atan Oren ∗, Guy Paz, Jacob Douek, Amalia Rosner, Keren Or Amar, Baruch Rinkevichsrael National Institute of Oceanography, P.O. Box 8030, Haifa 31080, Israel
r t i c l e i n f o
rticle history:eceived 18 December 2011eceived in revised form 11 June 2012ccepted 12 June 2012
Naturally occurring histocompatibility responses, following tissue-to-tissue allogeneic contacts, are com-mon among numerous colonial marine invertebrate taxa, including sponges, cnidarians, bryozoans andascidians. These responses, often culminating in either tissue fusions or rejections, activate a wide arrayof innate immune components. By comparing two allorejection EST libraries, developed from alloincom-patible challenged colonies of the stony coral Stylophora pistillata and the ascidian Botryllus schlosseri, werevealed a common basis for innate immunity in these two evolutionary distant species. Two prominentgenes within this common basis were the immunophilins, Cyclophilin A (CypA) and FK506-binding pro-tein (FKBP). In situ hybridizations revealed that mRNA expression of the coral and ascidian immunophilinswas restricted to specific allorecognition effector cell populations (nematoblasts and nematocytes inthe coral and morula cells in the ascidian). The expressions were limited to only some of the effector
cells within a population, disclosing disparities in numbers and location between naïve colonies andtheir immune challenged counterparts. Administration of the immunosuppression drug Cyclosporine-A during ascidian’s allogeneic assays inhibited both fusion and rejection reactions, probably throughthe inhibition of ascidian’s immunocytes (morula cells) movement and activation. Our results, togetherwith previous published data, depict an immunophilins-based immune mechanism, which is similarlyactivated in allogeneic responses of distantly related animals from sponges to humans.
Allorecognition, the ability to discriminate between conspe-ific self and non-self, has probably evolved over 600 millionears ago, concurrently with the development of multicellularity,llowing the demarcation of the very first multicellular organismsSrivastava et al. 2010). Colonial marine invertebrates, includ-ng taxa of sponges, cnidarians, bryozoans and ascidians, expressllorecognition proficiencies that exemplify high specificity andccuracy, including extensive allotypic diversity that in some cases,bide by strict mendelian inheritance rules (Scofield et al. 1982;rosberg 1988; Rosengarten and Nicorta 2011). The sessile lifetyle of these organisms and the frequently aggregated larvalettlements generate densely populated communities in whichntra-species encounters are common, usually resulting in tworchetypal allogeneic responses. The first is the morphological
usion of interacting genotypes and the formation of chimericalntities. The second is a destructive immune rejection response,arried out by specific effector cell-populations, which lead to the
physical separation between allogeneic parties. Its cellular originand its characterization vary between different phyla from archeo-cytes and granulated gray cells in sponges (Humphreys 1994),nematocytes in the cnidarians (Buss et al. 1984; Lange et al. 1989)to morula cells in the ascidians (Rinkevich et al. 1998; Ballarin et al.2001). In these phyla and in other invertebrate taxa, the activatedeffector cells reach and converge into allogeneic contacting areas,where they often discharge their harmful contents, culminating intissue damage, necrosis, and even whole organismal death (Busset al. 1984; Ballarin et al. 1998; Rinkevich 2005).
Based on the continuously extending genomic data and newmolecular tools now available, recent studies have attempted toscan invertebrates genomes, including Cnidaria and Urochordata(i.e. Miller et al. 2007), addressing the crux of innate immunity.Yet, little is known about the actual roles of the identified geneticelements in immune defense and allogeneic responses.
Here we compared allogeneic rejection transcriptomes in rep-resentatives of two disparate vertebrate phyla, the cnidarianStylophora pistillata, a branching coral, representing one of the
earliest multicellular animal phyla and the colonial tunicate Botryl-lus schlosseri, a urochordate, closely related to the vertebrates.This comparison revealed common expression patterns of spe-cific immune-related genes as well as shared functional attributes
xpressed during allogeneic rejection in the two species. Two genesrom the list, the immunophilins FK506-binding protein (FKBP)nd Cyclophilin A (CypA), exemplified parallel expressions in thellorejection effector cells; the coral’s nematocytes and the ascid-an’s morula cells. Cyclosporine A, an immunosuppression drug,
hich targets CypA, inhibited fusion and rejection in B. schlosseri.ased on these results and previous studies, we portrayed here both
unctional and molecular cross-phyla resemblances, indicating thexistence of a highly conserved innate immune mechanism in thenimal kingdom.
aterials and methods
llorecognition assays and the administration of Cyclosporine ACsA)
For allorecognition assays we used young S. pistillata colonies10–18 months old) originated from field collected planulae, asescribed (Oren et al. 2010) and laboratory-bred B. schlosseriolonies, offspring of founders collected from several coastal loca-ions (Oren et al. 2007). Allorecognition assays were performed onoth organisms as described (Rinkevich 1995).
In order to study the physiological effects of CsA on Botryllus allo-eneic interactions, allogeneic compatible and incompatible pairsf Botryllus colonies were submerged in 1 mg CsA in 100 ml sea-ater (10 �g/ml) just after initial tunic contacts were established
etween allogeneic pairs. As control, we used clones of the inter-cting genotypes (ramets), which were kept in 100 ml of seawatern identical conditions.
ight and electron transmission microscopy
Histological preparations were made on tissue samples takenrom allogeneic interacting and naïve Stylophora and Botryllusolonies. Samples were fixed in 4% formaldehyde for 2 h, embeddedn paraffin, serially cross-sectioned (5 �m) and stained by hema-oxylin and eosin. Observations were performed under Olympus×50 Upright microscope, equipped with Color View camera (Soft
maging System, Munster, Germany).For EM analyses, naïve and allogeneic interacting ramets were
xed in 2.5% glutaraldehyde in seawater and stored at 4 ◦C for 10ays. Next, they were washed twice in 0.2 M cacodylate bufferpH 7.2–7.4) and postfixed by 0.1% OsO4 in the same buffer for
h, at room temperature. After three buffer washes, samples wereehydrated by ethanol series and embedded in Epon 812. Zones of
nterest were selected and positioned properly in flat embeddingolds. Ultrathin sections of 80 nm were cut with Leica Ultracut
ultramicrotome. Finally, the sections were stained with uranylcetate and lead citrate. Grids were observed under a Hitachi 7500lectron microscope. Alternatively, thin sections of 1 �m weretained by a mixture of toluidine blue and azure II and observednder a Leica DME photonic microscope.
n situ hybridization
Interacting histoincompatible B. schlosseri and S. pistillata pairsf colonies and their naïve counterpart ramets were fixed for 2 hn 4% paraformaldehyde following 70% ethanol overnight, dehy-rated in 70% methanol, embedded in paraffin, and cut into 5 �mections. Total RNA was extracted, separately, from interactingairs and from corresponding naïve ramets (n = 4) by EpicentreasterPureTM RNA Purification kit (cat. no. MC85102, Madison,
inconsin, USA). The integrity of the total RNA was verified
y 1% agarose gel electrophoresis. First strand cDNA was syn-hesized by DNA synthesis kit (cat. no. K1622, Fermentas, MD,SA). Based on our EST database (Oren et al. 2007, 2010), we
y 218 (2013) 484– 495 485
designed primers that were used to obtain sense and antisenseDIG-labeled RNA probes. The probes were cloned into pdrive plas-mid (cat. no. 231124, Qiagen, Valencia, CA) and used as templatesfor synthesizing the appropriate DIG-labeled RNA probes (senseand antisense) using DIG RNA Labeling Kit (SP6/T7; Roche Diagnos-tics, Penzberg, Germany). Hybridization of probes to tissue sectionswas performed according to Breitschopf et al. (1992) for paraffin-embedded tissues. DIG-labeled RNAs on samples were observedusing anti-DIG antibody (cat. no. 11277073910, Roche, Mannheim,Germany), Nitro Blue Tetrazolium (cat. no. N5514, Sigma Aldrich, St.Louis, Missouri, USA) and 5-bromo-4-chloro-3-indolyl phosphatep-toluidine salt (cat. no. b0274, Sigma Aldrich, St. Louis, MO, USA)as substrates for peroxidase activity.
Quantitative RT PCR
RNA was extracted from three pairs of interacting Botrylluscolonies and their naïve counterparts as described in ‘In situhybridization’ section. First strand cDNA was synthesized in a totalvolume of 20 �l with the Thermo Scientific Verso cDNA synthesiskit. The PCR amplification was performed using designed sets ofprimers (IDT Inc.). The real time PCR mixture consisted of 40 nMcDNA sample, 70 nM of each primer and 12.5 �l of SYBR Greenmix (Abgene, Epsom, UK), in a final volume of 25 �l. RT PCR wascarried out in the GeneAmp 5700 PCR thermocycler (PE AppliedBiosystems, Foster City, CA) under the following conditions: 95 ◦Cfor 15 min followed by 40 cycles of 95 ◦C for 15 s, 60 ◦C for 30 sand 72 ◦C for 30 s. Amplification of cDNAs was performed in tripli-cates for each interacting pair and the relative abundance of eachselected mRNAs was normalized in reference to 18S rRNA (acces-sion no: AB211066). Data analysis was performed according toPfaffl (2001).
Results
S. pistillata and B. schlosseri allorejection repertoires
Many of the allogeneic interactions in S. pistillata and B. schlosseriresulted in incompatible responses. Stylophora histoincompatibleinteractions developed into necrotic fronts at contact areas (Amaret al. 2008; Oren et al. 2010; Fig. 1a) while those of Botryllus devel-oped cytotoxic necrotic lesions along the contact line betweenthe interacting colonies (points of rejection, PORs; reviewed inRinkevich 2005; Fig. 1b). The effector cells in Botryllus allore-jection are the morula cells; berry-shaped blood cells equippedwith several, transparent to yellowish-green vacuoles (approx. 2-�m in diameter), hosting the phenol-oxidase activity (Rinkevichet al. 1998; Ballarin et al. 2001; Fig. 1c and d). During the rejec-tion process, morula cells accumulated at the tips of interactingampullae (Fig. 1c) then infiltrated through the ampullar epithe-lium to the tunic matrix where they released their content anddegenerated, digested or phagocytized by circulating phagocytes(Fig. 1d).
Two subtraction libraries were prepared for the allogeneic rejec-tion processes in S. pistillata and in B. schlosseri, using the samemethodology (Oren et al. 2007, 2010). The libraries were basedon total RNA extracted from rejecting Botryllus and Stylophoracolonies and from their correlated genetic naïve clones. Duringthe subtraction protocol, ESTs unique to the rejecting clones wereenriched by selectively eliminating the ESTs that were sharedbetween the two extractions. The coral library contained 1760 high
quality sequences including 230 contigs and 1530 singlets (redun-dancy of 22.6%). The ascidian library contained 1693 high qualitysequences including 217 contigs and 1479 singlets (redundancy of21.6%). A blast analysis revealed that 51% of the ascidian sequences
486 M. Oren et al. / Immunobiology 218 (2013) 484– 495
Fig. 1. Stylophora and Botryllus allogeneic rejection. (a) Histoincompatible Stylophora colonies in late rejection response. Black arrowheads – rejection area; p – polyp. (b)R M of B– ing phv
spfioAwNrs
athta(1asPPbm
ejecting Botryllus colonies. z – zooids; amp – ampulla; POR – points of rejection. (c) E prophenol-oxidase containing vacuoles. (d) An activated morula cell in Botryllus beacuoles; pg – phagocyte. Scale bars: 4 �m.
howed high similarity (E-value ≤ 0.005) to database genes com-ared to only 28.5% in the coral library. One hundred and twentyour Botryllus sequences (14% of the total blast matches) weredentified as “immune-related” compared to 75 sequences (15%f the total blast matches) in the coral (Oren et al. 2007, 2010).s expected, no true elements of the adaptive immune systemere identified in the coral and the ascidian allorejection libraries.evertheless, the figures drawn are of complex innate immune
eactions, which took part in the genomic responses of both modelpecies.
