Figure 1.
GRA induces ILF in the colon, but not in the ileum of antibiotic treated mice.
Intestinal bacteria were ablated by daily oral gavage with an antibiotic cocktail daily for five days and then with the same cocktail in the drinking water for an additional five days prior to administration of GRA. The same cocktail was maintained in the drinking water through the course of the experiment. Animals infected with rotavirus strain EW were given 105 SD50 of virus inoculum by oral gavage. A) ileum; normal flora, B) ileum; antibiotic treated, C) colon; normal flora and D) colon; antibiotic treated. Tissue sections in (A) were stained for B cells (B220), T cells (CD3e) and dendritic cells (CD11c). Panel (B) does not include CD3e staining because T cells were not located in the absence of B220+ aggregate staining. Sections in C) and D) were strained for B cells, dendritic cells, T cells, and follicular dendritic cells (CD35). The labels on the left indicating treatments apply across rows for A–D. Magnification 10X.
Figure 2.
GRA induces ILF maturation in ileum and colon of C3H/HeJ mice.
Mice were administered GRA according the schedule described in the materials and methods. Animals infected with rotavirus strain EW were given 105 SD50 of virus inoculum by oral gavage. Tissue sections were stained for B cells (B220), dendritic cells (CD11c), T cells (CD3), and follicular dendritic cells (CD35). A) ileum and B) colon. Magnification 10X.
Figure 3.
GRA does not induce B cell recruitment to ILF in MyD88−/− mice.
Mice were administered GRA according the schedule described in the materials and methods. Animals infected with rotavirus strain EW were given 105 SD50 of virus inoculum by oral gavage. Tissue sections from the ileum (A) were stained for B cells (B220) and dendritic cells (CD11c). Panels for T cell (CD3a) and CD35 staining were negative and so not included in the figure. Tissue sections from the colon (B) were stained for B cells, T cells, and follicular dendritic cells. Magnification 10X.
Figure 4.
GRA activates NOD2-mediated reporter gene expression in vitro.
Cells were co-transfected with NOD2 expression plasmid pUNO-mNOD2 (InVivoGen), NFkB-luc-cis reporter and phRL renilla luciferase reporter plasmids. Twenty-four hours post-transfection, cells were treated with GRA and reporter gene expression was measured with Dual-Glo Luciferase Assay (Promega). Data are expressed as percent response relative to the NOD2 ligand muramyl dipeptide control set to 100%. Error bars represent standard error of the mean of three separate experiments. P<0.05.
Figure 5.
GRA does not induce B cell recruitment to ILF in NOD2−/− mice.
Mice were administered GRA according the schedule described in the materials and methods. Tissue sections were stained for B cells (B220), dendritic cells (CD11c), T cells (CD3), and follicular dendritic cells (CD35). A) ileum and B) colon. Magnification 10X.
Table 1.
GRA-induced gene expression in ileum and colon of select mouse models.