Class
Article
College
College of Agriculture and Applied Sciences
Department
Animal, Dairy, and Veterinary Sciences Department
Faculty Mentor
Heloisa Rutigliano
Presentation Type
Poster Presentation
Abstract
During pregnancy, the maternal immune system must be altered to allow the tolerance of a semi-allogeneic fetus. Our previous studies show a strong inflammatory response in the uterus of cows carrying somatic cell nuclear transfer (SCNT) pregnancies due to dysregulation in the proteins expressed by trophoblast cells. Between 30 and 90 days the rate of pregnancy loss is 50-100% for SCNT compared to 2-10% for artificial insemination (AI) pregnancies. Abnormal immunological crosstalk during placentation is a major cause of this loss in SCNT pregnancies. The trafficking of extracellular vesicles (EVs), membrane-bound cargo carriers, potentially represents a key form of crosstalk between the conceptus and endometrium at the fetal-maternal interface. The aims of this study are to determine the role of trophoblast-derived EVs in healthy bovine pregnancies established by AI and abortion-prone pregnancies established by SCNT. We hypothesize that EVs from SCNT pregnancies will stimulate T cells to express more pro-inflammatory mediators and to proliferate when stimulated compared to T cells treated with EVs from AI pregnancies.Pregnancies were established by AI or SCNT (n = 6/group) and collected at 42±3 days, a time of major embryonic loss in cattle. Placental tissue was digested and cultured in EV-depleted medium at a density of 500 cells/well for 21 days. EVs were isolated from trophoblast supernatant by size exclusion chromatography. Peripheral blood mononuclear cells (PBMCs) were collected from day 35-70 AI pregnant cows and isolated by density gradient centrifugation. PBMC populations were sorted using flow cytometry, plated at an average density of 5,000 cells/well (44-96 wells/cow), and treated with 107 to 105 EVs/well. Cells were cultured for 24 hours then frozen at -80°C. Reverse transcription quantitative polymerase chain reactions using primers for pro- and anti-inflammatory genes were performed on the collected cells using the high-throughput Fluidigm BioMark system. Expression was evaluated for 18 genes, including CD28, CTLA4, IFNG, IL1B, IL2, TXB21, and IL10. Experimental data were analyzed as a randomized block design using SAS® University Edition Version 3.8, where block was cow and experimental unit was the cell culture well. The interaction between treatment and block was investigated.Our data reveal changes in the relative gene expression level of several genes in CD4+ populations between AI EV-treated cells and SCNT EV-treated cells. As hypothesized, SCNT EVs upregulated the expression of pro-inflammatory genes, such as IFNG, IL2, and TXB21, compared to AI EVs, and AI EVs increased expression of anti-inflammatory genes, including CTLA4 and IL10, when compared to SCNT EVs. Interestingly, SCNT EVs also increased expression of anti-inflammatory gene CTLA4, and AI EVs enhanced pro-inflammatory expression of IL1B. This suggests that, rather than establishing either a solely anti-inflammatory or pro-inflammatory phenotype, EVs from both healthy and abortion-prone pregnancies may orchestrate a complex interplay between the expression of anti- and pro-inflammatory genes.
Location
Logan, UT
Start Date
4-12-2023 12:30 PM
End Date
4-12-2023 1:30 PM
Included in
The Role of Extracellular Vesicles in Immunomodulation During Bovine Pregnancy
Logan, UT
During pregnancy, the maternal immune system must be altered to allow the tolerance of a semi-allogeneic fetus. Our previous studies show a strong inflammatory response in the uterus of cows carrying somatic cell nuclear transfer (SCNT) pregnancies due to dysregulation in the proteins expressed by trophoblast cells. Between 30 and 90 days the rate of pregnancy loss is 50-100% for SCNT compared to 2-10% for artificial insemination (AI) pregnancies. Abnormal immunological crosstalk during placentation is a major cause of this loss in SCNT pregnancies. The trafficking of extracellular vesicles (EVs), membrane-bound cargo carriers, potentially represents a key form of crosstalk between the conceptus and endometrium at the fetal-maternal interface. The aims of this study are to determine the role of trophoblast-derived EVs in healthy bovine pregnancies established by AI and abortion-prone pregnancies established by SCNT. We hypothesize that EVs from SCNT pregnancies will stimulate T cells to express more pro-inflammatory mediators and to proliferate when stimulated compared to T cells treated with EVs from AI pregnancies.Pregnancies were established by AI or SCNT (n = 6/group) and collected at 42±3 days, a time of major embryonic loss in cattle. Placental tissue was digested and cultured in EV-depleted medium at a density of 500 cells/well for 21 days. EVs were isolated from trophoblast supernatant by size exclusion chromatography. Peripheral blood mononuclear cells (PBMCs) were collected from day 35-70 AI pregnant cows and isolated by density gradient centrifugation. PBMC populations were sorted using flow cytometry, plated at an average density of 5,000 cells/well (44-96 wells/cow), and treated with 107 to 105 EVs/well. Cells were cultured for 24 hours then frozen at -80°C. Reverse transcription quantitative polymerase chain reactions using primers for pro- and anti-inflammatory genes were performed on the collected cells using the high-throughput Fluidigm BioMark system. Expression was evaluated for 18 genes, including CD28, CTLA4, IFNG, IL1B, IL2, TXB21, and IL10. Experimental data were analyzed as a randomized block design using SAS® University Edition Version 3.8, where block was cow and experimental unit was the cell culture well. The interaction between treatment and block was investigated.Our data reveal changes in the relative gene expression level of several genes in CD4+ populations between AI EV-treated cells and SCNT EV-treated cells. As hypothesized, SCNT EVs upregulated the expression of pro-inflammatory genes, such as IFNG, IL2, and TXB21, compared to AI EVs, and AI EVs increased expression of anti-inflammatory genes, including CTLA4 and IL10, when compared to SCNT EVs. Interestingly, SCNT EVs also increased expression of anti-inflammatory gene CTLA4, and AI EVs enhanced pro-inflammatory expression of IL1B. This suggests that, rather than establishing either a solely anti-inflammatory or pro-inflammatory phenotype, EVs from both healthy and abortion-prone pregnancies may orchestrate a complex interplay between the expression of anti- and pro-inflammatory genes.