of April 24, 2012This information is current as http://www.jimmunol.org/content/188/9/4181doi:10.4049/jimmunol.11035092012; 2012;188;4181-4187; Prepublished online 4 AprilJ Immunol and Douglas F. AntczakMargaret M. Brosnahan, Donald C. Miller, Mackenzie Adams ) Chorionic GirdleEquus caballusthe Equine (IL-22 Is Expressed by the Invasive Trophoblast ofReferences http://www.jimmunol.org/content/188/9/4181.full.html#ref-list-1, 17 of which can be accessed free at:cites 52 articlesThis article Subscriptions http://www.jimmunol.org/subscriptions is online atThe Journal of ImmunologyInformation about subscribing to Permissions http://www.aai.org/ji/copyright.htmlSubmit copyright permission requests atEmail Alerts http://www.jimmunol.org/etoc/subscriptions.shtml/Receive free email-alerts when new articles cite this article. Sign up at Print ISSN: 0022-1767 Online ISSN: 1550-6606.Immunologists, Inc. All rights reserved.by The American Association ofCopyright ©2012 9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc., is published twice each month byThe Journal of Immunology on April 24, 2012www.jimmunol.orgDownloaded fromThe Journal of ImmunologyIL-22 Is Expressed by the Invasive Trophoblast of the Equine(Equus caballus) Chorionic GirdleMargaret M. Brosnahan, Donald C. Miller, Mackenzie Adams, and Douglas F. AntczakThe invasive trophoblast cells of the equine placenta migrate into the endometrium to form endometrial cups, dense accumulationsof trophoblast cells that produce equine chorionic gonadotropin between days 40 and 120 of normal pregnancy. The mechanisms bywhich the trophoblast cells invade the endometrium while evading maternal immune destruction are poorly defined. A gene ex-pression microarray analysis performed on placental tissues obtained at day 34 of gestation revealed a >900-fold upregulation ofmRNA encoding the cytokine IL-22 in chorionic girdle relative to noninvasive chorion. Quantitative RT-PCR assays were used toverify high expression of IL-22 in chorionic girdle. Additional quantitative RT-PCR analysis showed a striking increase in IL-22mRNA expression in chorionic girdle from days 32 to 35 and an absence of IL-22 expression in other conceptus tissues. Bio-informatic analysis and cDNA sequencing confirmed the predicted length of horse IL-22, which carries a 39 extension absent in IL-22 genes of humans and mice, but present in the cow and pig. Our discovery of IL-22 in the chorionic girdle is a novel finding, asthis cytokine has been previously reported in immune cells only. IL-22 has immunoregulatory functions, with primary action onepithelial cells. mRNA of IL-22R1 was detected in pregnant endometrium at levels similar to other equine epithelia. Based uponthese findings, we hypothesize that IL-22 cytokine produced by the chorionic girdle binds IL-22R1 on endometrium, serving asa mechanism of fetal-maternal communication by modulating endometrial responses to trophoblast invasion. The Journal ofImmunology, 2012, 188: 4181–4187.The mechanisms that enable feto-placental tissues to evadedestruction by the maternal immune system are a long-standing focus of scientific investigation. In the decadessince Medawar proposed the “fetus as allograft” model, reviewedby Billington (1), research has implicated a complex communi-cation between trophoblast, maternal immune cells, and endo-metrium. Examples include production of IL-4 and IL-10 (2),HLA-G, IDO (3), and complement-regulatory proteins (4) bytrophoblast, expression of RCAS1 by endometrium (3), andFOXP3+regulatory T cells at sites of trophoblast invasion (5).Migration and endometrial invasion are attributes of trophoblastcells in many species, with the binucleate equine chorionic girdle(CG) cells being one example of this invasive phenotype (6). Deepinvasion of trophoblast is thought to have played a role in humanevolution by facilitating development of the human brain, yet thisprocess also brings increased risk of immune-related placentaldysfunction and diseases such as pre-eclampsia (7). Some im-munomodulatory molecules (e.g., galectins) are proposed to haveevolved in tandem with specific forms of p lacentation (7, 8).Trophoblast cells may also be novel sources of molecules pro-duced by immune cells in adult organisms, as in the production ofmacrophage migration-inhibitory factor by human villous cyto-trophoblasts (9) and murine trophoblast giant cells (10).Using gene expression array analysis comparing invasive andnoninvasive equine trophoblast, we identified novel production ofthe immunomodulatory cytokine IL-22 by CG cells just prior totheir migration through the endometrium to form the binucleate,chorionic gonadotropin-producing endometrial cups (11). IL-22is a member of the IL-10 family of cytokines (12), and is involv edin mucosal immunity and the maintenance and repair of epithelia(13–16). Since its first description in 2000 in human and mouseT cells (17, 18), IL-22 has been documented exclusively in im-mune cells, including Th subsets (Th17, T h22), NK cells, andbovine gd T cells (13, 19–22).IL-22 acts upon a heterodimeric receptor composed of its pri-mary target IL-22R1 and IL-10R2 (23). This receptor is expressedon epithelial surfaces, including respiratory (24) and digestivetracts and skin (25). Binding of IL-22R1 by IL-22 activatestranscription factors STAT3, STAT1, or STAT5 (26), and regulatesgenes associated with innate immunity (27) and cellular differ-entiation, migration, and survival (28, 29). A second receptor, IL-22R2, is a soluble binding protein thought to block downstreamfunctions of IL-22 (30, 31). This study presents our initialmicroarray finding of IL-22 expression by CG cells, substantiatesand expands upon this using quantitative RT-PCR (qRT-PCR) andbioinformatics, and identifies potential targets expressing IL-22R1mRNA.Materials and MethodsAnimalsMares of various breeds, ages, and parity were bred by artificial insemi-nation to thoroughbred stallions using techniques previously described (32).All horses were owned by the Cornell Center for Equine Genetics andmaintained in a herd setting. Procedures were performed in accordancewith an animal care and use protocol approved by the Institutional AnimalCare and Use Committee of Cornell University.Baker Institute for Animal Health, College of Veterinary Medicine, Cornell Univer-sity, Ithaca, NY 14853Received for publication December 6, 2011. Accepted for publication February 19,2012.This work was
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