A Novel Dual-Color Luciferase Reporter Assay for Simultaneous Detection of Estrogen and Aryl Hydrocarbon Receptor Activation.

Consumers are exposed to a plethora of anthropogenic and natural substances that can act as agonists or antagonists for various transcription factors. Depending on the exposure and potency, such interactions can potentially lead to adverse health effects, particularly for substances with multiple molecular targets. The early detection of such interactions is thus of high toxicological interest. Here, we report on the development of a new cellular dual-color reporter assay that allows for time-resolved and quantitative recording of estrogen receptor (ER) and aryl hydrocarbon receptor (AHR) activation in living cells. Both receptors are known for their ligand promiscuity. Moreover, both receptor signaling pathways are interconnected by direct protein-protein interactions as well as by shared protein factors and the competition for ligands. The assay is based on two rare beetle luciferases that emit light in the red (SLR) and green (ELuc) spectrum and that have been stably inserted into human T-47D mammary carcinoma cells. The corresponding cell line is termed "XEER" and has been successfully subjected to proof-of-principle studies using prototypical ER and AHR ligands as well as various phytochemicals, xenobiotics, and extracts from various plastic products.

Toll like receptor 2 agonists lipoteichoic acid and peptidoglycan are able to enhance antigen specific IFNγ release in whole blood during recall antigen responses.

BACKGROUND: Interferon gamma release assays (IGRA) have been developed to support the diagnosis of diseases like tuberculosis, which lack robust serological test systems. IGRAs focus on cellular immunity especially memory T cells and thus complement serological testing. However, the low frequency of antigen-specific memory T cells in peripheral blood limits IFNγ production to minute amounts and constitutes a major challenge for downstream test systems. We hypothesized that certain toll like receptor (TLR) agonists might enhance IFNγ production in IGRAs after antigen challenge without inducing background cytokine production. In addition, we investigated the potential use of IL2 release after TLR agonist application as another surrogate marker in cytokine release assays. METHODS: 176 healthy controls (HC) were tested for IFNγ- and IL2-secretion in whole blood in the presence of different TLR agonists with and without antigen challenge by ELISA. The selected TLR agonists were lipopolysaccharide (LPS ≙ TLR4), lipoteichoic acid (LTA ≙ TLR2), peptidoglycan (PGN ≙ TLR2), zymosan (Zym ≙ TLR2 and 6), polyinosinic-polycytidylic acid (Poly I:C ≙ TLR3), flagellin (Fla ≙ TLR5), R848 (≙TLR7 and 8), loxoribine (Lox ≙ TLR7) and bropirimine (Bro ≙ TLR7). RESULTS: TLR2 agonists LTA and PGN increased IFNγ secretion after antigen challenge nearly twofold (740 vs. 443 pg/ml for LTA and 969 vs. 469 pg/ml for PGN, respectively) without eliciting higher background expression. TLR3 agonist Poly(I:C) and TLR5 agonist Fla also induced a twofold increase in IFNγ synthesis (2.230 vs. 1.085 pg/ml for Poly(I:C) and 518 vs. 278 pg/ml for Fla, respectively), but background expression was slightly increased (114 vs. 7 pg/ml for Poly(I:C) and 47 vs. 12 pg/ml for Fla, respectively). IL2 production was not increased after antigen challenge in the presence of LTA, PGN, Poly(I:C) or Fla. The agonists LPS, Zym, R848, Lox and Bro did not raise cytokine synthesis after antigen challenge or they generated high levels of cytokines by themselves. CONCLUSION: Of all tested agonists TLR2-specific LTA and PGN met the requirements to increase IFNγ synthesis in whole blood after challenge with recall antigens without heightening basal cytokine levels alone. Thus, they constitute a potential costimulating reagent for IGRAs. IL2 did not show any potential as a surrogate marker in cytokine release assays in combination with TLR agonists.