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REVIEW
published: 10 March 2021
doi: 10.3389/fimmu.2021.638725
Frontiers in Immunology | www.frontiersin.org 1March 2021 | Volume 12 | Article 638725
Edited by:
Stefan Jordan,
Charité – Universitätsmedizin
Berlin, Germany
Reviewed by:
Nan Gao,
The State University of New Jersey,
United States
Harry D. Dawson,
Agricultural Research Service (USDA),
United States
*Correspondence:
Prakash S. Nagarkatti
prakash@mailbox.sc.edu
Specialty section:
This article was submitted to
Mucosal Immunity,
a section of the journal
Frontiers in Immunology
Received: 07 December 2020
Accepted: 08 February 2021
Published: 10 March 2021
Citation:
Wisniewski PJ, Nagarkatti M and
Nagarkatti PS (2021) Regulation of
Intestinal Stem Cell Stemness by the
Aryl Hydrocarbon Receptor and Its
Ligands. Front. Immunol. 12:638725.
doi: 10.3389/fimmu.2021.638725
Regulation of Intestinal Stem Cell
Stemness by the Aryl Hydrocarbon
Receptor and Its Ligands
Paul J. Wisniewski, Mitzi Nagarkatti and Prakash S. Nagarkatti*
Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC, United States
Maintenance of intestinal homeostasis requires the integration of immunological and
molecular processes together with environmental, diet, metabolic and microbial cues.
Key to this homeostasis is the proper functioning of epithelial cells originating from
intestinal stem cells (ISCs). While local factors and numerous molecular pathways govern
the ISC niche, the conduit through which these processes work in concordance is the
aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, whose role in
immunoregulation is critical at barrier surfaces. In this review, we discuss how AhR
signaling is emerging as one of the critical regulators of molecular pathways involved
in epithelial cell renewal. In addition, we examine the putative contribution of specific
AhR ligands to ISC stemness and epithelial cell fate.
Keywords: gut epithelium, intestinal stem cells, aryl hydrocarbon receptor, aryl hydrocarbon receptor ligands,
morphogenetic pathways
INTRODUCTION
Maintenance of intestinal homeostasis is governed extensively by the integration of both molecular
and immunological processes. This integration is further mediated by the presence of enteric
microorganisms that colonize the gastrointestinal (GI) tract. The crosstalk between intestinal
microorganisms and the host in which they reside occurs at the gut mucosa, a specialized intestinal
tissue that represents one of the body’s most important interfaces with the environment. The gut
mucosa is comprised of the gut epithelium, a monolayer of epithelial cells that has critical functions
in avoiding self-digestion, contending with luminal contents without eliciting overt immune
responses and promoting self-tolerance (1). Due to its significance, the gut epithelium demonstrates
an astounding renewal capacity as the entire intestinal lining is replenished completely within 5
days (2–4). Homeostasis of the gut epithelium itself is maintained by an intestinal stem cell (ISC)
compartment that resides at the base of intestinal crypts, giving rise to specialized epithelial cell
lineages (5). As such, these ISCs are crucial for the renewal of the differentiated progeny that
comprise the gut epithelium. However, this rapid rate of renewal imposes greater demands on the
cellular hierarchy of the gut epithelium as well as a greater risk of developing intestinal malignancies
(6). What remains to be explored is the extent to which ISCs can be influenced by environmental
factors to maintain or restore intestinal homeostasis.
Of note is the modulation of immune responses from compounds derived from both
endogenous and exogenous sources via the aryl hydrocarbon receptor (AhR), a ligand-activated
transcription factor that integrates environmental, dietary, microbial and metabolic cues to control
transcriptional programs in a ligand-, cell- and context-specific manner (7). While there are some
recent reviews on the role of AhR in the regulation of inflammation through induction of anti-
inflammatory signaling involving IL-10, IL-22, prostaglandin E2, and Foxp3 (8), to the best of
our knowledge, there are no reviews on the role of AhR in ISC function and regulation. Using
Wisniewski et al. AhR Signaling and Intestinal Stemness
floxed Ahr to Villin-Cre mice, Metidji and colleagues have
recently shown AhR expression in intestinal epithelial cells
(IECs) to be critical for ISC homeostasis and gut barrier
integrity as it plays a dominant role in tempering Wnt
signals (9). Expression of tryptophan metabolizing enzyme,
indoleamine 2,3-dioxygenase 1 (IDO1), in IECs has also
shown to enhance differentiation of secretory cells and mucus
production in IEC-specific transgenic mice (mouse line pVil-
EGFP/IDO1) challenged with dextran sodium sulfate (DSS),
2,4,6-trinitrobenzene sulfonic acid (TNBS) or enteropathogenic
Escherichia coli (10). Because induction of IDO depends on
AhR expression and kynurenine produced by IDO acts as
an AhR agonist, these studies suggested that AhR promotes
intestinal homeostasis. Additionally, AhR has shown to sense
genotoxic compounds found in the diet and protect stem cells
against genotoxic stress through the induction of IL-22 by
innate lymphocytes (11). Together, these are a few examples
that highlight the extent to which AhR activation mediates the
regulation ISC stemness. In this review, we examine current
knowledge on how AhR activation can modulate ISC stemness
through essential signals of epithelial cell differentiation.
