In this study, we provide the first demonstration that steroid hormones affect TLR function. Specifically, E2
modulates TLR3 function through significant suppression of Poly I:C-induced proinflammatory and antiviral cytokine and chemokine production, which we have previously shown to be dependent on TLR3 in our system[26
]. The suppressive effect is not due to a decrease in the steady state levels of TLR3 mRNA or protein. However, the inhibition of function is hormone receptor-dependent, as demonstrated through the use of hormone receptor-positive and hormone receptor-negative cell lines and the hormone receptor antagonists, ICI 182, 780 and RU486.
The endometrial epithelial cell lines, AN3-CA, HEC-1-A, KLE, and RL95-2, and the breast adenocarcinoma cell line, MCF7, differentially express TLR3, ERA, ERB, PRA, and PRB mRNA. The levels of ERα mRNA in these cell lines differ quantitatively, with the MCF7 cells demonstrating the highest quantitative level of ERα mRNA and the HEC-1A cells not expressing ERα mRNA. All the cell lines utilized, with the exception of the HEC-1A cells, also express detectable levels of ERα protein. The expression of TLR3 is noteworthy because expression of TLR3 has not been documented in the HEC-1-A, KLE, or MCF7 lines. Additionally, MCF7 cells express TLR2, TLR5, and TLR6. The impact of hormones on signaling of other TLRs, including those expressed in the MCF7 and RL95-2 cells, has not been explored and will be the focus of future studies.
We found that E2
, but not P, significantly suppresses production of both proinflammatory and antiviral cytokines in Poly I:C-stimulated RL95-2 cells. Both E2
and P suppress production of cytokines and chemokines in KLE and MCF7 cells. Previous reports have demonstrated suppression of IL-6 mRNA by E2
in cytokine-stimulated and LPS-stimulated cells[2
], but other investigators have suggested that IL-6 production is not regulated by ovarian steroids[16
]. The data presented in our study suggests that regulation of the TLR3 signaling pathway by E2
results in suppression of secreted IL-6, as well as IL-8 and IP-10. The contradictory findings in the regulation of IL-6 by E2
may be due to variable expression of ER in the tissues under examination[17
]. Additionally, instability of hormone receptor expression in RL95-2 cells has been documented[37
]. In order to address these issues, hormone receptor expression was verified prior to all experiments in this study to eliminate the possibility of incorrect observations due to variable receptor expression. Furthermore, we have observed that E2
suppression of Poly I:C-stimulated cytokine production in RL95-2 cells occurs only when ERα is present.
Significant P effects were observed only in the MCF7 cells. Although RT-PCR results for the RL95-2 and MCF7 cells in Table demonstrate that both cell lines express PR, potential differences in quantitative PR expression may be present between the cell lines and this may account for the difference in P effects. However, other investigators have documented differential efficiency of the two PR isoforms dependent on cell context [41
]. Thus, we speculate that differences between specific cell lines and/or differences in co-activator and co-repressor expression may underlie the differences in P action in the two cell lines.
In addition to suppression of IL-6 and IL-8 production, we demonstrate suppression of IP-10 by E2
in Poly I:C-stimulated RL95-2 cells. E2
has been shown to suppress IP-10 function in murine mammary cells[44
]. However, suppression of IP-10 by E2
has not been previously documented in endometrial cells and tissues. Our findings are noteworthy due to the presence of TLR3, which recognizes viral dsRNA, in the endometrium and the role of IP-10 in the antiviral response. IP-10 may be important in recruitment of immune effector cells and activation of cell surface receptors essential for immune defense against viral pathogens, such as HIV, CMV, and HSV in the human endometrium[44
]. Thus, TLR3 activation and subsequent production of IP-10 following dsRNA stimulation may be a pivotal event in modulating endometrial events.
Although our previous finding of cyclic regulation of TLR3 in the endometrium suggested potential control of TLR3 expression and function by steroid hormones, we found that E2
does not suppress Poly I:C-induced cytokine and chemokine production by altering TLR3 mRNA or protein expression. Thus, our findings in the in vitro
model system suggest that E2
does not directly regulate TLR3 expression, but does regulate TLR3 function through interaction with ER. Previous findings suggested that maximal expression of TLR3 in the endometrium occurs in the mid and late secretory phases, a time when ERα protein in the endometrial epithelium is virtually undetectable [46
]. Thus, the suppressive action of E2
on cytokine expression would serve to suppress TLR3 action at times of low TLR3 expression. Inhibition of TLR3 function would not occur during times of maximal TLR3 expression due to lack of ERα. A similar mechanism of E2
inhibition of expression has been proposed for the β3 integrin subunit, which is expressed on endometrial epithelium in a pattern similar to that observed for TLR3, except with the β3 integrin subunit, E2
inhibits expression rather than function[49
The exact mechanism by which E2
regulates TLR3 function remains to be elucidated. Ray and colleagues and Galien and colleagues demonstrated an inhibition of the DNA-binding activity of the transcription factors NF-IL6 and NF-kB by the estrogen receptor, resulting in suppression of IL-6 gene expression[36
]. However, the mechanism of IL-8 and IP-10 suppression has not been explored. IP-10 production occurs upon activation of the transcription factor, interferon regulatory factor 3 (IRF3), rather than NF-κB. Studies examining interactions of IRF3 with ER have not been performed and will be necessary to determine if the nature of the suppressive effect of E2
on IP-10 production is similar to that of NF-κB and ER. Our future studies will be designed to determine the mechanism by which E2
regulates TLR3 signaling components.
Suppression of cytokines and chemokines by E2
was determined to be ER-dependent in this study. Hormone effects have been shown to be hormone receptor-dependent in other studies[51
]. TLR3-positive endometrial epithelial cell lines that do not express ERα, such as the HEC-1-A line, do not exhibit suppressed cytokine and chemokine production upon stimulation with Poly I:C. Additionally, treatment of TLR3-positive, ERα-positive cell lines with the ER antagonist, ICI 182, 780, restores levels of cytokine and chemokine production to vehicle only levels. All of the data presented in this study has been obtained using in vitro
experiments with endometrial epithelial cell lines. We have previously demonstrated that TLR3 is functional in primary endometrial epithelial cells[26
]. Since the response to E2
and P may be different in primary endometrial epithelial cells as compared to endometrial epithelial cell lines, future experiments will be performed using primary endometrial epithelial cells to address this question.
Our current and previous studies suggest a role for TLR3 in the endometrium. We have shown TLR3 expression to be cycle-dependent in primary endometrial epithelial cells and that the response to Poly I:C in our in vitro
system requires TLR3[26
]. This study demonstrates modulation of TLR3 function by the hormones of the endometrium. The cytokines and chemokines produced upon TLR3 ligation are important in normal endometrial functions and may be crucial in the pathogenesis of endometrial dysfunctions such as endometriosis [1
]. Recognition of viral dsRNA by TLR3 and the resulting production of inflammatory and antiviral cytokines and chemokines may be pivotal in the protection of the endometrium from pathogens. The cyclic regulation of TLR3 in the endometrium may allow protection against pathogens while maintaining a system of tolerance toward the embryo and preventing extensive tissue damage from the inflammatory response. It has been suggested that development of endometrial dysfunction is characterized by an increased inflammatory environment, allowing progression of disease[6
]. Thus, the influence of steroid hormones on TLR3 function, specifically the suppression of cytokines and chemokines produced upon stimulation of TLR3 by dsRNA, may be critical in the regulation, maintenance, and defense of the human endometrium.