Journal: The Journal of Experimental Medicine

Article Title: Type I interferons protect neonates from acute inflammation through interleukin 10–producing B cells

doi: 10.1084/jem.20062013

Figure Lengend Snippet: Analysis of IL-10 production by neonatal B cell subsets in response to various TLR agonists. (A) Phenotypic analysis of freshly isolated neonatal CD19 + B cells versus B cells cultured with or without CpG. (B) Neonatal splenic CD19 + B cells were sorted as CD5 + CD23 − , CD5 −/dim CD23 + , and CD5 − CD23 − subsets and stimulated with 1 μg/ml Pam3CSK4, 25 μg/ml Poly(I:C), 10 μg/ml LPS, 1 μg/ml R848, and 1 μg/ml CpG with or without 1 ng/ml IFN-β. Data are presented as mean values ± SD. (C and D) B6 neonates were injected i.p. with 100 μg CpG, and 3–6 h later, splenic B cells were either (C) sorted as CD19 + CD5 + and CD19 + CD5 − cells and cultured for 48 h or (D) stained intracellularly with anti–IL-10. (D) Continuous lines and shaded histograms show anti–IL-10 and control isotype antibodies, respectively, for B cell subsets and T cells (inset shows staining of PBS-injected neonates). The numbers in the gates in A, B, and D represent cell percentages.

Article Snippet: TLR2 ligand Pam3CSK4, TLR3 ligand Poly(I:C), TLR4 ligand LPS (from Escherichia coli 0111:B4), and TLR7 ligand R848 were purchased from Invivogen.

Techniques: Isolation, Cell Culture, Injection, Staining

Journal: The Journal of Experimental Medicine

Article Title: Type I interferons protect neonates from acute inflammation through interleukin 10–producing B cells

doi: 10.1084/jem.20062013

Figure Lengend Snippet: Neonatal B cells inhibit the production of proinflammatory cytokines in response to various TLR-Ls. (A and B) 5 × 10 5 B cells from B6 or IL-10 −/− neonatal mice were cultured with 5 × 10 4 Flt3L-pDCs (A) or GMCSF-cDCs in the presence of 1 μg/ml Pam3CSK4, 25 μg/ml Poly(I:C), 10 μg/ml LPS, 1 μg/ml R848, and 1 μg/ml CpG for 48 h. (A) IFN-α, (B) IFN-β, and (A and B) TNF-α, IL-12p70, and IL-10 were detected in supernatants by ELISA. NA, not applicable. No detectable inflammatory cytokine was produced by pDCs in response to Pam3CSK4, Poly(I:C), and LPS. Data are presented as mean values ± SD. (C) B6 neonates and adults were injected with GalN, together with TLR-L, and survival was monitored for 3 d. The following doses were used: 4 mg/kg Pam3CSK4, 2 mg/kg Poly(I:C), 4 mg/kg LPS, 2 mg/kg R848, and 4 mg/kg CpG. 0.35 g/kg GalN was used for Poly(I:C)-, LPS-, and CpG-injected groups, and 1 g/kg GalN was used for Pam3CSK4- and R848-injected groups.

Article Snippet: TLR2 ligand Pam3CSK4, TLR3 ligand Poly(I:C), TLR4 ligand LPS (from Escherichia coli 0111:B4), and TLR7 ligand R848 were purchased from Invivogen.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Produced, Injection

Journal: The Journal of Experimental Medicine

Article Title: Type I interferons protect neonates from acute inflammation through interleukin 10–producing B cells

doi: 10.1084/jem.20062013

Figure Lengend Snippet: Type I IFN signaling restricts the neonatal inflammation induced by CpG through IL-10–secreting B cells. (A) IL-12p40 was detected in the liver, spleen, and lungs of neonatal ( n = 3) and adult ( n = 4) 129sv or IFNAR −/− mice harvested 3 h after a 4-mg/kg CpG i.p. injection. The data shown are representative of three independent experiments. (B) WT and IFNAR −/− neonates and adults were injected i.p. with 1 g/kg GalN and 4 mg/kg CpG, and survival was monitored for 3 d. The cumulative results obtained in three to five experiments are shown. (C) WT and IFNAR −/− neonates were injected i.p. with 20 mg/kg CpG or left untreated. 3 h later, splenic CD5 + B cells, pDCs, and cDCs were analyzed for up-regulation of the CD69 and I-A b activation markers. Nontreated: WT (thin line) and IFNAR −/− (dotted line); CpG: WT (shaded area) and IFNAR −/− (bolded line). (D) WT and IFNAR −/− neonates were injected i.p. with 20 mg/kg CpG, 4 mg/kg Pam3CSK4, or PBS, and CD19 + cells were purified 3 h later. IL-10 was detected in cell culture supernatants by ELISA. (E) WT and IFNAR −/− neonatal or adult mice were injected i.p. with 20 mg/kg CpG, and 3 h later, spleen cells were recovered and cultured for 24 h. TNF-α was detected in supernatants by ELISA. (F) IL-10 −/− neonates received 8 × 10 6 neonatal B cells from WT, TLR9 −/− , or IFNAR −/− mice or adult WT B cells, or were left untreated. They were then challenged with 30 μg CpG. Survival was monitored for 7 d. ND, not detectable. *, P < 0.05 versus the other groups.

Article Snippet: TLR2 ligand Pam3CSK4, TLR3 ligand Poly(I:C), TLR4 ligand LPS (from Escherichia coli 0111:B4), and TLR7 ligand R848 were purchased from Invivogen.

Techniques: Injection, Activation Assay, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay