Izawa et al., 2007; Josien et al., 1999; Wong et al., 1997a) Delta12,14-prostaglandin J2 negatively regulates rankl gene expression in 106 30 and genetic and molecular analysis from nematodes to humans in- activated T lymphocytes: role of NF-kappaB and early growth response 107 transcription factors. J. Immunol. 178, 4039–4050. 108 31 dicates that programmed cell death is highly conserved (Ellis et al., Garceau, V., Smith, J., Paton, I.R., Davey, M., Fares, M.A., Sester, D.P., et al., 2010. Pivotal 109 32 1991), so it is therefore plausible that chRANKL may upregulate the advance: Avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and 110 33 levels of anti-apoptotic chicken Bcl-2 family members in BMDC and CSF-1 receptor genes and gene products. J. Leukoc. Biol. 87, 753–764. 111 34 BMM. Hikosaka, Y., Nitta, T., Ohigashi, I., Yano, K., Ishimaru, N., Hayashi, Y., et al., 2008. The 112 cytokine RANKL produced by positively selected thymocytes fosters medullary 113 35 thymic epithelial cells that express autoimmune regulator. Immunity 29, 114 36 5. Conclusion 438–450. 115 Hou, L., Hou, J., Yao, J., Zhou, Z., 2011. Effects of osteoprotegerin from transfection 116 37 of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts. Acta Vet. Scand. 53, 117 38 In summary, chRANKL, chRANK and chOPG mRNA expression has 21. 118 39 been analysed in different tissues and cells in the chicken. Expres- Ikeda, T., Kasai, M., Utsuyama, M., Hirokawa, K., 2001. Determination of three isoforms 119 of the receptor activator of nuclear factor-kappaB ligand and their differential 120 40 sion of chRANKL mRNA and its receptors was detected across a wide expression in bone and thymus. Endocrinology 142, 1419–1426. 121 41 range of tissues. Both of the receptors for chRANKL are differen- Ishida, T., Mizushima, S., Azuma, S., Kobayashi, N., Tojo, T., Suzuki, K., et al., 1996. 122 42 tially expressed in mature BMDC. The bioactivity of chRANKL has Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor 123 43 been analysed in both BMDC and BMM indicating conservation of protein that mediates signaling from an amino-terminal domain of the CD40 124 cytoplasmic region. J. Biol. Chem. 271, 28745–28748. 125 44 RANKL bioactivities between mammalian and chicken in BMDC but Izawa, T., Ishimaru, N., Moriyama, K., Kohashi, M., Arakaki, R., Hayashi, Y., 2007. 126 45 not completely in BMM. Crosstalk between RANKL and Fas signaling in dendritic cells controls immune 127 tolerance. Blood 110, 242–250. 128 46 Josien, R., Wong, B.R., Li, H.L., Steinman, R.M., Choi, Y., 1999. TRANCE, a TNF family 129 47 Acknowledgments member, is differentially expressed on T cell subsets and induces cytokine 130 48 production in dendritic cells. J. Immunol. 162, 2562–2568. 131 Kaiser, P., 2010. Advances in avian immunology–prospects for disease control: a 132 We would like to thank Lisa Rothwell for advice and assistance 49 review. Avian Pathol. 39, 309–324. 133 50 Q4 in this study. This work was largely funded by CEVA and sup- Kaiser, P., 2012. The long view: a bright past, a brighter future? Forty years of chicken 134 51 Q5 ported by the Biotechnology and Biological Sciences Research Council immunology pre- and post-genome. Avian Pathol. 41, 511–518. 135 Kaiser, P., Poh, T.Y., Rothwell, L., Avery, S., Balu, S., Pathania, U.S., et al., 2005. A genomic 136 52 Institute Strategic Programme grant. analysis of chicken cytokines and chemokines. J. Interferon Cytokine Res. 25, 137 53 467–484. 138 54 References Kim, M.H., Billiar, T.R., Seol, D.W., 2004. The secretable form of trimeric TRAIL, a potent 139 inducer of apoptosis. Biochem. Biophys. Res. Commun. 321, 930–935. 140 55 Knoop, K.A., Kumar, N., Butler, B.R., Sakthivel, S.K., Taylor, R.T., Nochi, T., et al., 2009. 141 56 Abdalla, S.A., Horiuchi, H., Furusawa, S., Matsuda, H., 2004a. Molecular cloning and RANKL is necessary and sufficient to initiate development of antigen-sampling 142 57 characterization of chicken tumor necrosis factor (TNF)-superfamily ligands, M cells in the intestinal epithelium. J. Immunol. 183, 5738–5747. 