Comparing the immune-related matches of the coral andscidian libraries resulted in several shared entries, includinghe following eight proteins and protein families (Table 1); (1)eat shock proteins 90 (HSP90s); (2) oxidative/other stress pro-eins including glutathione peroxidase, glutathione S-transferasend Cytochrome P450 (CYP450); (3) Pattern Recognition ReceptorsPRRs) including MBL Associated Serine Protease (MASP), Intelectin, complement proteins C1q/C1r and Rhamnose-binding lectins; (4)dhesion proteins including mucins, echinonectin, calreticulin andimilar orthologs for von Willebrand factor, Claudin and Spondin; (5)
roteases from the cathepsin family; (6) MyD88; (7) Poly ADP-Riboseolymerases (PARPs) and; (8) immunophilins including FK506-inding protein (FKBP) and Cyclophilin A. Overall, 74 similar blastatches were found in the immune-related categories of the coral
otryllus morula cells at the interacting ampullae. m – morula cells; black arrowheadsagocytized by a circulating phagocyte. m – morula cell; white arrowheads – empty
and the ascidian libraries, accounting for 37.2% of the total immune-related matches. Within these matches 43 (21.6% of the whole list)were exact matches.
However, above comparison has also yielded a number of funda-mental differences, including ascidian orthologs and gene-familiesthat were noticeably absent from the coral library. The most promi-nent groups were the tunicate HSP70s, the Trypsins and the Ficolins,containing at least five representatives, each. Whereas representa-tives of the first two families (HSP70s and Trypsins) were present incnidarians genomes (e.g. Tom et al. 1999; Putnam et al. 2007) theywere absent from our coral library. Other important gene-familiesabsent from the coral library were of Annexins (n = 4 representativesin the tunicate library), Selectins (n = 2) and two oxidative stressresponse genes, the Super Oxide Dismutase (SOD), known to specif-ically participating in Botryllus allorecognition (Ballarin et al. 1998,2002). There were also gene matches specific to the coral library;however, most of them could not be assigned to any significant genegroup. The few gene groups that were recognized solely among thecoral library immune-related matches included Metalloproteinases(n = 3), Calmodulins (n = 2) and Chitinases (n = 2). The expression of
chitinolytic enzymes in cnidarian immunity has recently been elu-cidated (Douglas et al. 2007).
Immune-related ESTs from each library were subjected to map-ping and annotation procedures using blast2go internet software
M. Oren et al. / Immunobiology 218 (2013) 484– 495 487
Table 1Similarities between B. schlosseri and S. pistillata allorejection transcriptomes (E-values of blast matches ≥ 0.001).
Protein name Botryllus Stylophora
Stress protein
Heat shock protein 90 alpha EE743591Heat shock protein 90 beta EE743602 GT564653, GT564680Heat-shock protein 90 EE743613, EE743624 GT564648Heat shock protein 70 EE743635, EE743646, EE743534,
EE743545, EE743556Heat shock protein 10 EE743564, EE743565Heat shock protein 20.5 GT564691Glutathione peroxidase EE743569 GT564704Cytochrome P450 EE743574, EE743575, EE743576 GT564676Microsomal glutathione S-transferase 3 variant EE743572Glutathione s-transferase protein 11 GT564662Microsomal glutathione S-transferase 1 GT564681Cu, Zn superoxide dismutase EE743567, EE743568Oxidation resistance protein 1 EE743570Putative 8-lipoxygenase–allene oxide synthasefusion protein
Von Willebrand factor like 1 EE743655, EE743654Von Willebrand factor like 2 EE743656Von Willebrand factor-like protein GT564708, GT564667, GT564718Integumentary mucin A.1 precursor EE743638 GT564684Mucin-like protein EE743634, EE743636Mucin TcMUCII, putative EE743637Mucin-4 [precursor] GT564699Echinonectin EE743644, EE743645, EE743647 GT564703, GT564713Calreticulin EE743593 GT564719Claudin-19 EE743639Claudin GT564702SCO-spondin EE743650Spondin 2 GT564656Spondin GT564692Bcam protein EE743628Scavenger receptor class B member 2 (LIMP II) EE743629Selectin, endothelial cell EE743631Selectin P EE743632Tumor necrosis factor-inducible protein 6 EE743633Annexin VII EE743640Annexin A7 EE743641Annexin isoform 1 EE743642Annexin 11a, isoform 2 EE743643SPARC EE743644Zonadhesin-like EE743649Notch like (ENSANGP00000010271 protein)Polydomain protein-like EE743652Tenascin-X EE743653Ci-META1 EE743536, EE743535
488 M. Oren et al. / Immunobiology 218 (2013) 484– 495
Table 1 (Continued)
Protein name Botryllus Stylophora
