Smad2 and Smad3 are redundantly essential for the suppression of iNOS synthesis in macrophages by regulating IRF3 and STAT1 pathways.

Although transforming growth factor (TGF)-β1 is a well-known immunosuppressive cytokine, little is known about the role of its downstream transcription factors, Smad2 and Smad3, in the suppression of macrophage activation. Previous studies have demonstrated that Smad3 is critical for the suppression of LPS-mediated inducible nitric oxide (NO) synthase (iNOS) induction, although the role of Smad2 remains to be investigated. In this study, we found that iNOS induction was enhanced in Smad2-deficient bone marrow-derived macrophages (BMDMs) and peritoneal macrophages in vitro and tumor-associated macrophages in vivo, compared with wild-type (WT) macrophages. However, TGF-β1 still suppressed iNOS induction in Smad2-deficient macrophages. In Smad2/3 double knockout (KO) (Smad2/3 DKO) BMDMs, LPS-mediated NO/iNOS induction was more strongly elevated than in Smad2 or Smad3 single KO BMDMs, and its suppression by exogenous TGF-β1 was severely impaired. These data suggest that Smad2 and Smad3 redundantly regulate iNOS induction. Similarly, the production of IL-6 and TNFα, but not IL-10 was augmented in Smad2/3 DKO BMDMs, suggesting that Smad2 and Smad3 also redundantly suppressed some cytokines production. In Smad2/3 DKO macrophages, TLR3- as well as TLR4-mediated IRF3 activation and IFN-β production were strongly augmented, which resulted in hyper STAT1 phosphorylation. Furthermore, IFN-β- and IFN-γ-induced iNOS induction in the absence of TLR signaling and STAT1 transcriptional activity were augmented in Smad2/3 DKO BMDMs. These results suggest that Smad2 and Smad3 negatively regulate iNOS induction in macrophages by suppressing multiple steps in the IRF3-IFN-β-STAT1 pathway.