143 58 CD30L and TNF-related apoptosis inducing ligand (TRAIL). J. Vet. Med. Sci. 66, Kong, Y.Y., Yoshida, H., Sarosi, I., Tan, H.L., Timms, E., Capparelli, C., et al., 1999. OPGL 144 59 643–650. is a key regulator of osteoclastogenesis, lymphocyte development and lymph- 145 60 Abdalla, S.A., Horiuchi, H., Furusawa, S., Matsuda, H., 2004b. Molecular study on node organogenesis. Nature 397, 315–323. 146 61 chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor- Lam, J., Nelson, C.A., Ross, F.P., Teitelbaum, S.L., Fremont, D.H., 2001. Crystal structure 147 62 associated factor-5. Vet. Immunol. Immunopathol. 98, 31–41. of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand 148 63 Anderson, D.M., Maraskovsky, E., Billingsley, W.L., Dougall, W.C., Tometsko, M.E., Roux, specificity. J. Clin. Invest. 108, 971–979. 149 64 E.R., et al., 1997. A homologue of the TNF receptor and its ligand enhance T-cell Locksley, R.M., Killeen, N., Lenardo, M.J., 2001. The TNF and TNF receptor 150 65 growth and dendritic-cell function. Nature 390, 175–179. superfamilies: integrating mammalian biology. Cell 104, 487–501. 151 66 Avery, S., Rothwell, L., Degen, W.D., Schijns, V.E., Young, J., Kaufman, J., et al., 2004. Luan, X., Lu, Q., Jiang, Y., Zhang, S., Wang, Q., Yuan, H., et al., 2012. Crystal structure 152 67 Characterization of the first nonmammalian T2 cytokine gene cluster: the cluster of human RANKL complexed with its decoy receptor osteoprotegerin. J. Immunol. 153 68 contains functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene 189, 245–252. 154

K.M.C. Sutton et al./Developmental and Comparative Immunology ■■ (2015) ■■–■■ 15

1 Mabbott, N.A., Donaldson, D.S., Ohno, H., Williams, I.R., Mahajan, A., 2013. Microfold Taguchi, Y., Gohda, J., Koga, T., Takayanagi, H., Inoue, J., 2009. A unique domain in 49 2 (M) cells: important immunosurveillance posts in the intestinal epithelium. RANK is required for Gab2 and PLCgamma2 binding to establish osteoclastogenic 50 3 Mucosal Immunol. 6, 666–677. signals. Genes Cells 14, 1331–1345. 51 4 Mast, J., Goddeeris, B.M., Peeters, K., Vandesande, F., Berghman, L.R., 1998. Takimoto, T., Takahashi, K., Sato, K., Akiba, Y., 2005. Molecular cloning and functional 52 5 Characterisation of chicken monocytes, macrophages and interdigitating cells characterizations of chicken TL1A. Dev. Comp. Immunol. 29, 895–905. 53 6 by the monoclonal antibody KUL01. Vet. Immunol. Immunopathol. 61, 343–357. Tregaskes, C.A., Glansbeek, H.L., Gill, A.C., Hunt, L.G., Burnside, J., Young, J.R., 2005. 54 7 Morris, A.E., Remmele, R.L., Jr., Klinke, R., Macduff, B.M., Fanslow, W.C., Armitage, R.J., Conservation of biological properties of the CD40 ligand, CD154 in a non- 55 8 1999. Incorporation of an isoleucine zipper motif enhances the biological activity mammalian vertebrate. Dev. Comp. Immunol. 29, 361–374. 56 9 of soluble CD40L (CD154). J. Biol. Chem. 274, 418–423. VanCott, J.L., Staats, H.F., Pascual, D.W., Roberts, M., Chatfield, S.N., Yamamoto, M., 57 10 Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355–365. et al., 1996. Regulation of mucosal and systemic antibody responses by T helper 58 11 Naismith, J.H., Sprang, S.R., 1998. Modularity in the TNF-receptor family. Trends cell subsets, macrophages, and derived cytokines following oral immunization 59 12 Biochem. Sci. 23, 74–79. with live recombinant Salmonella. J. Immunol. 156, 1504–1514. 60 13 Nakashima, T., Kobayashi, Y., Yamasaki, S., Kawakami, A., Eguchi, K., Sasaki, H., et al., Walsh, N.C., Alexander, K.A., Manning, C.A., Karmakar, S., Wang, J.F., Weyand, C.M., 61 14 2000. Protein expression and functional difference of membrane-bound and et al., 2013. Activated human T cells express alternative mRNA transcripts 62 15 soluble receptor activator of NF-kappaB ligand: modulation of the expression encoding a secreted form of RANKL. Genes Immun. 14, 336–345. 63 16 by osteotropic factors and cytokines. Biochem. Biophys. Res. Commun. 275, Wang, R., Zhang, L., Zhang, X., Moreno, J., Celluzzi, C., Tondravi, M., et al., 2002. 64 17 768–775. Regulation of activation-induced receptor activator of NF-kappaB ligand (RANKL) 65 18 Park, H.J., Park, O.J., Shin, J., 2005. Receptor activator of NF-kappaB ligand enhances expression in T cells. Eur. J. Immunol. 