Interleukin-1 beta treatment alters prostaglandin-related mRNA expression and cytokine abundance in equine endometrial culture

Abstract

Interleukin-1β (IL-1β) is a proinflammatory cytokine that modulates the immunoinflammatory response and induces prostaglandin synthesis. IL-1β has a variety of roles in the endometrium during inflammation, decidualization, pregnancy, and post-partum. It can cause changes in endometrial gene expression and may affect cytokine, chemokine, and prostaglandin-related pathways. The relationship between IL-1β, prostaglandins, and downstream inflammatory responses in the equine uterine environment is not fully known. Our objective was to determine the effect that IL-1β has on prostaglandin-related gene expression, as well as cytokine and chemokine abundance, in endometrial cell culture. Light breed mares (n=8) were examined daily by transrectal ultrasound until ovulation. An endometrial biopsy was collected at day 11 post ovulation, and used for primary endometrial cell culture. Cells were cultured in DMEM with 10% FBS. At 80% confluence, cells were either not supplemented or supplemented with: 100 nM Progesterone (Prog); 1 nM Estradiol-17β (Est); or 100 nM Prog and 1 nM Est (P+E). After 24 hours, cells were treated with recombinant equine IL-1β at either 10 ng/mL (Low) or 1000 ng/mL (High), or 0.2% DMSO as an untreated control. Twenty-four hours post-treatment, supernatant was collected, and a bead-based multiplex assay was used to quantify concentration of twelve cytokines and chemokines. Total RNA was extracted from the cells and mRNA expression was evaluatedfor 10 prostaglandin-related genes, using the ΔCT method with ACTB as the reference gene. Data analysis was performed using JMP Pro 16, using a restricted maximum likelihood model with treatment and supplementation as factors, and mare as a random factor. Treatment with IL-1β High resulted in increased levels of CCL2, CCL5, and CCL11 (p<0.001) in cell culture supernatant, as well as increased mRNA expression of PTGS2 (p<0.02), regardless of supplementation. Cells supplemented with Prog or P+E and treated with IL-1β High had increased expression of IL1R1, IL1RAP, PTGS1, and PTGES compared to non-supplemented cells (p<0.05). IL-1β High treatment increased LiF expression in cells supplemented with Prog or P+E compared to untreated cells (p<0.05). IL-1β likely acts to modulate inflammatory pathways in the endometrium, altering chemokine levels and activating expression of PTGS2, leading to changes in downstream prostaglandin synthesis and other related pathways. Progesterone appears to increase expression of several target genes when compared to non-supplemented cells. Progesterone has been shown to be involved in the suppression of several inflammatory pathways, and investigation into the ovarian steroid regulation of genes and proteins in the equine endometrium is warranted. Further studies on the effect of IL-1β on molecular pathways and prostaglandin regulation could lead to a greater understanding of the uterine environment in the pregnant and non-pregnant mare.