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Placenta 30, Supplement A, Trophoblast Research, Vol. 23 (2009) S26–S31 Contents lists available at ScienceDirect Placenta journal homepage: www.elsevier.com/locate/placenta Comparison of Immune Cell Recruitment and Function in Endometrium During Development of Epitheliochorial (Pig) and Hemochorial (Mouse and Human) Placentas B.A. Croy a, *, J. Wessels b, N. Linton b, C. Tayade b a b Department of Anatomy and Cell Biology, Queen’s University, Room 924 Botterell Hall, Kingston, ON K7L 3N6, Canada Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1 a r t i c l e i n f o a b s t r a c t Article history: Accepted 30 September 2008 The role of maternal immune cells in early implantation sites has received special attention from reproductive biologists because immune cells participate in tissue transplant rejection. During normal pregnancy, endometrial immune cells differ from those in blood by subset distribution and appear to be activated but non-destructive of conceptuses. The immune system evolved well before placental mammals. By comparing the regulation and functions of endometrial immune cells between species in two phylogenetic clades that model differently evolved placental types (pig (Sus scrofa) versus mouse (Mus musculus) and human (Homo sapiens)), we seek to understand how ‘‘non-self’’ trophoblast cells thrive in most pregnancies. Our studies suggest recruitment of specific immune cells to conceptusassociated endometrium and immune cell-promoted endometrial angiogenesis are of key importance for mammalian conceptus well-being. Ó 2009 Published by IFPA and Elsevier Ltd. Keywords: Angiogenesis Chemokine Chemokine decoy Dendritic cell Lymphocyte Trophoblast Uterine Natural Killer cell 1. INTRODUCTION The immune system evolved millions of years before placental mammals appeared (Fig. 1A) [1,2]. Components of innate immunity, including cells that resembled Natural Killer (NK) lymphocytes, molecules related to NK cell receptors and the major histocompatibility complex are estimated to have evolved >600 million years ago (Mya), prior to the first whole genome duplication. About 60 Mya afterwards, the recombinase activating genes (RAG) appeared that control somatic cell recombination in T and B lymphocytes, and thus adaptive immunity. Prior to this, antigen specific receptors, clonal selection and immunological memory would not have existed [3]. Evolution of placental mammals followed that of oviparous mammals and began w100 Mya. It is estimated that mouse (Mus musculus) placentation appeared w32 Mya, that of humans (Homo sapiens) w35 Mya and that of pigs (Sus scrofa) w55 Mya [4]. These relative relationships are shown in Fig. 1. Mammalian evolution would then result as reproductive strategies not discarded as incompatible with immune mechanisms and different immunological strategies may have evolved between species. * Corresponding author. Tel.: þ1 613 533 2859; fax: þ1 613 533 2566. E-mail address: [email protected] (B.A. Croy). 0143-4004/$ – see front matter Ó 2009 Published by IFPA and Elsevier Ltd. doi:10.1016/j.placenta.2008.09.019 Many investigators have addressed the functions of the maternal immune system during pregnancy in humans and mice and found considerable similarities. Both species belong to the molecular phylogenetic clade Euarchontoglires and are classified as having hemochorial placentation (Fig. 1B). That is, uterine epithelial cells are lost during conceptus implantation and an endometrial decidual response is induced. Many fewer immunological investigations have been reported on other species. We study the pig as an earlier placental mammal from a distinct molecular phylogenetic clade, the Laurasiatheria [4]. Pigs use epitheliochorial placentation. This means that epithelial cells are retained at the uterine lumen surface, there is no induction of endometrial stromal cell decidualization and that trophoblast does not invade maternal tissue but rather expands to cover a large maternal surface [5]. Here, our major immunological findings to date from studies of a commercial meat pig, the Yorkshire, are reviewed and contrasted with current understanding of murine and human implantation site immunology. Our major approaches have been real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) for comparisons of gene expression between endometrium and trophoblast from the same conceptus attachment site. In many cases, laser capture microdissection (LCM) was used to isolate immune cells or endometrial endothelial cells from histological sections prepared