Ci-META2 EE743538, EE743537Vwa1 protein EE743540Apolipophorins precursor EE743541Fibrinogen related domain variant 3 EE743542Fibrinogen related protein 12.1 precursor GT564697Integrin beta-1-binding protein 1 GT564686Cadherin-related tumor suppressor precursor GT564712Contactin-3 precursor GT564716
Proteases
Cathepsin A like (LOC494810 protein) EE743609Cathepsin C EE743610Cathepsin D EE743611 GT564678Cathepsin L EE743612, EE743614, EE743615 GT564646, GT564714Cathepsin B GT564723, GT564664Cathepsin Z GT564654Dipeptidyl peptidase 4 (T-cell activationantigen CD26)
EE743600
Peptidyl-dipeptidase A precursor EE743620Aspartyl aminopeptidase (LOC495491 protein) EE743616Predicted Ubiquitin specific protease 14 EE743617Legumain like (MGC64351 protein) EE743618Carboxypeptidase A2, pancreatic EE743619Cysteine protease GT564654Ubiquitin-specific-protease-3-like protein GT564645AFG3-like protein 2 GT564668Factor C
Interferon-inducible protein 16 EE743543Interferon regulatory factor like protein EE743544Interferon gamma-inducible protein 30 GT564659IFR1 protein (interferon-related protein) GT564695Interferon induced protein 2 GT564715Interleukin enhancer binding factor 3 EE743546NF-IL6 GT564657PMP1 protein precursor GT56471172 kDa type IV collagenase precursor GT564725Disintegrin metalloproteinase GT564665Matrix metallopeptidase 14 GT564669Matrix metalloproteinase-9 precursor GT564670Ilf2-prov protein GT564673Tumor necrosis factor superfamily, member5-induced protein 1
Calmodulin-like protein GT564651Calmodulin (CaM) GT564649
Programmed cell death
Poly [ADP-ribose] polymerase 1 (PARP-1) EE743550Poly [ADP-ribose] polymerase 14 (PARP-14) GT564671Death associated protein 1a EE743548 GT564674Caspase-like protein EE743549Similar to BAG-family molecular chaperoneregulator-1
GT564658
Programmed cell death 4b GT564701Anti-apoptotic protein NR13 (apoptosisregulator Nr-13)
GT564724
M. Oren et al. / Immunobiology 218 (2013) 484– 495 489
Table 1 (Continued)
Protein name Botryllus Stylophora
Proteasome activator subunit 3 GT564652
Tumor proteins
Tumor protein D52-like 2, isoform 2 EE743551tumor protein, translationally controlled 1 EE743552Gastric cancer antigen Zg14 EE743553B-cell CLL/lymphoma 7 protein family memberB
EE743554
Tumor differentially expressed 2-like GT564688
Proteasome-associatedproteins
LMP7-like protein EE743557a EE743555LAMP-3CG3455-PA GT56471726S proteasome non-ATPase regulatorysubunit
GT564710, GT564683
Proteosome subunit Y GT564696
Other
FK506-binding protein (FKBP) EE743559 GT564698, GT564655Cyclophilin A GO357570 GT564650Soluble immunoglobulin molecule homologue,IG BOTSC
EE743630
Helicase, lymphoid specific EE743560Esterase D\/formylglutathione hydrolase EE743561Putative senescence-associated protein EE743562Cold shock protein EE743563
a Gray scales indicate homology levels. Light gray: matches related to the same gene family. Dark gray: identical matches.
Binding
seq: 40 score: 30.23
Catalytic Activity
seq: 38 score: 9.97
ActivityHydrolase
seq: 27 score: 9.44
RibonucleotideAdenyl
Binding
seq: 11 score: 6.60
Protein Binding
seq: 24 score: 21.38
Peptidase Activity
seq: 23 score: 9.56ATP Binding
seq: 11 score: 11.00
Unfolded Protein
Binding
seq: 7 score: 7.00
BotryllusMolecular Function
StylophoraMolecular Function
Binding
seq: 17 score: 6.79Catalytic Activity
seq: 23 score: 4.86
Protein Binding
seq: 8 score: 6.40
ActivityHydrolase
seq: 19 score: 6.02
Metal Ion Binding
seq: 7 score: 5.16ATP Binding
seq: 6 score: 6.00Peptidase Activity
seq: 16 score: 8.30
Peptidase Activity,
acting on L-amino
acid peptides
seq: 10 score: 3.84
ActivityEndopeptidase
seq: 9 score: 5.40
Cysteine-type
ActivityEndopeptidase
seq: 5 score: 5.00
a. b.
Endopeptidase
Activity
seq: 13 score: 7.80
Serine-type Peptidase
Activity
seq: 10 score: 6.16
Serine-type
ActivityEndopeptidase
seq: 8 score: 8.00
Peptidase Activity-
acting on L-amino
acid peptides
seq: 19 Score: 9.27
RibonucleotideAdenyl
Binding
seq:6 score: 3.60
Cysteine-type
peptidase Activity
seq:5 score: 3.00
Fig. 2. Botryllus and Stylophora Gene Ontology (GO) charts of immune-related transcripts during allogeneic rejections. Using blast2go internet software, twelve significant(number of sequences ≥ 5 and score ≥ 3) “molecular function” sub ontology terms were identified for each organism of which nine were identical between the two charts.These GO terms were sorted into two root categories, the “binding” category and the “catalytic activity” category. The last contained cystein-type activity in S. pistillata vs.serine-type activity in B. schlosseri. Different levels of orange hues indicate significance according to GO term analysis. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of the article.)
