18b-Glycyrrhetinic Acid Delivered Orally Induces Isolated
Lymphoid Follicle Maturation at the Intestinal Mucosa
and Attenuates Rotavirus Shedding
Jay M. Hendricks, Carol Hoffman¤, David W. Pascual¤, Michele E. Hardy*
Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, United States of America
Glycyrrhizin, an abundant bioactive component of the medicinal licorice root is rapidly metabolized by gut commensal
bacteria into 18b-glycyrrhetinic acid (GRA). Either or both of these compounds have been shown to have antiviral, anti-
hepatotoxic, anti-ulcerative, anti-tumor, anti-allergenic and anti-inflammatory activity in vitro or in vivo. In this study, the
ability of GRA to modulate immune responses at the small intestinal mucosa when delivered orally was investigated.
Analysis of cytokine transcription in duodenal and ileal tissue in response to GRA treatment revealed a pattern of chemokine
and chemokine receptor gene expression predictive of B cell recruitment to the gut. Consistent with this finding, GRA
induced increases in CD19+B cells in the lamina propria and B220+B cell aggregates framed by CD11c+dendritic cells in
structures resembling isolated lymphoid follicles (ILF). Using a mouse model of rotavirus infection, GRA reduced the
duration of viral antigen shedding, and endpoint serum antibody titers were higher in GRA-treated animals. Together the
data suggest GRA delivered orally augments lymphocyte recruitment to the intestinal mucosa and induces maturation of B
cell-rich ILF independently of ectopic antigenic stimulus. These results provide further support a role for dietary ligands in
modulation of dynamic intestinal lymphoid tissue.
Citation: Hendricks JM, Hoffman C, Pascual DW, Hardy ME (2012) 18b-Glycyrrhetinic Acid Delivered Orally Induces Isolated Lymphoid Follicle Maturation at the
Intestinal Mucosa and Attenuates Rotavirus Shedding. PLoS ONE 7(11): e49491. doi:10.1371/journal.pone.0049491
Editor: Emiko Mizoguchi, Massachusetts General Hospital, United States of America
Received June 5, 2012; Accepted October 10, 2012; Published November 13, 2012
Copyright: ? 2012 Hendricks et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by PHS grant NCCAM AT004986. Additional support was provided by National Center for Research Resources (NCRR) grant
RR020185-09, National Institute of General Medical Sciences (NIGMS) grant GM103500-09, equipment from the Murdock Charitable Trust, and the Montana
Agriculture Experiment Station. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, United States of
Pharmacologically active constituents in extracts of the medic-
inal licorice root include glycyrrhizin (GA) and its aglycone
metabolite 18b-glycyrrhetinic acid (GRA). Both compounds have
been extensively studied for their effects on cellular physiology and
as immune system modulators in cultured cell lines, in small
animal models and in humans, with either or both demonstrating
anti-tumorgenic, anti-allergenic, anti-hepatotoxic, antiviral, anti-
ulcerative, or anti-inflammatory properties (reviewed in ).
Multiple mechanisms of activity have been proposed including
inductive or inhibitory effects on apoptosis, cytokine expression,
intracellular signaling pathways, transcription factor activation,
cellular membrane fluidity and modulation of oxidative stress [1–
6]. How or if these mechanisms function in vivo to account for the
ability of these compounds to attenuate pathology in infectious and
inflammatory diseases is not well understood.
GA has been shown to be beneficial in vivo in several systems. In
the clinical setting, intravenous administration of a commercial
formulation containing GA (Stronger Neo-MinophagenH) has
been used in Japan for .20 years to treat patients with chronic
viral hepatitis, with evidence of clinical improvement and re-
duction in progression to hepatocellular carcinoma [7–10].
Murine models of infectious and inflammatory diseases provide
further evidence for immune modulating or antimicrobial
properties of GA. GA reduces lethality associated with influenza
virus infection , and attenuates carrageenan-induced lung
injury , LPS-induced acute respiratory stress syndrome ,
and OVA-induced allergic asthma . In the gut, GA and
a formulation called Si-Ni-San containing GA, ameliorate in-
flammation-mediated pathology in a mouse model of colitis ,
and are associated with decreased expression of proinflammatory
cytokines IFN-c, IL-12, TNF-a, and IL-17, and increased
expression of anti-inflammatory cytokines IL-10 and TGF-b.
