Oxysterols Protect Epithelial Cells Against Pore-Forming Toxins.

Many species of bacteria produce toxins such as cholesterol-dependent cytolysins that form pores in cell membranes. Membrane pores facilitate infection by releasing nutrients, delivering virulence factors, and causing lytic cell damage - cytolysis. Oxysterols are oxidized forms of cholesterol that regulate cellular cholesterol and alter immune responses to bacteria.

Oxysterols protect bovine endometrial cells against pore-forming toxins from pathogenic bacteria.

Many species of pathogenic bacteria secrete toxins that form pores in mammalian cell membranes. These membrane pores enable the delivery of virulence factors into cells, result in the leakage of molecules that bacteria can use as nutrients, and facilitate pathogen invasion. Inflammatory responses to bacteria are regulated by the side-chain-hydroxycholesterols 27-hydroxycholesterol and 25-hydroxycholesterol, but their effect on the intrinsic protection of cells against pore-forming toxins is unclear.

is a Progesterone Target Gene in the Endometrium of Ruminants.

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate expression.

Subclinical endometritis in dairy cattle is associated with distinct mRNA expression patterns in blood and endometrium.

Cattle with subclinical endometritis (SCE) are sub-fertile and diagnosing subclinical uterine disease remains a challenge. The hypothesis for this study was that endometrial inflammation is reflected in mRNA expression patterns of peripheral blood leucocytes. Transcriptome profiles were evaluated in healthy cows and in cows with SCE using circulating white blood cells (WBC) and endometrial biopsy samples collected from the same animals at 45-55 days postpartum.

Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin.

Animal health depends on the ability of immune cells to kill invading pathogens, and on the resilience of tissues to tolerate the presence of pathogens. Trueperella pyogenes causes tissue pathology in many mammals by secreting a cholesterol-dependent cytolysin, pyolysin (PLO), which targets stromal cells. Cellular cholesterol is derived from squalene, which is synthesized via the mevalonate pathway enzymes, including HMGCR, FDPS and FDFT1.

Innate immunity and the sensing of infection, damage and danger in the female genital tract.

Tissue homeostasis in the female genital tract is challenged by infection, damage, and even physiological events during reproductive cycles. We propose that the evolutionarily ancient system of innate immunity is sufficient to sense and respond to danger in the non-pregnant female genital tract. Innate immunity produces a rapidly inducible, non-specific response when cells sense danger.

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case.

We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes.

Mechanisms linking bacterial infections of the bovine endometrium to disease and infertility.

Bacterial infections of the endometrium after parturition commonly cause metritis and endometritis in dairy cattle, and these diseases are important because they compromise animal welfare and incur economic costs, as well as delaying or preventing conception. Here we highlight that uterine infections cause infertility, discuss which bacteria cause uterine disease, and review the evidence for mechanisms of inflammation and tissue damage in the endometrium. Bacteria cultured from the uterus of diseased animals include Escherichia coli, Trueperella pyogenes, and several anaerobic species, but their causative role in disease is challenged by the discovery of many other bacteria in the uterine disease microbiome.

Enzyme linked immunosorbent assay for quantification of bovine interleukin-8 to study infection and immunity in the female genital tract.

Problem: The chemokine IL-8 recruits neutrophils to sites of infection, including the endometrium of the bovine uterus. However, quantification of bovine IL-8 often yields lower concentrations than for other species, which may reflect impaired innate immune responses by bovine cells or inaccurate measurement of IL-8 using the current human IL-8 ELISA method.

Method Of Study: An ELISA was developed and validated for detection of bovine IL-8.

Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression.

Subclinical endometritis (SCE) is an important postpartum disease in dairy cows, but conventional cytobrush diagnosis often gives imprecise results. The aim of this study was to analyze disease-associated changes in peripheral blood as potential diagnostic parameters. Cellular subpopulations of blood leukocytes from cows with or without SCE (45-55 days postpartum) were flow-cytometrically quantified.

Differential endometrial cell sensitivity to a cholesterol-dependent cytolysin links Trueperella pyogenes to uterine disease in cattle.

Purulent disease of the uterus develops in 40% of dairy cows after parturition, when the epithelium of the endometrium is disrupted to expose the underlying stroma to bacteria. The severity of endometrial pathology is associated with isolation of Trueperella pyogenes. In the present study, T.

Epithelial and stromal cells of bovine endometrium have roles in innate immunity and initiate inflammatory responses to bacterial lipopeptides in vitro via Toll-like receptors TLR2, TLR1, and TLR6.

Bacteria often infect the endometrium of cattle to cause endometritis, uterine disease, and infertility. Lipopeptides are commonly found among bacteria and are detected by the Toll-like receptor (TLR) cell surface receptor TLR2 on immune cells. Heterodimers of TLR2 with TLR1 or TLR6 activate MAPK and nuclear factor-κB intracellular signaling pathways to stimulate inflammatory responses.