490 M. Oren et al. / Immunobiology 218 (2013) 484– 495
Caeno rhabditi s ele gansDrosophila melano gasterNemat ostell a vec tensisStylopho ra pistill ataStrongylo cent rotu s purpu rat usCiona int estinali sBotryllu s schloss eri Danio rerioXenopu s laevi sGallus gallu sMus musculusHomo sapi ens
a
K I DKL Q I GV K RAE N - CV QKS RKGDQL HMHYT GT L - DGT EF DSS RT RN - E F T F T LGQGN I KGWDQGL NMCV GER I T I P H LGYGERG - AP K I P GNS V KF DVE L K I DR
R AQDL KVE V T PE V - CE QKS KNGDSL T MHY T GT L QADGKK F DSS F DRD - QPF T F QLGAGQ I KGWDQGL NMCV GEKRKL T I P Q LGYGDQG - AGNV P KAT L F DVE L N I GN
KVT KL Q I GV K VKD - CKR S VKGDSL HMHYT GKL E - DGT EF DSS I P RG - E PF VF T LGT GQ I KGWDQGL NMCAGEKRKL P S DMGYGDRG - AP K I P G AT L F EVE L K I GD
GV T I E V YK PE G - CT RKT KDGDL AMDYT GRL E KDGS VF DSS HKRQ - QT F DF TMGKGQ I QGWE GL KDMCV GEKRKL RVP AH LGYGDRG - AGDV P GANL F DVE L KE I KN
KSKKL Q I G I VE - CT RS RT GDRL SMHYT GKL E - DGT EF DSS I P RK - QT F DF T LGAGQ I KGWDQGL NMCE GEKRKL P S N LGYGDRG - S P K I P G AT F E VE L K I NR
A L E KL Q I G I QK I P KE CT QKS KK GDT L SMHYT GV K - DGE F DSS L KRG - QPF EF K LGAGQ I GGWDQGL GMC I GEKRKL P H LGYGDQ G - AGGK I P G AT L F T VE L K I NG
K VDKL Q I GV KR VE - CERKSGS GDV DMHYT GT L E - DGSKF DSS RDRD - T PF T F T LGQGY I KGWDKGL GMCE GERR KL K I P S DMGYGDRG - S P K I P G AT F DVE L K I KD
- K L Q I G I KK R VDN - CP I KS RKGDV NT HY T GKL E - DGT EF DSS I P RN - QPF T F T LGT GQ I KGWDQGL GMCE GEKRKL P S E LGYGDRG - AP K I P G AT L K -
GRKKL Q I GVKK R VE N - CP V KS RKGDT L HMHY T GKL E - DGT EF DSS I P RN - QPF T F T LGT GQ I KGWDQGL GMCE GEKRKL P S E LGYGDRG - AP K I P G AT F E VE L K I ER
AE V K I E V HL PE S - CS P KS KK GDL NAHY DGF L AS NGS KF YCS RT QNDGHPKWF V GVGQ I KG LD I AMMNMCP GEKRKV P S LA YGQ GY AQGK I P NAT F E I E L Y AV NK
GKRKL Q I GVK R VDH - CP I KS RKGDV HMHY T GKL E - DGT EF DSS L P QN - QPF VF S LGT GQ I KGWDQGL GMCE GEKRKL P SE LGYGERG - A P K I P G AT L F E VE L K I ER
GKRKL Q I GV K R VDH - CP I KS RKGDV HMHY T GKL E - DGT EF DSS L P QN - QPF VF S LGT GQ I KGWDQGL GMCE GEKRKL P S E LGYGERG - A P K I P G AT L F E VE L K I ER
Caeno rhabditi s ele gansDrosophila melano gasterNemat ostell a vec tensisStylopho ra pistill ataStrongylo cent rotu s purpu rat usCiona int estinali sBotryllu s schloss eri Danio rerioXenopu s laevi sGallus gallu sMus musculusHomo sapi ens
b
L
K
K
K
GGES IYGE KF PDE NF KEKHT GP GVL
GGKS I YGNKF P DE NF E L KHT GS G L
GGKS IYGAKF ADE NF NL KHT GP G L
GGKS IYGE KF ADE NF - L KHT GKG L
GGKS I YGE KF ADE NF T L KHT E P GTL
GGKS IYGAKF ADE NF KL KHEGP GYL
GGKS IYGNKF P DE NF T L KHT GP G L
GGKS IYGP RF P DE NF KL KHT GP G L
GGKS I YGNKF ADE NF T L KHT GP G L
GGKS IYGE KF ADE NF LKHT GP G L
GGRS IYGE KF EDE NF LKHT GP G L
GGKS IYGE KF ED E NF LKHT GP G L
I
I
I
I
I
I
I
I
I
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SMANAGP NT NGSQF LCT V KT E WLDGKHV F GRV E GLD KAV E S - NGS QS GKPVKDCM ADCGQ
SMANAGANT NGS QF I CT V KT AWLDNKHV F GEV E GLD KK I E S - Y GS QS GKTS KK I ANSGS
SMANAGPGT NGSQF LCT AKTS WLDGKHV F GSV KDGMD KK I E K - V GS DS GKTS KK ADSGE
SMANAGPNT NGSQF I CS - KT P WLDGKHV F GSV E GS E QMN D - V GD- S GKTS KP L ADCGQ
SMANAGPNT NGSQF I CT AV TS WLDGKHV F GKV AQGSD KK V E S - Y GS ES GKTS KK I ADCGQ
SMANAGKDT NGS QF I TT V KTS WLDGKHV F GK I DGMD KA I E AT QT GAQDRPVKDV ADCGE
SMANAGPNT NGSQF I CT AKT AWLDGKHT V F GS I E GMD KK I E A - V GS QS GKTS KK V T V AASGQ
SMANAGV NT NGS QF I CT AKT E WLDGRHV F GSV KE GMD RKV E A - L GS RS GRT AQR I S I T DCGE
SMANAGANT NGSQF I CT AKTS WLDGKHV F GQV E GMD KTMD R - L GS QS GKP SKK V T NSGQ
SMANAGPNT NGS QF I CT AKT E WLDGKHV F GRV KE GMN E AME R - CGS KDGKTS KQ I T I SNCGQ
SMANAGP NT NGS QF I CT AKT E WLDGKHV F GKV KE GMN E AME R - F GS RNGKTS KK I T I SDCGQ
Fig. 3. Multiple alignments for Cyclophilin A and FK506-binding protein sequences from S. pistillata, B. schlosseri and ten selected database sequences. (a) Cyclophilin Aalignment including a significant signature of Cyclophilin A, B and H-like cyclophilin-type peptidylprolyl cis-trans isomerase (PPIase) domains (marked in a box). (b) FK506BPa the boa reade
((abafai“lbfaiBl
Im
r(lavGata(tscat
lignment including a significant signature of FKBP-type PPIase domain (marked incid content. (For interpretation of the references to color in this figure legend, the
