Effect of repeated intravenous lipopolysaccharide infusions on systemic inflammatory response and endometrium gene expression in Holstein heifers

Abstract

This study aimed to evaluate the effect of repeated intravenous lipopolysaccharide (LPS) infusions in nonlactating heifers on (1) the systemic proinflammatory state as measured by biomarkers in blood and plasma, and (2) endometrial gene expression of candidate transcripts on d 15 of gestation. Our hypothesis was that target transcripts related to a major functional group would be negatively modified in the preimplantation endometrium by the LPS treatments. In the first experiment (n = 13), a systemic proinflammatory state [defined as increased plasma concentrations of tumor necrosis factor (TNF)-α and haptoglobin for 2 wk] was established using 2 different sequential LPS infusion protocols. In the second experiment, heifers (n = 22; 11 mo of age) had their time of ovulation synchronized by a modified Ovsynch protocol and were enrolled in 1 of 2 treatments: control (CON; n = 11), which received sterile saline solution i.v., and LPS treatment (LPS; n = 11), submitted to repeated i.v. LPS injections (0.10, 0.25, 0.50, 0.75, 1.00, and 1.25 µg/kg) starting 2 d after artificial insemination (AI; d 0) and then every other day until d 15 after AI. At each LPS injection, rectal temperatures were measured hourly for 6 h. Blood samples were collected from d -1 to d 13 for analyses of progesterone, TNF-α, and haptoglobin in plasma, along with white blood cell (WBC) count and differential analysis. On d 15, endometrium tissue biopsies were taken and kept at -80°C until quantitative real-time PCR analysis of 30 target transcripts related to the immune system, adhesion molecules, and endometrium receptivity. Data were checked for normality and analyzed by repeated-measures ANOVA using PROC UNIVARIATE and PROC MIXED of SAS (SAS Institute Inc., Cary, NC). After each LPS injection, temperature was greater in the first 4 h in the LPS group compared with CON. Both TNF-α and haptoglobin increased in the LPS treatment with a significant treatment by day interaction. Total leukocyte count did not differ between treatments, but the differential count increased for neutrophils, band cells, and monocytes, and decreased for lymphocytes and eosinophils in LPS compared with CON. Progesterone concentrations in plasma did not differ between treatments during the experimental period. Out of 30 target genes analyzed, 3 transcripts were differentially expressed: indoleamine 2,3-dioxygenase (IDO; fold-change = 0.48) and pentraxin-3 (PTX3; fold-change = 0.38) were downregulated, whereas myxovirus-resistance protein (MX1; fold-change = 2.85) was upregulated in the LPS group. Sequential LPS injections were able to induce a prolonged systemic proinflammatory state, but effects on gene expression were limited to transcripts associated with the immune system. These results suggest that a mechanism for subfertility is linked to a proinflammatory state in dairy heifers.

Keywords: endometrium; gene expression; heifer; inflammation; lipopolysaccharide.