Sustained anti-inflammatory effects of TGF-β1 on microglia/macrophages

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3 days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7 dpr when transforming growth factor-beta 1 (TGF-beta 1) expression was robustly increased. After transient incubation with TGF-beta 1 for 24 h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72 h after removal of TGF-beta 1. The sustained TGF-beta 1 effects were not attributable to microglia-derived endogenous TGF-beta 1, as revealed by TGF-beta 1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-beta 1 receptor-selective blocker SB525334. After incubation with TGP-beta 1 for 24 h and its subsequent removal, LPS-induced phosphorylation of I kappa B kinases (IKKs), I kappa B degradation, and NF kappa B nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-beta 1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3 dpr, whereas it was almost disappeared on 7 dpr. The findings suggest that abundantly produced TGF-beta 1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced I kappa B degradation.