Journal: PLoS Pathogens
Article Title: NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis
Figure Lengend Snippet: IL-1β-dependent IL-10 production mediated by nucleotide-binding oligomerization domain (NOD) 2 enhances C5a generation by suppressing CD55 expression on Ly6-G + cells during sepsis. (A) CD55 and CR1/2 expression on gated F4/80 − Ly6-G + peritoneal cells from WT, Nod2 −/− , and Nod2 −/− mice injected with recombinant IL-1β or IL-10 was estimated 24 h after CLP (mean fluorescence intensity [MFI] of CD55 expression in the panels). (B) CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from Il-10 −/− or Il-10 −/− mice injected with recombinant IL-1β was estimated 24 h after CLP (MFI of CD55 expression in the panels) (C) To block IL-10 receptor engagement in vivo , anti-IL10 receptor mAbs were i.p. injected into WT and Nod2 −/− mice administered recombinant IL-10 during CLP-induced sepsis. CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from these mice 24 h after CLP was evaluated. (D) The levels of CD55 expression on F4/80 − Ly6-G + peritoneal cells were compared in WT, Nod2 −/− , IL-10 −/− , and Il-1r −/− mice 24 h after CLP. (A–D) anti-CD55 mAb (lines) and control IgG (diagrams filled with gray) were used. (E) Peritoneal cells from WT and Nod2 −/− mice 24 h after CLP were blotted for Bb factor. (F) Peritoneal cells from WT and Nod2 −/− mice 12 h after CLP were incubated with RPMI media containing 10% WT mouse serum for 24 h. (G and H) To evaluate the effect of CD55 on C5a generation in vivo , WT, Nod2 −/− , (G) or Nod2 −/− mice given recombinant IL-10 (H) were i.p. injected with soluble CD55 12 h after CLP. Serum and peritoneal C5a levels and the survival percentages of these mice were measured during CLP-induced sepsis ( a P = 0.0124, log-rank test; WT [n = 8], Nod2 −/− [n = 8 ], and soluble CD55-injected WT [n = 6 ] or Nod2 −/− mice [n = 8 ] in G, a P = 0.0024, log-rank test; Nod2 −/− mice [n = 8], Nod2 −/− mice injected with recombinant IL-10 [n = 8] or recombinant IL-10 and soluble CD55 [n = 6] in H). *P
Article Snippet: Antibodies against phosphor–p38, p38 (Cell Signaling Technology, MA, USA), phosphor-Rip2 (Thermo scientific, Rockford, USA), Rip2 (Santa Cruz Biotechnology, CA, USA), Bb (Santa Cruz Biotechnology), NOD2 (Santa Cruz Biotechnology), and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Thermo scientific) were used.
Techniques: Binding Assay, Expressing, Mouse Assay, Injection, Recombinant, Fluorescence, Blocking Assay, In Vivo, Incubation