from conceptus attachment sites. Relative gene B.A. Croy et al. / Placenta 30, Supplement A, Trophoblast Research, Vol. 23 (2009) S26–S31 S27 2.2. Endometrial immune cells in early pig pregnancy Fig. 1. Illustration of the estimated evolutionary time scale in millions of years (Myr) for development of the immune system and placental mammals (left) and for the phylogenetic clades of mammals (right). Self–non-self discrimination was the first immune trait to evolve. It used innate immune processes such as evolution of transplantation antigens (MHC) and receptor (R)-bearing Natural Killer (NK) cells. Appearance of the duplicated Recombinase activating genes (RAG1, RAG2) permitted somatic recombination and evolution of adaptive immunity. RAG1 and RAG2 are used by T and B lymphocytes to produce their wide ranges of exquisitely specific antigen surface receptors. expression in these pure populations of cells was compared with that in the endometrial and trophoblast biopsies. 2. FUNCTIONAL MICRODOMAINS ARE PRESENT WITHIN THE PORCINE GESTATIONAL UTERUS 2.1. Gene expression is not uniform throughout the porcine conceptus attachment site Pregnancy is 114 days in commercial pigs. Blastocysts begin to attach at gestation day (gd)12 on the mesometrial side of the uterus but several days elapse before firm attachment is achieved [6]. At this time, porcine blastocysts produce oestradiol (E2), interferon (IFN)-gamma and -delta [7]. These are not major known products of mouse or human blastocysts [8,9]. At gd19, we examined the levels of transcription of endometrial IFNG, tumour necrosis factor-alpha (TNF) and vascular endothelial growth factor (VEGFA), products of murine and human uterine Natural Killer (uNK) cells. In comparison to mesometrial samples from virgin gilts (female pigs at puberty), gd19 mesometrial tissue had significantly elevated transcription of these three genes [10]. In anti-mesometrial endometrium, only transcription of TNF was increased. A greater increase in TNF occurred mesometrially. Thus, positionally-defined microdomains of immune gene expression occur in porcine endometrium during conceptus attachment. The mouse blastocyst attaches on the opposite side of the uterus (anti-mesometrial), inducing there, decidualization and changes in gene transcription. Lateral regions of the uterus then decidualize and finally the mesometrial region [11] where, in mice and pigs, the suspensory mesentery serves as a conduit for the uterine vessels that will supply placentae. No decidualization occurs between mouse implantation sites. We examined gene expression in mesometrial pig endometrium between gd19 conceptus attachment sites. Neither IFNG nor VEGFA transcription was elevated above that in non-pregnant tissue but TNF transcripts were equivalent in abundance between and at attachment sites [10]. This suggests gestational TNF transcription in porcine endometrium may be induced endocrinologically rather than by conceptus attachment. Oestrous cycles and pregnancy modify immune cell subsets in the porcine endometrium and uterine epithelium [12,13]. Again, this is similar to mice, humans and many other mammals. We observed that pregnancy induced mild skewing in these subsets with enrichment of uNK cells and their transient expression of cytotoxicity from gd12 that was abruptly terminated at gd28 [14,15]. Porcine uNK cells show association with uterine glands as seen in humans [16] but not mice, and with blood vessels, as seen in both humans and mice [16,17]. Porcine uNK cells are found below the luminal epithelium, absent in humans and mice as well as in the endometrial stroma, equivalent to the uNK cell-rich decidua basalis of humans and mice. Uterine NK cells become abundant rapidly during decidualization in mice (post-implantation) and humans (late secretory phase of each menstrual cycle and early pregnancy) but cytotoxic activity is not easily demonstrated ex vivo [16,17]. Recent work using multiparous sows of a different meat breed and earlier study time points suggests uNK cells are lost from pig attachment regions and move into deeper endometrial regions [13]. This would position the porcine uNK cell population closer to the uNK cell positions found in mice. We reported that presence of a blastocyst was essential to shift lymphocyte subsets in pigs [18]. This is not true in mice or humans. In mice, artificial deciduomata induce differentiation of numerous uNK cells, likely via initiation of synthesis of interleukin-15 (IL15), an essential viability factor for the progenitors of NK cells [19–21]. In women, ectopic pregnancy