(143) Bovine Conceptus Secreted Products Modulate Peripheral Blood Cytokine and Chemokine Release in Vitro.

Zachary K. Seekford¹; Thainá Minela¹; Brette Poliakiwski¹; Damon J. Smith¹; Izadora S. de Souza¹; Reinaldo F. Cooke¹; Carlos A. Rodriguez³; Kiril M. Dimitrov³; Fuller W. Bazer¹; Gregory A. Johnson²; Ky G. Pohler¹

¹Department of Animal Science, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX USA

²Department of Veterinary Integrative Biosciences, School of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX USA

³Texas A&M Veterinary Medical Diagnostic Laboratory, Texas A&M AgriLife, College Station, TX USA

Abstract Text:

Significant pregnancy loss occurs during the first 60 days of gestation in cattle. While many of these losses occur during the first 30 days of gestation, another period of loss occurs later in embryonic and placental development. Throughout gestation, there are dynamic changes in bidirectional communication between the conceptus (embryo/fetus and associated placental membranes) and endometrium. This signaling includes conceptus secreted products such as interferon tau (IFNT), prostaglandins (PG) F2α, PGE2, and pregnancy associated glycoproteins (PAGs). During early placental attachment (24–35 days), mono- and multinucleated trophectoderm cells begin secreting PGF2α, PGE2, and PAGs that are detectable in maternal blood. There are clear associations between reduced concentrations of PAGs in maternal blood and pregnancy failure. While the explicit functions of PAGs have yet to be elucidated, PAGs may mediate immune tolerance of the conceptus by modulating maternal immune function. We hypothesized that conceptus secreted products would alter cytokine and chemokine release by peripheral blood leukocytes. Nulliparous cycling beef heifers (n = 5; 303.3 ± 21.3 kg) underwent estrus synchronization. Corpora lutea size, concentrations of progesterone in plasma, and complete counts of leukocytes were measured 15 days after ovulation. Whole blood collected 15 days after ovulation was then subjected to in vitro stimulation with either medium alone, phorbol 12-myristate 13-acetate and ionomycin (CellStim), 500 pg/mL recombinant ovine IFNT (IFNT), 150 pg/mL PGF2α (PGF), 10 pg/mL PGE2 (PGE), or 10 ng/mL pregnancy specific protein B (PAG) for 24 h. After the 24 h challenge, blood was centrifuged for isolation of conditioned plasma and the concentration of interferon gamma (IFNG), interleukin (IL) -1A, IL-1B, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-36RA, IP-10, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, tumor necrosis factor A (TNFA), and vascular endothelial growth factor A (VEGFA) were quantified using a Milliplex magnetic bead assay. The absolute and relative numbers of neutrophils, lymphocytes, monocytes, eosinophils, and basophils fell within the normal range for healthy cattle. Average corpora lutea size and concentration of progesterone in plasma were 15.4 ± 3.8 mm² and 8.17 ± 4.7 ng/mL, respectively. CellStim increased (P < 0.01) the secretion of IFNG, IL-1A, IL-1B, IL-8, MIP-1α, MIP-1β, and VEGFA relative to medium alone. Whole blood treated with PGF and IFNT tended to reduce (P = 0.072; P = 0.102) the secretion of MIP-1α by 57.5 and 57.9 percent, respectively, compared to medium alone. Whole blood challenged with PGF tended to reduce (P = 0.091) the secretion of TNFA by 53.5 percent compared to medium alone. The secretion of VEGFA was reduced by 68.4 percent (P = 0.037) for whole blood challenged with IFNT compared to PAG (29.9 ± 1.25 pg/mL vs. 43.8 ± 1.25 pg/mL, respectively). Whole blood challenged with PAG increased (P = 0.007) the secretion of MIP-1β by 5.9-fold compared to medium alone (22.6 ± 1.5 pg/mL vs. 3.8 ± 1.5 pg/mL, respectively). Collectively, these results indicate that conceptus secreted products alter the release of cytokines and chemokines from peripheral blood leukocytes. Thus, as conceptus products such as PAGs interact with endometrial resident immune cells, they may be altering cytokine and chemokine secretion within the microenvironment to recruit immune cells to the endometrium; potentially supporting endometrial remodeling and angiogenesis, both essential for successful establishment of pregnancy.