THE INTESTINAL STEM CELL
COMPARTMENT
While several populations of ISCs have been described, the
driving force of epithelial cell renewal and tissue repair are the
fast-cycling crypt base columnar (CBC) stem cells marked by
a leucine-rich-repeat containing G-protein coupled receptor 5
(LGR5) (2,12,13). These ISCs, or LGR5-positive (+) CBC
stem cells, divide daily and reside at the crypt base (14)
(Figure 1A). Due to the limited space of intestinal crypts, ISCs
undergo ‘neutral competition’ in which half are pushed out of
the ISC niche at random to the above transit-amplifying (TA)
compartment where they then become committed progenitor
cells (14,15). Immediately preceding TA cells is a slow dividing
‘reserve stem cell’ or position 4/ +4 cell population, counting
the adjacent cells from the crypt base, that replenishes the
pool of active stem cells under normal circumstances or fully
differentiates into epithelial cells in the advent of a disrupted
LGR5+compartment such as during acute inflammation (16,
17). What governs this variable stem cell activity and states of
competency are niche-derived signals, such as growth factors and
wingless-related integration site (Wnt) ligands, from neighboring
Paneth cells within the gut epithelium and from subepithelial
mesenchymal cells, including the rare winged helix transcription
factor Foxl1 expressing (Foxl1+) telocytes which maintain
intestinal crypt cell proliferation and promote homeostatic
renewal of the gut epithelium, as well as the recently identified
CD34+Gp38+mesenchymal cells which rapidly respond to
intestinal injury and produce a myriad of factors involved in
ISC maintenance and tissue repair (18–21). ISCs are therefore
subjected to and directed in activity by a host of proximal signals
that encompass the ISC niche. What orchestrates the generation
of new epithelial cells from ISCs and their subsequent functional
specialization in tandem with ISC niche-derived signals are
several molecular pathways. Among these are the Wnt/β-catenin,
Notch, Hedgehog and bone morphogenic protein (BMP), as well
as the epidermal growth factor receptor (EGFR) and ephrin (Eph)
pathways which direct ISC proliferation and cell positioning (21).
Here, we provide an overview of select signals relevant to AhR
activation (Figure 1B).
AHR SIGNALING AND REGULATION
The AhR is a basic helix-loop-helix (bHLH) ligand-dependent
transcription factor that responds to a variety of ligands due to
its malleable ligand-binding site and is the only member of the
bHLH superfamily of transcription factors that can be activated
by ligands (22). Signaling of the AhR involves a central PER-
ARNT-SIM (PAS) domain that is involved in DNA recognition,
ligand binding, and chaperone interactions which are critical
for ensuing transcriptional events (Figure 2). In its inactivated
form, the AhR resides in the cytoplasm within a chaperone
complex comprised of heat-shock protein 90 (Hsp90), p23,
X-associated protein 2 (XAP2), and AhR-associated protein 9
(ARA9) (23). Hsp90 preserves a conformational state of the
AhR that prevents unsolicited translocation into the nucleus
and allows binding of a ligand, while the phosphoprotein
p23 facilitates the interaction between the AhR and Hsp90
(24,25). XAP2 regulates AhR turnover and ARA9 augments
AhR signaling by increasing available binding sites and by
increasing the amount of cytosolic AhR (26,27). Upon binding
of a ligand, the AhR undergoes structural modifications that
expose nuclear localization sequences in which two adjacent
protein kinase C sites become phosphorylated (28–30). Once
translocated, AhR dissociates from its chaperone complex as
AhR receptor nuclear translocator (ARNT) replaces Hsp90
forming a heterodimer (23). This AhR-ARNT heterodimer
binds to cis elements of DNA that contain aryl hydrocarbon
responsive elements (AhREs, also known as xenobiotic- or
dioxin-response elements). These regulatory elements containing
the core sequence 5’-TNGCGTG-3’ can be found in the promoter
regions of numerous target genes including cytochrome P450
enzymes such as CYP1A1, which metabolizes AhR ligands,
thereby suppressing its activation (31). Once bound to AhREs,
this complex acts as a transcriptional complex that can
alter transcriptional activity and chromatin structure through
histone acetyltransferase and methyltransferase activity (32).
AhR activity is tightly controlled by two primary mechanisms
in which the first involves proteolytic degradation 4 h after
the ligand-bound AhR has associated with AhREs and is then
exported from the nucleus (33). The second involves the AhR
repressor protein (AhRR) which is structurally analogous to the
AhR but does not require a ligand to translocate into the nucleus
and interacts with ARNT. It is upregulated upon AhR activation
and therefore acts as a transcriptional repressor (34).
