John V. Fahey

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.

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.

A new strategy to understand how HIV infects women : identification of a window of vulnerability during the menstrual cycle

Although 85% of new HIV cases are due to sexual transmission from men to women little attention is being paid to the immune system in the female reproductive tract (FRT) and to how it meets the conflicting challenges of protecting from pathogens and permitting procreation. As a new approach we have tried to envision how HIV evades FRT mucosal immune protection and have been led to the unexpected conclusion that in a normal menstrual cycle there is a window of vulnerability (7-10 days following ovulation) in which the potential for viral infectivity in the FRT is enhanced. During that period aspects of the innate humoral and cell-mediated immune systems are suppressed by sex hormones to optimize conditions for procreation. Suppression occurs in the upper (Fallopian tubes uterus endocervix) and lower (ectocervix and vagina) FRT and coincides with the recruitment of potentially infectable cells and upregulation of coreceptors essential for viral uptake. Implications of these findings are that the entire FRT is a potential target for HIV infection immune cells and antibodies in blood are not surrogate markers for immune protection in the FRT and immune protection against HIV will require an understanding of the hormone-induced regulation of humoral cellmediated and innate immune systems throughout the FRT. (excerpt)

Prostaglandin inhibition of T-cell proliferation is mediated at two levels

Abstract While numerous investigators have established that E-series prostaglandins inhibit proliferation of lectin- or antigen-activated T cells, the mechanism by which this effect is mediated remained poorly defined. It was recently shown that T-cell replication is mediated by a soluble factor (T-cell growth factor, TCGF), produced by antigen- or lectin-stimulated T cells. Thus, inhibition of T-cell replication by prostaglandins could be controlled either at the level of TCGF production or at the subsequent step of actual proliferation. We have found that differentiated cytotoxic T lymphocytes harvested from TCGF-dependent culture were, indeed, significantly sensitive to as little as 1 n M PGE 1 or PGE 2 ; 1000 n M PGE 1 or PGE 2 reduced TCGF-dependent T-cell proliferation by greater than 43% in all cases. In addition, PGE 1 and PGE 2 suppressed lectin-induced production of TCGF to remarkably similar concentration-dependent levels. Thus, the prostaglandin-E-mediated suppression of lectin-initiated T-cell proliferation could be traced to an inhibition of both the production and action of the T-cell-specific mitogen, TCGF. Since TCGF is produced by one T-cell subpopulation and acts on different T-cell subpopulations including both T helper and T cytotoxic cells, the consequences of prostaglandin-E inhibition of TCGF may have broad effects on cellular and humoral immune responses.

Sex Hormone Regulation of Innate Immunity in the Female Reproductive Tract: The Role of Epithelial Cells in Balancing Reproductive Potential with Protection against Sexually Transmitted Pathogens

Citation Wira CR, Fahey JV, Ghosh M, Patel MV, Hickey DK, Ochiel DO. Sex hormone regulation of innate immunity in the female reproductive tract: the role of epithelial cells in balancing reproductive potential with protection against sexually transmitted pathogens. Am J Reprod Immunol 2010

Innate and adaptive immunity at mucosal surfaces of the female reproductive tract: stratification and integration of immune protection against the transmission of sexually transmitted infections

This review examines the multiple levels of pre-existing immunity in the upper and lower female reproductive tract. In addition, we highlight the need for further research of innate and adaptive immune protection of mucosal surfaces in the female reproductive tract. Innate mechanisms include the mucus lining, a tight epithelial barrier and the secretion of antimicrobial peptides and cytokines by epithelial and innate immune cells. Stimulation of the innate immune system also serves to bridge the adaptive arm resulting in the generation of pathogen-specific humoral and cell-mediated immunity. Less understood are the multiple components that act in a coordinated way to provide a network of ongoing protection. Innate and adaptive immunity in the human female reproductive tract are influenced by the stage of menstrual cycle and are directly regulated by the sex steroid hormones, progesterone and estradiol. Furthermore, the effect of hormones on immunity is mediated both directly on immune and epithelial cells and indirectly by stimulating growth factor secretion from stromal cells. The goal of this review is to focus on the diverse aspects of the innate and adaptive immune systems that contribute to a unique network of protection throughout the female reproductive tract.

Effect of Menstrual Status on Antibacterial Activity and Secretory Leukocyte Protease Inhibitor Production by Human Uterine Epithelial Cells in Culture

The objective of this study was to examine the production of antibacterial factor(s) by uterine epithelial cells from pre- and postmenopausal women. Apical rinses from polarized epithelial cells recovered from women at the proliferative and secretory stages of the menstrual cycle were equally effective in killing Staphylococcus aureus and Escherichia coli, but those from postmenopausal women were not. Secretory leukocyte protease inhibitor (SLPI) concentrations of apical washes from premenopausal women were significantly higher than those obtained from postmenopausal women. SLPI production correlated with bactericidal activity with respect to menstrual status and time in culture. Anti-SLPI antibody significantly decreased bactericidal activity of premenopausal epithelial cell rinses. The endometrial epithelial cell line HEC-1A did not have a bactericidal effect, nor did it produce SLPI. In contrast, HEC-1B cells produced SLPI and a factor that inhibited bacterial growth. These results indicate that menstrual status (pre- vs. postmenopausal) influences the production of SLPI and bactericidal activity by uterine epithelial cells.

Anti-HIV Activity in Cervical-Vaginal Secretions from HIV-Positive and -Negative Women Correlate with Innate Antimicrobial Levels and IgG Antibodies

Background We investigated the impact of antimicrobials in cervicovaginal lavage (CVL) from HIV(+) and HIV(−) women on target cell infection with HIV. Since female reproductive tract (FRT) secretions contain a spectrum of antimicrobials, we hypothesized that CVL from healthy HIV(+) and (−) women inhibit HIV infection. Methodology/Principal Findings CVL from 32 HIV(+) healthy women with high CD4 counts and 15 healthy HIV(−) women were collected by gently washing the cervicovaginal area with 10 ml of sterile normal saline. Following centrifugation, anti-HIV activity in CVL was determined by incubating CVL with HIV prior to addition to TZM-bl cells. Antimicrobials and anti-gp160 HIV IgG antibodies were measured by ELISA. When CXCR4 and CCR5 tropic HIV-1 were incubated with CVL from HIV(+) women prior to addition to TZM-bl cells, anti-HIV activity in CVL ranged from none to 100% inhibition depending on the viral strains used. CVL from HIV(−) controls showed comparable anti-HIV activity. Analysis of CH077.c (clone of an R5-tropic, mucosally-transmitted founder virus) viral inhibition by CVL was comparable to laboratory strains. Measurement of CVL for antimicrobials HBD2, trappin-2/elafin, SLPI and MIP3α indicated that each was present in CVL from HIV(+) and HIV(−) women. HBD2 and MIP3α correlated with anti-HIV activity as did anti-gp160 HIV IgG antibodies in CVL from HIV(+) women. Conclusions/Significance These findings indicate that CVL from healthy HIV(+) and HIV(−) women contain innate and adaptive defense mechanisms that inhibit HIV infection. Our data suggest that innate endogenous antimicrobials and HIV-specific IgG in the FRT can act in concert to contribute toward the anti-HIV activity of the CVL and may play a role in inhibition of HIV transmission to women.

Trappin-2/Elafin: a novel innate anti-human immunodeficiency virus-1 molecule of the human female reproductive tract

Trappin‐2/Elafin is a serine protease inhibitor that plays a major role as an anti‐inflammatory mediator at mucosal surfaces. In addition, Trappin‐2/Elafin has antibacterial activity against Gram‐positive and Gram‐negative bacterial and fungal pathogens. In this study we examined the production of Trappin‐2/Elafin by epithelial cells from the human upper and lower female reproductive tract as well as its activity as an anti‐human immunodeficiency virus (HIV)‐1 molecule. We found that primary uterine, Fallopian tube, cervical and ectocervical epithelial cells produce Trappin‐2/Elafin constitutively and that production of Trappin‐2/Elafin is enhanced following stimulation with Poly(I:C), especially by the uterine cells. Given the presence of Trappin‐2/Elafin in the reproductive tract, we tested the ability of recombinant Trappin‐2/Elafin to inhibit HIV‐1, an important sexually transmitted pathogen. We found that recombinant Trappin‐2/Elafin was able to inhibit both T‐cell‐tropic X4/IIIB and macrophage‐tropic R5/BaL HIV‐1 in a dose‐dependent manner. The inhibitory activity was observed when virus was incubated with Trappin‐2/Elafin but not when Trappin‐2/Elafin was added to cells either before infection or after infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV‐1 and Trappin‐2/Elafin. Additionally, we measured the levels of secreted Trappin‐2/Elafin in cervico‐vaginal lavages (CVL) from both HIV‐positive and HIV‐negative women and found that average levels of secreted Trappin‐2/Elafin were higher in the CVL from HIV‐negative women, although the values did not reach statistical significance. We also found that women at the secretory phase of the menstrual cycle produced more Trappin‐2/Elafin in CVL relative to women at the proliferative phase of the menstrual cycle. Our data suggest that Trappin‐2/Elafin might be an important endogenous microbicide of the female reproductive tract that is protective against HIV‐1.