IFN-I signaling regulates Ly6C^hi monocytes recruitment and activation.

<p>(A) Expression of Ly6C and CD11c on GM-CSF-differentiated BM-DCs at day 8 of culture (dot plot). BM-DCs of the indicated genotypes were stimulated with heat-inactivated Ca. After 24 h, CCL2, CCL7, KC, and MIP-2 release was determined by ELISA or a multiplex bead array system. Data presented show the mean ± SEM of three independent experiments. (B–H) Mice of the indicated genotype were injected with a lethal dose of 1×10⁵ cfus Ca. At indicated time points, blood, BM and kidneys were collected. (B) Ca cfus in BM were determined and expressed as cfus/mouse bones (n = 3–5 mice per group). Each symbol represents one mouse; horizontal bars indicate the calculated median. (C) BM from day 1-infected mice was isolated and placed in culture. At indicated time points, CCL2 release into the media was measured (n = 3 mice per group). (D) Expression of Ly6C and CD11b on blood leukocytes at day 5 of Ca infection in WT versus <i>Ifnar1^−/−</i> mice. Inflammatory monocytes were gated in R1. (E) Quantification of cells in the R1 gate at different time points post infection. Depicted are percent inflammatory monocytes of total WBCs (left) and absolute cell numbers in ×10⁷/l blood (right). (n = 3–5 mice per group) (F–G) Kidney leukocytes were enriched and characterized by multi-label flow cytometry. (F) Expression of Ly6C and CD11b on kidney leukocytes at day 1 of Ca infection. Inflammatory monocytes are gated in R2 (dot plot). Expression of the inflammatory DC surface markers CCR2, CD11c, MHC II, and PDCA1 by cells in the R2 gate. Solid lines, staining of R2 cells of day 1-infected mice; shaded histograms, for CCR2: staining with isotype control antibody, for CD11c and MHCII: staining of CD11c⁻MHCII⁻ neutrophils, for PDCA1: staining of R2 cells of uninfected mice. Data presented are representatives of three independent experimental repeats (total n = 11–14 mice per group). (G) Kidney leukocytes at day one of infection were stained intracellularly for iNOS. Graphs show iNOS⁺ cells (green) overlayed on total CD45⁺ leukocytes (black). Bar diagram shows the quantification of iNOS⁺ cells in percent of total CD11b⁺ cells. Data presented show the mean ± SEM of two independent experiments (n = 8–9 mice per group). (H) Kidney total RNA was analysed for gene expression of iNOS. Data presented show the mean ± SEM of three independent experiments (n = 7–12 mice per group).