32, 1090–1098. 66 19 the activity of macrophages as antigen presenting cells. Exp. Mol. Med. 37, Wong, B.R., Josien, R., Lee, S.Y., Sauter, B., Li, H.L., Steinman, R.M., et al., 1997a. TRANCE 67 20 524–532. (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF 68 21 Pasparakis, M., Alexopoulou, L., Episkopou, V., Kollias, G., 1996. Immune and family member predominantly expressed in T cells, is a dendritic cell-specific 69 22 inflammatory responses in TNF alpha-deficient mice: a critical requirement for survival factor. J. Exp. Med. 186, 2075–2080. 70 23 TNF alpha in the formation of primary B cell follicles, follicular dendritic cell Wong, B.R., Rho, J., Arron, J., Robinson, E., Orlinick, J., Chao, M., et al., 1997b. TRANCE 71 24 networks and germinal centers, and in the maturation of the humoral immune is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun 72 25 response. J. Exp. Med. 184, 1397–1411. N-terminal kinase in T cells. J. Biol. Chem. 272, 25190–25194. 73 26 Rossi, S.W., Kim, M.Y., Leibbrandt, A., Parnell, S.M., Jenkinson, W.E., Glanville, S.H., Wong, B.R., Josien, R., Lee, S.Y., Vologodskaia, M., Steinman, R.M., Choi, Y., 1998. The 74 27 et al., 2007. RANK signals from CD4(+)3(−) inducer cells regulate development TRAF family of signal transducers mediates NF-kappaB activation by the TRANCE 75 28 of Aire-expressing epithelial cells in the thymic medulla. J. Exp. Med. 204, receptor. J. Biol. Chem. 273, 28355–28359. 76 29 1267–1272. Wu, Z., Rothwell, L., Young, J.R., Kaufman, J., Butter, C., Kaiser, P., 2010. Generation 77 30 Schiano de Colella, J.M., Barbarat, B., Sweet, R., Gastaut, J.A., Olive, D., Costello, R.T., and characterization of chicken bone marrow-derived dendritic cells. Immunology 78 31 2008. Rank ligand stimulation induces a partial but functional maturation of 129, 133–145. 79 32 human monocyte-derived dendritic cells. Eur. Cytokine Netw. 19, 81–88. Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M., Mochizuki, S., et al., 80 33 Schneider, K., Kothlow, S., Schneider, P., Tardivel, A., Gobel, T., Kaspers, B., et al., 2004. 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/ 81 34 Chicken BAFF–a highly conserved cytokine that mediates B cell survival. Int. osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. 82 35 Immunol. 16, 139–148. Acad. Sci. U.S.A. 95, 3597–3602. 83 36 Schoppet, M., Henser, S., Ruppert, V., Stubig, T., Al-Fakhri, N., Maisch, B., et al., 2007. 37 Osteoprotegerin expression in dendritic cells increases with maturation and is 84 38 NF-kappaB-dependent. J. Cell. Biochem. 100, 1430–1439. Uncited references Q6 85 39 Seshasayee, D., Wang, H., Lee, W.P., Gribling, P., Ross, J., Van Bruggen, N., et al., 2004. 40 A novel in vivo role for osteoprotegerin ligand in activation of monocyte effector 86 41 function and inflammatory response. J. Biol. Chem. 279, 30202–30209. Hou, W.S., Van Parijs, L., 2004. A Bcl-2-dependent molecular timer regulates the 87 42 Simonet, W.S., Lacey, D.L., Dunstan, C.R., Kelley, M., Chang, M.S., Luthy, R., et al., 1997. lifespan and immunogenicity of dendritic cells. Nat. Immunol. 5, 583–589. 88 43 Osteoprotegerin: a novel secreted protein involved in the regulation of bone Williamson, E., Bilsborough, J.M., Viney, J.L., 2002. Regulation of mucosal dendritic 89 44 density. Cell 89, 309–319. cell function by receptor activator of NF-kappa B (RANK)/RANK ligand 90 45 Smith, C.A., Farrah, T., Goodwin, R.G., 1994. The TNF receptor superfamily of cellular interactions: impact on tolerance induction. J. Immunol. 169, 3606–3612. 91 46 and viral proteins: activation, costimulation, and death. Cell 76, 959–962. Yun, T.J., Chaudhary, P.M., Shu, G.L., Frazer, J.K., Ewings, M.K., Schwartz, S.M., et al., 92 47 Staines, K., Young, J.R., Butter, C., 2013. Expression of chicken DEC205 reflects the 1998. OPG/FDCR-1, a TNF receptor family member, is expressed in lymphoid cells 93 48 unique structure and function of the avian immune system. PLoS ONE 8, e51799. and is up-regulated by ligating CD40. J. Immunol. 161, 6113–6121. 94