INTERACTION BETWEEN THE AHR AND
SELECT MOLECULAR SIGNALS OF ISC
HOMEOSTASIS
At present, an increasing volume of evidence indicates that
the AhR is a pleiotropic regulator of molecular processes that
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Wisniewski et al. AhR Signaling and Intestinal Stemness
FIGURE 1 | Overview of the intestinal stem cell compartment and molecular cascades related to AhR signaling. (A) Active and quiescent stem cells reside at the crypt
base in both the small and large intestine. Local morphogenetic factors produced from small intestinal Paneth and subepithelial mesenchymal cells regulate ISC
(Continued)
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Wisniewski et al. AhR Signaling and Intestinal Stemness
FIGURE 1 | activity. As stimulated ISCs migrate upward into the transit amplifying compartment, they then become committed differentiated cells. A brush border
resides at the apical surface of epithelial cells in the small intestine which maximizes the absorptive surface area. In contrast, both Paneth cells and a brush border are
absent in the colon. While the absence of Paneth cells in the colon results in decreased antimicrobial proteins, the colon employs other mechanisms to maintain
intestinal homeostasis. LGR5 leucine-rich-repeat containing G-protein coupled receptor 5, CBC crypt base columnar. (B) Numerous molecular pathways orchestrate
epithelial cell fate in concordance with local morphogenetic factors within the ISC. Key proteins in each signaling pathway (blue) interact with transcription factors
(brown) to regulate gene transcription. Upstream of this, additional kinases (orange) modulate these pathways. Both Wnt/β-catenin and Notch signaling play a pivotal
role in regulating the differentiation of either secretory or absorptive epithelial cell types. Together with EGF/MAPK and JAK/STAT signaling as well as others, these
pathways work in tandem to regulate ISC stemness. Akt protein kinase B, APC adenomatous polyposis coli, Bcat beta catenin, CK casein kinase, CSL
CBF1/SU/LAG1, Cyto cytokine, DKK Dickkopf, DSL Delta/Serrate/LAG2 transmembrane ligands, DVL Disheveled, EGF epidermal growth factor, EGFR epidermal
growth factor receptor, γgamma secretase, GAP GTPase-activated protein, GRB2 growth factor receptor-bound protein 2, GSK glycogen synthase kinase, HES1
hairy and enhancer of split 1, JAK/STAT janus kinase/signal transducer and activator of transcription, MAML Mastermind-like, MATH1 Protein atonal homolog 1,
mTOR mammalian target of rapamycin, NICD Notch intracellular domain, PI3k phosphoinositide 3-kinase, PKCalpha protein kinase c alpha, RNF43 ring finger 43,
Rspo respondins, RTK receptor tyrosine kinase, SHP2 Src homology 2 phosphatase 2, SOCS suppressors of cytokine signaling, SOS son of sevenless, TCF/LEF
T-cell factor/lymphoid enhancer-binding factor, Ub ubiquination, ZNRF3 zinc and ring finger 3.
FIGURE 2 | The AhR signaling pathway. Induction of AhR signaling requires binding of a ligand, allowing the AhR complex to translocate into the nucleus and initiate
transcriptional events. Gene products such as the AhR repressor protein (AhRR) and the cytochrome P450 enzyme CYP1A1 suppress AhR signaling by acting as a
direct antagonist to the AhR (dotted line) or by metabolizing AhR ligands (red arrow). AhREs aryl hydrocarbon responsive elements, ARA9 AhR-associated protein 9,
ARNT AhR receptor nuclear translocator, HATs histone acetyltransferases, HMTs histone methyltransferases, Hsp90 heat-shock protein 90, XAP2 X-associated
protein 2.
extend beyond its historical role as a xenobiotic sensor. In
particular is its emerging role in immune development and
function at barrier surfaces including the skin, respiratory tract
and GI tract (35). In addition, AhR activation may contribute to
intestinal homeostasis by regulating ISC stemness and progeny
through morphogenetic signals and others as summarized here
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Wisniewski et al. AhR Signaling and Intestinal Stemness
FIGURE 3 | Summary of AhR signaling and its ligands on aspects of ISC homeostasis. Activation of the AhR regulates the Wnt, Notch and EGFR/MAPK signaling
pathways in ISCs. In addition, specific AhR ligands synergize with key stemness pathways within various cell types to exert unique and beneficial effects. FICZ
tryptophan derivative 6-formylindolo (3, 2-b) carbazole, I3C indole-3-carbinol, IALD indole-3-aldehyde, IL-10R1 interleukin-10 receptor 1, IPA indole-3-propionic acid,
GC goblet cell, KN kynurenine, Qu quercetin, Res resveratrol.
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Wisniewski et al. AhR Signaling and Intestinal Stemness
(Figure 3). As so eloquently defined by Aponte and Caicedo,
stemness in this regard combines the ability of ISCs to perpetuate
its lineage, to give rise to differentiated epithelial cells, and to
interact with its environment to maintain a balance between
quiescence, proliferation and regeneration (36).
Wnt/β-catenin
The canonical Wnt/β-catenin pathway plays a pivotal role in the
establishment of tissue architecture during development and in
homeostasis of adult tissues (37). In the intestine, it is critical
for the proliferation and maintenance of ISCs as well as for the
differentiation of goblet cells (GCs) (38,39). Here, β-catenin is an
essential cytoplasmic signal transducer (40). Given its role in the
maintenance of intestinal homeostasis, aberrant Wnt/β-catenin
signaling has shown to be a hallmark of colorectal cancer (CRC)
development characterized by a loss of the tumor suppressor
adenomatous polyposis coli (APC) and hyperactivation of
Wnt/β-catenin signals (41). In contrast, a putative mechanism
by which AhR activation regulates the Wnt/β-catenin pathway
has recently been determined to be through the activity of
E3 ubiquitin ligases RNF43 and ZNRF3 which target Wnt
frizzled receptors for degradation in ISCs, thereby inhibiting
Wnt signaling (42). Specifically, selective ablation of the AhR or
overexpression of CYP1A1 in IECs potentiated ISC proliferation
(as indicated by Ki67 expression) and inflammation-induced
tumorigenesis (9). This was accompanied by a reduced RNF43
and ZNRF3 expression, and a concomitant increase in β-catenin
and Wnt target gene expression (9). These results show that AhR
deficiency or the degradation of its ligands in IECs promotes
the hyperactivation of Wnt signaling due to a selective defect
in the induction of key negative pathway regulators. In turn,
this highlights the potential of physiological AhR signals to
temper Wnt responsiveness in ISCs. Using an in vitro model of
wound healing, Kasai et al. have further shown that disruption of
adherens junctions by Spinner Modification (S-MEM) in Caco-
2 cells enhances the interaction between β-catenin and AhR
but not with TCDD treatment (an AhR agonist) as evidenced
by immunoprecipitation and that the ablation of β-catenin
by siRNAs enhances the induction of CYP1A1 mRNA with
S-MEM or TCDD treatment (43). This suggests that while
ablation of AhR may potentiate Wnt signals, ablation of β-catenin
may then potentiate AhR signals in response to tissue injury
though direct degradation of β-catenin by ligand-activation of
the AhR remains questionable. As recently demonstrated, only
one primary association between β-catenin and the AhR in vitro
could be found after failing to induce β-catenin degradation
by AhR activation with various AhR ligands in multiple cell
lines, that β-catenin does enhance AhR-mediated transcriptional
activation (44). As such, the most significant discrepancy is
whether the above interaction is assessed in vivo or in vitro.
Consequently and as the findings of Kasai et al. suggest, that
despite TCDD not enhancing the interaction between β-catenin
and the AhR, local factors following tissue injury may act as
endogenous AhR ligands to then temper Wnt signals which could
explain the hyperactivation of Wnt signals in vivo following
IEC-specific ablation of AhR.
EGFR-MAPK/ERK and Notch
Epidermal growth factor (EGF) is an extracellular ligand
produced from neighboring Paneth and subepithelial
mesenchymal cells that plays a pivotal role in intestinal growth
as it potentiates cell survival and ISC proliferation through
downstream Mitogen-activated protein kinase (MAPK) signals
(45–47). MAPKs are vital signaling molecules that influence a
broad range of cellular processes including proliferation and
differentiation in IECs (47). Together with the Wnt/β-catenin
pathway, MAPK signaling governs ISC stemness as well as
their differentiation into TA cells (48). Of the many MAPK
signaling pathways, the Ras/Raf/MEK/ERK system is the best
characterized which culminates in the terminal phosphorylation,
and thus activation, of the MAPKs ERK1 and ERK2 (Figure 2B).
MAPK/ERK signaling is potentiated by the Src homology 2 (SH2)
phosphatase 2 (SHP2), a ubiquitously expressed cytoplasmic
phosphotyrosine (pY) phosphatase whose target substrate is
the EGF receptor (EGFR) which is a transmembrane receptor
tyrosine kinase (RTK) (49). Deletion of SHP2 in IECs results in
a decreased ERK phosphorylation (50) whereas its activation
confers resistance to dextran sulfate sodium (DSS)-induced
colitis and Citrobactoer rodentium (C. rodentium) infection
through the MAPK/ERK pathway (51). At present, the extent to
which AhR activation works in tandem with SHP2-MAPK/ERK
signaling to promote intestinal homeostasis remains largely
unknown; however, its role in potentiating MAPK/ERK signals
must be highlighted. Independent of SHP2, AhR activation by
the tryptophan derivative 6-formylindolo (3, 2-b) carbazole
(FICZ) has indeed shown to ameliorate DSS-induced colitis and
exclusively promote the MAPK/ERK-dependent differentiation
of GCs (52). Importantly, this selectivity for GCs occurs in
parallel with a suppression of Notch signals as indicated by a
down-regulation of the Notch intracellular domain (NICD)
which is released upon Notch activation (Figure 1B). Like
the Wnt/β-catenin pathway, Notch signaling has a profound
effect on intestinal development as it regulates ISC stemness
and epithelial cell fate (53,54). In this regard, Notch signaling
suppresses the differentiation of GCs and its actions may
therefore be countered by the activation ERK as previously
described (51). Taken together, these findings suggest that
AhR-MAPK/ERK signaling promotes intestinal homeostasis by
selecting for the differentiation of GCs.
