Downregulation of BAFF-RFP in Nphs after S . typhimurium infection.

(A and B) WT and BAFF-RFP +/- mice were infected i.p. with 500 CFU S . typhimurium , BM and splenic cell subsets were analyzed by flow cytometry. (C-E) BM Nphs were isolated from WT and BAFF-RFP +/- mice and stimulated for 24 hrs with live S . typhimurium or bacterial lysates in vitro . (A) BAFF-RFP expression in BM and splenic Nphs are shown as representative histograms from three independent experiments. Numbers in the plots indicate the percentage of BAFF-RFP + cells. (B) Graphs show the percentage of BAFF-RFP + cells ( left panels ) and BAFF-RFP mean fluorescence intensity (MFI) expressed as BAFF-RFP/WT ratio ( right panels ) and summarize data from three independent experiments (n = 6–10). Dotted lines show percentage of RFP + cells background in WT control. (C) BAFF-RFP expression of unstimulated BM Nphs and BM Nphs stimulated with live S . typhimurium (1:10 MOI). Data are shown as duplicate representative histograms from three independent experiments. (D) Flow cytometry data show BAFF-RFP expression as a BAFF-RFP/WT ratio of the MFI and are from one representative experiment of three independent experiments. (E) Baff mRNA expression analyzed by quantitative PCR and shown as arbitrary units relative to 18S. Data show one representative experiment of two independent experiments. (C-E). In vitro experiments with purified BM Nphs were done pooling 6–10 mice per group. In D and E, G-CSF plus GM-CSF were used as positive controls. (D and E). Bar graphs show means ± SEM; * p <0.05, ** p <0.01, **** p <0.0001, as determined by One-Way Anova with Holm-Sidak multiple comparisons test. (ST refers to S . typhimurium ).