http://www.blast2go.org). Molecular function Gene OntologyGO) charts were produced separately for the coral and for thescidian, each containing twelve sub ontology terms (Fig. 2a and). The two charts revealed striking similarity both in GO termsnd in their relative strength (scoring and number of sequencesor each term). Both analyses contained the functions “binding”nd “catalytic activity” in the highest graph sub-level. The “bind-ng” term in both charts consisted of “protein binding” and theadenyl ribonucleotide binding” terms. The “binding” lower chartevels included “ATP binding” in both charts with “unfolded proteininding” term for Botryllus (Fig. 2a) and “metal ion binding” termor Stylophora (Fig. 2b). Both charts contained “peptidase activitycting on l-amino acid peptides” sub-term in the “catalytic activ-ty” branch, consisting of “serine-type endopeptidase activity” forotryllus (Fig. 2a) and “cystein-type endopeptidase activity” for Sty-
ophora (Fig. 2b).
nvertebrates FKBP and CypA are homologues to their vertebrateatches
BLASTX algorithm screens of the two ESTs rejection librariesevealed immunophilin true matches to FK506-binding proteinFKBP) and of Cyclophilin A (CypA). The library sequences of Sty-ophora FKBP (termed SP-FKBP; acc: GT564655; E-value = 6e−64)nd Botryllus FKBP (termed BS-FKBP; acc. EE743559; E-alue = 8e−67) and of Stylophora CypA (termed SP-CypA; acc:T564650; E-value = 2e−44) and Botryllus CypA (termed BS-CypA;cc. GO357570; E-value = 3e−58) were aligned to each other ando additional ten representative genebank sequences of FKBPnd CypA using the ClustalW application in the BioEdit softwarehttp://bioedit.software.informer.com; Fig. 3a and b). This mul-iple alignment tool showed the amino acid sequences in both
equences were significantly similar to the database genes andontained the conserved domains of those gene families (Fig. 3and b). Both the coral and the ascidian FKBPs contained the FKBP-ype peptidylprolyl cis-trans isomerase domain (PPIase) and both
xes). The stars indicate metal ion-dependent adhesion sites. Colors indicate aminor is referred to the web version of the article.)
the coral and the ascidian Cyclophilin A sequences contained theCyclophilin A, B and H-like cyclophilin-type PPIase domains. Theidenticality in the amino acid sequences between BS-FKBP and theother FKBP sequences tested ranged between 48 and 75% while inthe coral it ranged between 47 and 60%. The CypAs showed highersimilarity with 70–85% identical AA for Botryllus and 64–79% forStylophora (Table 2).
The expression patterns of the coral and ascidianimmunophilins were tested using in situ hybridization (ISH) onhistological sections of naïve and alloimmune rejecting Stylophoraand Botryllus colonies. In the Stylophora system, both SP-CypA andSP-FKBP were specifically expressed in the ectodermal layer of thepolyp’s actinopharynx (Fig. 4) as compared to the sense control(Fig. 4c). SP-FKBP expression was limited to large nematocytesfound in low-densities in the inner epithelium (Fig. 4d–f). Thenumbers of SP-FKBP expressing cells were enhanced signifi-cantly during rejection responses (Fig. 4d) as compared to very fewstained cells in histological sections from naïve colonies (Fig. 4e andf). SP-CypA was expressed in a dense layer of small nematoblastslocated in the basal layer of the ectoderm (Fig. 4g and h) with noapparent expression changes during alloimmune rejection. qPCRexpression quantification for SP-FKBP and SP-CypA that was pre-viously performed by us showed significant upregulation of bothgenes (+2.2; SD = 0.5; +2.7; SD = 0.5; Oren et al. 2010). In the Botryl-lus system, both BS-CypA and BS-FKBP expressions were restrictedto part of the morula cells population (Fig. 5b–e) as compared tothe sense controls (Fig. 4c). BS-CypA and BS-FKBP positive cellswere recorded throughout the blood system, including ampullae,vasculature, blood sinuses and along the endostyle niches. Higherconcentrations of expressing cells were observed in the interactingampullae of rejecting colonies as compared to naïve ampullae(∼80 vs. ∼50% respectively; Fig. 5b–e). However, qPCR analyses
performed on the whole interacting ramets showed in rejectingsubclones a general decrease in expression levels of both genes ascompared to naïve subclones (−2.6; SD = 1.4, −2.3; SD = 2.1, in log2respectively).