sites are devoid of decidua and uNK cells but both are found within the uterus as long as the ectopic gestation continues [22]. Thus, progesterone (P4) has more obvious effects on endometrial immune cells in mice and humans than in pigs. This effect appears to be indirect as lymphocytes in murine implantation sites do not express progesterone receptor (PGR) [23]. Studies in humans have suggested that immature dendritic cells (DC) recognized by anti- CD209/DC-SIGN have a contact relationship with uNK cells in early decidua [24,25]. We found that antihuman CD209 marked a relatively rare cell of stellate morphology in virgin and in gd20 and gd50 pig endometrium. These DC-like cells frequently localized with blood vessels but appeared to have random positional relationships with lymphocytes [26]. 2.3. Chemokines and chemokine decoy receptors at the maternal–fetal interface Chemokines are small cytokine molecules important in the positioning of immune cells, endothelial progenitors cells and other cell types within developing tissues. There are >40 known chemokines that are subclassified structurally. An alternative classification is whether a chemokine contributes to recruitment of cells that promote inflammation or to cells that promote homeostasis. The former are more common. Most chemokines bind to more than one signalling receptor but also to non-signalling, decoy receptors [27]. Three of these are known and reported to be expressed by mouse and human trophoblast. Decoy receptors D6 and DARC bind and target the degradation of inflammatory chemokines [28] while Chemocentryx decoy receptor (CCX CKR) targets degradation of homeostatic chemokines (Fig. 2) [29,30]. Only two chemokines are known that are not ligands of the characterized decoy receptors. These are CCL1 and CXCL12. Decoy function is required in vivo to resolve inflammation [31,32]. We addressed the expression of chemokine decoy receptors in porcine attachment site endometrial biopsies and in sitematched trophoblast [33]. We successfully amplified porcine D6 and CCX CKR (GenBank accession numbers: DQ437505.1 and EU168334.1) but were unsuccessful using multiple primer pairs, in amplifying DARC, a gene not yet sequenced in pigs. Both D6 S28 B.A. Croy et al. / Placenta 30, Supplement A, Trophoblast Research, Vol. 23 (2009) S26–S31 Fig. 2. A list chemokines that bind to the decoy receptor molecules D6, DARC and CCX CKR. Some inflammatory chemokines bind to both D6 and DARC. and CCX CKR were expressed by porcine trophoblast. Both genes were also expressed at more abundant levels by co-localized endometrium during early pregnancy [33]. We also established that at least one ligand for all three known chemokine decoy receptors is found in porcine endometrium and trophoblast [33]. D6 and CCX CKR were demonstrated at gd20 and 50 by IHC using cross-reacting, anti-human reagents. Fig. 3 shows a photomicrograph of gd50 porcine trophoblast dual labelled for D6 and its ligand CCL4 (previously called MIP-1-beta, Act2 or Scya4). Expression of both D6 and CCL4 by trophoblasts and by endometrium suggests that intraepithelial as well as stromal positioning of cells chemoresponsive to CCL4 (i.e., those expressing CCR5), is co-regulated by D6. Pregnancies have been studied in mice genetically ablated for D6 [34]. Although implantation site morphology is slightly altered in this strain, large litters are carried to term. If pregnant D6 null mice are challenged by immunological protocols that provoke abortion (lipopolysaccharide or human anti-phospholipid antibodies), they are exquisitely sensitive to pregnancy failure. Thus, D6 is considered a non-redundant, conceptus-sparing molecule in pregnant mice [27,34]. Commercial meat pigs show two waves of spontaneous fetal loss. Rescue experiments indicate these are genetically normal conceptuses with full term potential (unpublished data). We did not find transcriptional changes in maternal or fetal D6 during porcine peri-attachment or mid pregnancy losses. In contrast, mid pregnancy loss was associated with both maternal Fig. 3. Photomicrographs of D6 decoy receptor expression (A), expression of CCL4, a D6 ligand (B) and an overlay to display receptor/ligand co-expression (C) in healthy gd50 paraffin-embedded porcine trophoblast. Sections were stained with rat anti-human D6 (R&D Systems) and anti-mouse and rat CCL4 (eBioscience Inc.) antibodies. D6: green; CCL4: red. Scale bar is 400 mm. Inset (D) is negative control. Background reactivity is non-specific binding to RBCs. Scale bar is 200 mm. B.A. Croy et al. / Placenta 30, Supplement A, Trophoblast Research, Vol. 23 (2009) S26–S31 S29 and fetal elevation in CCX CKR [33]. This suggests that removal of both maternal and fetal signals for homeostasis is a pathway contributing to fetal death in pigs. CCX CKR has not been studied in the endometrium of other species, leaving undefined the relevance of this strategy to pregnancy failure in other mammals. lymphocytes always made significant contribution to HIF1A abundance in endometrium. However, at sites of conceptus arrest, lymphocytes did not transcribe HIF1A, suggesting perhaps their early participation in blocking the maternal neoangiogenesis required to support a conceptus. 3. IMMUNE CELL FUNCTIONS WITHIN PORCINE CONCEPTUS ATTACHMENT SITES 3.2. Endometrial lymphocytes and DCs are angiogenic 3.1. Endometrial lymphocytes are sensors of their microenvironment Pattern recognition is a more primitive method of immune cell recognition than is use of antigen specific receptors. One of the major families of pathogen-associated recognition pattern molecules (PAMPs) is the Toll-like receptor (TLR) family, originally described in invertebrates [35]. TLRs signal in a manner analogous to IL1B and several TLRs play key roles in innate immune responses. Immature DC and monocytes/macrophages have the most abundant expression of TLRs. TLRs and other pattern recognition receptors have been described at the human and mouse maternal– fetal interface where they are postulated to prevent microbial infections of implantation sites [36]. First trimester human villous and extravillous trophoblasts express TLR2 and TLR4. TLR1 and TLR6 can form heterodimers with TLR2 and be co-localized with it [37]. Human endometrial cells also express TLRs, particularly lumen epithelial cells and uNK cells. The latter do not appear to respond directly to TLR ligands but become activated to produce IFNG by TLR-responding accessory cells [38]. TLR expression is less well studied at the mouse maternal–fetal interface. Sobill and her colleagues examined TLR transcription and function in cultured mouse uterine epithelial cells and found consistent expression of TLR1–6 and sporadic expression of TLR7–9 [39]. In random-bred mice, uterine expression of TLR2, 3, 4 and 9 was reported by Gonzalez et al., between midgestation and term in comparison to non-cycle-timed, virgin uterus. Transcripts for these four TLRs increased gradually as pregnancy progressed. Placenta also expressed these molecules dynamically with TLR4 decreasing significantly in late gestation. Western blotting and IHC verified transcription of the receptors [40]. Using LPS challenge of late gestation, inbred mice, Salminen et al, found induction of uterine and placental TLR2 transcription but no change in that of TLR4 [41] while Zhang and colleagues report outcomes in DBA-mated CBA females indicative of functional endometrial TLR3 [42]. Based upon these reports, we cloned TLRs1–10 for Sus scrofa [43] and asked whether the TLRs expressed by human trophoblast (TLR1, 2, 4, 6) are expressed at the porcine maternal–fetal interface. TLR2 expression was not found but porcine trophoblast and endometrium expressed TLR1, 4 and 6. Levels of TLR1 and TLR6 were similar in both trophoblasts and endometrium at both gd20 and 50. In contrast, peri-attachment TLR4 was greater in endometrium than in trophoblast while at midgestation, TLR4 transcripts had become more abundant in trophoblast than in endometrium. CD209/DCSIGNþ cells were among the porcine endometrial cell types expressing TLR4 and TLR6 but they did not express TLR1 [44]. Activation of TLR4 and other TLRs in mouse macrophages induces the expression of VEGF [45]. Another molecular sensor that appears to have a key role in human and mouse implantation sites is hypoxia inducible factor (HIF1) [46–48]. We examined porcine HIF1A expression in virgin endometrium and peri-implantation stage maternal and fetal tissues. Levels of transcription rose gradually within attachment sites (gd15, 19, 21, 23) from basal levels found in virgin endometrium and there were always relatively more endometrial than trophoblastic transcripts [10]. Transcripts from endometrial lymphocytes dissected at healthy attachment sites suggested that The process of enlargement of maternal vascular supply to placentas is different between pigs and mice. In pigs, expansion of the sub-epithelial capillary network is the major event while in mice, as in humans, structural and functional changes to spiral arteries are key [17,49]. VEGFA is the key molecule maintaining endothelial cell viability and driving their proliferation to initiate angiogenesis. To study porcine attachment sites, primers for VEGFA, Placenta growth factor (PGF) and VEGFRI and RII were used on samples from virgin, gd20 and gd50 gilts. Lymphocytes had a much greater abundance of VEGFA transcripts at all three time points than endometrial endothelial cells and than trophoblast from matched pregnancy sites [50]. PGF transcription was at similar abundance in lymphocytes and in endothelial cells in virgin endometrium but, during pregnancy, endothelium had more transcripts than lymphocytes and both had more transcripts than trophoblast. Lymphocytes preferentially expressed transcripts for VEGFRI rather than VEGFRII, the reverse of trophoblast. IHC confirmed expression of these four molecules [49,50]. Conceptus arrest was associated with a combined transcriptional loss of VEGFA and gain of PGF in lymphocytes and relatively constant receptor transcription. PGF has a decoy function for VEGFA that may enhance the reduction of endothelial cell viability achieved through loss of VEGFA transcription [51]. In human and mouse implantation sites, lymphocytes are reported to produce VEGFA, PGF and other angiogenic molecules [52–54]. Disruption of this pathway has been associated with human gestational complications, particularly pre-eclampsia ([55]. In mice, genetic loss of VEGFA is an embryonic lethal while loss of PGF has more minor, non-lethal effects [56]. 3.3. Endometrial lymphocytes transcribe FasL, Fas and pro-inflammatory type 1 cytokines Maturation and activation of lymphocytes leads to transcription of death pathway effector molecules and inflammatory cytokines. To address whether lymphocytes in virgin porcine endometrium and at conceptus attachment sites between gd15 and 50 were fully differentiated, activated cells, analyses of transcripts for FASLG, FAS, IFNG and TNF were undertaken. Basal transcription of both FASLG and FAS in lymphocytes from virgin uteri was not altered at gd15 but levels had begun to rise by gd19 and were significantly higher by gd 21 [50]. From gd6, mouse uNK cells express FASLG in their cytoplasmic granules and surface FAS [57]. Both molecules are also reported as functional in human uNK cells [58–60]. Pregnancy induced a gain in porcine lymphocyte transcription of IFNG but not of TNF unless the conceptus was experiencing midgestation arrest [50]. Interestingly, trophoblast showed much greater transcription of TNF than endometrium or endometrial endothelium or lymphocytes in healthy attachment sites. From day 6, mouse uNK cells release IFNG [61] and, by gd8, TNF [62]. The latter cytokine is granule localized [62]. Human uNK cells transcribe both molecules early during normal pregnancy [58]. 4. SUMMARY The biology of uterine and placenta attachment site specific lymphocytes in pigs appears to be very similar to that in mice and humans. To date, only minor differences in usage of TLRs have been S30 B.A. Croy et al. / Placenta 30, Supplement A, Trophoblast Research, Vol. 23 (2009) S26–S31 identified as distinct. Co-ordinated maternal and fetal elevation of CCX CKR, the decoy receptor responsible for degradation of homeostatic chemokines, during midgestation conceptus arrest, has been uniquely described but remains to be investigated in other species. Given the more ancient phylogenetic development of porcine placentation versus human and mouse, a more primitive porcine usage of immune cells might have been expected. The clade separation of these species also appears to be without effect, although blastocyst attachment site positions and placental structure differ greatly. Endometrial immune cells for the species we compared as samples from two of the four molecularly-defined clades of placental evolution appear to be environmental sensing cells able to participate in the process of ‘‘non-hard-wired’’ angiogenesis at individual placenta attachment sites. This convergence is consistent with the concept that placental evolution was accompanied by modification of the functional programs of an established primitive immune system in ways that increase the success of placental reproduction. 5. CONFLICT OF INTEREST The authors do not have any potential or actual personal, political, or financial interest in the material, information, or techniques described in this paper. Acknowledgements These studies have been supported by NSERC, OMAFRA, Ontario Pork, Agriculture and Agri-Food Canada, Bioniche Life Science, Inc. and the Canada Research Chairs’ Program. We thank Mr Kota Hatta for assistance in preparation of the illustrations. 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