Patricia A. Pioli

Innate and adaptive immunity in female genital tract: cellular responses and interactions

Summary:  The mucosal immune system in the female reproductive tract (FRT) has evolved to meet the unique requirements of dealing with sexually transmitted bacterial and viral pathogens, allogeneic spermatozoa, and the immunologically distinct fetus. Analysis of the FRT indicates that the key cells of the innate and adaptive immune systems are present and functionally responsive to antigens. Acting through Toll‐like receptors in the Fallopian tubes, uterus, cervix, and in the vagina, epithelial cells, macrophages, natural killer cells, and neutrophils confer protection through the production of chemokines and cytokines, which recruit and activate immune cells, as well as bactericidal and virucidal agents, which confer protection at times when adaptive immunity is downregulated by sex hormones to meet the constraints of procreation. The overall goal of this paper is to define the innate immune system in the FRT and, where possible, to define the regulatory influences that occur during the menstrual cycle that contribute to protection from and susceptibility to potential pathogens. By understanding the nature of this protection and the ways in which innate and adaptive immunity interact, these studies provide the opportunity to contribute to the foundation of information essential for ensuring reproductive health.

Differential expression of Toll-like receptors 2 and 4 in tissues of the human female reproductive tract.

ABSTRACT Toll-like receptor (TLR) signal transduction is a central component of the innate immune response to pathogenic challenge. Although recent studies have begun to elucidate differences in acquired immunity in tissues of the human female reproductive tract, there is a relative paucity of work regarding innate defense mechanisms. We investigated TLR mRNA and protein expression in tissues of the human female reproductive tract. Constitutive mRNA expression of TLRs 1 to 6 was observed in fallopian tubes, uterine endometrium, cervix, and ectocervix. Furthermore, transcripts of the signaling adapter MyD88 and the accessory molecule CD14 were also detected in all tissues assayed. Quantitative analysis of TLR2 mRNA levels revealed highest expression of this molecule in fallopian tube and cervical tissues, followed by endometrium and ectocervix. In contrast to TLR2, TLR4 expression declined progressively along the tract, with highest expression in the upper tissues (fallopian tubes and endometrium), followed by cervix and ectocervix. In addition to mRNA, protein expression of TLR2 and TLR4 was also documented in these tissues. These data suggest that TLRs are differentially expressed in distinct compartments of the female reproductive tract and may provide insight regarding the regulation of inflammation and immunity within the tract.

Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163

CD163, a monocyte and macrophage‐specific surface glycoprotein, which is increased by interleukin‐10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia‐associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI‐0 indicate that a metalloproteinase is responsible for LPS‐mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Estradiol Suppresses NF-κB Activation through Coordinated Regulation of let-7a and miR-125b in Primary Human Macrophages

Previous findings suggest that 17β-estradiol (estradiol) has a suppressive effect on TNF-α, but the mechanism by which estradiol regulates TNF-α expression in primary human macrophages is unknown. In this article, we demonstrate that pretreatment of human macrophages with estradiol attenuates LPS-induced TNF-α expression through the suppression of NF-κB activation. Furthermore, we show that activation of macrophages with LPS decreases the expression of κB-Ras2, an inhibitor of NF-κB signaling. Estradiol pretreatment abrogates this decrease, leading to the enhanced expression of κB-Ras2 with LPS stimulation. Additionally, we identified two microRNAs, let-7a and miR-125b, which target the κB-Ras2 3′ untranslated region (UTR). LPS induces let-7a and inhibits miR-125b expression in human macrophages, and pretreatment with estradiol abrogates these effects. 3′UTR reporter assays demonstrate that let-7a destabilizes the κB-Ras2 3′UTR, whereas miR-125b enhances its stability, resulting in decreased κB-Ras2 in response to LPS. Our data suggest that pretreatment with estradiol reverses this effect. We propose a novel mechanism for estradiol inhibition of LPS-induced NF-κB signaling in which κB-Ras2 expression is induced by estradiol via regulation of let-7a and miR-125b. These findings are significant in that they are the first to demonstrate that estradiol represses NF-κB activation through the induction of κB-Ras2, a key inhibitor of NF-κB signaling.

Pivotal Advance: Activation of cell surface Toll‐like receptors causes shedding of the hemoglobin scavenger receptor CD163

The hemoglobin scavenger receptor (HbSR) CD163 is a monocyte/macrophage‐specific glycoprotein that binds and facilitates uptake of haptoglobin‐hemoglobin (Hp‐Hb) complexes, which are rapidly formed in the circulation upon hemolysis of red blood cells. Hemolysis can be caused by a diverse range of infectious agents and provides pathogens a source of iron to enhance their survival and replication. Previous work demonstrated that lipopolysaccharide (LPS) activates monocytes to cleave cell‐bound HbSR into a soluble mediator that retains the capacity to bind Hp‐Hb complexes. We report that blocking LPS activation of Toll‐like receptor 4 prevents LPS‐mediated shedding of CD163. Furthermore, activation of two other cell surface Toll‐like receptors (TLR), TLR2 and TLR5, induces shedding of the HbSR from human monocytes. In contrast, treatment of monocytes with intracellular TLR3, TLR7, and TLR9 agonists failed to cause HbSR shedding, suggesting that this shedding event is selective to cell surface TLR activation. These data demonstrate that the soluble HbSR is released from monocytic cells in response to TLR signaling as an acute innate immune response to extracellular pathogen infections.

Up-regulation of human monocyte CD163 upon activation of cell-surface Toll-like receptors

The hemoglobin (Hb) scavenger receptor, CD163, is a cell‐surface glycoprotein that is expressed exclusively on monocytes and macrophages. It binds and internalizes haptoglobin‐Hb complexes and has been implicated in the resolution of inflammation. Furthermore, the regulation of CD163 during an innate immune response implies an important role for this molecule in the host defense against infection. LPS, derived from the outer membrane of Gram‐negative bacteria, activates TLR4 to cause acute shedding of CD163 from human monocytes, followed by recovery and induction of surface CD163 to higher levels than observed on untreated monocytes. We now report that the TLR2 and TLR5 agonists—Pam3Cys and bacterial flagellin—have similar effects on CD163 surface expression. Up‐regulation of CD163 following treatment of human PBMC with TLR2, TLR4, and TLR5 agonists parallels increased production of IL‐6 and IL‐10, and neutralization of IL‐6 and/or IL‐10 blocks CD163 up‐regulation. Furthermore, simultaneous stimulation of TLR2 or TLR5 in combination with TLR4 activation results in enhanced up‐regulation of CD163. It is notable that exogenous recombinant IFN‐γ (rIFN‐γ) suppresses cell‐surface, TLR‐mediated IL‐10 production as well as CD163 up‐regulation. Sustained down‐regulation of CD163 mediated by rIFN‐γ can be partially rescued with exogenous rIL‐10 but not with exogenous rIL‐6. This divergent regulation of CD163 by cytokines demonstrates that human monocytes react differently to infectious signals depending on the cytokine milieu they encounter. Thus, surface CD163 expression on mononuclear phagocytes is a carefully regulated component of the innate immune response to infection.

Estradiol Regulates Expression of Estrogen Receptor ERα46 in Human Macrophages

Background Monocytes and macrophages are key innate immune effector cells that produce cytokines and chemokines upon activation. We and others have shown that 17β-estradiol (E2) has a direct role in the modulation of monocyte and macrophage immune function. However, relatively little is known about the ability of E2 to regulate isoform expression of estrogen receptors (ERs) in these cells. Methodology/Principal Findings In this study, we quantify expression of ERα and ERβ in human monocytes and macrophages. We also show for the first time that the N-terminal truncated ERα variant, ERα46, is expressed in both cell types. Promoter utilization studies reveal that transcription of ERα in both cell types occurs from upstream promoters E and F. Treatment with E2 induces ERα expression in macrophages but has no effect on ERβ levels in either cell type. During monocyte-to-macrophage differentiation, ERα is upregulated in a time-dependent manner. Previous studies by our group demonstrated that E2 treatment attenuates production of the chemokine CXCL8 in an ER-dependent manner. We now show that ERα expression levels parallel the ability of E2 to suppress CXCL8 production. Conclusions/Significance This work demonstrates for the first time that human macrophages predominantly express the truncated ER variant ERαp46, which is estradiol-inducible. This is mediated through usage of the ERα F promoter. Alternative promoter usage may account for tissue and cell type-specific differences in estradiol-induced effects on gene expression. These studies signify the importance of ERα expression and regulation in the ability of E2 to modulate innate immune responses.

Lipopolysaccharide-Induced IL-1β Production by Human Uterine Macrophages Up-Regulates Uterine Epithelial Cell Expression of Human β-Defensin 2

The uterine endometrium coordinates a wide spectrum of physiologic and immunologic functions, including endometrial receptivity and implantation as well as defense against sexually transmitted pathogens. Macrophages and epithelial cells cooperatively mediate innate host defense against bacterial invasion through the generation of immunologic effectors, including cytokines and antimicrobial peptides. In this study, we demonstrate that stimulation of peripheral blood monocytes and uterine macrophages with bacterial LPS induces the production of biologically active proinflammatory IL-1β. High doses of estradiol enhance LPS-induced IL-1β expression in an estrogen receptor-dependent manner. Furthermore, both peripheral blood monocyte- and uterine macrophage-derived IL-1β induce secretion of antimicrobial human β-defensin 2 by uterine epithelial cells. These data indicate dynamic immunologic interaction between uterine macrophages and epithelial cells and implicate a role for estradiol in the modulation of the immune response.

Cross‐linking of FcγR triggers shedding of the hemoglobin‐haptoglobin scavenger receptor CD163

CD163, the hemoglobin (Hb)‐haptoglobin scavenger receptor, is a monocyte/macrophage‐restricted member of the scavenger receptor, cysteine‐rich family of proteins. In addition to being expressed on the cell surface, a soluble form of CD163 has also been reported. Like tumor necrosis factor α (TNF‐α), surface CD163 is proteolytically cleaved from the plasma membrane in response to lipopolysaccharide (LPS) stimulation. As cross‐linking of the Fcγ receptor (FcγR) is similarly known to induce TNF‐α shedding, the effect of FcγR stimulation on CD163 shedding was investigated. We found that FcγR stimulation resulted in a rapid release of surface CD163 into the supernatant that was blocked by inhibitors of protein kinase C and tyrosine kinases. Although LPS and FcγR stimulation in short‐term cultures suppressed CD163 mRNA expression, long‐term cultures of monocytes treated with LPS—but not with a FcγR cross‐linking reagent—resulted in an interleukin‐10‐dependent recovery of surface CD163 expression. These studies suggest that the presence of immune complexes in infection or autoimmunity may radically alter the nature of CD163‐dependent monocyte/macrophage processes. This may be particularly important in disease states in which immune complexes and high levels of free Hb are present, such as in autoimmune hemolytic anemia, transfusion reactions, or infections by hemolytic bacteria.

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.