M. Oren et al. / Immunobiology 218 (2013) 484– 495 491
Table 2FKBP and CypA homologies of S. pistillata and B. schlosseri (in bold) and ten other distantly related animals. Lower diagonal numbers indicate FKBPs AA identities (% out of102 AA); upper diagonal numbers indicate CypA AA identities (% out of 87 AA).
yclosporine A inhibits Botryllus morula cells activation duringllorecognition
Botryllus colonies in the process of either fusion or rejection,ere exposed to the immunosuppresor agent Cyclosporine A (CsA),nder dose level of 10 �g/ml, a non-toxic concentration, as wasonducted in a sponge experiment (Sabella et al. 2007). Adminis-ration of CsA upon first allogeneic contacts between the colonies’unics blocked fusion and rejection responses (Fig. 6a–f), revealingully extended peripheral ampullae (Fig. 6d and f) as in controls.
ith only initial tunic–tunic fusion outcomes, none of the otherarly allogeneic responses (e.g. morphological changes in interact-ng blood vessels, stretched-out ampullae, increased blood flow,nd above all, the immediate [within 2 h] recruitment of morulaells toward tips of interacting ampullae; Tanaka 1973; Scofield andagashima 1983; Rinkevich et al. 1998) was observed (Fig. 6a–f).he interacting colonies (compatible and incompatible pairs, n = 4,ach) remained unaffected for the entire 48 h duration of thexperiment, while corresponding ramet-control pairs (n = 4) cul-inated in full rejections or fusions, 6–24 h from the onset of
airing, exhibiting late interaction states at the end of the 48 heriod, including advanced tissue fusion in the compatible pairsnd POR formation in incompatible pairings (Fig. 6a–f). Followingrug removal, affected colonies resumed normal routes for allo-eneic responses, climaxed with fusion/rejection outcomes. Theecovery time (onset of the first fused blood vessel, in fusion and therst seen POR, in rejection) ranged between 12 h and 48 h for allo-eneic compatible pairs and between 20 h and 3 days for rejectingairs.
Administering CsA at advanced alloimmune stages (6–18 h fromairing), following the completion of initial interaction signs, suchs fused tunics, ampullae contacts and morula cells recruitmentt contact area, did not halt fusion or rejection processes, as theyontinued in the same pace as the controls. However the rejectionrocess resulted in fainter and smaller PORs compared to controlsn = 3).
iscussion
Allogeneic interactions in sessile colonial marine invertebratesre often regarded as “natural transplantations” because they shareimilar morphological features with the organ/tissue transplan-ation scenarios in humans and other vertebrates. However, noomology has been found so far between invertebrates and ver-ebrates allorecognition genes, not even between those of differentnvertebrates species (Dishaw and Litman 2009; Rosengarten and
icorta 2011). Such is the case of the enigmatic Fu/HC system of. schlosseri (Rinkevich et al. 2012) which controls allorecognitionutcomes during natural interactions between Botryllus geno-ypes (Oka and Watanabe 1960). Neither of these invertebrates’
76 69 70 87 88 50 *** 9872 70 71 88 89 51 99 ***
candidates showed any homology as so far to known databasegenes. Similarly, the recently characterized Hydractinia alr1–alr2system (Nicorta et al. 2009) has not yet revealed any homologyto any other organism. However, while allorecognition regulatorsappeared unique for any specific animal species studied or groups,downstream immune system effector arms could share com-mon elements (Rosengarten and Nicorta 2011), as demonstratedhere. Previously, by employing allogeneic-based EST subtractionlibraries, we revealed that histocompatibility processes in the uro-chordate B. schlosseri and in the coral S. pistillata activated a widearray of vertebrate-like immune system genes (Oren et al. 2007,2010). In order to search for common immune gene candidatesparticipating in allorejection, we compared the relevant orthologsof the two libraries. As much as 37% similar genebank homologiesand about 75% common molecular function GO-terms were identi-fied. These results suggested that some elements in the allogeneicimmune response share a common and ancient evolutionary root.The differences between the ascidian’s and the coral’s rejectionlibraries reflect the distant positions of both taxa (Anthozoa vs. Uro-chordata) on the evolutionary tree. The ascidian rejection librarycomprises true orthologs of the HSP70s, trypsins (serine proteases),ficolins, annexins and selectins gene groups not represented in thecoral rejection library, suggesting that they may have acquired theirroles in allogeneic immunity later in evolution. Using the GO-termanalysis tools, we have identified a significant functionality shiftingin the “peptidase activity” ontology, from cysteine-type peptidaseactivity in the coral to serine-type peptidase activity in the ascid-ian library. This variation may be important to our understandingof the evolution of invertebrate immunity.
From the commonly expressed alloimmune repertoire, wechose to focus on two highly conserved genes, FKBP and CypA of theimmunophilins group, because of their association with immuno-suppression processes in the vertebrates (Schreiber 1991). Usingmultiple alignment comparison against database top matches andmotif search, we identified the respective transcripts with the high-est certainty.
The vertebrates’ immunophilins FKBP and CypA were his-torically discovered based on their ability to bind specificimmunosuppressant molecules of fungal and bacterial origins.The cyclophilins (Cyps) bind cyclosporine A (CsA), while FK506-binding proteins (FKBPs) bind FK506 (tacrolimus) and rapamycin(sirolimus). These interactions create protein–drug complexes thatinhibit the initiation of T-cells activation cascades, resulting inimmunosuppression (Liu et al. 1991; Marks 1996). In natural condi-tions immunophilins function as chaperons, exhibit peptidylprolylcis-trans (PPIase) isomerase activity and participate in biological
processes other then immunosuppression (reviewed in Barik 2006;Lücke and Weiwad 2011).