GA-induced anti-inflammatory cytokine expression also was
demonstrated in a gut ischemia-reperfusion model .
In contrast to GA, less in vivo data are available for GRA.
Despite less direct evidence for in vivo activity, GA is rapidly
metabolized into GRA , and it is likely that some of the
immune modulating effects of GA are attributable to its primary
metabolite. Studies have shown intraperitoneal administration of
GRA to mice in a model of visceral leshmaniasis results in reduced
parasite burden , and repeated subcutaneous administration of
GRA abrogates lung pathology associated with Staphylococcal
pneumonia . In addition, we recently have shown that GRA
reduces lesion size and virulence gene expression in a mouse
model of MRSA skin infection . Taken together, these studies
provide evidence that GA and GRA modulate immune responses
PLOS ONE | www.plosone.org1November 2012 | Volume 7 | Issue 11 | e49491
to a variety of infectious agents, and regulate cell stress responses
in chronic inflammatory environments, suggesting potential of
these purified compounds to be used as therapeutics or immune
adjuvants. There are little data however, that address whether
these compounds have similar activity when taken orally, and
whether purified compounds or crude extracts commonly used as
dietary supplements affect host defense responses through this
route of administration.
In this study, potential mechanisms of immune system
modulating activity of orally administered GRA were investigated.
Analysis of cytokine gene expression in small intestinal tissue
following administration of GRA revealed a specific pattern of
chemokine and chemokine receptor gene expression that was
predictive of B cell recruitment to the gut mucosa. Increases in
CD19+B cells in the small intestinal lamina propria were observed
in GRA-treated mice, and histological analyses identified B220+B
cell clusters with morphology and cell content consistent with
structures of isolated lymphoid follicles (ILFs). The ability of GRA
to induce lymphoid tissue maturation independently of ectopic
antigenic stimulus suggests GRA affects immune cell responses in
the gut and activates signaling pathways favorable to modulation
of mucosal B cell populations. Using the adult mouse model of
rotavirus infection, we further show that GRA shortened the
duration of viral antigen shedding, suggesting the changes in gene
expression and lymphocyte recruitment to the intestine induced by
GRA likely is functionally relevant in enteric virus infection.
Materials and Methods
All animal experiments were performed according to the NIH
Guidelines for Care and Use of Animals, with approval from the
Montana State University Institutional Animal Care and Use
Committee (Protocol number 2011-44).
Compounds and Virus
Glycyrrhizin (GA) and 18b-glycyrrhetinic acid (GRA) were
purchased from Sigma-Aldrich. Stock solutions were prepared to
a concentration of 100 mg/mL in DMSO (vehicle) and aliquots
were stored at 280uC. Stock solutions were diluted to working
concentrations in calcium-magnesium free phosphate-buffered
saline (PBS), and tested for endotoxin with the Limulus
Amoebocyte Lysate Assay (Associates of Cape Cod, Inc). The
final concentration of endotoxin in the working stock was ,0.025
Murine rotavirus strain EW was prepared and maintained in
intestinal homogenates harvested from neonatal mice as previously
Animal Dosing and Infections
Four to six week old male C57BL/6 mice were obtained from
Jackson Laboratories. Fifty mg/kg of GRA or vehicle only was
administered by oral gavage according to the timetable dictated by
the experiment. For infection studies, mice were administered 105
shedding dose 50 (SD50) of EW in a volume of 100 mL by oral
gavage, or 100 mL of intestinal homogenate prepared from
uninfected neonatal mice. Fecal samples were collected daily.
Animals were euthanized at the conclusion of the experiments to
harvest intestinal tissue for RNA isolation, flow cytometry, and
Following oral administration of GRA, one cm sections of either
duodenum or ileum were dissected and stored in RNAlater
(Qiagen) at 4uC for a minimum of 18 hrs. All sections were devoid
of Peyer’s Patches. RNA was extracted with the RNeasy system
(Qiagen) and quantified with a Nanodrop 1000 (Fisher Scientific).
Cytokine transcripts were measured with the SABiosciences
Mouse Inflammatory Cytokine Array (PAMM-011A) or Custom
Mouse RT2ProfilerTM. Custom arrays included Cxcr5, Ccl19,
Ccl21b, Cxcl13, Lta, Ltb, Ccr6, Ccr7, Ccr9, Ifng, and Il10. One
mg of RNA was reverse transcribed with RT2First Strand kit
(SABiosciences) following the manufacturer’s instructions. PCR
reactions were performed on an Realplex 4 s (Eppendorf ).
Reaction conditions consisted of 95uC for 10 minutes, followed by
40 cycles of 95uC for 15 seconds, and 60uC for one minute. Data
from a minimum of three mice per group were combined and are
expressed as fold-change over vehicle-treated animals. Fold-
changes .2 were scored as significant.
Harvesting and Analysis of Intestinal Cell Populations
At the indicated times post-dosing and/or post-infection, cells
from the Peyer’s Patches (PPs), mesenteric lymph nodes (MLNs),
and small intestinal lamina propria (LP) were isolated as previously
described . Antibodies used for staining and analysis by flow
cytometry included: anti-CD4 A488, anti-CD8 PE, anti-CD19
PE-cy7, anti-CD69 eF605, anti-CD127 PE-cy5, anti-CD185 PE,
and anti-CD8a AF700, all from eBiosciences. Anti-CD138 PE and
anti-CD11c APC were from BD Biosciences. Flow cytometry was
performed on a BD LSR flow cytometer using FacsDIVA software
and data were analyzed with FlowJo software.
ELISAs for Fecal Rotavirus Antigen Shedding and Anti-
rotavirus Serum Antibody
ELISA for fecal rotavirus antigen detection was performed as
previously described . Fecal samples were diluted 10-fold w/v
in TNC (50 mM Tris, 150 mM NaCl, 5 mM CaCl2) containing
0.05% Tween-20 and protease inhibitors (25 mM leupeptin,
1.5 mM aprotinin, 1 mM benzamidine, 30 mM pepstatin A).
Flat-bottom 96-well plates were coated with a monoclonal
antibody to rotavirus structural protein VP6 (A6M)  diluted
in carbonate/bicarbonate buffer overnight at room temperature.
50 mL fecal suspension was added to the wells and plates were
incubated for one hour at 37uC. Anti-rotavirus SA11 antibody was
added to the wells and incubated for one hour at 37uC, followed
by HRP-conjugated goat anti-rabbit antibody.
To detect serum antibody to rotavirus , 96 well plates were
coated with anti-SA11 antibody overnight. SA114F stock virus was
treated with 25 mM EDTA for 20 minutes, then added to the
wells and incubated for one hour at 37uC. Serial dilutions of serum
samples were added to the wells and incubated for an additional
hour at 37uC. Reactions for both the fecal antigen ELISA and the
serum antibody ELISA were developed with TMB Microwell
Peroxidase(KPL) for10 minutes,
1 M H3PO4. Absorbance at a wavelength of 450 nm was
measured on a VersaMax Microplate Reader (Molecular Devices).
Small intestine was dissected and separated into duodenum,
jejunem and ileum. The lumen of each section was rinsed with
PBS, and then infused with OCT. The sections were coiled into
a cryomold with the proximal end at the center, covered with
OCT and snap frozen in liquid nitrogen. Five mm thick sections
were mounted on Superfrost slides (Fisher), and fixed with 75%
acetone/25% ethanol for five minutes, air dried and then stained
with antibodies to B220 (A488), CD11c (PE), and CD3e (PE), all
from eBiosciences. Images were captured on a Nikon Eclipse i80
GRA Induces ILF Formation
PLOS ONE | www.plosone.org2November 2012 | Volume 7 | Issue 11 | e49491
fluorescent microscope. Mean fluorescence intensity (MFI) was
measured with NIH Image/ImageJ software (http://rsb.info.nih.
GRA Induces Transcription of a Specific Pattern of Genes
Encoding Chemokine Receptors and Corresponding
Ligands in the Small Intestine
The ability of orally delivered GRA to modulate immune
system activity at the gut mucosa initially was analyzed by
measuring cytokine gene expression in small intestinal tissue. Mice
were administered GRA or vehicle alone by oral gavage. Ten
hours post-treatment, total RNA was extracted from sections of
the gut taken ,one cm from the gastroduodenal junction, or the
ileum, and changes in cytokine gene transcription were measured
by RT-qPCR using a Mouse Inflammatory Cytokine array. In the
initial experiment, ten genes were up-regulated in GRA-treated
mice .2-fold over vehicle-treated controls. These genes, listed in
Table 1, plus one additional gene of interest, CCL21b, were
chosen to design custom arrays. The same pattern of up-regulated
chemokine and chemokine receptor transcripts was observed in
multiple repetitions of the experiment, and was similar regardless
of whether RNA was extracted from duodenal or ileal tissue.
GRA-induced transcripts included chemokine receptor CXCR5
and its ligand CXCL13, receptor CCR7 and its ligands CCL19
and CCL21b, and receptors CCR6 and CCR9. Increased
transcription of genes encoding the ligands for CCR6 and
CCR9 (CCL20 and CCL25, respectively) did not meet the
established cut-off of .2-fold change, although CCL20 was
moderately up-regulated in the original full array (1.6 fold, data
not shown). Lymphotoxin A (Lta) and lymphotoxin B (Ltb) also
were up-regulated in GRA-treated animals. IFN-c and IL-10 were
moderately increased in the first experiment, but induction was
not consistent between multiple experiments. Some variability
between mice in terms of the presence or absence of a response
was observed. However, the pattern was reproducible with respect
to both the transcripts that were induced and the relative
magnitude of expression in all mice that responded, and these
genes never were up-regulated in vehicle-treated controls.
Expression of genes encoding these chemokine receptors and
their corresponding ligands is consistent with signals known to be
required for lymphocyte recruitment to the intestinal mucosa, and
with formation and maturation of B cell-rich isolated lymphoid
follicles (ILF, ([24,25], see below).
To test whether enteric rotavirus infection affected induction of
these genes by GRA, mice were infected for 18 hours with murine
rotavirus strain EW prior to administration of GRA. The same
pattern of gene expression was observed (Table 1), indicating virus
replication does not modulate the signal-inducing activity of GRA
early post-infection. These results suggest GRA likely has a direct
effect on specific cellular targets in the small intestinal mucosa that
results in coordinated chemokine and receptor gene expression.
Immune Cell Populations Induced in MLNs and PPs by
The observed pattern of chemokine and receptor gene
expression led us to examine the effects of GRA on immune cell
populations at mucosal inductive sites. Mice were administered
GRA or vehicle and infected with EW or mock-infected. Animals
were sacrificed nine days post-infection and immune cell
populations in the PPs and MLNs were analyzed by flow
cytometry (Figure 1). The percentage of CD4+T cells increased
in GRA-treated, uninfected mice compared to vehicle-treated
controls in the MLNs, but not in the PPs. In the PPs, CD8+T cells
were significantly increased in GRA-treated, infected mice relative
to vehicle-treated, infected mice. CD8+T cells also appeared to
increase in the MLNs in GRA-treated, uninfected mice compared
to vehicle-treated animals, but this increase did not score as
significant. These data suggest GRA may have an effect on T cell
accumulation in these inductive tissues, particularly CD8+T cells
in PP of infected mice. Analysis of myeloid cell populations in
GRA- or vehicle-treated, infected animals showed significant
differences in dendritic cell (DC) subsets CD11chighand CD11clow,
as well as macrophage (CD11b+) cell populations in the MLNs.
The only significant difference observed in the PPs was CD11b+
cells in GRA treated, uninfected mice.
A striking difference in the CD138+population was observed
between mice given GRA and mice administered vehicle. CD138
(syndecan-1) is expressed on pre-B and immature B cells in the
bone marrow, absent on circulating B cells, and re-expressed on
plasma cells . GRA-treated mice had a significantly higher
percentage of CD138+cells than vehicle-treated mice both in the
MLNs and the PPs (Figure 1). This difference was not observed in
GRA-treated infected mice, likely overshadowed by influx of
lymphocytes into these tissues in response to virus infection. To
investigate this further and determine the kinetics of the initial
response, mice (uninfected) were gavaged with GRA or vehicle,
and MLNs and PPs were harvested 24 and 48 hours post-
treatment (Figure 2). CD138+cells were increased in both tissues
by 48 hours in animals given GRA, but not in animals given
vehicle, suggesting GRA affects B cell differentiation in these
mucosal inductive sites.
