Tumor Necrosis Factor Superfamily: Expression and Regulation at the Maternal-conceptus Interface in Pigs

Inkyu Yoo,Yoon Chul Kye,Jisoo Han,Soohyung Lee,Wonchul Jung,Tae Sub Park,Cheol-Heui Yun,Hakhyun Ka 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

For the establishment of successful pregnancy the maternal immune system must tolerate the implanting semi-allogenic conceptus, but the mechanism underlying this process is not fully understood in pigs. Among many factors, members of the tumor necrosis factor superfamily (TNFSF) have been considered as important immune regulators in cell-mediated immunity. TNFSF members bind to their responsive receptor containing cytoplasmic death domain to induce apoptosis in immune cells. Action of TNFSF members at the during pregnancy has been studied in some species including humans, mice and cows, suggesting that the TNFSF-induced apoptosis of activated maternal immune cells at the maternal-conceptus interface may be one of the mechanisms against rejection of semi-allogenic fetus. However, the expression and function of TNFSF members at the maternal-conceptus interface have not fully understood in pigs. Thus, to initiate the study on the role of TNFSF members for the establishment of pregnancy, we determined the expression of the TNFSF members, CD40 ligand (CD40LG), FAS ligand (FASLG), TNFα and TNF-related apoptosis inducing ligand (TRAIL; TNFSF10) in the endometrium and conceptus tissues during pregnancy in pigs. Real-time RT-PCR analysis showed that CD40LG, FASLG, TNFα and TNFSF10 mRNAs were expressed in the uterine endometrium during the estrous cycle and pregnancy. Levels of CD40LG, FASLG, TNF-α and TNFSF10 mRNA in endometrial tissues significantly increased on Day 15 of pregnancy, and levels of FASLG, TNF-α and TNFSF10 were also high on Day 60 of pregnancy and decreased thereafter. Immunohistochemical analysis showed that CD40LG and TNFSF10 proteins were localized mainly to luminal epithelial (LE) cells on Day 15 of pregnancy and amniotic membrane during late pregnancy, while FASLG protein was localized to LE cells during Day 30 to term and glandular epithelial cells during the estrous cycle and pregnancy. To understand the regulatory mechanism of endometrial CD40LG, FASLG and TNFSF10 expression by conceptus-derived cytokines, we treated endometrial tissues with increasing doses of interferon-γ (IFNG) and interferon-δ (IFND) and found that IFNG increased the expression of CD40L, TNFα and TNFSF10 mRNA, but not FASLG mRNA, and IFND increased TNFSF10 mRNA. To further understand the role of TNFSF10 on apoptosis of immune cells at the maternal-conceptus interface, we measured cell death of peripheral blood mononuclear cells by uterine epithelial cells expressing TNFSF10, and found that TNFSF10-expressing epithelial cells decreased survival of immune cells, especially of myeloid lineage. These results showed that CD40LG, FASLG, TNFα and TNFSF10 were expressed in a cell-type and stage-specific fashion in the endometrium during pregnancy, suggesting that CD40LG, FASLG, TNF and TNFSF10 may play an important role in the establishment of pregnancy by regulating the maternal immune response at the maternal-conceptus interface in pigs.

Tumor Necrosis Superfamily: Expression and Regulation at the Maternal- Conceptus Interface in Pigs

Inkyu Yoo,Jisoo Han,Hwanhee Jang,Soogil Chae,Soohyung Lee,Hakhyun Ka 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

For the establishment of successful pregnancy the maternal immune system must tolerate the implanting semi-allogenic conceptus, but the mechanism underlying this process is not fully understood in pigs. Among many factors, members of the tumor necrosis factor superfamily (TNFSF) have been considered as important immune regulators in cell-mediated immunity. TNFSF members bind to their responsive receptor containing cytoplasmic death domain to induce apoptosis in immune cells. Action of TNFSF members at the maternal-fetal interface during pregnancy has been studied in some species including humans, mice and cows, suggesting that apoptosis of activated maternal immune cells in the uterine endometrium is a defense mechanism against rejection of semi-allogenic fetus. However, the expression and function of TNFSF members at the maternal-conceptus interface in pigs have not fully understood. Thus, to initiate the study on the role of TNFSF members for the establishment of pregnancy, we determined the expression of CD40 ligand (CD40LG), FAS ligand (FASLG) and TNFrelated apoptosis inducing ligand (TRAIL; TNFSF10) in the uterine endometrium and conceptus tissues during pregnancy in pigs. We obtained endometrial tissues from day (D) 12 and D15 of the estrous cycle, and D12, D15, D30, D60, D90, and D114 of pregnancy, conceptus tissues on D12, D15, and chorioallantoic tissues on D30, D60, D90, and D114 of pregnancy in pigs. Real-time RT-PCR analysis showed that CD- 40LG, FASLG and TNFSF10 mRNAs were expressed in the uterine endometrium during the estrous cycle and pregnancy. Levels of CD40LG, FASLG and TNFSF10 mRNA in endometrial tissues were significantly increased on D15 of pregnancy and levels of FASLG and TNFSF10 were also high on D60 of pregnancy and decreased thereafter. Chorioallantoic tissues also expressed CD40LG, FASLG and TNFSF10 mRNA during mid- to late pregnancy. Immunohistochemical analysis showed that CD40LG and TNFSF10 proteins were mainly localized to luminal epithelial (LE) cells on D15 of pregnancy and amniotic membrane during late pregnancy, while FASLG protein was localized to LE cells during D30 to term and glandular epithelial cells during the estrous cycle and pregnancy. To understand the regulatory mechanism of endometrial CD- 40LG, FASLG and TNFSF10 expression by interferon gamma (IFNG), which is pro- duced by the implanting conceptus, we treated endometrial tissues with increasing doses of IFNG and found that IFNG increased the expression of FASLG and TNFSF10 mRNA, but not CD40LG mRNA. These results indicate that CD40LG, FASLG and TNFSF10 are expressed in a cell-type and stage-specific fashion in the uterine endometrium and chorioallantoic tissues during pregnancy, suggesting that CD40LG, FASLG and TNFSF10 may play an important role in the establishment of pregnancy by regulating the maternal immune response at the maternal-conceptus interface in pigs.

