Measurement of the Influence of Androstenediol on TLR Signaling

Treatment of mice with the steroid hormone androstenediol (AED) protects against infection with influenza, herpes, and coxsackie viruses. Additionally, antibody responses against influenza and tetanus vaccination are amplified by AED treatment. Macrophages recognize viruses using toll-like receptor 3 (TLR3) and TLR7. After interaction with their ligands, TLRs drive NF-&kappaB transcriptional activity resulting in TNF-&alpha synthesis. Somewhat unique to TLR3 and TLR7 is the transcription of the anti-viral type I IFNs (IFN&alpha/&beta) mediated by IRF3 and IRF7 respectively. Objectives: Because AED protected against viral infection, we hypothesized that AED would enhance transcription of those genes driven by TLR3 and TLR7. However, we first had to establish a model in which we could specifically activate TLR3 or TLR7 and measure the influence of our experimental conditions on TLR-dependent gene expression. Methods: Cultures of the mouse macrophage cell line, RAW264.7, were treated with a dose curve of poly(I:C) to determine the optimal concentration of this TLR3 agonist. Alternatively, cells were treated with imiquimod to determine the optimal concentration of this TLR7 agonist. After 2-hour incubation, total RNA was extracted from cultures, and real-time RT-PCR was used to quantify the expression of TNF-&alpha as an NF-&kappaB-dependent gene; IFN&beta was quantified as a measure of IRF3/IRF7-dependent gene expression. Results: The data show that as little as 2.0&mug/ml was sufficient to activate TLR3 and drive TNF-&alpha gene expression above vehicle controls. An optimal 30-fold induction of TNF-&alpha RNA expression was obtained with 20 &mug/ml Poly(I:C). For TLR7 stimulation, an optimal 10-fold induction of IFN&beta RNA expression was obtained with 3.6&mug/ml imiquimod. Conclusions: We have established a cell culture model where we can assess the influence of androstenediol on either TLR3- or TLR7-dependent signaling in macrophages. This model will enable us to delineate the mechanism by which AED enhances anti-viral immunity.

Supported by NIH/NIDCR T32 DE014320-04