Generation of Tollip^−/− mice. (A) Organization of the mouse Tollip gene, targeting vector, and mutated allele. Filled boxes denote exons (1-6). The arrow and asterisks denote the translational initiator ATG and terminator TAA sequences, respectively. (B) Analysis of genomic DNA by PCR using primers spanning the regions indicated in panel A. (C) Western blot analysis of cellular extracts from Tollip^+/+ and Tollip^−/− MEFs or 293T cells transfected with VSV TollipΔN (amino acids 47 to 274) or full-length VSV Tollip using antibodies to Tollip (upper panel) or VSV (middle panel). To confirm equal loading the same blot was reprobed with antitubulin antibodies (lower panel).

IRAK degradation occurs normally in Tollip^−/− cells. (A) Tollip-deficient (−/−) or wild-type (+/+) MEFs were stimulated with IL-1β (20 ng/ml) for the indicated times. Cell lysates were prepared and immunoblotted with anti-IRAK-1 or anti-IκBα antibodies. (B) Tollip-deficient (−/−), wild-type (+/+), or MyD88-deficient (−/−) MEFs were stimulated with the indicated concentrations of IL-1β for 15 min. Cell lysates were prepared and immunoblotted with various antibodies as indicated. As a loading control in these experiments, the same membrane was reprobed with antibodies against tubulin. NT, not treated.

Tollip-deficient mice produce less IL-6 and TNF-α mRNA and protein upon LPS and IL-1β stimulation. (A) Tollip^−/− or wild-type mice were injected intravenously with 1 μg of IL-1β (five mice/group) or 1 μg of LPS (three mice/group), and the production of IL-6 in the serum was measured at 2 h by ELISA. (B) BMDM were treated with 100 ng of IFN-γ/ml and stimulated with the indicated concentrations of IL-1β, and the production of IL-6 in the supernatant was measured by ELISA 24 h later. (C) BMDM or BMDC were stimulated with the indicated concentrations of LPS, and the production of IL-6 and TNF-α in the supernatant was measured by ELISA 24 h later. The experiments in panels B and C were repeated at least twice in triplicates with similar results. Significant differences are indicated by an asterisks (✽, P < 0.04). (D) Tollip-deficient (−/−) or wild-type mice (+/+) were injected intraperitoneally with a lethal dose of LPS (E. coli O111:B4) at a dose of 40 mg/kg (body weight) to induce septic shock. The serum concentrations of IL-6, TNF-α, and IL-12p40 for all mice/group (n = 9) were measured by ELISA 3 h after injection, and the data shown are averages for each group. Animals were then monitored every 12 h for moribund state and then sacrificed. (E) Peritoneal macrophages or DC were stimulated with 10 or 100 μg of poly(I-C)/ml, and the production of IL-6, TNF-α, and RANTES in the supernatant was measured by ELISA 24 h later. DC from wild-type (+/+) and Tollip-deficient (−/−) mice were stimulated with LPS (1 μg/ml) or poly(I-C) (100 μg/ml) for 24 h. Cell lysates were prepared and immunoblotted as indicated. (F) Wild-type and Tollip^−/− MEFs were stimulated with IL-1β (5 ng/ml) or LPS (100 ng/ml) for the indicated times, and the IL-6 or TNF-α mRNA levels were determined by quantitative real-time RT-PCR analysis. ✽, not detected.

Analysis of IL-1β-, LPS-, and PGN-triggered signaling cascades. (A) MEFs (A) or bone marrow-derived macrophages (B) from wild-type (+/+) and Tollip-deficient (−/−) mice were stimulated with LPS (1 μg/ml), PGN (10 μg/ml), or IL-1β (50 ng/ml) for the indicated times. Cell lysates were prepared and immunoblotted with various antibodies as indicated. Shown are representative experiments, repeated at least three times with similar results. (C) Peritoneal macrophages from wild-type (+/+) and Tollip-deficient (−/−) mice were stimulated with LPS at the indicated concentrations. Cell lysates were prepared and immunoblotted with the indicated antibodies. (D) MEFs were transfected with 500 ng of a pLuc (a NF-κB-dependent reporter plasmid) and 20 ng of phRLTK (Renilla luciferase). The cells were stimulated for 6 h with IL-1β (20 ng/ml), LPS (1 μg/ml), or TNF-α (100 ng/ml) and lysed, and the luciferase activities determined by using the dual-luciferase reporter assay system. Values shown are from data obtained in two transfection experiments. NT, nontreated.

TLR- and IL-1β-dependent activation of immune cells is not impaired in the absence of Tollip. (A) Mice were injected intravenously with 1 μg of LPS, and 6 h later splenic DCs were analyzed by flow cytometry for the indicated activation markers. Histograms represent data for F4/80^−/low CD11c⁺ gated cells. (B) Splenocytes were incubated with LPS at the indicated concentrations, and proliferation was monitored 48 h later by pulsing the cells overnight with [³H]thymidine. (C) Splenic B cells were isolated by using B220⁺ MACS beads and incubated with 5 μg of LPS/ml or 10 μg of CpG/ml for 48 h. Surface expression of MHC II was then monitored by flow cytometry. (D) Splenocytes were plated in triplicate in medium containing 10 ng of IL-12/ml, 20 ng of IL-18/ml, or both. The production of IFN-γ in the supernatant was measured by ELISA 72 h later. (E) Thymocytes were incubated in medium containing 20 U of IL-2/ml, 10 ng of IL-1β/ml, and 2.5 μg of ConA/ml as indicated for 3 days. The cell proliferation was measured by pulsing the cells overnight with [³H]thymidine. The data are averages of quadruplicates and are representative of three independent experiments.