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Effects of local lipopolysaccharide administration on the expression of Toll-like receptor 4 and pro-inflammatory cytokines in uterus and oviduct of rabbit does

Menchetti, Laura, Barbato, Olimpia, Filipescu, Iulia Elena, Traina, Giovanna, Leonardi, Leonardo, Polisca, Angela, Troisi, Alessandro, Guelfi, Gabriella, Piro, Federica, Brecchia, Gabriele
Theriogenology 2018 v.107 pp. 162-174
Gram-negative bacteria, Toll-like receptor 4, animal models, antimicrobial peptides, blood sampling, chemokines, domestic animals, epithelial cells, females, gene expression, genes, histology, humans, inflammation, innate immunity, interleukin-1beta, lipopolysaccharides, messenger RNA, oviducts, protein synthesis, quantitative polymerase chain reaction, rabbits, reproductive performance, reverse transcriptase polymerase chain reaction, secretion, sodium chloride, staining, stromal cells, tumor necrosis factor-alpha, uterus
Inflammation of the uterus and oviduct is associated with reduced reproductive performance in humans and domestic animals. Toll-like receptors are expressed in various immune and non-immune cells and play a crucial role in innate immunity. Toll-like receptor – 4 (TLR4) can detect lipopolysaccharide (LPS) from Gram-negative bacteria leading to the secretion of pro-inflammatory cytokines, chemokines, antimicrobial peptides and other inflammatory mediators. To investigate the effects of a local inflammation on the expression levels of TLR4 and pro-inflammatory cytokines, 12 female rabbits received an intracervical infusion with either saline solution endotoxin-free (carrier, 2 mL; n = 6) or LPS (500 μg diluted in 2 mL of saline solution; n = 6). Blood samples were performed at 0, 30, 60 and 90 min and 2,4,6 and 24 h after treatment to evaluate interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) plasma concentrations. Animals were sacrificed 24 h post-treatment. The uterus and oviducts were immediately collected. The gene expression and protein levels of TLR4 and pro-inflammatory cytokines were detected by quantitative real-time PCR (qRT-PCR) and immuno-histochemical assay, respectively. Our study showed that the intracervical administration of LPS induced local inflammation given that the animals showed no clinical signs, the histological samples revealed signs of inflammation and plasma levels of pro-inflammatory cytokines were unchanged compared to the control. LPS produced an increase in the TLR4 mRNA expression levels in the uterus with respect to the control (P < 0.05). In LPS-treated rabbits the gene expression of IL-1β was higher in the uterus and oviducts and TNF-α only in the oviduct (P < 0.05) as compared to the control. The immuno-histochemical assay showed that TLR4, IL-1β and TNF-α were expressed in the reproductive tissues of the rabbit. Moreover, after the LPS stimulation the stromal cells of the uterus exhibited a higher staining for TLR4 (P < 0.05) and the epithelial cells of the oviduct for TNF-α and IL-1β (P < 0.05) with respect to the control. These results suggest that (1) TRL4, IL-1β and TNF-α are expressed in uterus and oviducts of the doe, and (2) LPS up-regulates the gene and protein expression of TLR4 and pro-inflammatory cytokines in uterus and oviducts. Therefore, the rabbit could be a useful animal model for studying the local mechanisms involved in reproductive dysfunctions caused by subclinical infections.