Myd88fl fl

Littermate mice were co-housed randomly regardless of their genotype during different diet regimen and additional nutritional supplementation. However, during stool transfer experiments, mice were separated and co-housed according to their genotypes to avoid cross contamination. During treatment with antibiotics, mice were supplemented with a mix composed of 0.04 g ampicillin, 0.02 g vancomycin, 4.3 ml neomycin, 8.6 ml metronidazol dissolved in 200 ml drinking water. During transfer experiments, following antibiotic treatment for one week, 7 weeks old mice were colonized with fresh stool pellets (9×10⁶ bacteria) from mice fed on HFD (that were fed on HFD for already 24 weeks). GOS (Bi2muno) (5.5 gr) was provided in 150 ml drinking water, which was refreshed every 2 days. Sodium butyrate (Aldrich, 303410) (50 μg/g mouse in 100 μl H₂O) and arabinogalactan (Fluka, A-09788) (0.01 g/g mouse in 100 μl H₂O) were provided orally three times a week. K-ras^G12Dint mice were further crossed to Myd88^fl/fl and Myd88^−/− animals (Jackson Laboratory, Bar Harbor, ME USA). During adoptive transfer experiments, six weeks old K-ras^G12Dint mice were irradiated (9 Gy) and 2×10⁶ bone marrow cells from Myd88^−/− or wildtype mice were transferred by tail vein injection to recipients. The procedures were approved by the Regierung von Oberbayern.

C57BL/6 (B6) mice homozygous for loxP flanked caspase-8 allele (Casp8^fl/fl) (21 (link)) were crossed with mice expressing Cre under control of the CD11c promoter (Cre^CD11c, Jackson Laboratory, Alexander Chervonsky), generating Cre^CD11cCasp8^fl/fl mice. PCR on FACS-sorted splenic conventional DC populations (B220⁻CD11c⁺CD8⁻ and B220⁻CD11c⁺CD8⁺) from Cre^CD11cCasp8^fl/fl mice showed deletion of caspase-8 but not in plasmacytoid DCs (CD11c^intermediatePDCA-1⁺B220⁺), lymphocytes or macrophages (Supplemental Figure 1). Cre^CD11cCasp8^fl/fl BMDCs showed caspase-8 deletion (Supplemental Figure 1). OT-II/RAG^−/− and B6.CD45.1 were purchased (Jackson Laboratory). RIPK3^−/− (Genentech), IRF3^−/− (a gift from Mike Diamond), IRF7^−/− (a gift from Mike Diamond), MyD88^fl/fl (Jackson Laboratory) mice were bred to Cre^CD11cCasp8^fl/fl generating RIPK3^−/−Cre^CD11cCasp8^fl/fl, IRF3^−/−Cre^CD11cCasp8^fl/fl, IRF7^−/−Cre^CD11cCasp8^fl/fl and MyD88^fl/flCre^CD11cCasp8^fl/fl mice. Real-time PCR performed by Transnetyx on FACS-sorted splenic conventional DC populations from MyD88^fl/flCre^CD11cCasp8^fl/fl mice showed caspase-8 and MyD88 deletion (Supplemental Figure 1). Female mice were used in all studies. Proteinuria was assessed using uristix (Siemens). Transnetyx performed genotyping. Experiments were approved by Northwestern University IACUC.