Fig. 5.

Endothelial Nox2 limits lipopolysaccharide (LPS)-induced transcription of toll-like receptor (TLR4) via nuclear factor (NF)-κB and reduces sepsis mortality. (A–D) Human umbilical vein endothelial cells (HUVEC) were pre-incubated with gp91ds-tat (30 μmol/L) or scrambled-tat (sc-tat, 30 μmol/L) and then stimulated with lipopolysaccharide (LPS, 200 ng/mL). (A) TLR4 mRNA levels 8 h after LPS stimulation. (B) Presence of p65 NF-κB subunit (green) in the nucleus (blue), 30 min after LPS stimulation. Scale bars, 25 μm. (C) Luciferase reporter assay for NF-κB activation after LPS stimulation. RLU: relative lumen units. Graphs and images represent mean ± SEM of three independent experiments performed in triplicate. ∗P < 0.05 for indicated comparisons. Kruskal–Wallis followed by Dunn's multiple comparison test. (D) Sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 wild-type (WT, n = 10) or TLR4-deficient (TLR4^-/-, n = 7), or TLR2-deficient (TLR2^-/-, n = 5), or TLR9-deficient (TLR9^-/-, n = 5) mice. Some animals were pretreated with the Nox2 inhibitor apocynin (200 mg/kg, s.c.) All mice were treated with antibiotics (ATB, ertapenem sodium, 30 mg/kg, i.p.) after surgery. Survival was evaluated up to 7 days after surgery. ∗P < 0.05 compared to WT mice submitted to CLP and treated with ATB. ⁺P < 0.05 compared to WT mice submitted to CLP and treated with ATB plus apocynin. Log-rank (Mantel–Cox) test.