91 Treatment of bovine endometrial explants with interleukin-1 beta increases the relative abundance of transcripts for pro-inflammatory cytokines

Previous work in cattle has demonstrated that exposure of endometrial cells or tissue to sperm or seminal plasma increases the abundance of the pro-inflammatory cytokine interleukin-1 β (IL-1β). Two experiments were conducted to determine the effect of treatment of bovine endometrial explants with IL-1β on subsequent changes in the transcript abundance of other pro-inflammatory cytokines. For Exp. 1, endometrial explants were collected from abattoir-derived uteri of mature beef cows at various stages of the oestrous cycle using an 8 mm punch biopsy. Harvested explants were randomly assigned to one of six treatments consisting of complete medium (RPMI 1640 plus 10% [v/v] fetal bovine serum, 1% [v/v] penicillin-streptomycin, and 1% [v/v] amphotericin B) supplemented with 0, 0.1, 1, 10, 100, or 1,000 ng/mL recombinant bovine IL-1β. Explants were cultured at 37°C in a humidified atmosphere with 5% CO₂ for 24 h. After treatment, explants were harvested, placed in lysis buffer, and then stored at −20°C until subsequent analysis. The experiment included seven biological replicates. Exp. 2 was performed as described for Exp. 1, except that explants were collected from abattoir-derived uteri assessed to be between Day 0 and 5 of the oestrous cycle. Additionally, explants were randomly assigned to treatment with rbIL-1β for three timepoints: 2, 6, and 24 h. Explants were harvested from culture at the respective timepoints, placed into RNAlater, and stored at −20°C until subsequent analysis. The experiment included eight biological replicates. For both experiments, RNA was extracted and then submitted to qRT-PCR to assess the relative abundance of six gene transcripts: IL1R1, IL1B, IL6, IL8, CSF2, and TNFA. Relative expression of the genes of interest was calculated using the 2^−DCt method with GAPDH as the reference. Data were analysed by least-squares analysis of variance. In Exp. 1, the relative abundance of IL1B was increased (P < 0.05) following treatment with 1, 10, and 100 ng/mL rbIL-1β. Moreover, treatment with 10 ng/mL rbIL-1β increased (P < 0.05) abundance of IL-6 and CSF2. There was no effect of rbIL-1β treatment on the relative abundance of IL1R1, IL8, or TNFA. In Exp. 2, abundance of IL1B was increased (P < 0.05) following treatment with 100 and 1,000 ng/mL rbIL-1β, irrespective of timepoint. Similar results were obtained for IL6, with 10, 100, and 1,000 ng/mL rbIL-1β increasing (P < 0.05) abundance, regardless of timepoint. Abundance of IL8 was increased (P < 0.05) following treatment with 1, 10, 100, and 1,000 ng/mL rbIL-1β, but the effect was only observed following 6 h of treatment. There was also a treatment × timepoint interaction for the abundance of TNFA. Treatment with 100 and 1,000 ng/mL rbIL-1β increased (P < 0.05) abundance of TNFA at 2 and 6 h, but not at 24 h. Treatment of endometrial explants with rbIL-1β altered the abundance of several pro-inflammatory cytokines, including IL1B, IL6, IL8, CSF2, and TNFA. Further work is necessary to elucidate the role of IL-1β during the early post-mating period.