Granulosa cells from emerged antral follicles of the bovine ovary initiate inflammation in response to bacterial pathogen-associated molecular patterns via Toll-like receptor pathways.

Bacterial infections of the uterus or mammary gland commonly perturb ovarian antral follicle growth and function, causing infertility in cattle. Cells of the innate immune system use Toll-like receptors (TLRs) TLR2, TLR4, and TLR5 to recognize pathogen-associated molecular patterns (PAMPs) of bacteria, leading to production of inflammatory mediators, such as IL-1beta, IL-6, and IL-8. The present study examined whether granulosa cells from emerged antral follicles have functional responses to typical bacterial PAMPs.

Phenotypic and functional heterogeneity of bovine blood monocytes.

Murine and human peripheral blood monocytes are heterogeneous in size, granularity, nuclear morphology, phenotype and function. Whether and how bovine blood monocytes follow this pattern was analyzed in this study. Flow cytometrically, classical monocytes (cM) CD14⁺CD16⁻, intermediate monocytes (intM) CD14⁺ CD16⁺ and nonclassical monocytes (ncM) CD14⁺ CD16⁺ were identified, with cM being the predominant subset (89%).

Explants of intact endometrium to model bovine innate immunity and inflammation ex vivo.

Problem: Bacterial infections commonly cause bovine endometritis and infertility via innate immune pathways. However, mechanistic studies using isolated cells or chopped tissue may be compromised by the disruption of endometrial architecture and release of damage-associated molecular patterns. So, this study aimed to establish an ex vivo model of intact bovine endometrium to study innate immunity and inflammation.

Toll-like receptor expression and function in the COV434 granulosa cell line.

Problem: Bacterial infections perturb ovarian follicle function, despite the lack of immune cells such as macrophages within healthy ovarian follicles. This study examined whether the granulosa cells that line ovarian follicles could, like macrophages, use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns (PAMPs) and initiate inflammation.

Method Of Study: The COV434 human granulosa and THP-1 macrophage cell lines were used to determine the expression of TLRs and measure the production of cytokines, chemokines and estradiol in response to the PAMPs lipopolysaccharide, peptidoglycan, lipoteichoic acid and flagellin from bacteria.

Innate immunity in the human endometrium and ovary.

Problem: Microbial infections of the upper female genital tract perturb the function of the endometrium and ovary. Defense of these tissues is predominantly dependent on innate immunity. This review gives a perspective on innate immunity in the non-pregnant upper female genital tract of women.

Toll-like receptor 4 mediates the response of epithelial and stromal cells to lipopolysaccharide in the endometrium.

Background: Ascending infections of the female genital tract with bacteria causes pelvic inflammatory disease (PID), preterm labour and infertility. Lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria. Innate immunity relies on the detection of LPS by Toll-like receptor 4 (TLR4) on host cells.

The chemokine IL8 is up-regulated in bovine endometrial stromal cells by the BoHV-4 IE2 gene product, ORF50/Rta: a step ahead toward a mechanism for BoHV-4 induced endometritis.

Postpartum infections of the endometrium and metritis are common causes of delayed conception and infertility in cattle. These infections are characterized by inflammation of the endometrium and secretion of the chemokine interleukin 8 (IL8), which attracts granulocytes to the endometrium. Bovine herpesvirus 4 (BoHV-4) is tropic for the endometrium and the only virus consistently associated with postpartum metritis.

Isolation and characterization of bovine herpesvirus 4 (BoHV-4) from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome.

Background: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with a Worldwide distribution in cattle and is often isolated from the uterus of animals with postpartum metritis or pelvic inflammatory disease. Virus strain adaptation to an organ, tissue or cell type is an important issue for the pathogenesis of disease. To explore the mechanistic role of viral strain variation for uterine disease, the present study aimed to develop a tool enabling precise genetic discrimination between strains of BoHV-4 and to easily manipulate the viral genome.

Bovine endometrial stromal cells display osteogenic properties.

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone.

Bacterial infection of endometrial stromal cells influences bovine herpesvirus 4 immediate early gene activation: a new insight into bacterial and viral interaction for uterine disease.

Experimental infection with the gamma-herpesvirus bovine herpesvirus 4 (BoHV-4) rarely establishes disease, yet BoHV-4 is commonly associated with uterine disease in cattle. Uterine disease involves co-infection with bacteria such as Escherichia coli, which stimulate the production of prostaglandin E(2) (PGE(2)) by endometrial cells. BoHV-4 replication depends on immediate early 2 (IE2) gene transactivation and, in the present study, PGE(2), E.

Bovine herpesvirus 4 is tropic for bovine endometrial cells and modulates endocrine function.

Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the gamma-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells.