Todd M. Schaefer

Toll-like receptor (TLR) expression and TLR-mediated cytokine/chemokine production by human uterine epithelial cells.

The objective of this study was to examine the expression of toll‐like receptors (TLRs) by the uterine epithelial cell line ECC‐1 and to determine if stimulation of the expressed TLRs induces changes in cytokine and/or chemokine secretion. The expression of TLR1 to TLR9 by ECC‐1 cells was demonstrated by reverse transcription polymerase chain reaction, with only TLR10 not being expressed. Stimulation of ECC‐1 cells using agonists to TLR2, TLR4 and TLR5 induced the expression of the chemokines interleukin‐8 (IL‐8) and monocyte chemotactic protein‐1 (MCP‐1), as well as the pro‐inflammatory cytokine IL‐6, and occurred in a dose‐dependent manner. In response to zymosan and flagellin, pathogen‐associated molecular patterns (PAMP) that are recognized by TLR2 and TLR5 respectively, ECC‐1 cells secreted significantly more IL‐8, MCP‐1 and IL‐6 than in response to other TLR agonists. In contrast, agonists to TLR3, TLR7, and TLR9 had no effect on the secretion of the 13 cytokines or chemokines analysed. These results indicate that uterine epithelial cells are important sentinels of the innate immune system. Further it indicates that all but one of the known TLRs are expressed by ECC‐1 cells and that stimulation through specific TLRs mediates changes in the expression of key chemokines and pro‐inflammatory cytokines that aid in the defence of the uterus against potential pathogens.

Innate Immunity in the Human Female Reproductive Tract: Antiviral Response of Uterine Epithelial Cells to the TLR3 Agonist Poly(I:C)

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human β-defensin-1 and -2 (HBD1 and HBD2), IFN-β, and the IFN-β-stimulated genes myxovirus resistance gene 1 and 2′,5′ oligoadenylate synthetase. The expression of TLR1–9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-α, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1β. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-β mRNA expression 70-fold and myxovirus resistance gene 1 and 2′,5′ oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express β-defensins, IFN-β, and IFN-β-stimulated genes that can have a direct inhibiting effect on viral replication.

Effect of Toll‐Like Receptor (TLR) Agonists on TLR and Microbicide Expression in Uterine and Vaginal Tissues of the Mouse

Epithelial cells lining the uterine lumen are the first line of defense against pathogenic microbes. The objective of this study was to examine the expression of Toll‐like receptors (TLRs), defensins and secretory leukocyte protease inhibitor (SLPI) in the mouse uterus and vagina and in primary uterine epithelial cells and to determine whether TLR agonists induce TLR and defensin expression.

IL-1β-Mediated Proinflammatory Responses Are Inhibited by Estradiol via Down-Regulation of IL-1 Receptor Type I in Uterine Epithelial Cells

The objective of this study was to examine the effects of sex hormones on IL-1β-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human β-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1β in the presence of estradiol or progesterone. Estradiol inhibited the IL-1β-mediated mRNA expression and secretion of human β-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1β-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10−7 to 10−10 M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1β-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell’s ability to respond to IL-1β. These results suggest that the inhibitory effect of estradiol on IL-1β-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.

ORIGINAL ARTICLE: CCL20/MIP3α is a Novel Anti‐HIV‐1 Molecule of the Human Female Reproductive Tract

Problem  CCL20/MIP3α is a chemokine for immature dendritic cells as well as an antibacterial against gram‐positive and gram‐negative bacteria. The role of CCL20/MIP3α as an antiviral is unknown. In this study, we have examined the production of CCL20/MIP3α by epithelial cells from the upper female reproductive tract as well as its activity as an antiviral molecule.

Migration Inhibitory Factor Secretion by Polarized Uterine Epithelial Cells is Enhanced in Response to the TLR3 Agonist Poly (I:C)

Uterine epithelial cells produce cytokines that stimulate leukocytes in response to a microbial insult. The goals of this study were to determine if uterine epithelial cells produce the pro‐inflammatory cytokine macrophage migration inhibitory factor (MIF), and to see if toll‐like receptor (TLR) agonists stimulate MIF secretion.

Sex hormone modulation of human uterine epithelial cell immune responses

Sexually transmitted infections are a major worldwide public health problem affecting millions of people. A number of bacteria, fungi, viruses, and protozoa can infect reproductive tissues, resulting in varying degrees of pathology ranging from little discomfort to death. The female reproductive tract has evolved innate and adaptive immune mechanisms that protect from microbial infection, thereby reducing infection and disease. Central to this protection are the epithelial cells that line the female reproductive tract. In the uterus, columnar epithelial cells provide a physical barrier to microbial infection, possess toll-like receptors that detect pathogens and secrete a number of constitutive and induced factors that directly or indirectly hinder infection. For example, uterine epithelial cells secrete peptides that destroy pathogenic microbes. In addition, epithelial cells produce chemokines and cytokines that attract and activate innate immune cells and serve as a link to the adaptive immune system. Further, uterine epithelial cells serve as a conduit for secretory antibodies to enter the lumen and can present antigen to T cells. These protective mechanisms contribute to an environment in the uterus that is generally considered sterile, unlike the environment in the lower female reproductive tract. The uterine environment is in constant flux due to the concentration changes in sex hormones that occur in preparation for reproduction. The sex hormones estrogen and progesterone alter the local immune system to prepare for conception, influence how well the immune system will tolerate antigenic sperm and a semi-allogeneic fetus and yet provide a network of protective immune mechanisms against microbial pathogens. Understanding how sex hormones influence uterine epithelial cell function will provide a basis for immune protection in the uterus.

Effect of oestradiol and pathogen-associated molecular patterns on class II-mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

Cells of the female reproductive tract (FRT) can present antigen to naive and memory T cells. However, the effects of oestrogen, known to modulate immune responses, on antigen presentation in the FRT remain undefined. In the present study, DO11.10 T‐cell antigen receptor transgenic mice specific for the class II MHC‐restricted ovalbumin (OVA) 323–339 peptide were used to study the effects of oestradiol and pathogen‐associated molecular patterns on antigen presentation in the FRT. We report here that oestradiol inhibited antigen presentation of OVA by uterine epithelial cells, uterine stromal cells and vaginal cells to OVA‐specific memory T cells. When ovariectomized animals were treated with oestradiol for 1 or 3 days, antigen presentation was decreased by 20–80%. In contrast, incubation with PAMP increased antigen presentation by epithelial cells (Pam3Cys), stromal cells (peptidoglycan, Pam3Cys) and vaginal cells (Pam3Cys). In contrast, CpG inhibited both stromal and vaginal cell antigen presentation. Analysis of mRNA expression by reverse transcription PCR indicated that oestradiol inhibited CD40, CD80 and class II in the uterus and CD40, CD86 and class II in the vagina. Expression in isolated uterine and vaginal cells paralleled that seen in whole tissues. In contrast, oestradiol increased polymeric immunoglobulin receptor mRNA expression in the uterus and decreased it in the vagina. These results indicate that antigen‐presenting cells in the uterus and vagina are responsive to oestradiol, which inhibits antigen presentation and co‐stimulatory molecule expression. Further, these findings suggest that antigen‐presenting cells in the uterus and vagina respond to selected Toll‐like receptor agonists with altered antigen presentation.

1142280946 Effect of estradiol and PAMPs on class II mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

Problem:  Antigen presenting cells (APC) in the female reproductive tract play important roles in innate immune defense and activation of the adaptive immune responses. The objective of this study was to examine the effects of estradiol and PAMP on antigen presentation in the female reproductive tract.