GRA Induces CD19+B Cell Recruitment to the LP
To test how the timing of GRA dosing affected B and T cell
populations in mucosal inductive sites as well as in the LP effector
site, mice were treated either one day pre-infection and one day
post-infection (or mock-infection) as before, or every other day for
the course of the experiment. In the MLNs, significant increases in
the CD8+T cell population in GRA-treated, uninfected mice
relative to vehicle-treated controls were observed (Figure 3). There
Table 1. GRA-induced changes in cytokine expression in
Ccr9 2.62.2 1.3
Cxcl13 4.24.6 7.1
Il10 2.6 1.61.3
Ccl21bND 3.6 2.3
Representative data are shown for RNA isolated from duodenal or ileal tissue.
Data shown for duodenal tissue are from the initial full array. Data from ileal
sections from uninfected and EW infected mice were obtained with the custom
array. Data are presented as fold-increase over mock-treated controls. ND – not
GRA Induces ILF Formation
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were no differences in CD4+or CD8+T cell populations between
the different dosing schedules. In PPs, there were no significant
differences in CD4+T cells between GRA-treated and vehicle-
treated uninfected or infected animals, except the overall
percentages in infected mice were somewhat higher. In contrast,
CD8+T cells in the PPs markedly increased in GRA-treated,
Figure 1. Immune cell populations modulated by GRA in uninfected and rotavirus -infected mice. C57Bl/6 mice (n=5 per group) were
administered GRA or vehicle alone orally one day pre-infection with 105SD50of murine rotavirus strain EW, and then one day post-infection. Cells
isolated from the MLNs and PPs were analyzed for changes in B cells (CD19), T cells (CD4 and CD8), their activation (CD69); and dendritic cells
(CD11chighand CD11clow), macrophages (CD11b), and plasma cells (CD138). *p,0.05, **p,0.01. Error bars are SEM.
Figure 2. CD138+cells are increased in MLNs and PPs 48 hours post-treatment (hpt) with GRA. Mice (n=3 mice per group) were
administered GRA or vehicle by oral gavage, and cell populations in the MLNs and PPs were analyzed with antibodies to CD11c, CD11b, and CD138.
**p,0.01. Error bars are SEM.
GRA Induces ILF Formation
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infected animals compared to animals given vehicle, as before. A
similar trend was observed in uninfected animals, although this
difference was not as great. These data further support the idea
that GRA impacts CD8+T cells in mucosal inductive tissues.
Importantly, changes in CD8+T cells were not enhanced by
repeated GRA doses, suggesting the initial signaling events
induced by GRA are of key importance and result in a sustained
cellular immune response.
LP lymphocytes also were analyzed to determine whether orally
delivered GRA could influence immune cell populations at
a mucosal effector site. A profound increase in CD19+B cells
was observed in the LP of GRA-treated mice (Figure 3).
Importantly, this increase was observed in both uninfected and
infected animals, and there were no differences between the dosing
schedules. These data, interpreted in the context of the gene
expression data, suggest GRA induces B cell recruitment to the
small intestinal mucosa, and does so in the absence of ectopic
GRA Induces Formation of B220+B Cell Clusters
ILFs consist of a single B cell follicle surrounded by DC with few
scattered T cells [27,28]. In an experiment prompted by gene
expression data and demonstrated increases in CD19+ cells in the
LP, mice were administered GRA or vehicle, and then mock-
infected or infected with rotavirus to determine if ILFs were
induced by GRA. GRA was given one day pre-infection and then
again one day post-infection, as before. Intestinal sections were
prepared one day following the second dose of GRA, or nine days
post-infection and stained for detection of B cells (B220), DCs
(CD11c), or T cells (CD3).
In ileal sections harvested one day after the second dose of
GRA, obvious B220+cell clusters surrounded by CD11c+cells,
and a few CD3+T cells were observed (Figure 4). DAPI staining
further indicated that villi containing these B220+cell clusters were
shorter and broader than surrounding villi, and typical LP
structure was displaced. This cell composition and the morphology
of villi where they are located are consistent with mature ILF
[27,29]. These structures were not present in tissues from vehicle-
treated mice. Instead, small areas of CD11c+cells with few B220+
cells were routinely visible. Estimates of changes in B220+cell
density between GRA-treated and vehicle-treated animals made
by measuring mean fluorescent intensity indicated a DMFI .7
fold between the two groups. The smaller structures in vehicle-
treated mice may be indicative of immature ILF [28,29], and
suggest GRA induces ILF maturation and ectopic antigenic
stimulus is not required.
In sections harvested from infected mice at the early time point,
B220+cells were increased in GRA-treated mice relative to
vehicle-treated mice (DMFI .3 fold, Figure 4). B220+cells also
appeared increased in GRA-treated infected mice at nine days, at
which time the infection was resolved, although the difference was
Figure 3. GRA induces CD19+cell accumulation in the lamina propria in uninfected and rotavirus infected mice. Mice (n=5 mice per
group) were administered GRA or vehicle by oral gavage, and then mock-infected or infected with EW. Two dosing schedules were used: 1) GRA or
vehicle alone was administered one day pre-infection and then one post-infection (pre/post), or 2) every two days through the course of infection.
Nine days post-infection, cell populations isolated from the MLNs, PPs and LP were analyzed by flow cytometry for changes in B (CD19+) and T (CD4+
and CD8+) cells. *p,0.05, **p,0.01. Error bars are SEM.
GRA Induces ILF Formation
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not as great (DMFI .1.5 fold, Figure 5). These data suggest
rotavirus infection induces ILF which has not been observed
before, and that GRA might augment the B cell response in the
Oral Administration of GRA Reduces the Duration of
Rotavirus Antigen Shedding
We reported that GRA inhibits rotavirus replication in cell
culture [23,30]. The ability of GRA to attenuate virus replication
in vivo was tested. In the adult mouse model of rotavirus infection,
the magnitude of replication is measured by fecal antigen shedding
[20,31]. Mice were administered GRA or vehicle by oral gavage
one day pre-infection with EW, and then again one day post-
infection. The day of onset and magnitude of virus shedding was
not different between GRA-treated and vehicle-treated animals
(Figure 6A). However, virus shedding in mice that received GRA
was resolved one day earlier, and on the last positive day was
reduced by approximately 50%. Viral antigen was not detectable
in either group at day nine post-infection. GRA treatment did not
result in lower amounts of fecal antigen, but shortened the
duration of virus shedding, suggesting an effect of GRA on the
immune response to infection instead of a direct effect on virus
replication. This idea is supported by the observation that anti-
rotavirus serum antibody titers were statistically higher in GRA-
treated animals relative to controls, although the difference was
small (Figure 6B). Anti-rotavirus fecal IgA titers were not different
between treated and untreated animals (data not shown).
GRA Enhances Accumulation of CD3+T Cells in the PPs of
Rotavirus-infected Mice Early Post-infection
PPs from infected mice treated with GRA or vehicle from the
same experiment described above harvested at the early time point
were evaluated for changes in B220, CD11c, and CD3 expression.
There were marked increases in CD3+T cells in the PPs of
infected mice administered GRA compared to vehicle-treated or
uninfected mice (Figure 7), suggesting GRA enhances T cell
accumulation in mucosal inductive sites in response to infection,
a correlation supported by flow cytometry (Figures 1 and 3).
Figure 4. GRA induces formation of B220+aggregates in uninfected and rotavirus-infected mice. Mice (n=3 mice per group) were
administered GRA or vehicle by oral gavage, and infected or mock infected with EW. In these mice, GRA or vehicle was administered one day pre-
infection and then again one day post-infection. Ileal sections were prepared one day after the second GRA dose 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.
GRA Induces ILF Formation
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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 [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 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 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, 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 was
Figure 5. GRA enhances formation of B220+aggregates in rotavirus-infected mice at 9dpi. Mice (n=3 mice per group) were administered
GRA or vehicle by oral gavage, and infected or mock infected with EW. In 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.
Figure 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.
GRA Induces ILF Formation
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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
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
[32,35–38]. 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. AhR2/2mice 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 peptido-
glycan 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.
Conceived and designed the experiments: MEH JMH. Performed the
experiments: JMH CH. Analyzed the data: JMH CH MEH DWP.
Contributed reagents/materials/analysis tools: CH DWP. Wrote the
paper: MEH JMH.
Figure 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.
GRA Induces ILF Formation
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PLOS ONE | www.plosone.org9 November 2012 | Volume 7 | Issue 11 | e49491