Transcriptional response of the bovine endometrium and embryo to endometrial polymorphonuclear neutrophil infiltration as an indicator of subclinical inflammation of the uterine environment.

The aim of the present study was to analyse the effect of subclinical endometritis on endometrial and embryonic gene expression. A total of 49 cows at either Day 0 or Day 7 of the oestrous cycle (62-83 days post partum) following superovulation were classified as having subclinical endometritis (SE-0, SE-7) or a healthy endometrium (HE-0, HE-7) on the basis of endometrial cytological evaluation. Endometrial samples and associated embryos were subjected to global transcriptome analysis using the Bovine GeneChip (Affymetrix, Santa Clara, CA, USA) and aberrant transcript profiles were observed in SE-0 and SE-7 cows. At Day 0, 10 transcripts were found to be differentially expressed in endometrial samples. Specifically, the PDZK1, PXDN, DDHD2, GPLD1 and SULT1B1 genes were downregulated, whereas the PKIB, LOC534256, BT29392, LYZ and S100A14 genes were upregulated in SE-0 cows. Similarly, 11 transcripts were found to be differentially regulated on Day 7. Of these, GNPTG, BOLA-DQA5, CHD2, LOC541226, VCAM1 and ARHGEF2 were found to be downregulated, whereas PSTPIP2, BT236441 and MGC166084 were upregulated in SE-7 cows. Accordingly, endometrial health status affected the number of flushed, transferable embryos. In all, 20 genes were differentially regulated in blastocysts derived from HE-7 and SE-7 cows. Of these, GZMK, TCEAL4, MYL7, ADD3 and THEM50B were upregulated, whereas NUDCD2, MYO1E, BZW1, EHD4 and GZMB were downregulated. In conclusion, endometrial polymorphonuclear neutrophil infiltration as an indicator of subclinical endometritis is associated with changes in endometrial gene expression patterns, including genes involved in cell adhesion and immune modulation. Consequently, subclinical endometritis affects gene expression in embryos, including the expression of genes related to membrane stability, the cell cycle and apoptosis.

Lipopolysaccharide priming enhances expression of effectors of immune defence while decreasing expression of pro-inflammatory cytokines in mammary epithelia cells from cows.

BACKGROUND: Udder infections with environmental pathogens like Escherichia coli are a serious problem for the dairy industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of immune stimulants such as lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes/macrophages are known to develop tolerance towards the endotoxin LPS (endotoxin tolerance, ET) as adaptation strategy to prevent exuberant inflammation.We have recently observed that infusion of 1 µg of LPS into the quarter of an udder effectively protected for several days against an experimentally elicited mastitis. We have modelled this process in primary cultures of mammary epithelial cells (MEC) from the cow. MEC are by far the most abundant cells in the healthy udder coming into contact with invading pathogens and little is known about their role in establishing ET. RESULTS: We primed primary MEC cultures for 12 h with LPS (100 ng/ml) and stimulated three cultures either 12 h or 42 h later with 107/ml particles of heat inactivated E. coli bacteria for six hours. Priming-related alterations in the global transcriptome of those cells were quantified with Affymetrix microarrays. LPS priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (ß-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1, TGM3) as well as mediators of the sentinel function of MEC (CCL5, CXCL8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL1B, IL6, TNF-α) and immune effectors (NOS2, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL1B and of IRF7 as key to this modulation. CONCLUSION: LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells.

Lipopolysaccharide pretreatment of the udder protects against experimental Escherichia coli mastitis.

Exposure to pathogen-associated molecular patterns such as LPS can cause an immune refractory state in mammals known as endotoxin tolerance (ET), resulting in a decreased inflammatory response after pathogen contact. This ET concept was used to reduce the severity of an experimentally-induced clinical mastitis. Cows were pretreated with 1 µg LPS per udder quarter and challenged 72 h (group L72EC) or 240 h (group L240EC) later with 500 CFU Escherichia coli. Pretreated animals showed no leukopenia after challenge, no (L72EC), or only slightly (L240EC), elevated body temperature and significantly reduced systemic and local clinical scores compared with cows that were not pretreated. Whereas an increase of milk somatic cell count after the E. coli challenge was abrogated in L72EC animals, it was significantly delayed in the L240EC group. In both pretreated groups the bacterial load in milk was markedly reduced. Based on the expression of inflammation-related genes in lobulo-alveolar mammary tissue, the tolerizing effect of LPS pretreatment is based on the inhibited up-regulation of inflammatory (TNF-α, IL-6, CXCL8, CCL20) and anti-inflammatory genes (IL-10, IRAK-M). These findings indicate that the concept of ET may be usefully applied as mastitis prophylaxis facilitating a rapid response to microbial infection and avoiding dysregulated inflammation.

Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium.

Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.

Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle.

Uterine microbial disease affects half of all dairy cattle after parturition, causing infertility by disrupting uterine and ovarian function. Infection with Escherichia coli, Arcanobacterium pyogenes, and bovine herpesvirus 4 causes endometrial tissue damage. Toll-like receptors on endometrial cells detect pathogen-associated molecules such as bacterial DNA, lipids, and lipopolysaccharide (LPS), leading to secretion of cytokines, chemokines, and antimicrobial peptides. Chemokines attract neutrophils and macrophages to eliminate the bacteria, although persistence of neutrophils is associated with subclinical endometritis and infertility. Cows with uterine infections are less likely to ovulate because they have slower growth of the postpartum dominant follicle in the ovary, lower peripheral plasma estradiol concentrations, and perturbation of hypothalamic and pituitary function. The follicular fluid of animals with endometritis contains LPS, which is detected by the TLR4/CD14/LY96 (MD2) receptor complex on granulosa cells, leading to lower aromatase expression and reduced estradiol secretion. If cows with uterine disease ovulate, the peripheral plasma concentrations of progesterone are lower than those in normal animals. However, luteal phases are often extended in animals with uterine disease, probably because infection switches the endometrial epithelial secretion of prostaglandins from the F series to the E series by a phospholipase A2-mediated mechanism, which would disrupt luteolysis. The regulation of endometrial immunity depends on steroid hormones, somatotrophins, and local regulatory proteins. Advances in knowledge about infection and immunity in the female genital tract should be exploited to develop new therapeutics for uterine disease.

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

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.