Most 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 
. 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 
. Analysis of cell populations associated with ILF formation in transgenic mice engineered to express CXCL13 in intestinal epithelial 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 
. In addition to the role of CXCL13 in ILF expansion and relevant to the gene expression induced by GRA, TLR activated LTi-like express LTα1LTβ2 to interact with the LTβR on stromal cells, which in turn release cytokines including DC recruitment ligands CCL19 and CCL21, that together with other signals including IL-22, result in maturation of ILF and a T cell independent B cell response 
. 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 
. 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 was observed 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 luminal stimuli, and their development and maturation are dependent on both dietary ligands and post-gestational acquisition of gut microbiota 
. A critical and required role for the aryl hydrocarbon receptor (AhR) in regulating ILF maturation recently has been reported 
. AhR is a ligand-activated transcription factor responsive to environmental signals including xenobiotics, dietary and endogenous ligands 
. AhR activation results in signaling and gene expression patterns that regulate multiple physiological processes including detoxification, immune cell modulation and maintenance of metabolic homeostasis. AhR−/−
mice or mice fed a diet deficient in AhR ligands do not develop ILF, and ILF are restored by addition of an AhR ligand to deficient diets 
. Studies to determine whether GRA or other components of licorice extract act through the AhR and thus introduce a new ligand for this receptor are ongoing.
ILF are dynamic structures particularly responsive to changes in gut flora, and play a central role in regulating IgA production that controls commensal populations 
. The dependence of ILF formation on the composition of the microbiota puts forth the intriguing possibility that GRA alters the composition of the bacterial population in the gut. Recognition of bacterial peptidoglycan by pattern recognition receptor NOD1 in epithelial cells also is required for optimal ILF formation, 
, putting forth an alternative hypothesis that GRA activates signaling pathways controlled by NOD1 and TLR, thus offering an explanation for the rapid gene induction. Whether GRA, GA or crude licorice root extracts affect the interplay between gut tissue and the microbiota that could be responsible for some of the immune system modulating effects that have been attributed to these compounds warrants investigation.
Oral administration of GRA to mice one day prior to and one day after infection with rotavirus did not affect the onset or magnitude of fecal antigen shedding, but shedding resolved more than one day sooner compared to untreated animals. The lack of a difference between onset and magnitude of virus replication supports the idea that effects of GRA in the infected mouse are immune-mediated, as administration of GRA was associated with accelerated clearance. Whether the reduction in the duration of shedding is a direct result of ILF maturation is under investigation. Notably, GRA induced CD19+ cell accumulation in the LP, and ILF formation in the LP of both uninfected and infected mice, suggesting GRA affects signaling pathways that drive lymphocyte recruitment, and can occur independently of virus infection. ILF regulate IgA production to maintain intestinal homeostasis as well as to respond effectively to pathogens. A defined role for these ILF in rotavirus clearance remains to be determined. GRA also had an effect on expansion of T cells in the PP early post-infection, suggesting GRA is pleotropic in its ability to modulate immune cell activity. Detailed mechanisms by which GRA induces these responses at the gut mucosa, including identification of target cells currently are under investigation.