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
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