Immunophilins evolutionary origin is primal (Trandinh et al.1992). In sponges, the earliest existing metazoans, treated with
492 M. Oren et al. / Immunobiology 218 (2013) 484– 495
Fig. 4. In situ hybridization for S. pistillata immunophilins: (a) H&E staining of the Stylophora cross-section at the middle part of the polyp. Gvc – gastrovascular cavity. (b)H&E staining of the ectoderm area. n – nematocytes; sp – spirocytes; sc – secretory cells. (c) SP-FKBP sense control. (d) SP-FKBP expression in Stylophora actinopharynxduring rejection, in nematocytes (red arrowheads). (e and f) SP-FKBP expression in naïve Stylophora actinopharinx (cross-sections 100× and 400×, respectively).Expressionwas confined to only few nematocytes (red arrowhead). en – expressing nematocytes, n – nematocytes; z – symbiotic zooxanthella. (g and h) SP-CypA expression duringrejection in Stylophora actinopharynx, within the dense layer of nematoblasts located at the basal ectoderm. nb – nematoblast Scale bars: 50 �m. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of the article.)
M. Oren et al. / Immunobiology 218 (2013) 484– 495 493
Fig. 5. In situ hybridization for B. schlosseri immunophilins. (a) BS-CypA sense control. m – morula cell; por – point of rejection. (b and c) BS-CypA expression in Botryllus,c ly staic . (d) Nd naïv
nFMStS
ross-sections, 400×. (b) Naïve ampulla. About half of the morula cells are positiveells are positively stained. (d and e) BS-FKBP expression in Botryllus ampullae, 400×uring rejection. Most of the morula cells are positively stained (approx. 80%). nm –
on-toxic concentrations of the human immunosuppressant drugsK506 and CypA significantly affected allorecognition responses.
üller et al. (2001) showed that treating histoincompatible
uberites domuncula sponge ramets with FK506 will cause themo fuse instead of rejecting each other as they would normally do.imilar tests with autographs showed no effect on their fusion.
ned. (c) Morula cells aggregate during rejection. Most (approx. 80%) of the morulaaïve ampulla. About half of the morula cells are positively stained. (e) Morula cells
e morula cells; em – immunophilins expressing morula cells. Scale bars: 5 �m.
In the sponge Microciona prolifera, CsA blocked both rejection andfusion responses, without turning rejection outcomes into fusions,
through the inhibition of immunocytes (gray cells) movements intorejection sites (Sabella et al. 2007). Likewise, our experiments withB. schlosseri, resulted in CsA inhibition of both rejection and fusionresponses, through the inhibition of ascidian immunocytes (morula
494 M. Oren et al. / Immunobiology 218 (2013) 484– 495
Fig. 6. Inhibition of B. schlosseri allogeneic responses by cyclosporine A. (a) Control fused Botryllus colonies revealing allogeneic tissues that are completely merged. (b) Fusionassay using clones of the same genotypes as in sub-figure ‘a’, administrated with 10 �g/ml CsA. Fusion is inhibited. (c) Early interaction signs between histoincompatiblecontrol Botryllus colonies. Ampullae are stretched out, filled with morula cells. (d) Same genotypes as in sub-figure ‘c’ treated with 10 �g/ml CsA. No allogeneic interaction isobserved. (e) Control rejecting Botryllus colonies. PORs (white arrowhead) are formed along the contact line. (f) Botryllus rejection assay using clones of the same genotypesa f:) blaa
ctrttl
s in sub-figure ‘e’, treated with 10 �g/ml CsA. Rejection is inhibited. Subfigures (c–m – ampulla, z – zooid. Scale bars: 100 �m.
ells) movement to interacting ampullae. We have also found thathe mRNA expression of the coral and ascidian immunophilins was
estricted to the coral’s and the ascidian’s allorecognition effec-or cells (nematocytes and morula cells, respectively). Moreover,he mRNA expressions of the two genes, in both organisms, wereimited to only some of the allorecognition effector cells, further
ck arrowhead indicates the contact line between colonies. ia – interacting ampulla;
displaying dissimilarities in numbers and locations between naïveand rejecting subclones. These expression patterns may reflect dif-
ferent modes of activation, resembling the different modes of Tlymphocytes activation in the vertebrate immune system.
The cumulative data (including the results of this work) on theimmunophilins-based immunosuppression in sponges, ascidians
M. Oren et al. / Immunobiology 218 (2013) 484– 495 495
Table 3Common findings about immunophilins and immunophilins-associated immunosuppression in four distantly related phyla during allogeneic responses (Marks 1996; Mülleret al. 2001; Sabella et al. 2007; this study). ‘?’ = unknown; ‘
√’ = exists.
Organism (phylum/group) True immunophilins orthologs Immunophilins expressing cells Target of CsA Outcomes of immunosuppressionby FK506 and CsA
nd humans, the immunophilins homology and their homologuexpression in cnidarians and ascidians allorecognition effector cellssummarized in Table 3), indicate that the immunocytes activa-ion mechanism during allogeneic immune responses may share aommon evolutionary origin in these remotely affiliated organisms.he remarkable similarities exist at all levels of the process andnclude similar inhibitory effect of the immunosuppressive agentsK506 and CsA, similar profiling of the target cells and parallelxpression patterns of corresponding peptides (FKBP and CypA).e have not yet characterized in the coral and the ascidian any
ther vertebrate-like element (in addition to the immunophilins)f the T-cells activation cascade, therefore any analogy to the ver-ebrate cascade should be carefully considered. Nevertheless, this
olecular and functional comparative study provides strong evi-ences for the existence of highly conserved mechanism associatedith histocompatibility responses throughout the animal kingdom.
cknowledgements
We thank Elizabeth Moiseeva for excellent histology and Marie-ine Escande for EM preparation. This study was supported by
arine Genomics Europe Network of Excellence (EDD Node) andy the Israel Science Foundation (1342/08 and 68/10).
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