Pro-inflammatory Cytokines and Their Receptors: Expression and Regulation in the Uterine Endometrium during the Estrous Cycle in Pigs

Yoo, Inkyu,Kim, Minjeong,Han, Jisoo,Jang, Hwanhee,Choi, Sun-Ho,Ka, Hakhyun The Korean Society of Embryo Transfer 2016 한국동물생명공학회지 Vol.31 No.4

Pro-inflammatory cytokines, interleukin-$1{\beta}$(IL1B), IL6, and tumor necrosis factor-alpha (TNF), are known to play important roles in regulating the endometrial function in the uterus during the estrous cycle and pregnancy in several species. However, the expression and function of these cytokines and their receptors in the uterine endometrium during the estrous cycle have not been studied in pigs. Thus, this study determined the expression and regulation of IL1B, IL6, TNF and their respective receptors, IL1R1, IL1RAP, IL6R, GP130, TNFRSF1A, and TNFRSF1B during the estrous cycle in pigs. To analyze levels of each gene expression in the uterine endometrium we obtained from endometrial tissues on Days 0, 3, 6, 9, 12, 15, and 18 of the estrous cycle. Real-time RT-PCR analysis showed that levels of IL1B, IL1RAP, IL6R, GP130, TNF, TNFRSF1A, and TNFRSF1B mRNAs were highest on Day 15 or 18 of the estrous cycle, which corresponds to the proestrus period. Levels of IL1R1 were highest on Day 0, while levels of IL6 were biphasic with high levels on Day 6 and Day 15. The abundance of IL1B, IL6, IL6R, and TNF mRNAs was decreased by progesterone, while levels of GP130 were increased by progesterone in endometrial tissue explants. These results showed that expression of pro-inflammatory cytokines and their receptors changed stage-specifically during the estrous cycle and regulated by progesterone in the uterine endometrium in pigs, suggesting that these pro-inflammatory cytokines may be involved in the regulation endometrial function during the estrous cycle in pigs.

Analysis of The CD40/CD40L System in The Uterine Endometrium During Pregnancy in Pigs

Inkyu Yoo,Jisoo Han,Minjeong Kim,Hwanhee Jang,Soogil Chae,Suhyung Lee,Hakhyun Ka 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10

For the establishment and maintenance of successful pregnancy the maternal immune system must tolerate semi-allogenic fetus during pregnancy. Several mechanisms explaining immune tolerance have been proposed. Among those, it has been suggested that the CD40/CD40L system is involved in immune tolerance in several tissues. However, expression and function of CD40/CD40L in the maternal-fetal interface during pregnancy have not been studied in pigs. Thus, this study determined expression and localization of CD40 and CD40L in the uterine endometrium during pregnancy in pigs. We obtained uterine endometrial tissue samples from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Quantitative real-time PCR analysis showed that levels of CD40L mRNA expression during pregnancy increased on D15 of pregnancy and decreased thereafter whereas levels of CD40 mRNA was highest on D30 of pregnancy. Localization of CD40 and CD40L proteins by immunohistochemistry showed that CD40 was localized to vascular endothelial cells with strongest signal intensity on D15 of pregnancy, and CD40L was localized to luminal epithelial cells on D15 of pregnancy and amniotic membrane during mid- to late pregnancy. To determine the effect of IFNG on CD40 and CD40L expression, we took advantage of endometrial explant culture using tissues from D12 of the estrous cycle, and found that CD40 was up-regulated by IFNG in a dose-dependent manner. These results showed that CD40 and CD40L were expressed in the uterine endometrium in a cell-type and stage-specific fashion during pregnancy, and IFNG induced CD40, indicating that the CD40/CD40L system may be important for establishment and maintenance of pregnancy in pigs. [Supported by the Next Generation BioGreen21 Program (#PJ01110301), Rural Development Administration]

