Estrogen Receptor Alpha Binding to ERE is Required for Full Tlr7- and Tlr9-Induced Inflammation

Abstract

We previously found that a maximum innate inflammatory response induced by stimulation of Toll-like receptors (TLRs) 3, 7 and 9 requires ERα, but does not require estrogen in multiple cell types from both control and lupus-prone mice. Given the estrogen-independence, we hypothesized that ERα mediates TLR signaling by tethering to, and enhancing, the activity of downstream transcription factors such as NFκB, rather than acting classically by binding EREs on target genes. To investigate the mechanism of ERα impact on TLR signaling, we utilized mice with a knock-in ERα mutant that is unable to bind ERE. After stimulation with TLR ligands, both ex vivo spleen cells and bone marrow-derived dendritic cells (BM-DCs) isolated from mutant ERα ("KIKO") mice produced significantly less IL-6 compared with cells from wild-type (WT) littermates. These results suggest that ERα modulation of TLR signaling does indeed require ERE binding for its effect on the innate immune response.

Keywords: DCs; ERα; TLRs.

Figure 1

(A) Twenty mice (n = 8 WT, 12 KIKO) were sacrificed at 18 weeks. Spleens from KIKO mice were smaller and there was a significant difference in spleen cell counts between WT and KIKO mice. In a subset of animals (n = 4 WT, 5 KIKO), femurs were harvested and bone marrow cells were counted. There was a trend toward reduced numbers in the KIKO mice that did not reach significance. (B) Ex vivo spleen cells (n = 4 WT, 7 KIKO) and cultured BM-DCs (n = 4 WT, 5 KIKO) were stimulated overnight (18h) with 200umol loxoribine (TLR 7 ligand) or 1ug/mL CpG DNA (TLR9 ligand) under estrogen-free media conditions. TLR stimulation resulted in increased IL-6 production that was significantly decreased in media from KIKO mice compared to wild-type mice.