Journal: Arthritis & Rheumatology (Hoboken, N.j.)
Article Title: Toll‐Like Receptor 9 Deficiency Breaks Tolerance to RNA‐Associated Antigens and Up‐Regulates Toll‐Like Receptor 7 Protein in Sle1 Mice
Figure Lengend Snippet: Regulation of antibody production by Toll‐like receptor 9 (TLR‐9) in Sle1 mice. A and B , B cell (B220+) activation ( A ) and proliferation ( B ) in splenocytes from young (8–10‐week‐old) Sle1 and Sle1 TLR‐9 −/− mice that were left untreated (media), stimulated with R848 (0.01 μg/ml), or stimulated with lipopolysaccharide (LPS; 1 μg/ml). B cell activation was measured by flow cytometry after 24 hours of stimulation and is shown as the percentage of CD69+ cells. B cell proliferation was measured according to the 5,6‐carboxyfluorescein succinimidyl ester dilution after 72 hours of stimulation. C , TLR‐7 expression, measured by intracellular flow cytometry, in mouse splenic B220+ B cells. TLR‐7–deficient Sle1 mice ( Sle1 TLR‐7 −/− ) (n = 4) and fluorescence minus one (FMO) samples were used as negative controls. MFI = median fluorescence intensity. D , Levels of IgG subtypes, measured by Luminex, in Sle1 and Sle1 TLR‐9 −/− mouse culture supernatants collected after 96 hours of incubation. Cultures were left untreated, stimulated with R848, or stimulated with LPS. E , Expression of surface IgG (sIgG) (IgG1/IgG2a/IgG2b/IgG3) on freshly isolated B220+CD19+ splenocytes from Sle1 and Sle1 TLR‐9 −/− mice. F and G , TLR‐7 expression and frequencies of splenic CD138+ plasma/plasmablasts ( F ) and CD11b+ dendritic cells (DCs) ( G ) in Sle1 and Sle1 TLR‐9 −/− mice. In A , B , and D , bars show the mean ± SEM from 2–3 independent experiments (n = 9–15 mice per group). Data were assessed by multiple t ‐tests, and statistical significance was corrected using the Holm‐Sidak method. In C , E , F , and G , data are from 1 representative experiment with 8–10‐week‐old mice (n = 8 Sle1 and 5 Sle1 TLR‐9 −/− mice). Circles represent individual mice; horizontal lines and error bars show the mean ± SEM. Significance was determined by Student's t ‐test. * = P
Article Snippet: They were plated at 1.5 × 106 cells/ml in round‐bottomed 96‐well plates in the presence of either CpG‐B (ODN 1826), R848, or LPS‐EB Ultrapure (all from InvivoGen) at the indicated concentrations.
Techniques: Mouse Assay, Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Luminex, Incubation, Isolation