THE CONTRIBUTION OF AHR LIGANDS TO
ISC HOMEOSTASIS
Tryptophan Metabolites
Tryptophan is an essential amino acid and is a precursor for
several bioactive molecules, especially serotonin; however, only
a small percentage of tryptophan is metabolized into serotonin.
Instead, ∼95% of tryptophan is metabolized into kynurenine
(KN) which plays a critical role in cellular energy production
following its eventual conversion into nicotinamide adenine
dinucleotide (NAD+) through the kynurenine pathway (KP)
(55,56). What remains of the KN pool under physiological
conditions is converted into kynurenic acid (KA) (56) and
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Wisniewski et al. AhR Signaling and Intestinal Stemness
both metabolites are potent AhR ligands (57,58). Interestingly,
KN may regulate epithelial cell fate through the AhR. Of note
are recent findings demonstrating that both tryptophan and
KN promote GC differentiation in HT-29 cells as determined
by Muc2 gene expression (59). Analyses confirmed that both
inhibition of KN synthesis by 1-Methlytryptophan (1-MT) and
inhibition of AhR signaling by its antagonist α-naphthoflavone
suppresses Muc2 gene expression, suggesting a loose connection
between AhR activation and KN synthesis in the production of
GCs. Importantly however, while KN was shown to increase the
protein expression of β-catenin relative to NICD (Wnt vs. Notch
signals), these effects were dependent on the media in which
the cells were grown (i.e., DMEM vs. RPMI) which can vary
in amino acid and glucose content (59). Interestingly, an early
report by Park et al. has indicated that AhR is highly expressed in
LGR5+stem cells in the small intestine and that administration
of its potent ligand FICZ, a tryptophan derivative generated
by ultraviolet B irradiation (60), inhibits the development of
intestinal organoids in a concentration-dependent manner in
vitro as indicated by significant reduction in absolute numbers
of organoids and slightly reduces Paneth cells in the small
intestine with a concomitant reduction in crypt length and a
reduction in colonic crypt length in vivo (61). It was also found
that FICZ reduced the protein expression of active β-catenin
in organoids derived from small intestinal crypts (perhaps due
to a loss of morphogenetic factors produced from crypt Paneth
cells) though increased the gene expression of the transcription
factor ATOH1, which promotes the differentiation of secretory
lineages from ISCs, as well as altered the gene expression of
other morphogenetic pathway markers (61). Though no changes
in GC number were observed following FICZ administration,
the observed increase of ATOH1 expression highlights the
putative role of FICZ in promoting the differentiation of GCs
as previously shown (52). In all, these findings suggest that
KN promotes the differentiation of GCs in tandem with AhR
activation but that these actions may be dependent on additional
local factors such as other amino acids. In addition, FICZ
modulates multiple morphogenetic pathways and its effect on
epithelial cell fate may be consistent with KN in promoting
the differentiation of GCs but the suppression of Paneth cells
and thus the extent to which FICZ modulates differentiation
of secretory lineages warrants further investigation. Further, the
selective reduction in Paneth cell number may reflect region-
specific effects of FICZ on epithelial cell fate.
Microbiota-Derived
The gut microbiota encompasses a diverse array of microbial
taxa that colonize the full length of the GI tract, consisting of
approximately 3.8 ×1013 cells in total (62). The majority of the
gut microbiota is harbored in the colon and modulates its host’s
physiology by the production of microbiota-derived metabolites
that act upon multiple organ systems through various “host-
microbe metabolic axes” (63). These metabolites include (but are
not limited to) tryptophan catabolites and short-chain fatty acids
(SCFAs) originating from the bacterial fermentation of dietary
protein and soluble fiber (64,65). These metabolites serve as AhR
ligands of varying affinities and may affect ISC stemness.
Tryptophan Catabolites
As mentioned above, much of dietary tryptophan is metabolized
into KN through the KP as the majority of ingested protein is
digested and absorbed in the small intestine (66). Depending
on total intake however, excess protein and amino acids (6–
18 g/day) may reach the colon and become accessible to the
resident gut microbiota (67). While there are bacteria that
specialize in the proteolytic fermentation of dietary protein, the
degradation of tryptophan appears to be a ubiquitous function
shared among several bacterial species that reside throughout the
GI tract (65,68). Most notably is the ability of the gut microbiota
to convert tryptophan into indole and indole derivatives via
the enzyme tryptophanase (TnaA) (69,70). To date, research
indicates that a variety of both Gram-positive and -negative
bacteria are capable of producing large amounts of indole and
consequently, that indole acts as a significant signaling molecule
within microbial communities having been implicated in the
control of diverse aspects of bacterial physiology as reviewed
elsewhere (71). Given its importance in shaping the ecological
landscape and physiology of the gut microbiota, bacteria-derived
indole and its derivatives have a significant impact on host gut
physiology and health. While several derivatives of indole exist,
here we focus on the AhR ligands indole-3-aldehyde (IALD) and
indole-3-propionic acid (IPA) and their prospective contribution
to ISC stemness as these two ligands have shown to directly
impact ISC stemness to date.
Among the many aspects of immune development that
AhR signaling plays a role in, notably is its impact on innate
lymphoid cells (ILCs). ILCs are a heterogenous population of
immune cells that are non-T and non-B lymphocytes which
lack antigen-specific receptors and are hence activated through
cytokine signaling (72). ILCs have distinct groups that express
transcription factors and produce signature cytokines including
group 3 ILCs (ILC3s), which release interleukin (IL)-22 upon
AhR activation (73). This AhR-IL-22 axis expressed in ILC3s
is critical for the maintenance of intestinal homeostasis as
AhR deficiency in RORγt+ILCs increases susceptibility to
C. rodentium infection due to a lack of IL-22 production
(74). Likewise, AhR deficiency in mice causes an increase
in Th17 cells and an expansion of commensal segmented
filamentous bacteria (SFB) due to a concomitant reduction
in IL-22 (75). Further, haplodeficiency of RORγt with genetic
ablation of AhR spontaneously induces colitis indicating the
importance of RORγt in maintaining the ILC3 compartment
and subsequent IL-22 production in tandem with AhR activation
(75). Additional findings confirm that treatment with IL-22
increases ISC stemness both in vivo and ex vivo as well as reduces
intestinal pathologies associated with graft-versus-host disease
(76). In this same study, it was also found that STAT3 activation
was crucial for both organoid formation and IL-22 mediated
epithelial regeneration highlighting the importance of JAK/STAT
signaling in ISC stemness. While evidence also illustrates a
Notch-AhR-IL-22 axis which regulates colon tissue homeostasis
through the development of IL-22 producing ILCs (77–79), an
earlier report has shown that IALD produced primarily from
Lactobacillus reuteri (L. reuteri) increases the production of
IL-22 in indoleamine 2,3-dioxygensase 1 (IDO1) deficient mice,
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Wisniewski et al. AhR Signaling and Intestinal Stemness
conferring antifungal resistance and mucosal protection when
challenged with Candida albicans (C. albicans) or DSS (80). As
expected, these beneficial effects were not observed in AhR-
deficient mice emphasizing the AhR-dependent release of IL-22
(80). In addition, a more recent study explored the protective
effect of L. reuteri on the integrity of the gut mucosa in an attempt
to elucidate the therapeutic benefits of Lactobacilli often found
in yogurt (81). The authors reported that L. reuteri upregulated
IL-22 production and stimulated ISC regeneration (as indicated
by an increase in LGR5+stained cells) in both organoid/LPL
co-cultures and in mice which was also observed with IALD
administration. Lastly and similar to the findings of Lindemans
et al. (76), the secretion of IL-22 by LPLs stimulated with
L. reuteri or IALD increased the phosphorylation of STAT3 both
in vivo and ex vivo. Together, these findings suggest that IALD
derived from Lactobacilli plays a pivotal role in the production
of IL-22 within AhR-expressing immune cells and that through
the AhR-IL-22 axis, promotes ISC regeneration and epithelial
restitution which is dependent on STAT3 activation.
IL-10 is a potent anti-inflammatory cytokine whose
significance is well established in IBD. This important cytokine
signals through the IL-10 receptor ligand-binding subunit
(IL-10R1) and is induced during inflammation to suppress the
production of proinflammatory mediators in IECs (82). To
date, studies indicate that IL-10 regulates mucin biosynthesis
in GCs and that IL-10 is critical for Paneth cell development
and function (83,84). While the direct effect of IL-10 on IEC
function and development is less explored, these findings suggest
that IL-10 may have an influence on secretory epithelial cells.
Nevertheless, a recent report has shown that both IALD and
IPA induce IL-10R1 expression in vitro and that this induction
requires AhR signaling as the ablation of its dimeric partner
ARNT prevented the indole-dependent induction of IL-10R1
(85). Moreover, only wild-type Escherichia coli (E. coli) were able
to generate IALD and IPA, and thus induce epithelial IL-10R1.
Collectively, these results indicate a putative role of IL-10
signaling in secretory epithelial cell function and development,
and that the microbiota-derived indole derivates IPA and IALD
augment the therapeutic effects of IL-10 via AhR signals in the
preservation of mucosal homeostasis.
Short-Chain Fatty Acids
SCFAs are one of the major end products of microbial
fermentation and are formed from carbohydrate, protein and
glycoprotein precursors by anaerobic bacteria (86). Principal
SCFAs are acetate, propionate and butyrate in which all are
important sources of carbon and energy for host tissues (87).
These organic acids are absorbed through the gut mucosa and can
modulate host energy homeostasis through interactions between
chemosensory enteroendocrine cells (87,88). Interestingly,
butyrate is a critical energy source for colonocytes (89) and
exhibits therapeutic effects like that of other AhR agonists
including induction of Treg cells, anti-inflammatory responses
as well as the induction of IL-22 (90–93). While recent
data show that all three SCFAs enhance AhR responsiveness
in vitro primarily as histone deacetylase (HDAC) inhibitors
(94), additional findings demonstrate that butyrate can activate
AhR signaling independent of its role as an HADC inhibitor
suggesting that it is a direct AhR ligand as well (95). As
recently reviewed (96), studies that have investigated the effect
of butyrate on ISCs are discrepant, however. For instance, as
butyrate is a primary energy source for colonocytes (89), it can
facilitate ISC proliferation through gluconeogenesis (97) and
improved microcirculation by dilating colonic resistance arteries
(98). In contrast, butyrate has shown to suppress colonic stem
cell proliferation by HDAC inhibition and Foxo3 regulation, a
transcription factor that governs cell proliferation and longevity
(99). While disagreements remain, studies to date overall posit
that butyrate regulates ISC proliferation in the colon and
controls the differentiation of GCs. Still however, the extent
to which butyrate regulates ISC stemness via AhR activation
remains elusive.
Plant-Derived
Given the ubiquitous influence of AhR signaling in the
maintenance of barrier surfaces and its ability to ligate numerous
ligands, the efficacy of natural AhR ligands in the treatment of
inflammatory disorders in murine models has been extensively
explored. Of note are the phytochemicals quercetin, resveratrol
and indole-3-carbinole (I3C). At present, there are no studies
that have examined the effects of these flavonoids in IBD
patients but the therapeutic aspects thereof have been extensively
studied due to their potent antioxidant and anti-inflammatory
properties (100). While each indeed has potent therapeutic effects
in the treatment of experimental IBD (101), recent evidence
suggests that these effects extend beyond modulation of immune
responses and inflammation in which maintenance of ISC
stemness may also be a benefit.
Quercetin is an abundant polyphenol found in many natural
foods including fruits, vegetables, and nuts (102). Like quercetin,
resveratrol is a polyphenol best known to be enriched in the
skins and seeds of red grapes used to make red wine (103). Due
to their low affinity, both are corroborated to be indirect AhR
ligands and control AhR responsiveness by inhibiting the actions
of CYP1A1 which prevents the metabolic turnover of the potent
AhR agonist FICZ (104). As shown above, multiple signaling
pathways, including MAPKs and canonical Wnt/β-catenin
cascades, regulate cellular turnover of the intestinal epithelium.
Expectedly, oncogenic mutations inducing the hyperactivation
of both pathways perturb intestinal homeostasis and result in
intestinal malignancies. Of note are the oncogenic mutations
of K-Ras (KRAS) within the EGFR-MAPK/ERK pathway which
has shown to be involved in CRC development (105,106).
Resveratrol has been found to possess a broad-spectrum of health
benefits including anti-cancer activities (107) and findings by
Saud et al. specify that resveratrol acts directly to suppress KRAS
expression (108). Using a conditional knockout model of APC
in mice supplemented with resveratrol, the authors determined
that resveratrol inhibits tumor growth and proliferation which
is accompanied by a reduction in LGR5, KRAS and nuclear
β-catenin expression. Interestingly, mRNA levels of KRAS did
not change with resveratrol but instead, an 80% increase in
the expression of the miRNA miR-96 was observed. As miR-
96 has shown to regulate the translation of KRAS mRNA (109,
Frontiers in Immunology | www.frontiersin.org 8March 2021 | Volume 12 | Article 638725
Wisniewski et al. AhR Signaling and Intestinal Stemness
110), the authors concluded that the mechanism through which
resveratrol confers its therapeutic effects is the post-translational
modification of KRAS by miRNAs. Similarly, recent findings
of Damiano and colleagues suggest that the therapeutic effects
of quercetin are also enacted via the MAPK/ERK pathway
particularly as it relates to GC function (111). In human
intestinal GC-like LS174T and Caco-2 cells, the authors observed
a significant increase in MUC2 and MUC5AC expression in
both cell lines following exposure to quercetin and that these
effects were dependent on the induction of both MAPK/ERK and
protein kinase C alpha (PKCα) signals. PKC is a family of lipid-
sensitive serine/threonine protein kinases that regulate various
cellular functions including cell proliferation, differentiation,
migration, adhesion and apoptosis (112). Importantly, PKCα
activity is a strong agonist of ERK signaling via Ras activation
and works in parallel to regulate cell cycle withdrawal in IECs
(113). Taken together, these studies suggest that resveratrol
and quercetin promote intestinal homeostasis through opposing
directions of the same signaling cascade; resveratrol regulates
cell proliferation by inhibiting Wnt and MAPK/ERK signals
(via the suppression of KRAS) whereas quercetin modulates
the biosynthesis of mucins in intestinal GCs via the activation
of MAPK/ERK and PKCαsignals. Regarding AhR signaling,
quercetin may confer its effects on intestinal GCs indirectly
by allowing the FICZ-AhR-MAPK/ERK axis discussed above to
ensue whereas resveratrol may exert its therapeutic effects via the
induction of miR-96 which has shown to be regulated by the AhR
in the lung (114).
I3C is a breakdown product of glucobrassicin, a sulfur-
containing compound that is rich in cruciferous vegetables such
as broccoli and cabbage and is converted primarily into 3,3′-
diindolylmethane (DIM) due to the acidic environment in the
stomach upon digestion (115). I3C has shown much promise
in the treatment of IBD as we have recently demonstrated
that it prevents colitis via the induction of IL-22 (116), further
highlighting the importance of the AhR-IL-22 axis in intestinal
homeostasis. With regard to ISC stemness, a recent report by
Park et al. further associates I3C-AhR induction with both Wnt
and Notch signals in the regulation of GC differentiation (117).
Similar to their earlier report using FICZ (61), administration
of I3C by oral gavage inhibited the development of intestinal
organoids in an AhR-dependent manner as indicated by a
decrease in the proliferation of both ISCs and TA cells.
RNA expression analyses of lineage specific genes in cultured
organoids further concluded that I3C directly impacts the
development of GCs, Paneth cells and enterocytes such that
I3C increases MUC2 and lysozyme expression but decreases
intestinal alkaline phosphatase (IAP) expression. In addition, and
in contrast to their previous report, GCs were increased in I3C-
treated mice. Given the preferential increase of genes related
to secretory epithelial cell types, additional analyses confirmed
that I3C indeed potentiates Wnt but suppresses Notch signals
as evidenced by an increase in β-catenin and a decrease in
Notch protein expression as well as in HES1 RNA expression,
a transcription factor activated downstream of Notch signaling
which suppresses ATOH1. While these findings suggest that
I3C potentiates Wnt signaling, it may be context dependent
as Metidji et al. have demonstrated that dietary I3C tempers
Wnt hyperactivity in VillinCreR26LSL−Cyp1a1mice co-challenged
with azoxymethane (AOM)/DSS by enhancing the expression
of ZNRF3 and RNF43 (9). This discrepancy might be due to
mode of administration of I3C as purified diets may potentiate
differential effects on intestinal health in comparison to normal
chow (118). In sum, these findings posit that I3C plays a direct
role in the development of ISCs via the AhR perhaps in a
context-specific manner to maintain intestinal homeostasis and
indicate that this regulation is likely mediated by both Wnt and
Notch signals.
CONCLUSIONS
The ISC niche is complex and is the epicenter from which
all intestinal epithelial cells arise. The fate of these stem cells
and the function of their differentiated progeny are driven by
varying local factors whose actions are coordinated through
numerous signaling cascades that blend to govern ISC stemness.
To add to this complexity, evidence reported herein highlights
the extensive integration of AhR activation by various AhR
ligands in the regulation of such pathways associated with
ISC stemness. What proves challenging moving forward is
addressing the promiscuous nature of AhR signaling itself.
To remedy this, animal studies that investigate the effects of
AhR deficiency in a cell-specific manner together with global
ablation could provide more insight into the exact mechanisms
through which the AhR exerts its effects. In addition, mode of
dietary ligand administration (refined diets vs. intraperitoneal
injection vs. oral gavage) should be strongly considered as
each method could differentially affect experimental outcomes.
Altogether, while much of the responses from AhR activation
are context- and cell-dependent, the present findings illustrate
the ubiquitous effects of AhR signaling in the maintenance
of the ISC niche. What remains to be explored further is
the extent to which both the mucosal immune system and
the induction of molecular cascades in epithelial cells work in
tandem with the AhR to regulate ISC stemness and epithelial
cell fate.
AUTHOR CONTRIBUTIONS
PW: wrote the manuscript and designed the figures. MN:
provided extensive input regarding the focus and organization
of the manuscript. PN: provided extensive editing and additional
content to the manuscript. All authors contributed to the article
and approved the submitted version.
FUNDING
This work was supported by NIH Grants P01AT003961,
R01AI123947, R01AI129788, R01ES030144, and P20GM103641
to PN and MN.
Frontiers in Immunology | www.frontiersin.org 9March 2021 | Volume 12 | Article 638725
Wisniewski et al. AhR Signaling and Intestinal Stemness
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