Stat6 Activation by IL-4 Downregulates Toll-Like Receptor-4 Expression

Abstract

RATIONALE: Toll-like receptor 4 (TLR4), part of the bacterial lipopolysaccharide (LPS) receptor, is an important bridge between innate and adaptive immunity. Interleukin-4 (IL-4) and STAT6, which are increased in atopic patients, may modulate TLR4. METHODS: U-937 monocytic cells were incubated with IL-4 and mRNA was quantified by real-time PCR and surface expression measured by flow cytometry. Cells were transfected with a vector containing STAT6 gene to evaluate over expression of this transcription factor on TLR4 expression. Cells were also incubated with Tyrphostin AG490 to inhibit STAT6 phosphorylation. Different TLR4 promoter constructs were made to determine the IL-4 DNA responsive elements. A STAT6 chromatin immunoprecipitation assay was performed to evaluate STAT6 binding sites. RESULTS: IL-4 inhibited TLR4 mRNA and protein surface expression early but the effect was lost over time. STAT6 over expression enhanced the inhibition and prolonged it. IL-4 inhibition was abolished upon pre-incubation with the tyrosine kinase inhibitor Tyrphostin AG490. Specific DNA sequences upstream of the TLR4 promoter were determined to be necessary for STAT6 to inhibit transcriptional activation. STAT6 was found to bind to a specific site upstream of TLR4 gene when monocytes were incubated with IL-4. CONCLUSIONS: Our findings demonstrate that IL-4, through STAT6 activation and binding, can modulate TLR4 expression. Therefore, Th2 cytokines may have an impact on the LPS responsiveness of cells.