A robust TH17 response is detected in the CNS (central nervous system) of T-bet deficient animals, which is stronger in frequency and magnitude than in control animals, suggesting that T-bet acts as a suppressor TH17 lineage commitment. We have shown that T-bet silences the expression of RORgt in developing TH17 cells by blocking the transcriptional activity of Runx1, a positive regulator of RORgt gene expression. Runx1 also acts as a co-activator and together with RORgt induces the expression of the Il17a and Il17f genes. Therefore, the binding of T-bet to Runx1 will deplete the pool of free Runx1 available for the formation of transcriptionally active Runx1/RORgt complexes in TH17 cells. These data suggest that if a naive CD4+ is to be differentiated into a TH17 cell, it has to repress T-bet expression. The mechanism by which T-bet is silenced in developing TH17 cells remains obscure. Two recent studies have demonstrated that T-bet expression in TH1 cells is regulated post-transcriptionally by the microRNA miR-29b [Immunity (2011) 35:169-181; Nature Immunology (2011) 12:861-869]. Our preliminary data using Dicer-deficient TH17 cells suggest that T-bet expression is negatively regulated by microRNAs in this TH subset. The goal of this project was to identify which microRNA is responsible for suppressing T-bet expression in TH17 cells. We performed microRNA microarrays on TH cells that express T-bet (such as TH0 and IL-23 treated TH17 cells) and in TH17 cells, which do not express T-bet. Differential analysis revealed several microRNAs that were expressed at high levels in TH17 cells and diminished in TH cells expressing T-bet. Furthermore, bioinformatics analysis revealed the presence of binding sites in the T-bet 3?-UTR specific for these microRNAs. Collectively, these results suggest that the selected microRNAs are good candidates for the silencing of T-bet expression during TH17 differentiation. To directly address this question, we will over-express the candidate microRNAs in TH1 cells, which normally have high levels of T-bet, and investigate whether the presence of these microRNAs in TH1 cells have a negative effect on T-bet expression. If this turns out to be the case, we will silence these microRNAs in TH17 cells and investigate whether this manipulation will affect TH17 differentiation or T-bet expression in IL-17A producing cells. In addition, we will mutate specific microRNA binding sites in the T-bet 3?-UTR region and determine whether these microRNAs act as direct regulators of T-bet expression, or they act indirectly by affecting signaling pathways that result in T-bet induction (such as IFN-g signaling pathway, for example) The results from this study will reveal the basic understanding of the regulatory mechanisms that act to constrain the T-bet specific pathway that promotes the development of the opposing TH1 lineage.
NIH ExPorter
NIH ExPorter