Sesame Oil

All mice used in these studies were on the C57BL/6 background. C57BL/6 mice were purchased from The National Cancer Institute (Frederick, MD) or The Jackson Laboratory (Bar Harbor, MN). OTI T cell receptor transgenic mice^{42 (link)}, Math1^fl/fl mice^{13 (link)}, Math1^PGRCre mice^{43 (link)}, CD11c^YFP transgenic mice^{20 (link)}, CX₃CR1^GFP mice^{44 (link)}, and ROSA^dtomato mice^{45 (link)}, mice were purchased from The Jackson Laboratory. Myd88^−/− mice^{46 (link)}, a gift from Dr. Akira (Osaka University, Osaka, Japan), were bred onto the C57BL/6 background by the Speed Congenics Facility of the Rheumatic Diseases Core Center, and maintained in house. Transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the villin promoter (vil-Cre-ERT2) mice ^{47 (link)} were a gift from Sylvie Robine (Institut Curie, Paris, France). Math1^fl/fl mice were bred to vil-Cre-ERT2 mice to generate mice with inducible depletion of GCs following deletion of Math1 in villin expressing cells, referred to as Math1^iΔvil mice. To deplete GCs, Math1^iΔvil mice mice were injected with 100 µg tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) dissolved in sunflower seed oil with 20% ethanol (Sigma-Aldrich) i.p. daily for 4 days. Three days after the final injection, mice were placed on antibiotics for one week and then analyzed for GAP formation by two-photon as described below. Colon tissue was sectioned and stained for goblet cells with alcian blue to ensure goblet cells were knocked out. To generate Math1 reporter mice, Math1^PGRCre mice, which express a progesterone inducible Cre under the control of the Math1 locus, were bred with ROSA^dtomato mice, referred to as dtom^iMath1 mice. To label colonic GCs, dtom^iMath1 mice were injected with 200 µg RU486 (mifepristone, Caymen Chemicals, Ann Arbor, MI, USA) dissolved in sesame oil (Acros Organics, Sigma-Aldrich) i.p. every day for 7 days. To delete Myd88 signaling from GCs, Myd88^fl/fl mice were bred to Math1^PGRcre mice, referred to as to Myd88^iΔMath1 mice. To induce deletion of Myd88 in GCs, Myd88^iΔMath1 mice were injected with 200 µg RU486 i.p. on day 0 and day 2, and analyzed on day 4. Myd88^fl/fl Math1^WT littermate mice injected with RU486 were used for controls. Gnotobiotic mice were obtained from the Washington University Digestive Disease Research Core Center murine models core. Animals, other than gnotobiotic mice, were housed in a specific-pathogen-free facility and fed routine chow diet. In some experiments mice were treated with a combination of antibiotics (Abx); ampicillin (1 g/L), metronidazole (1 g/L), neomycin (1 g/L), and vancomycin (500 mg/L) in drinking water for 10 days. Animals were 6 to 16 weeks of age at the time of analysis. Animal procedures and protocols were carried out in accordance with the institutional review board at Washington University School of Medicine.