Expression of genes associated with immunity in the endometrium of cattle with disparate postpartum uterine disease and fertility

Abstract

Background: Contamination of the uterine lumen with bacteria is ubiquitous in cattle after parturition. Some animals develop endometritis and have reduced fertility but others have no uterine disease and readily conceive. The present study tested the hypothesis that postpartum cattle that develop persistent endometritis and infertility are unable to limit the inflammatory response to uterine bacterial infection.

Methods: Endometrial biopsies were collected several times during the postpartum period from animals that were subsequently infertile with persistent endometritis (n = 4) or had no clinical disease and conceived to first insemination (n = 4). Quantitative PCR was used to determine the expression of candidate genes in the endometrial biopsies, including the Toll-like receptor (TLR 1 to 10) family of innate immune receptors, inflammatory mediators and their cognate receptors. Selected proteins were examined by immunohistochemistry.

Results: The expression of genes encoding pro-inflammatory mediators such as interleukins (IL1A, IL1B and IL6), and nitric oxide synthase 2 (NOS2) were higher during the first week post partum than subsequently. During the first week post partum, there was higher gene expression in infertile than fertile animals of TLR4, the receptor for bacterial lipopolysaccharide, and the pro-inflammatory cytokines IL1A and IL1B, and their receptor IL1R2. The expression of genes encoding other Toll-like receptors, transforming growth factor beta receptor 1 (TGFBR1) or prostaglandin E2 receptors (PTGER2 and PTGER4) did not differ significantly between the animal groups. Gene expression did not differ significantly between infertile and fertile animals after the first week postpartum. However, there were higher ratios of IL1A or IL1B mRNA to the anti-inflammatory cytokine IL10, during the first week post partum in the infertile than fertile animals, and the protein products of these genes were mainly localised to the epithelium of the endometrium.

Conclusion: Cattle may maintain fertility by limiting the inflammatory response to postpartum bacterial infection in the endometrium during the first week after parturition.

Figure 1

Endometrial expression of CD45, TLR4, CD14, and MD-2. Expression of mRNA encoding CD45, TLR4, CD14, and MD-2 in endometrial biopsies collected from infertile (closed bar) and fertile animals (open bar), during Periods 1 and 2. RNA was isolated from biopsies, reverse transcribed and analysed by quantitative PCR for the mRNA encoding pan-leukocyte marker, CD45 and components of the LPS receptor complex, TLR4, CD14 and MD-2. *P < 0.05 compared with infertile animals, within the period. Numerical values are presented as the mean + SEM.

Figure 2

Endometrial expression of TLR1, TLR2 and TLR6. Expression of mRNA encoding TLR1, TLR2 and TLR6 in endometrial biopsies collected from infertile (closed bar) and fertile animals (open bar), during Periods 1 and 2. RNA was isolated from biopsies, reverse transcribed and analysed by quantitative PCR for the mRNA encoding bacterial lipoprotein receptors, TLR1, TLR2 and TLR6. Numerical values are presented as the mean + SEM.

Figure 3

Endometrial expression of TLR3, TLR5, TLR7, TLR9, TLR10 and NOD1. Expression of mRNA encoding TLR3, TLR5, TLR7, TLR9, TLR10 and NOD1 in endometrial biopsies collected from infertile (closed bar) and fertile animals (open bar), during Periods 1 and 2. RNA was isolated from biopsies, reverse transcribed and analysed by quantitative PCR for the mRNA encoding intracellular receptors TLR3, TLR7, TLR9 and NOD1, for the flagellin receptor TLR5, and for TLR10 that has unknown function. Numerical values are presented as the mean + SEM.

Figure 4

Endometrial expression of IL1A, IL1B and IL1R2. Expression of mRNA encoding IL1A, IL1B and IL1R2 in endometrial biopsies collected from infertile (closed bar) and fertile animals (open bar), during Periods 1 and 2. RNA was isolated from biopsies, reverse transcribed and analysed by quantitative PCR for the mRNA encoding the pro-inflammatory cytokine isoforms IL1A and IL1B and corresponding receptor, IL1R2. *P < 0.05 compared with infertile animals, within the Period. Numerical values are presented as the mean + SEM.

Figure 5

Endometrial expression of IFN-a, IL6, TNF and NOS2. Expression of mRNA encoding IFN-a, IL6, TNF and NOS2 in endometrial biopsies collected from infertile (closed bar) and fertile animals (open bar), during Periods 1 and 2. RNA was isolated from biopsies, reverse transcribed and analysed by quantitative PCR for the mRNA encoding the pro-inflammatory mediators IFN-a, IL6, TNF and NOS2. Numerical values are presented as the mean + SEM.

Figure 6

Endometrial expression of IL10, TGFBR1, PTGER2 and PTGER4. Expression of mRNA encoding IL10, TGFBR1, PTGER2 and PTGER4 in endometrial biopsies collected from infertile (closed bar) and fertile animals (open bar), during Periods 1 and 2. RNA was isolated from biopsies, reverse transcribed and analysed by quantitative PCR for the mRNA encoding the anti-inflammatory mediator IL10, and the receptors for the anti-inflammatory mediators transforming growth factor beta 1 (TGFBR1) and prostaglandin E₂ (PTGER2 and PTGER4). Numerical values are presented as the mean + SEM.

Figure 7

Ratio of IL1A and IL1B to IL10 expression. Ratio of expression of mRNA encoding IL1A or IL1B to IL10. The relationship between the expression of mRNA encoding IL1A or IL1B to IL10 was determined for infertile (closed bars) and fertile animals (open bars) using numerical values obtained by Q-PCR.

Figure 8

Endometrial expression of TNF, IL-6, TLR4, IL-10, IL-1 alpha, IL-1 beta and cytokeratin protein. Immunohistochemical localization of TNF, IL-6, TLR4, IL-10, IL-1 alpha, IL-1 beta and cytokeratin in the endometrium of postpartum cows. 5 μm sections of Formalin-fixed paraffin-embedded endometrial biopsies were examined using the primary antibodies described in Methods with Alexa Fluor 555 secondary antibodies, mounted with DAPI Vectashield. Representative images of TNF, IL-6, TLR4, IL-10, IL-1 alpha, IL-1 beta and cytokeratin immunoreactive protein and an isotype IgG control in endometrial tissue from animals week 1 post partum are shown. The scale bar is 100 μm.