Class II Trans Activator and Major Histocompatibility Complex Class II Molecules: Expression at the Maternal-Conceptus Interface in Pigs

Inkyu Yoo,Jisoo Han,Soogil Chae,Soohyung Lee,Hakhyun Ka 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

For the successful pregnancy maternal immune response must be regulated to tolerate the semi-allogenic conceptus. In adaptive immunity, major histocompatibility complex class II (MHC class Ⅱ) molecules play critical role in presenting foreign antigens to the other immune cells. During early pregnancy in pigs, various types of endometrial cells, including epithelial cells, vascular endothelial cells, stromal cells, and infiltrated immune cells are activated by conceptus-derived interferon gamma (IFNG) and express immune regulatory molecules. Therefore, we determined the expression and regulation of MHC class II molecules, swine leukocyte antigen (SLA) -DM, -DO, -DR and -DQ and their transcription coactivator CIITA at the maternal-conceptus interface. We obtained endometrial tissue samples from day (D) 12 and D15 of the estrous cycle and D12, D15, D30, D60, D90 and D114 of pregnancy. Quantitative real-time PCR anaysis showed that mRNA levels of all genes dramatically increased on D15 of pregnancy. To determine the effect of interferon-gamma (IFNG) on SLAs and CIITA expression, we took advantage of explant culture and found that all of SLAs and CIITA were up-regulated by IFNG in a dose-dependent manner. Immunohistochemistry showed that CIITA protein was localized to vascular endothelial cells and stroma near the luminal epithelium on D15 of pregnancy. SLA-DQ protein is localized to stromal leukocytes and vascular endothelial cells during the estrous cycle and early pregnancy with the highest signal intensity on D15 of pregnancy. These results indicate that CIITA and MHC class II molecules are expressed in the uterine endometrium in a cell-type and stage-specific fashion during pregnancy, and may play a critical role in regulation of maternal immune response to support the establishment and maintenance of pregnancy at the maternal- conceptus interface in pigs.

Analysis of IFNG-regulated genes in the uterine endometrium during early pregnancy in pigs

Inkyu Yoo,Yohan Choi,Heewon Seo,Jisoo Han,Minjeong Kim,Hakhyun Ka 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8

The implantation process in pigs is initiated when the conceptus begins secretion of estrogen, the signal for maternal recognition of pregnancy, and cytokines including interleukin-1β(IL1B), interferon delta (IFND) and interferon gamma (IFNG). Our previous study showed that IFNG receptors, IFNGR1 and IFNGR2, were expressed in the uterine endometrium during the estrous cycle and early pregnancy. However, the molecular and cellular mechanism of IFNG in the uterine endometrium in pigs is poorly understood. To determine the role of IFNG on the uterine endometrium during the implantation period, we took advantage of RNA-Seq analysis using explant tissues treated with IFNG in the presence of estrogen and progesterone, and found that many genes including CXCL9, CXCL10, CXCL11, IDO1, IL15, IL15RA, TNFSF10 (TRAIL), and WARS were up-regulated by IFNG. Additional analysis in the uterine endometrial tissues from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy determined the expression of these IFNG-regulated genes in pigs by quantitative real-time PCR Results showed that expression of CXCL9, CXCL10, and IDO1 dramatically increased on D15 of pregnancy, and expression of CXCL11 and TNFSF10 was high during mid- to term pregnancy. These results indicate that IFNG regulates immune-associated genes in the uterine endometrium in a stage-specific fashion during pregnancy, and may play a critical role to support the establishment and maintenance of pregnancy at the fetomaternal interface in pigs.

Class II Transactivator Expression in the Uterine Endometrium during Pregnancy in Pigs

Inkyu Yoo,Jisoo Han,Minjeong Kim,Hwanhee Jang,Hakhyun Ka 한국동물생명공학회(구 한국동물번식학회) 2015 발생공학 국제심포지엄 및 학술대회 Vol.2015 No.10

Analysis of IL6 and Its Receptor Expression at the Maternal-Conceptus Interface during Pregnancy in Pigs

Inkyu Yoo,Yohan Choi,Jisoo Han,Minjeong Kim,Hwanhee Jang,Hakhyun Ka 한국동물생명공학회(구 한국동물번식학회) 2014 Reproductive & Developmental Biology(Supplement) Vol.38 No.2s

Analysis of CIITA Expression in the Uterine Endometrium during Pregnancy in Pigs

Inkyu Yoo,Yohan Choi,Heewon Seo,Hakhyun Ka 한국동물생명공학회(구 한국동물번식학회) 2013 Reproductive & Developmental Biology(Supplement) Vol.37 No.2s

Analysis of Indoleamine-2,3-Dioxygenase 1 (IDO1) in the Uterine Endometrium during Pregnancy in Pigs

Inkyu Yoo,Yohan Choi,Heewon Seo,Jisoo Han,Minjeong Kim,Hakhyun Ka 한국동물생명공학회(구 한국동물번식학회) 2013 발생공학 국제심포지엄 및 학술대회 Vol.2013 No.1