A Hepatic Protein, Fetuin-A, Occupies a Protective Role in Lethal Systemic Inflammation

Abstract

BackgroundA liver-derived protein, fetuin-A, was first purified from calf fetal serum in 1944, but its potential role in lethal systemic inflammation was previously unknown. This study aims to delineate the molecular mechanisms underlying the regulation of hepatic fetuin-A expression during lethal systemic inflammation (LSI), and investigated whether alterations of fetuin-A levels affect animal survival, and influence systemic accumulation of a late mediator, HMGB1.Methods and FindingsLSI was induced by endotoxemia or cecal ligation and puncture (CLP) in fetuin-A knock-out or wild-type mice, and animal survival rates were compared. Murine peritoneal macrophages were challenged with exogenous (endotoxin) or endogenous (IFN-γ) stimuli in the absence or presence of fetuin-A, and HMGB1 expression and release was assessed. Circulating fetuin-A levels were decreased in a time-dependent manner, starting between 26 h, reaching a nadir around 24–48 h, and returning towards base-line approximately 72 h post onset of endotoxemia or sepsis. These dynamic changes were mirrored by an early cytokine IFN-γ-mediated inhibition (up to 50–70%) of hepatic fetuin-A expression. Disruption of fetuin-A expression rendered animals more susceptible to LSI, whereas supplementation of fetuin-A (20–100 mg/kg) dose-dependently increased animal survival rates. The protection was associated with a significant reduction in systemic HMGB1 accumulation in vivo, and parallel inhibition of IFN-γ- or LPS-induced HMGB1 release in vitro.ConclusionsThese experimental data suggest that fetuin-A is protective against lethal systemic inflammation partly by inhibiting active HMGB1 release.

Conditional ablation of HMGB1 in mice reveals its protective function against endotoxemia and bacterial infection

“…4C). We also observed that upon bacterial infection of macrophages in vitro, there was a noticeable accumulation of HMGB1 in the cytosol, which also occurs upon LPS stimulation (36,37) (Fig. 4D).…”

Serum Amyloid A Stimulates PKR Expression and HMGB1 Release Possibly through TLR4/RAGE Receptors

“…RAW 264.7 macrophages, U937 monocytes and primary macrophages were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. Adherent macrophages or monocytes were gently washed with, and cultured in, DMEM before stimulating with LPS (0.5 μg/mL), HMGB1 (0.5 μg/mL) or human SAA, in the absence or presence of PKR inhibitors or SAA-neutralizing IgGs for 16 h. Subsequently, the cell-conditioned culture media were analyzed respectively for levels of HMGB1, nitric oxide and other cytokines by Western blotting analysis, the Griess reaction and cytokine antibodies arrays as previously described (38,39).…”

“…To evaluate the role of SAA in lethal sepsis, Balb/C mice (male, 7-8 wks, 20-25 g) were subjected to lethal endotoxemia or sepsis induced by cecal ligation and puncture (CLP) as previously described (38,39,42). Briefly, the cecum of Balb/C mice was ligated at 5.0 mm from the cecal tip and then punctured once with a 22-gauge needle.…”

Carbenoxolone Blocks Endotoxin-Induced Protein Kinase R (PKR) Activation and High Mobility Group Box 1 (HMGB1) Release

“…Both RAW 264.7 cells and primary macrophages were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. Adherent macrophages were gently washed with, and cultured in, DMEM before stimulating with LPS (0.5 μg/mL) in the absence or presence of CBX or purinergic P2X 7 R antagonists (oATP, 50 μmol/L; BBG, 0.5 μmol/L; KN62, 0.5 μmol/L) for 16 h. Subsequently, the cell-conditioned culture media were analyzed for levels of HMGB1, nitric oxide (NO) and other cytokines by Western blotting analysis, the Griess reaction, ELISA, and cytokine antibodies arrays as described previously (36)(37)(38).…”