Elucidation of the Molecular Mechanisms by Which Inflammatory and Anti-Inflammatory Monokines Regulate Interferon (IFN)- γ Production

Pro-inflammatory monocyte-derived cytokines (monokines) such as interleukin (IL)-12, IL-15 and IL-18 work in concert to quickly induce IFN- γ in natural killer (NK) cells, while the anti-inflammatory monokine transforming growth factor beta 1 (TGF-β1) can suppress NK cell IFN- γ production. Here, we provide new molecular evidence as to how these monokines carry out their opposing functions. In the human NK-92 cell line we show that Smad proteins, mediators of TGF- β1 signaling, directly inhibit IFN- γ production. Using chromatin immunoprecipitation (ChIP), we found that both Smad2 and Smad4 associate with the IFN- γ promoter in the proximal region but not in other regions, such as exon 4. Luciferase reporter assays in 293T cells indicated that Smad2 alone, which does not possess a DNA binding domain, had little effect on the inhibition IFN-γ of promoter activity. In contrast, Smad3 and Smad4, both of which have DNA binding activity, dramatically suppressed IFN-γ promoter activity. Co-transfection of Smad2 and Smad4, or Smad3 and Smad4, in 293T cells resulted in synergistic repression. Interestingly, the Smad proteins also inhibited the transactivation of the IFN-γ promoter by T-BET, a recently discovered transcription factor controlling IFN- γ production in mouse T cells and NK cells. This was consistent with our data showing that TGF-β1 inhibited T-BET expression in human NK cells. Using IFN-γ promoter deletion constructs, we mapped the region responsible for Smad-mediated inhibition to the -204 proximal promoter, where Smad binding elements are enriched. In parallel, using micro-array analysis, western-blotting and semi-quantitative PCR in human primary NK cells and the NK-92 cell line, we demonstrated that the combination of the pro-inflammatory monkines IL-12 with IL-15 or IL-18 was able to antagonize multiple components of TGF- β1 signaling, including: 1) inhibition of the expression and the phosphorylation of Smad2; 2) suppression of Smad3 expression; and 3) down-regulation of the expression of the TGF- β receptor II subunit. Moreover, although these monokines had no effect on Smad4 expression, using ChIP assay we observed that Smad4 dissociated from the proximal IFN-γ promoter upon co-stimulation of NK cells with IL-12 and IL-18. In summary, our data show that TGF- β1 suppresses IFN- γ production via the direct binding of Smad proteins to the proximal IFN-γ promoter and via inhibition of T-BET, while the pro-inflammatory cytokines serve to inhibit or down modulate TGF- μ signaling components and reverse Smad binding to the proximal IFN-γ promoter. Thus, we suggest that NK cell production of IFN-γ is controlled by the integration of anti-inflammatory and pro-inflammatory monokine signals, leading to reciprocal antagonism.