F O C U S O N H o m e os tat i c I m m un e R e s p ons e s

REVIEWS
Intestinal epithelial cells: regulators
of barrier function and immune
homeostasis
Lance W. Peterson1 and David Artis1,2
Abstract | The abundance of innate and adaptive immune cells that reside together with
trillions of beneficial commensal microorganisms in the mammalian gastrointestinal
tract requires barrier and regulatory mechanisms that conserve host–microbial
interactions and tissue homeostasis. This homeostasis depends on the diverse functions
of intestinal epithelial cells (IECs), which include the physical segregation of commensal
bacteria and the integration of microbial signals. Hence, IECs are crucial mediators of
intestinal homeostasis that enable the establishment of an immunological environment
permissive to colonization by commensal bacteria. In this Review, we provide a
comprehensive overview of how IECs maintain host–commensal microbial relationships
and immune cell homeostasis in the intestine.

Inflammatory bowel disease

(IBD). A chronic condition of
the intestine characterized
by severe inflammation
and mucosal destruction.
The most common forms of
IBD in humans are ulcerative
colitis and Crohn’s disease,
which have both distinct and
overlapping pathological and
clinical characteristics.
Mucins
Heavily glycosylated proteins
that are the major component
of the mucus that coats
epithelial barrier surfaces.
1
Department of Microbiology
and Institute for Immunology,
Perelman School of Medicine,
University of Pennsylvania.
2
Department of Pathobiology,
School of Veterinary
Medicine, University of
Pennsylvania, Philadelphia,
Pennsylvania 19104, USA.
e‑mails: lancep@mail.med.
upenn.edu;
dartis@mail.med.upenn.edu
doi:10.1038/nri3608
Specialized epithelial cells constitute barrier surfaces that
separate mammalian hosts from the external environ-
ment. The gastrointestinal tract is the largest of these
barriers and is specially adapted to colonization by
commensal bacteria that aid in digestion and markedly
influence the development and function of the mucosal
immune system. However, microbial colonization car-
ries with it the risk of infection and inflammation if epi-
thelial or immune cell homeostasis is disrupted. Key to
the coexistence of commensal microbial communities
and mucosal immune cells is the capacity to maintain the
segregation between host and microorganism. The intes-
tinal epithelium accomplishes this by forming a physical
and biochemical barrier to commensal and pathogenic
microorganisms. Furthermore, intestinal epithelial cells
(IECs) can sense and respond to microbial stimuli to
reinforce their barrier function and to participate in the
coordination of appropriate immune responses, ranging
from tolerance to anti-pathogen immunity. Thus, IECs
maintain a fundamental immuno­regulatory function
that influences the development and homeostasis of
mucosal immune cells.
The association between increased bacterial trans­
location and risk of developing inflammatory bowel disease
(IBD) suggests a central role for dysregulated epithelial
barrier function in either the aetiology or the pathol-
ogy of intestinal inflammation and IBD1. Increasing
evidence also indicates that the loss of intestinal barrier
function contributes to systemic immune activation,
which promotes the progression of chronic viral infec-
tions, including infection with HIV and hepatitis virus2,3,
and metabolic disease4,5. Furthermore, host–microbial
interactions that occur at the IEC barrier contribute to a
broad range of extra-intestinal autoimmune and inflam-
matory diseases, including type 1 diabetes, rheumatoid
arthritis and multiple sclerosis6–9. Hence, a comprehen-
sive understanding of the barrier and immunoregulatory
properties of IECs could aid in the development of new
strategies to prevent and treat multiple human infectious,
inflammatory and metabolic diseases.
The topics of commensal bacterial diversity, microbial
regulation of immune cell development and host–viral
interactions in the intestine have been reviewed exten-
sively elsewhere10–14. Therefore, in this Review, we dis-
cuss the role of IECs in promoting intestinal homeostasis
through the segregation and regulation of commensal
microorganisms and the host immune system. Recent
advances in the understanding of the barrier, microbial-
sensing and immunoregulatory functions of IECs are
reviewed, with a particular focus on their relationship to
intestinal health and disease. We discuss the barrier func-
tion maintained by IEC-derived mucins and anti­microbial
proteins, the pathways through which IECs regulate
innate and adaptive immune cells present at the intes-
tinal barrier and the contribution of IEC recognition of
microbial colonization to IEC function and homeostasis.

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R E V IE W S
Small intestine
Commensal
bacteria
sIgA
AMPs
Goblet
cell
Paneth
cell
Crypts
Tubular invaginations of the
intestinal epithelium. Lining
the base of the crypts are
small intestinal Paneth cells,
which produce numerous
antimicrobial proteins, and
stem cells, which continuously
divide to give rise to the entire
intestinal epithelium.
Villi
Projections of the intestinal
epithelium into the lumen
of the small intestine that
have an outer layer
consisting of mature,
absorptive enterocytes,
mucus-secreting goblet cells
and enteroendocrine cells.
Pluripotent intestinal
epithelial stem cells
(Pluripotent IESCs).
Tissue-resident stem cells
that undergo continuous
self-renewal and are
responsible for regenerating
all lineages of mature
intestinal epithelial cells,
including enterocytes,
enteroendocrine cells,
goblet cells and Paneth cells.
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Follicle-associated epithelium Colon
Apoptotic
IECs
Mucus
Second-
layer
TFF3 mucus
M cell
Mucus
Enteroendocrine
cell
Enterocyte
Stromal B cell
cell
Lymphoid
follicle
IESC
Macrophage DC
Figure 1 | The IEC barrier.  Intestinal epithelial cells (IECs) form a biochemical and physical barrier that maintains
segregation between luminal microbial communities and the mucosal immune system. The intestinal
Nature Reviewsepithelial stem
| Immunology
cell (IESC) niche, containing epithelial, stromal and haematopoietic cells, controls the continuous renewal of the
epithelial cell layer by crypt-resident stem cells. Differentiated IECs — with the exception of Paneth cells — migrate
up the crypt–villus axis, as indicated by the dashed arrows. Secretory goblet cells and Paneth cells secrete mucus and
antimicrobial proteins (AMPs) to promote the exclusion of bacteria from the epithelial surface. The transcytosis and
luminal release of secretory IgA (sIgA) further contribute to this barrier function. Microfold cells (M cells) and goblet
cells mediate transport of luminal antigens and live bacteria across the epithelial barrier to dendritic cells (DCs),
and intestine-resident macrophages sample the lumen through transepithelial dendrites. TFF3, trefoil factor 3.
IEC regulation of barrier function digestive function. The luminal secretion of mucins
The intestinal epithelium is the largest of the body’s and antimicrobial proteins (AMPs) by goblet cells and
mucosal surfaces, covering ~400 m2 of surface area Paneth cells, respectively, establishes a physical and
with a single layer of cells organized into crypts and biochemical barrier to microbial contact with the epi-
villi (FIG. 1) . This surface is continually renewed by thelial surface and underlying immune cells17,18 (FIG. 1).
pluripotent intestinal epithelial stem cells (pluripotent Collectively, the diverse functions of IECs result in
IESCs) that reside in the base of crypts, where the pro- a dynamic barrier to the environment, which protects
liferation, differentiation and functional potential of the host from infection and continuous exposure to
epithelial cell progenitors is regulated by the local stem potentially inflammatory stimuli.
cell niche15,16 (BOX 1). Although the majority of cells
bordering the intestinal lumen are absorptive entero- Keeping the bugs at bay — IEC secretory defences. The
cytes, which are adapted for metabolic and digestive secretion of highly glycosylated mucins into the intesti-
function, the diversity of functions that the intestinal nal lumen by goblet cells creates the first line of defence
epithelium carries out is reflected by the presence of against microbial encroachment. The most abundant
additional specialized IEC lineages. of these mucins, mucin 2 (MUC2), plays an essential
Secretory IECs, including enteroendocrine cells, part in the organization of the intestinal mucous layers
goblet cells and Paneth cells, are specialized for main- at the epithelial surface of the colon19. The importance
taining the digestive or barrier function of the epithe- of mucin production by goblet cells is emphasized by
lium. Enteroendocrine cells represent a link between the the spontaneous development of colitis and the predis-
central and enteric neuroendocrine systems through position to inflammation-induced colorectal cancers
the secretion of numerous hormone regulators of observed in MUC2‑deficient mice20,21. Additional goblet
© 2014 Macmillan Publishers Limited. All rights reserved
 F O C U S O N H o m e os tat i c I m m un e R e sRpEons eS
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Box 1 | The IESC niche
Along the crypt–villus axis of the epithelium, pluripotent intestinal epithelial
stem cells (IESCs) residing in the base of crypts give rise to a transit-amplifying
population of cells that undergo rapid proliferation and differentiation into the
various intestinal epithelial cell (IEC) subsets. Terminally differentiated cells — with
the exception of Paneth cells — migrate up the crypt–villus axis until they are lost
from the epithelial layer. For this process to be maintained, epithelial stem cells
must be able to undergo repeated rounds of replication and possess the capacity
for continuous self-renewal16. Recent advances in stem cell biology have identified
markers of IESCs that have contributed to the understanding of epithelial
self-renewal and differentiation16,183–185.
The patterning and distribution of proliferating crypt units in the intestine depend
on paracrine signalling between the epithelium and the underlying mesenchyme.
A balance between bone morphogenetic protein signals and antagonists, such as
noggin and gremlin, provides a niche for proliferating stem cells while limiting ectopic
crypt formation15. IESCs further rely on signalling through both the WNT–β‑catenin
and the Notch pathways for promoting self-renewal and directing differentiation
towards secretory versus non-secretory lineage IEC fates16.
The responsiveness of epithelial progenitors to external regulation in settings of
inflammation or infection remains less well understood. In particular, how immune
system-mediated signalling integrates into the homeostatic pathways described
above or acts through alternative pathways for altering stem cell function is poorly
defined. However, several recent studies have given insight into the regulation of
WNT–β‑catenin signalling by the pro-inflammatory cytokines interferon‑γ and tumour
necrosis factor, offering an example of how immune signalling and homeostatic
pathways for regulating the stem cell niche can converge186,187. Furthermore,
cell-intrinsic mechanisms of integrating host–commensal microorganism interactions
into IEC homeostasis have been recently described188.
cell-derived products, such as trefoil factor 3 (TFF3) and
resistin-like molecule‑β (RELMβ), further contribute to
the regulation of a physical barrier in the intestine. TFF3
Autophagy
A cellular process by which
provides structural integrity to mucus through mucin
cytoplasmic organelles and crosslinking and acts as a signal that promotes epithe-
macromolecular complexes lial repair, migration of IECs and resistance to apopto-
are engulfed by double sis22,23. RELMβ functions to promote MUC2 secretion,
membrane-bound vesicles
regulate macrophage and adaptive T cell responses
for delivery to lysosomes
and subsequent degradation. during inflammation and, in the setting of nematode
This process is involved in infection, directly inhibit parasite chemotaxis24,25.
constitutive turnover of Intestinal barrier function is further reinforced by
proteins and organelles and is the secretion of AMPs by IECs. Enterocytes are capa-
central to cellular activities that
maintain a balance between
ble of producing some AMPs, such as the C‑type lec-
the synthesis and breakdown tin regenerating islet-derived protein IIIγ (REGIIIγ),
of various proteins. throughout the small intestine and colon. By contrast,
Paneth cells are uniquely adapted for the secretion of
Unfolded protein response
many additional AMPs, including defensins (cryptdins
(UPR). A response that
increases the ability of the in mice), cathelicidins and lysozyme, in the crypts of
endoplasmic reticulum to the small intestine18,26. These AMPs disrupt highly con-
fold and translocate proteins, served and essential features of bacterial biology, such as
decreases the synthesis of surface membranes, which are targeted by pore-forming
proteins, causes the arrest of
the cell cycle and promotes
defensins and cathelicidins, and Gram-positive cell wall
apoptosis. peptidoglycans, which are targeted by C‑type lectins18,27.
This strategy enables the broad regulation of both com-
Plasma cells mensal and pathogenic bacteria and limits resistance
Terminally differentiated cells
of bacteria to antimicrobial responses. Regional vari-
of the B cell lineage that
secrete large amounts of ation in AMP production exists along the longitudinal
antibodies. axis of the intestinal tract 28. Although further analysis
is required, this distribution may reflect anatomically
Lamina propria restricted host–commensal bacteria interactions that
Connective tissue that
underlies the epithelium of the
drive the differential regulation of IEC responses or
mucosa and contains stromal serve to shape heterogeneity in the composition and
and haematopoietic cells. localization of microbial communities.
NATURE REVIEWS | IMMUNOLOGY VOLUME 14 | MARCH 2014 | 143
© 2014 Macmillan Publishers Limited. All rights reserved
Paneth cell- and enterocyte-derived REGIIIγ has
recently been described as a mediator of host–microbial
segregation in the gut 29. Similar to the function and
regulation of MUC2 in the colon, REGIIIγ acts to
exclude bacteria from the epithelial surface of the
small intestine, and its production is dependent on
IEC-intrinsic recognition of commensal microbial sig-
nals29. Interactions between AMPs, including REGIIIγ,
and mucins lead to concentrated antimicrobial activity
at the epithelial surface30. Thus, the combined func-
tions of secretory IECs seem to limit the quantity and
diversity of live bacteria that can reach the epithe-
lial surface or interact with the underlying mucosal
immune system.
The importance of maintaining the health of secre-
tory IECs is reflected in human IBD and models of
murine intestinal inflammation, in which genetic
defects in autophagy and the unfolded protein response
(UPR) disrupt the function of Paneth and goblet cells
and promote disease susceptibility 31–37. Autophagy
in IECs has been shown to act in an innate immune
capacity to limit the dissemination of invasive bacteria
passing through the epithelium38, but it also supports
the packaging and exocytosis of Paneth cell granules33.
When autophagy is disrupted in mice they become sus-
ceptible to a form of experimental colitis33. Notably, this
susceptibility is dependent on exposure to a common
strain of an enteric virus (murine norovirus), providing
an example of the compound genetic and environmen-
tal interactions that contribute to disease pathogene-
sis36. Disruption of UPR genes results in endoplasmic
reticulum stress in secretory cells and spontaneous
intestinal inflammation34. Notably, disruption of either
autophagy or the UPR leads to the compensatory
engagement of the other, supporting a model in which
the two are interrelated39. Furthermore, the engage-
ment of these pathways by Paneth cells is required for
maintaining intestinal homeostasis in mice, and their
combined absence leads to the development of a spon-
taneous disease resembling human Crohn’s disease39.
These findings, coupled with genetic evidence from
patients with IBD for the role of autophagy and the
UPR in disease pathogenesis31,32,34,37, support an impor-
tant link between the disruption of Paneth cell function
and the potential origins of intestinal inflammation.
Finally, IECs directly transport secretory immu-
noglobulins across the epithelial barrier. Following
their production by plasma cells in the lamina propria,
dimeric IgA complexes are bound by the polymeric
immunoglobulin receptor (pIgR) on the basolateral
membrane of IECs and actively transcytosed into
the intestinal lumen 40. The collaboration between
IgA-secreting B cells and IECs provides an adaptive
immune component to the epithelial barrier that reg-
ulates commensal bacterial populations to maintain
IEC and immune cell homeostasis41–43. Future studies
to better understand how mucus, AMP and secretory
immunoglobulin dynamics can be regulated to sup-
port barrier function will enable the development of
therapeutic interventions for preserving intestinal
homeostasis.
R E V IE W S
Box 2 | IEC tight junctions and turnover
Below the mucous layers, intestinal epithelial cells (IECs) form a continuous physical
barrier. Tight junctions connect adjacent IECs and are associated with cytoplasmic
actin and myosin networks that regulate intestinal permeability. In the setting of
inflammatory bowel disease (IBD), dysregulation of these interactions, mediated by
tumour necrosis factor signalling and by myosin light chain kinase activity, leads
to IEC cytoskeletal rearrangements that disrupt tight junctions and increase
permeability189,190. These findings suggest that IEC tight junctions could be important
targets for enhancing the integrity of the intestinal barrier in IBD.
As the IEC barrier is continuously renewed, the turnover of IECs provides an additional
challenge to the maintenance of epithelial continuity. Recent studies have described
pathways by which adjacent cells seal potential voids created during the extrusion of
either apoptotic or live cells from the single-cell layer191,192. As dysregulated epithelial
cell turnover and apoptosis are associated with intestinal inflammation, the contribution
of these mechanisms to the limiting of barrier breaches and further inflammation is of
relevance to our understanding of epithelial cells as an efficient physical barrier.
Although increased intestinal permeability has been correlated with IBD1,193,194,
it remains unclear whether the loss of barrier function is a cause or a consequence of
intestinal inflammation in human disease. Evidence from mouse models with genetic
defects in tight-junction-associated proteins suggests that disruption of barrier
function alone is not always sufficient to cause disease195,196. Notably, in mice with a
deletion of the tight-junction protein junctional adhesion molecule A, the secretion
of commensal bacteria-specific IgA can compensate for the loss of barrier function
and limit disease severity following chemically induced colitis195. Thus, compensatory
immune mechanisms can act to protect against the development of colitis, even in the
setting of barrier disruption, supporting a multi-hit model of disease susceptibility195.
Sampling of luminal contents by IECs. Despite the bar-
rier function supported by IECs (BOX 2), the intestinal
epithelium includes specialized adaptations that conflict
with the concept of complete segregation between host
immune cells and microorganisms. Specialized IECs,
called microfold cells (M cells), mediate the sampling
Peyer’s patches of luminal antigens and intact microorganisms for pres-
Groups of lymphoid aggregates entation to the underlying mucosal immune system44.
located in the submucosa of
These specialized IECs are concentrated in the follicle-
the small intestine that contain
many immune cells, including associated epithelium overlaying the luminal surface of
B cells, T cells and dendritic intestinal lymphoid structures, including Peyer’s patches
cells. They have a luminal and isolated lymphoid follicles44,45.
barrier consisting of specialized Although nonspecific uptake and transcytosis of anti-
epithelial cells, called microfold
cells, which sample the lumen
gens represents a well-established mechanism of sampling
and transport antigens. by M cells, it has recently been demonstrated that more
efficient mechanisms of receptor-mediated transport also
Pattern-recognition exist. The surface glycoprotein GP2 acts as a receptor for
receptors
the bacterial pilus protein FimH, and the M cell-mediated
(PRRs). Receptors that
recognize structures shared transport of the pathogen Salmonella enterica across the
by foreign microorganisms epithelial barrier depends on GP2–FimH interaction46.
or endogenous molecules This suggests that M cells are capable of both specific
associated with pathogenesis. receptor-mediated microbial uptake and nonspecific
Signalling through these
receptors promotes
antigen uptake from the intestinal lumen. Although the
tissue-specific innate immune active transport of luminal contents across the epithelial
responses including the barrier was thought to be a unique function of M cells, it
production of cytokines. was recently shown that small-intestinal goblet cells also
contribute to this process through the delivery of soluble
Toll-like receptor
(TLR). An evolutionarily luminal antigens to subepithelial dendritic cells (DCs)47.
conserved pattern-recognition Although both M cells and goblet cells seem to be capable
receptor located at the cell of antigen delivery to the lamina propria, the functional
surface or at intracellular importance and contribution of these two pathways to
membranes. The natural
ligands of TLRs are conserved
the development of anti-pathogen responses or to the
molecular structures found in maintenance of immune tolerance remains incompletely
bacteria, viruses and fungi. understood.
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© 2014 Macmillan Publishers Limited. All rights reserved
In addition to luminal antigen sampling by IECs,
subepithelial mononuclear phagocytes, through inter-
actions with IECs, sample luminal contents through
transepithelial dendrites 48,49 (discussed below). The
adaptation of the epithelial barrier for the sampling of
luminal contents accommodates limited and controlled
bacterial and antigen translocation to direct appropriate
tolerogenic or anti-pathogen responses. The influence
of these transport pathways on the immune response is
not well understood, but distinct pathways of acquiring
antigens may influence the context in which immune
cells interpret microbial signals50. Furthermore, trans-
port through these pathways may alter bacteria and
antigens to enable controlled transport of antigens to
be differentiated from dysregulated bacterial transloca-
tion50. Harnessing the functions of IECs in this sampling
process holds promise for the development of mucosal
vaccines and the regulation of intestinal inflammation.
IECs — sentinels in intestinal homeostasis
Central to the capacity of IECs to maintain barrier
and immunoregulatory functions is their ability to act
as frontline sensors for microbial encounters and to
integrate commensal bacteria-derived signals into anti­
microbial and immunoregulatory responses (FIG. 2). IECs
express pattern-recognition receptors (PRRs) that enable
them to act as dynamic sensors of the microbial envi-
ronment and as active participants in the directing of
mucosal immune cell responses (see Supplementary
information S1 (table)). Members of the Toll-like recep-
tor (TLR)51, NOD-like receptor (NLR)52,53 and RIG‑I‑like
receptor (RLR) families54,55 provide distinct pathways for
the recognition of microbial ligands or endogenous sig-
nals associated with pathogenesis. Unlike sterile sites in
the body where recognition of foreign microorganisms
initiates highly inflammatory cascades, the abundance
of symbiotic commensals in the intestine necessitates
that IECs maintain a state of altered responsiveness
(discussed below). Although the study of PRR pathways
in haematopoietic cells has mostly focused on their
pro-inflammatory properties in antigen-presenting and
effector immune cell populations, their role in regulating
tissue homeostasis and immune tolerance has emerged
as a major component of their function in IECs (see
Supplementary information S1 (table)).
The homeostatic role of microbial recognition by IECs.
Evidence of a role for PRRs in the protection against
intestinal inflammation and repair of epithelial dam-
age emerged from studies of mice deficient in TLRs
and signalling adaptors or depleted of key commensal
microorganisms56. Landmark work by Medzhitov and
colleagues demonstrated, through the use of TLR- and
MYD88‑deficient and broad-spectrum antibiotic-treated
mice, that commensal bacteria-derived signals contrib-
ute to epithelial homeostasis and repair in a model of
chemically induced colitis using dextran sodium sulphate
(DSS)56. This and other studies defined beneficial roles of
IEC-intrinsic TLR signalling that include the expression
of cytoprotective heat-shock proteins, epidermal growth
factor receptor ligands56,57, and TFF3 (REF. 58), and the
 F O C U S O N H o m e os tat i c I m m un e R e sRpEons eS
V IE W s

a Commensal bacteria
b AMPs Mucin

TFF3

TLR9
(apical) ROS

Endosome TLR3, Tight

TLR7, junction
TLR8

MYD88 TRIF
Ub Heat-shock
Ub proteins
Ub
Iκ B IκB IKKγ
IKKα IKKβ
p50 p65
NLRP3,
NLRP6, NF-κB
NLRC4

Pro-caspase 1 NF-κB NF-κB

p50 p65 p50 p65

NOD1,
Inflammasome NOD2
RIP2
FRMPD2

IL-1β EGFR
and IL-18 TLR2, TLR4, APRIL, BAFF, IL-25,
TLR5 or TLR9 retinoic acid, TGFβ EGFR ligands
and TSLP

NOD-like receptor
(NLR). A pattern-recognition
receptor located in the cytosol.
NLRs recognize a wide range of
foreign structures and patterns
associated with pathogenesis.
Some members of this family
form multiprotein complexes
known as inflammasomes,
which regulate the processing
and secretion of
pro-inflammatory cytokines.
RIG‑I‑like receptor
(RLR). A pattern-recognition
receptor located in the cytosol
that responds to viral RNA.
Dextran sodium sulphate
(DSS). A large polysaccharide
that causes epithelial injury
and inflammation in the
intestinal tract and is
commonly used in models of
experimentally induced colitis
for studying the response to
intestinal injury.
Figure 2 | Microbial recognition promotes IEC health and function.  a | Pattern-recognition receptors (PRRs),
including intestinal epithelial cell (IEC)-expressed Toll-like receptors (TLRs) and NOD-like receptors (NLRs), recognize
conserved microbial-associated molecular motifs and pathogen-specific virulence properties. Nature
TLRsReviews | Immunology
recruit the signalling
adaptors MYD88 and TIR-domain-containing adaptor protein inducing interferon‑β (TRIF) on ligation to signal molecules
via nuclear factor‑κB (NF‑κB), p50 and p65 subunit activation and the mitogen-activated protein kinase (MAPK) pathway
(not shown). Nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 signal through receptor-interacting
protein 2 (RIP2) to activate NF‑κB and MAPKs, whereas other IEC-expressed NLRs, including NOD-, LRR- and pyrin
domain-containing 3 (NLRP3), NLRP6 and NOD-, LRR- and CARD-containing 4 (NLRC4), form inflammasome complexes
with pro-caspase 1 for the cleavage and activation of interleukin‑1β (IL‑1β) and IL‑18. Polarized expression of PRRs by IECs
at either the apical or basolateral membrane may contribute to the discrimination between commensal and pathogen
microbial signals. For example, signalling through surface or endosomal TLR9 at the apical pole of IECs promotes the
inhibition of NF-κB signalling, whereas TLR signalling from the basolateral pole promotes NF-κB activation. b | Microbial
recognition is integrated by IECs. This promotes cell survival and repair (mediated by trefoil factor 3 (TFF3), heat-shock
proteins and epidermal growth factor receptor (EGFR) ligand expression), barrier function (mediated by increased mucin
and antimicrobial peptide (AMP) producton) and immunoregulatory responses (mediated by a proliferation-inducing
ligand (APRIL), B cell-activating factor (BAFF), IL-25, retinoic acid, transforming growth factor‑β (TGFβ) and thymic
stromal lymphopoietin (TSLP)), FRMPD2, FERM and PDZ domain-containing 2; IκB, inhibitor of NF‑κB; IKK, IκB kinase;
ROS, reactive oxygen species; Ub, ubiquitin.
enhanced integrity of apical tight-junction complexes59 (IκB) kinase (IKK) complex or NF‑κB essential modula-
(FIG. 2). Furthermore, IEC-specific deletion of elements tor (NEMO), results in enhanced DSS-induced or spon-
necessary for the activation of the transcription factor taneous colitis60,61. These studies establish an essential
complex nuclear factor-κB (NF‑κB) downstream of TLR role for TLRs, in addition to other NF‑κB signalling
signalling in mice, including the inhibitor of NF‑κB pathways, in epithelial homeostasis and repair.

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Nuclear factor-κB
(NF‑κB). A family of
transcription factors important
for pro-inflammatory and
anti-apoptotic responses that
are activated by the ubiquitin-
dependent degradation of
their respective inhibitors,
members of the inhibitor of
NF‑κB (IκB) family. This process
is mediated by the kinases, IκB
kinase 1 (IKK1) and IKK2.
Inflammasomes
Multiprotein complexes that
contain a member of the
NOD-like receptor family,
adaptor proteins and the
protease caspase 1. These
complexes regulate the
catalytic processing and
secretion of pro-inflammatory
cytokines, including
interleukin‑1β (IL‑1β) and
IL‑18.
Reactive oxygen species
(ROS). Chemically reactive
molecules containing oxygen
that, when produced in
large amounts, have
pro-inflammatory and
antimicrobial effects.
Physiological levels of ROS
have been shown to regulate
cellular signalling pathways.
146 | MARCH 2014 | VOLUME 14 www.nature.com/reviews/immunol
As additional families of TLRs, such as the NLRs and
RLRs, have been ascribed roles in regulating inflamma-
tory immune cell responses, they have also been shown
to be important in IECs for the regulation of intestinal
homeostasis52–55. The identification of nucleotide-bind-
ing oligomerization domain 2 (NOD2), an NLR family
member that recognizes bacterial muramyl dipeptide
(MDP), as the first genetic susceptibility locus for
Crohn’s disease has fuelled interest in the role of this
PRR and the related protein NOD1 in both immune
cells and the intestinal epithelium37,62,63. Moreover,
inflammasomes formed by caspase 1 and NLRs, includ-
ing IEC-expressed NOD-, LRR- and pyrin domain-
containing 3 (NLRP3), NLRP6, NLRP12 and NOD-,
LRR- and CARD-containing 4 (NLRC4), have a complex
influence over inflammation and epithelial repair, as dem-
onstrated by both pathological and protective roles in con-
stitutive knockout mouse models52,53,64 (see Supplementary
information S1 (table)). As NLRs are expressed by sev-
eral cell populations in the intestine, conditional knock-
out models will be required to elucidate the precise
haematopoietic, epithelial and stromal contributions of
these PRRs during inflammation and repair.
Finally, reactive oxygen species (ROS) produced in
response to commensal or pathogenic bacteria have
a role in IEC-intrinsic signalling that acts to promote
epithelial repair, independently of their microbicidal
effects65,66. Through the inactivation of cellular redox-
sensitive tyrosine phosphatases, ROS promote the form­
ation by IECs of focal adhesions, which are necessary
for cell migration and wound healing 65,66. Strikingly,
these findings show remarkable symmetry with studies
in Drosophila melanogaster, in which ROS also promote
epithelial homeostasis, suggesting an evolutionarily
conserved role for ROS in mediating protective effects
of commensal microorganism-dependent cellular
responses67–69.
The protective effects of microbial recognition by
IECs may come at a cost. Although commensal micro-
bial signals are protective in settings of tissue damage or
infection, they can drive tumorigenesis and cancer when
homeostatic responses become dysregulated60,70. Epithelial
cell-intrinsic TLR, MYD88 and NF‑κB signalling have all
been implicated in promoting tumour development and
progression in multiple genetic70–73 and inflammation-
induced60,72,74,75 mouse models of colorectal cancer. The
convergence between PRR signalling and pro-oncogenic
signalling pathways could partly explain the tumorigenic
effects of microbial stimulation. The stabilization of key
oncogenic proteins, such as MYC, has been shown to be
promoted by MYD88 signalling 71. Furthermore, NF‑κB
can enhance WNT signalling in terminally differentiated
IECs to promote their dedifferentiation into stem cell‑like
tumour initiators73.
Paradoxically, some NLR signalling pathways protect
against tumorigenesis, partly through the regulation of
cell death and proliferation in damaged or transformed
IECs76–80 and through the regulation of tissue repair
responses mediated by interleukin‑18 (IL‑18) signal-
ling 53,81,82. The complexity of the multiple roles of micro-
bial recognition by IECs serves to further highlight the
© 2014 Macmillan Publishers Limited. All rights reserved
delicate nature of the balance that exists between homeo-
stasis and inflammation and its importance in maintain-
ing healthy host–microorganism symbiosis.
Specialized regulation of PRR pathways in IECs. The
proximity of IECs to an abundance of luminal microbial
signals necessitates specialized mechanisms for maintain-
ing altered or hyporesponsive PRR signalling in response
to commensal bacteria-dependent stimuli83,84. In support
of this, IECs express negative regulators of PRR-dependent
pro-inflammatory signalling 75,83,85,86 (see Supplementary
information S1 (table)). The disruption of these regula-
tory pathways or constitutive activation of NF‑κB pre-
dispose mice to dysregulated epithelial homeostasis and
exaggerated inflammation72,75,85,87. Furthermore, it has
been appreciated that commensal bacteria-dependent
production of ROS by IECs can attenuate the activation
of NF‑κB, broadly tolerizing IECs to microbial stimula-
tion through PRR signalling 88,89. Although additional
mechanisms exist for the negative regulation of PRR sig-
nalling pathways90, in most cases the extent to which they
are active in IECs and their contributions to intestinal
homeostasis remain to be determined.
In addition to maintaining the hyporesponsiveness
of IECs, innate immune pathways must differentiate
between signals derived from commensal and patho-
genic microorganisms for the scaling of an appropriate
inflammatory response91. The polarized nature of the
intestinal epithelium allows for the anatomical segrega-
tion of PRRs (FIG. 2). In vitro and in vivo models demon-
strate differential responsiveness of IECs to apical versus
basolateral stimulation with multiple TLR ligands92–94.
For example, although basolateral exposure of IECs to
TLR9 ligands results in canonical activation and nuclear
translocation of NF‑κB, apical exposure results in a
net inhibitory effect through the stabilization of IκB94.
This apical signal induces tolerance to subsequent TLR
stimulation, demonstrating a unique adaptation for the
cross-tolerance of microbial recognition pathways and
a differential response to microbial signals based on
anatomical location94 (FIG. 2).
This concept of subcellular segregation and polarized
distribution of TLRs has been translated to the regula-
tion of additional PRR pathways95,96. Through a series
of elegant genetic screens, FERM and PDZ domain-
containing 2 (FRMPD2) — which is a positive regula-
tor of NOD2‑mediated NF‑κB activation in response to
MDP recognition — was recently identified to act as a
scaffold protein that promotes basolateral membrane
localization and selective basolateral activation through
interactions with the leucine-rich repeat (LRR) domain
of NOD2 (REF. 96) (FIG. 2). Common Crohn’s disease-
associated variants of NOD2 contain mutations in this
LRR domain. These NOD2-mutant proteins were shown
in vitro to lack the ability to interact with FRMPD2, to
colocalize at the basolateral membrane of epithelial cells
and to respond to stimulation with NOD2 ligands62,63,96.
These studies give insight into the mechanism of NOD2
dysfunction associated with IBD and how IECs may
spatially regulate the activation of PRR signals at the
intestinal barrier.

Viability-associated PAMPs
(Vita-PAMPs). Members of a
special class of pathogen-
associated molecular patterns
recognized by the innate
immune system to signify
microbial life. These patterns
differentiate dead and living
microorganisms to allow for
scaling of appropriate immune
responses based on the level of
threat the microbial signals
represents.
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Finally, mechanisms by which IECs may break their
relative tolerance to microbial signals in settings of path-
ogen infection are poorly defined. In contrast to sterile
sites in the body, control of inflammation in the intes-
tine may be more adapted to relying on the recognition
of ‘danger’ signals associated with pathogenesis, rather
than on the presence of microbial signals alone97. The
recognition of danger has been proposed to be medi-
ated through the detection of properties associated with
microbial viability, termed viability-associated PAMPs
(vita-PAMPs), that distinguish living pathogens from
inert microbial debris, as well as through the detection
of conserved virulence factors of pathogens, such as bac-
terial secretion systems and toxins that penetrate into the
cellular cytosol91,98. Although these mechanisms for scal-
ing microbial threats have been studied and identified in
phagocytes and antigen-presenting cells, their function
and relevance in IECs are less well understood.
Commensal microorganism-dependent regulation of
barrier function. In addition to the homeostatic role of
microbial recognition by IECs, the intestinal epithelium
acts as an essential integrator of environmental signals for
the regulation of microbial colonization, barrier function
and mucosal immune responses. As previously discussed,
the production of an apical mucous layer, the secretion
of broadly targeted AMPs and the transcytosis of secre-
tory IgA contribute to epithelial barrier function. Reduced
mucous layer thickness in germ-free mice can be reversed
by treatment with TLR ligands, indicating that commen-
sal bacteria-dependent signals regulate mucus produc-
tion by goblet cells17. Similarly, the expression of many
epithelial cell-derived AMPs is enhanced by, or dependent
on, the presence of commensal microbial signals18,29,99–101.
As cells with specialized antimicrobial function, Paneth
cells play a particularly important part in the regulation
of AMP production through cell-intrinsic expression of
MYD88 and NOD2 (REFS 100,101).
The transport of IgA across the epithelial barrier
is regulated, in part, by the expression of pIgR on the
basolateral membrane of IECs, which is promoted by
MYD88- and NF‑κB‑dependent signalling in response
to commensal microbial signals40,102. Finally, the integ-
rity of tight junctions and transepithelial permeability
are regulated by commensal microbial signals, including
TLR2‑dependent redistribution of the tight-junction pro-
teins to apical cell–cell contacts59. Thus, the ability of IECs
to sense their microbial surroundings has an integral role
in regulating their barrier function.
Regulation of immune cells by IECs
IECs produce numerous immunoregulatory signals that
are necessary for tolerizing immune cells, limiting steady-
state inflammation and directing appropriate innate and
adaptive immune cell responses against pathogens and
commensal bacteria. Many of these responses depend
on the translation of commensal bacteria-derived sig-
nals by IECs to mucosal immune cells. The produc-
tion of the cytokines thymic stromal lymphopoietin
(TSLP)103–105, transforming growth factor‑β (TGFβ)104,106
and IL‑25 (REF. 107) and the B cell-stimulating factors
© 2014 Macmillan Publishers Limited. All rights reserved
a proliferation-inducing ligand (APRIL; also known
as TNFSF13) and B cell-activating factor (BAFF; also
known as TNFSF13B)108,109 by IECs is promoted by com-
mensal bacteria via PRR signalling (FIG. 2). We discuss
below the immunoregulatory functions of IECs, describ-
ing their contribution to the priming of adaptive immune
cell responses, regulation of innate effector responses and
homeostasis of adaptive immune cell function in the
intestinal environment.
Mononuclear phagocytes and antigen presentation. IECs
exert their influence over the priming of both cellular and
humoral adaptive immune responses via a continuous
dialogue with antigen-presenting mononuclear phago-
cytes (FIG. 3). IEC-derived TSLP, TGFβ and retinoic acid,
produced in response to commensal bacteria-derived sig-
nals, promote the development of DCs and macrophages
with tolerogenic properties, including the production
of IL‑10 and retinoic acid103,104,110. Considerable hetero­
geneity exists among intestinal mononuclear phagocytes,
the classification of which has been previously compli-
cated by conflicting nomenclature, as well as phenotypical
and functional plasticity in settings of inflammation111.
However, two distinct populations that have been
characterized are the pre‑DC‑derived CD11c+CD103+
DCs and monocyte-derived CD11clowF4/80+CX3CR1hi
intestine‑resident macrophages112–115.
CD103+ DCs act as migratory antigen-presenting cells
and upon activation traffic to secondary lymphoid tis-
sues, including the mesenteric lymph nodes and Peyer’s
patches, carrying with them antigenic material and live
bacteria for presentation to adaptive immune cells116,117.
Influenced by their previous interactions with IECs at the
intestinal barrier, these migratory DCs promote immune
tolerance through the differentiation of forkhead box P3
(FOXP3+) regulatory T cells by a TGFβ- and retinoic
acid‑dependent mechanism116,118,119. Furthermore, the
production of retinoic acid by IEC-conditioned CD103+
DCs is responsible for the imprinting of gut-homing
properties on T cells, allowing for the targeting of recircu-
lating mature cells to the original site of antigen encoun-
ter in the intestinal lamina propria120–124. Thus, in addition
to promoting naive T cell maturation based on antigen
specificity, CD103+ DCs relay the original context of
antigenic encounter at the intestinal epithelial barrier.
In contrast to CD103+ DCs, CX3CR1hi intestine-
resident macrophages lack migratory properties in the
steady state and instead persist in close physical contact
with IECs, where they act as avid phagocytes to medi-
ate clearance of pathogens and commensal bacteria
that traverse the epithelial barrier 116,125. Their expres-
sion of tight-junction proteins allows the formation of
trans­epithelial dendrites that penetrate into the lumen
of the intestine for sampling of exogenous antigens48,125.
Reflecting the functional dependence of these cells on
the epithelium, the extension of these trans­epithelial
dendrites is initiated by TLR signalling, not in myeloid
cells themselves, but in IECs49. The CX3CR1hi intestine‑
resident macrophage population has also been impli-
cated in the maintenance of mucosal tolerance, as they
have been shown to promote the survival and local
R E V IE W S

Innate immune regulation Adaptive immune regulation sIgA

Commensal
bacterium TLA SEMA7A

IL-25, TSLP
TSLP,
IL-33, IL-25 TGFβ, APRIL,
IFNγ, IEL IL-7,
TSLP RA BAFF
TNF IL-15
IL-13, IL-1β,
amphiregulin IL-17, IL-23 IL-12
IL-22
IL-10

IgA+ plasma cell

IL-25
ILC2 ILC3 ILC1

TSLP TReg cell

DC
Lamina propria
TCR RA,
MHC TGFβ

Macrophage Monocyte
Naive T cell TReg cell
Basophil Type 2
progenitor MPP
IL-10, Direct IEC effect
Mast cell Basophil B cell RA,
TGFβ Indirect IEC effect
Peyer’s patch Immune response
or mesenteric Differentiation
lymph node Basophil

Figure 3 | IECs regulate innate and adaptive immunity.  Intestinal epithelial cell (IEC)-derived cytokines interleukin‑25
Nature progenitors
(IL‑25) and thymic stromal lymphopoietin (TSLP) elicit the expansion and differentiation of basophil Reviews | Immunology
and
multipotent progenitor type 2 (type 2 MPP) cells, respectively. IL‑25, IL‑33 and TSLP stimulate group 2 innate lymphoid
cells (ILC2s), whereas IL‑25 suppresses innate lymphoid cell subset 1 (ILC1) and ILC3 function by limiting macrophage
production of pro-inflammatory cytokines IL‑1β, IL‑12 and IL‑23. IECs condition dendritic cells (DCs) and macrophages
towards a tolerogenic phenotype through the production of TSLP, transforming growth factor‑β (TGFβ) and retinoic acid
(RA). These DCs promote the differentiation of naive CD4+ T cells into regulatory T (TReg) cells and the maturation of B cells
into IgA-secreting plasma cells. Mucosal cell-derived DCs also imprint a gut-homing phenotype on primed B cells and
T cells through the production of RA. After trafficking to the intestine, TReg cells are expanded in number by macrophages
that are conditioned to produce IL‑10 by TSLP-mediated stimulation and through contact-dependent interactions with
IEC-expressed semaphorin 7A (SEMA7A). The production of a proliferation-inducing ligand (APRIL) and B cell-activating
factor (BAFF) by IECs and by TSLP-stimulated macrophages and DCs promotes class-switch recombination and
the production of IgA by B cells in the intestinal lamina propria. IEL, intra-epithelial lymphocyte; IFNγ, interferon‑γ;
sIgA, secretory IgA; TCR, T cell receptor; TLA, thymus leukaemia antigen; TNF, tumour necrosis factor.

Innate lymphoid cells
(ILCs). A group of innate
immune cells that are
lymphoid in morphology and
developmental origin, but lack
properties of adaptive B cells
and T cells such as recombined
antigen-specific receptors.
They function in the
regulation of immunity, tissue
homeostasis and inflammation
in response to cytokine
stimulation.
expansion of previously primed regulatory T cells 126.
CX3CR1hi macrophages promote tolerance in the intes-
tinal lamina propria through the production of IL‑10,
which leads to suppression of inflammatory cytokine
production by colitogenic T cells and promotion of
regulatory T cell function127,128. IECs maintain this
tolerogenic function through their production of solu-
ble factors, such as TSLP, TGFβ and retinoic acid103,104,110,
as well as through contact-dependent interactions
involving IEC expression of the integrin ligand sema-
phorin 7A, which induces IL‑10 expression by CX3CR1hi
macrophages and promotes intestinal homeostasis129.
IECs also play an important part in the induction of
T helper 2 (TH2) cell responses during helminth infec-
tion. In this setting, the IEC-derived cytokines TSLP
and IL‑25 promote the expansion and differentiation of
haematopoietic progenitor cells towards mononuclear
and myeloid cell phenotypes that promote the develop-
ment of type 2 cytokine responses at mucosal sites130–133.
These cells include a distinct population of basophil pro-
genitors and a population of multipotent progenitor cells,
which undergo extramedullary haematopoiesis and rep-
resent an innate link between IEC-derived signals and the
polarization of TH2 cell immune responses to helminths
and allergens132,133.
Innate lymphocyte function. In addition to the myeloid
cell and granulocyte populations, a recently identified
innate immune cell population of innate lymphoid cells
(ILCs) plays a crucial part in intestinal immune homeo-
stasis. ILCs lack properties of adaptive lymphocytes, such
as recombined antigen-specific receptors134. They are
found at barrier surfaces, including mouse and human
lung 135, skin136 and intestine137, where they function

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© 2014 Macmillan Publishers Limited. All rights reserved


Natural killer cells
(NK cells). A subset of innate
lymphoid cells originally
defined on the basis of their
cytolytic activity against
tumour targets but now
recognized to serve a broader
role in host defence and
inflammation through the
production of cytokines.
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as regulators of tissue homeostasis, inflammation and
early innate response to infection. ILCs are regulated,
in part, by epithelial cell-derived immunoregulatory
signals (FIG. 3). ILCs display phenotypical and functional
heterogeneity, which has been reviewed extensively
elsewhere134,138–140. ILCs are characterized by their devel-
opmental requirements and differential cytokine expres-
sion into group 1, group 2 and group 3 ILCs, which share
functional similarities with the adaptive CD4+ TH1, TH2
and TH17 cell populations, respectively.
Group 1 ILCs include classical natural killer cells
(NK cells) and innate lymphoid cell subset 1 (ILC1)
cells, and are characterized by the production of the
TH1 cell‑associated cytokines interferon-γ (IFNγ) and
tumour necrosis factor (TNF) in response to IL‑12
and/or IL‑15 (REF. 140). Although NK cells can directly
kill target cells through cytotoxic activity, other ILC1s are
limited to cytokine production in response to stimula-
tion. Although these ILC1s have a less well-understood
function than NK cells, several recent reports suggest
a possible role in mediating intestinal inflammation in
murine colitis models and human IBD141,142.
Group 2 ILCs (collectively termed ILC2s) produce the
TH2 cell‑associated cytokines IL‑5 and IL‑13 (REF. 140).
These factors contribute to an early innate response to
intestinal helminth infection and invoke a protective
epithelial response, including goblet cell hyperplasia and
enhanced mucus secretion143–145. Furthermore, ILC2s pre-
sent in the lung promote airway hyperresponsiveness or
tissue repair in mouse models of allergy and influenza
virus infection146–149. This suggests that ILC2s may have
analogous functions in the intestine, perhaps during food
allergy or wound repair; however, evidence for these roles
has yet to be described. The proliferation and activation
of ILC2s is supported by the predominantly epithelial
cell-derived cytokines IL‑25, IL‑33 and TSLP143–145,150.
The contribution of microbial stimulation to these sig-
nals reinforces the idea of the epithelium as an integrator
of environmental signals for the regulation of immune
cell function103,104,107.
Finally, group 3 ILCs produce TH17 and TH22 cell-
associated cytokines, including IL‑17A and IL‑22, in
response to stimulation by IL‑23 (REF. 140). This group
includes ILC3s, as well as lymphoid tissue inducer (LTi)
cells, which have a well-established role in secondary
lymphoid tissue organogenesis, mediated by interactions
with stromal cells during embryonic development 151.
IL‑22 has an important role in protecting the intestinal
epithelium following injury or infection by bacterial
pathogens152,153. In addition, ILC3‑derived IL‑22 sup-
ports the anatomical containment of gut-associated
lymphoid tissue-resident commensal bacteria and the
protection of IESCs in models of graft-versus-host dis-
ease154–156. These tissue-protective functions of IL‑22 are
balanced by detrimental effects in certain inflammatory
settings and in the initiation of inflammation-induced
cancer 82,156,157. Collectively, these studies demonstrate
the context-dependent nature of IL‑22 function. By con-
trast, ILC3‑derived IL‑17 is thought to have a primarily
pro-inflammatory effect in the intestine and has been
implicated in both mouse colitis and human IBD158–160.
© 2014 Macmillan Publishers Limited. All rights reserved
IECs play an indirect part in the regulation of ILC3s
in response to commensal bacteria-derived signals. For
example, IEC-derived IL‑25 leads to the suppression of
IL‑23 production by macrophages and decreased IL‑22
production by ILC3s161. By contrast, commensal bacteria-
dependent signals have also been shown to stimulate the
production of IL‑7 by IECs162, which supports the pro-
duction of IL‑22 by ILC3s through the stabilization of the
transcription factor retinoid-related orphan receptor-γt
(RORγt; encoded by RORC)162,163. These seemingly con-
flicting roles for IECs in regulating ILC3 function in
response to commensal bacterial stimulation may be
explained by heterogeneity among intestinal ILCs and by
differential targeting of cell types capable of producing
pro-inflammatory versus tissue-protective cytokines139.
Although the function of ILCs has been appreciated
in numerous mouse models, the importance and relative
contribution of these cells to inflammation in settings
of human disease remain incompletely defined. Future
work in this field will be required to further characterize
the heterogeneity and tissue-specific functions of these
cells, elaborate our understanding of their contribu-
tions to human disease and develop means of clinically
targeting their protective or detrimental functions.
Tissue-resident T cells. Following priming by intestine-
derived antigen-presenting cells in secondary lymphoid
tissues, conventional effector T cells recirculate through
the body before settling in the intestine, where they exert
their tolerogenic or inflammatory effect on the local
environment (FIG. 3). Here, mature T cells are subject to
the direct influence of IECs for their functional main-
tenance and survival in the lamina propria. Specialized
cells known as intraepithelial lymphocytes (IELs) exist
in intimate contact with the IEC layer, and bidirectional
interactions between IELs and IECs maintain immune
homeostasis at the intestinal barrier 164–166. IELs display an
activated phenotype and include conventional T cells, as
well as subsets of cells expressing a restricted repertoire
of T cell receptor specificities and specialized properties,
including γδ T cells and NKT cells165,167. Recent studies
have advanced the understanding of the developmental
origin of these cells and the functions that they have at
the intestinal barrier 165. These include the demonstration
that committed CD4+ T cells can undergo transcriptional
reprogramming when they become IELs to develop a
distinct phenotype resembling that of CD8+ cytotoxic
T cells168. Although the influence of the local environ-
ment in promoting this developmental change has not
been explored, the intimate interactions that these cells
have with IECs suggest that epithelial cell-derived signals
may promote their maintenance and function.
Tissue-resident conventional T cells primed to act
as rapidly responsive effectors are important during
on­going inflammation and infection, as well as for the
protection of the mucosal barrier against future chal-
lenge. This is thought to be particularly important
in CD8+ T cell-dependent memory responses169. As such,
CD8+ T cells with a tissue-resident memory phenotype
are uniquely enriched among αβ T cells present in the
intestinal IEL compartment of mice and humans169,170.
R E V IE W S

Class-switch recombination
(CSR). The process by which
proliferating B cells rearrange
their DNA to switch from
expressing IgM (or another
class of immunoglobulin) to
expressing a different
immunoglobulin heavy-chain
constant region, thereby
producing antibody with
different effector functions.
These tissue-resident memory T (TRM) cells interact
with IECs through CD103 (also known as αEβ7 integ-
rin), which binds the adhesion molecule E-cadherin on
IECs171,172. This may promote retention of these and other
cells at the intestinal epithelium.
Mouse IECs were recently demonstrated to contrib-
ute to the refinement of the CD8+ TRM cell pool in favour
of high-affinity precursors that allow for a more efficient
memory response to secondary mucosal challenge173.
This occurs through the contact-dependent selective
expansion and survival of high-affinity or high-avidity
CD8+ T cell populations expressing homodimers of the
co-receptor subunit CD8α (known as CD8αα+ IELs),
which interact with the IEC-expressed MHC class I‑like
molecule, thymus leukaemia antigen (TLA) 173 .
Understanding how such memory cell populations are
maintained is of particular interest in the design of effi-
cient vaccines against pathogens that invade mucosal
surfaces. Strategies have been explored for generating
CD8+ TRM cells with protective effects at extra-intestinal
sites174,175. Through an improved understanding of how
IELs are maintained within the intestinal epithelium,
we can hope to improve vaccine strategies for prevent-
ing infections with pathogens such as HIV and enteric
viruses176.
IgA-secreting plasma cells. The maturation of naive
B cells into mature IgA-secreting plasma cells through
heavy chain class-switch recombination (CSR) depends
on priming by mucosal DCs carrying antigen and live
bacteria from the intestinal epithelium117,177. Similar to
the priming of a T cell mucosal phenotype, these DCs
are conditioned by IEC-derived signals to promote IgA
class switching and a gut-homing phenotype through
the production of nitric oxide (NO), IL‑10 and retinoic
acid, in conjunction with TGFβ signalling 117,124,178 (FIG. 3).
In the presence of a cognate CD4+ T cell response, T cell
expression of CD40L acts as a necessary signal for B cell
CSR. In the absence of help from T cells, CSR can occur
through the stimulation of B cells by APRIL and BAFF,
and signalling through transmembrane activator and
CAML interactor (TACI) and BAFF receptor (BAFFR;
also known as TNFRSF13C)108,109,179,180. This process is
directly supported by IECs through the production of
APRIL and BAFF in response to commensal bacteria-
induced NF‑κB signalling 108,109. Moreover, IECs induce
APRIL and BAFF production by mucosal DCs through
TSLP signalling, which acts to amplify the effect on B cell
stimulation108,109. This pathway is of clinical relevance to
the most prevalent human primary immunodeficiencies,
common variable immunodeficiency and IgA deficiency,
in which a subset of patients have mutations in the gene
encoding the TACI receptor that lead to defects in CSR
and IgA production181,182.
Concluding remarks
Collectively, the studies highlighted in this Review dem-
onstrate the diverse and multifaceted roles that IECs
have in the continuous maintenance of intestinal home-
ostasis. Through secretory epithelial cell responses and
the maintenance of a continuous cell layer, IECs effec-
tively sustain a physical and biochemical barrier between
hosts and their environment. As cells forming a uniquely
adapted barrier surface, IECs actively respond to their
local environment through regulatory mechanisms that
earn IECs recognition as central mediators of microbial
and immune homeostasis in the intestine. As much of
what is understood of IEC function has been derived
from studies using mouse models, a future challenge
lies in the translation of this understanding into human
systems and the development of novel therapeutics for
targeting the pathways that contribute to human health.

1. Mankertz, J. & Schulzke, J.‑D. Altered permeability
in inflammatory bowel disease: pathophysiology and
clinical implications. Curr. Opin. Gastroenterol.
23, 379–383 (2007).
2. Brenchley, J. M. et al. Microbial translocation is a
cause of systemic immune activation in chronic HIV
infection. Nature Med. 12, 1365–1371 (2006).
3. Sandler, N. G. et al. Host response to translocated
microbial products predicts outcomes of patients
with HBV or HCV infection. Gastroenterology 141,
1220–1230 (2011).
4. Cani, P. D. et al. Metabolic endotoxemia initiates
obesity and insulin resistance. Diabetes 56,
1761–1772 (2007).
5. Amar, J. et al. Intestinal mucosal adherence and
translocation of commensal bacteria at the early
onset of type 2 diabetes: molecular mechanisms and
probiotic treatment. EMBO Mol. Med. 3, 559–572
(2011).
6. Wen, L. et al. Innate immunity and intestinal
microbiota in the development of type 1 diabetes.
Nature 455, 1109–1113 (2008).
7. Lee, Y. K., Menezes, J. S., Umesaki, Y. &
Mazmanian, S. K. Colloquium paper:
Proinflammatory T‑cell responses to gut
microbiota promote experimental autoimmune
encephalomyelitis. Proc. Natl Acad. Sci. USA 108,
4615–4622 (2010).
8. Berer, K. et al. Commensal microbiota and myelin
autoantigen cooperate to trigger autoimmune
demyelination. Nature 479, 538–541 (2011).
9. Wu, H.‑J. et al. Gut-residing segmented filamentous
bacteria drive autoimmune arthritis via T helper 17
cells. Immunity 32, 815–827 (2010).
10. Kamada, N., Seo, S.‑U., Chen, G. Y. & Núñez, G.
Role of the gut microbiota in immunity and
inflammatory disease. Nature Rev. Immunol. 13,
321–335 (2013).
11. Hooper, L. V., Littman, D. R. & Macpherson, A. J.
Interactions between the microbiota and the immune
system. Science 336, 1268–1273 (2012).
12. Honda, K. & Littman, D. R. The microbiome in
infectious disease and inflammation. Annu. Rev.
Immunol. 30, 759–795 (2012).
13. Maynard, C. L., Elson, C. O., Hatton, R. D. &
Weaver, C. T. Reciprocal interactions of the
intestinal microbiota and immune system.
Nature 489, 231–241 (2012).
14. Moon, C. & Stappenbeck, T. S. Viral interactions
with the host and microbiota in the intestine.
Curr. Opin. Immunol. 24, 405–410 (2012).
15. Crosnier, C., Stamataki, D. & Lewis, J. Organizing
cell renewal in the intestine: stem cells, signals
and combinatorial control. Nature Rev. Genet. 7,
349–359 (2006).
16. Van der Flier, L. G. & Clevers, H. Stem cells,
self‑renewal, and differentiation in the intestinal
epithelium. Annu. Rev. Physiol. 71, 241–260 (2009).
17. Kim, Y. S. & Ho, S. B. Intestinal goblet cells and mucins
in health and disease: recent insights and progress.
Curr. Gastroenterol. Rep. 12, 319–330 (2010).
18. Gallo, R. L. & Hooper, L. V. Epithelial antimicrobial
defence of the skin and intestine. Nature Rev.
Immunol. 12, 503–516 (2012).
19. Johansson, M. E. V. et al. The inner of the two
Muc2 mucin-dependent mucus layers in colon is
devoid of bacteria. Proc. Natl Acad. Sci. USA 105,
15064–15069 (2008).
20. Velcich, A. et al. Colorectal cancer in mice
genetically deficient in the mucin Muc2. Science 295,
1726–1729 (2002).
21. Van der Sluis, M. et al. Muc2‑deficient mice
spontaneously develop colitis, indicating that MUC2 is
critical for colonic protection. Gastroenterology 131,
117–129 (2006).
22. Taupin, D. R., Kinoshita, K. & Podolsky, D. K.
Intestinal trefoil factor confers colonic epithelial
resistance to apoptosis. Proc. Natl Acad. Sci. USA 97,
799–804 (2000).
23. Dignass, A., Lynch-Devaney, K., Kindon, H., Thim, L. &
Podolsky, D. K. Trefoil peptides promote epithelial
migration through a transforming growth factor
β-independent pathway. J. Clin. Invest. 94, 376–383
(1994).
24. Artis, D. et al. RELMβ/FIZZ2 is a goblet cell-specific
immune-effector molecule in the gastrointestinal tract.
Proc. Natl Acad. Sci. USA 101, 13596–13600 (2004).
25. