These mice, GRA or vehicle was administered one day pre-infection and then again one day post-infection. Ileal sections were prepared nine days post-infection, and then were stained for the detection of B cells (B220), DCs (CD11c), and T cells (CD3). Arrows indicate ILF-containing villi; arrowheads indicate adjacent ILF-absent villi. Magnification = 206. doi:10.1371/journal.pone.GNF-7 site 0049491.gDiscussionMost in vivo studies that describe immune system modulating activity of licorice root-derived compounds GA and GRA have used intraperitoneal, subcutaneous, or intravenous routes of administration either in human patients or in animal models. Exceptions include studies of the effects of GA, GRA or other bioactive components of licorice in mouse models of allergy, or oral and gastric ulcerative lesions [1,13,14]. There are no studies of which we are aware that analyze extended immunomodulatory gene expression and cellular responses induced at the gut mucosa upon oral delivery of GRA. The pattern of chemokine and chemokine receptor transcript expression induced by GRA is consistent with that described for immune cell recruitment to the gut and maturation of ILFs [24,25]. Analysis of cell populations associated with ILF formation in transgenic mice engineered to express CXCL13 in intestinalepithelial cells indicated a mechanism of CXCL13-mediated recruitment of B cells, as well as lymphoid tissue inducer (LTi)-like and NK cells to the gut mucosa [24]. In addition to the role of CXCL13 in ILF expansion and relevant to the gene expression induced by GRA, TLR activated LTi-like express LTa1LTb2 to interact with the LTbR on stromal cells, which in turn release cytokines including DC recruitment ligands 
CCL19 and CCL21, that together with other signals including IL-22, 18055761 result in maturation of ILF and a T cell independent B cell response [32,33]. GRA-induced gene expression data HIV-RT inhibitor 1 supplier presented here thus are consistent with the roles of CXCL13, CXCR5, CCR6, CCR7, CCL19, CCL21, and LTA/LTB in ILF formation [24,25,32,34]. That the pattern of GRA-mediated transcript induction is functionally relevant is further supported by increases in CD19+ cells in the LP, and presence of mature ILF in GRA-treated mice. The same pattern of gene expression induced by GRA wasFigure 6. GRA reduces the duration of rotavirus antigen shedding. C57Bl/6 mice (n = 5 mice per group) were administered 50 mg/kg GRA or vehicle alone by oral gavage one day pre-infection and then one day post-infection. A) Fecal samples from individual mice were collected and rotavirus antigen was detected by ELISA. B) Total endpoint anti-rotavirus serum antibody titers were measured by ELISA. Error bars are SEM; p = 0.03. doi:10.1371/journal.pone.0049491.gGRA Induces ILF FormationFigure 7. GRA induces T cell expansion in PPs of rotavirus-infected mice. PP tissue sections from EW-infected mice from the experiment described in the legend to Figure 4 were stained for the detection of B cells (B220), DCs (CD11c), and T cells (CD3). Magnification = 10x. doi:10.1371/journal.pone.0049491.gobserved when mice were given the parent compound GA (data not shown). Although rapidly metabolized by gut commensal bacteria, GA is present in the natural licorice extract in the highest concentration. It will be important to investigate whether genes up-regulated by purified GRA and cell recruitment to the gut also are modulated by crude extract commonly used a dietary supplement. ILFs arise from precursor cryptopatches upon.These mice, GRA or vehicle was administered one day pre-infection and then again one day post-infection. Ileal sections were prepared nine days post-infection, and then were stained for the detection of B cells (B220), DCs (CD11c), and T cells (CD3). Arrows indicate ILF-containing villi; arrowheads indicate adjacent ILF-absent villi. Magnification = 206. doi:10.1371/journal.pone.0049491.gDiscussionMost in vivo studies that describe immune system modulating activity of licorice root-derived compounds GA and GRA have used intraperitoneal, subcutaneous, or intravenous routes of administration either in human patients or in animal models. Exceptions include studies of the effects of GA, GRA or other bioactive components of licorice in mouse models of allergy, or oral and gastric ulcerative lesions [1,13,14]. There are no studies of which we are aware that analyze extended immunomodulatory gene expression and cellular responses induced at the gut mucosa upon oral delivery of GRA. The pattern of chemokine and chemokine receptor transcript expression induced by GRA is consistent with that described for immune cell recruitment to the gut and maturation of ILFs [24,25]. Analysis of cell populations associated with ILF formation in transgenic mice engineered to express CXCL13 in intestinalepithelial cells indicated a mechanism of CXCL13-mediated recruitment of B cells, as well as lymphoid tissue inducer (LTi)-like and NK cells to the gut mucosa [24]. In addition to the role of CXCL13 in ILF expansion and relevant to the gene expression induced by GRA, TLR activated LTi-like express LTa1LTb2 to interact with the LTbR on stromal cells, which in turn release cytokines including DC recruitment ligands CCL19 and CCL21, that together with other signals including IL-22, 18055761 result in maturation of ILF and a T cell independent B cell response [32,33]. GRA-induced gene expression data presented here thus are consistent with the roles of CXCL13, CXCR5, CCR6, CCR7, 
CCL19, CCL21, and LTA/LTB in ILF formation [24,25,32,34]. That the pattern of GRA-mediated transcript induction is functionally relevant is further supported by increases in CD19+ cells in the LP, and presence of mature ILF in GRA-treated mice. The same pattern of gene expression induced by GRA wasFigure 6. GRA reduces the duration of rotavirus antigen shedding. C57Bl/6 mice (n = 5 mice per group) were administered 50 mg/kg GRA or vehicle alone by oral gavage one day pre-infection and then one day post-infection. A) Fecal samples from individual mice were collected and rotavirus antigen was detected by ELISA. B) Total endpoint anti-rotavirus serum antibody titers were measured by ELISA. Error bars are SEM; p = 0.03. doi:10.1371/journal.pone.0049491.gGRA Induces ILF FormationFigure 7. GRA induces T cell expansion in PPs of rotavirus-infected mice. PP tissue sections from EW-infected mice from the experiment described in the legend to Figure 4 were stained for the detection of B cells (B220), DCs (CD11c), and T cells (CD3). Magnification = 10x. doi:10.1371/journal.pone.0049491.gobserved when mice were given the parent compound GA (data not shown). Although rapidly metabolized by gut commensal bacteria, GA is present in the natural licorice extract in the highest concentration. It will be important to investigate whether genes up-regulated by purified GRA and cell recruitment to the gut also are modulated by crude extract commonly used a dietary supplement. ILFs arise from precursor cryptopatches upon.