Animals were maintained in pathogen-free housing and experiments were carried out in accordance to federal, state, and institutional guidelines. Colitis was induced by administration of 3% (w/v) Dextran Sulfate Sodium (36–50 kDa, MP Biomedicals; Santa Ana, CA, USA) in drinking water for 7 days, followed by 3 days with normal water. CD4+ cells were purified from pooled lymph nodes and spleens of WT or gene-deficient DO11.10+/− Rag2−/− mice using magnetic beads (>96% purity). A total of 3–5 × 105 were intravenously injected per recipient in 400 μL PBS. Where noted, recipient
mice were treated with 500 μg of anti-IL-4 mAb on days 0, 3, and 6 (Clone: 11B11). For immunizations, WT donor T cells were transferred into Balb/c mice and, 24 h later, these were intravenously injected with 1–2 × 105 congenic, INK 128 mw bone marrow-derived dendritic cells (BM-DCs) that were preactivated with LPS and loaded with Ova peptide (1 μg/mL each).
For sOva Rag2−/− hosts, single cell suspensions were made from peripheral lymph nodes and restimulated overnight with Ova-pulsed BM-DCs (5:1 lymphocyte to DC ratio). For immunized hosts, CD4+ cells were purified from pooled LNs and spleens, then restimulated overnight (10:1 T to DC ratio). For DSS colitis experiments, single cell suspensions were made from mesenteric lymph nodes (mLNs) and restimulated overnight with platebound antiCD3 mAb (10 ug/mL, clone 145–2C11). All cultures were treated with Brefeldin A (10 μg/m) for the final 2 h, fixed, permiabilized, and stained with anti-CD4 and anti-DO11.10 in combination with anti-cytokine and HIF inhibitor or anti-TF antibodies. Gating strategies for
all intracellular flow cytometry experiments are shown in Supporting Information Fig. 1. Cell sorting was used to purify CD4+ DO11+ CD44high cells from adoptively transferred hosts (5–10 × 104 cells/group). Naïve, CD4+ DO11+ CD25− CD44low controls were purified from DO11.10 Rag2+/+ mice. Real-time PCR protocol and primer sequences have been reported ZD1839 in vivo . Data are presented as fold induction (n > 1) or reduction (n < 1) compared to naïve controls (n = 1). Student's t-test was used to quantify statistical deviation. In all figures, error bars denote standard deviation and asterisks represent significant differences (p < 0.05). The authors thank Dr. Abul Abbas (UCSF) for mice, reagents, and advice. We also thank Dr. J. O'Shea (NIH) and members of the O'Shea laboratory for discussions. Research supported by NIH grant RO1AI64677 and a minority supplement to A.V.V. (PA-05-015). The authors declare no financial conflict of interests. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. "
“The pattern-recognition molecules mannan-binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL-associated serine proteases (MASPs).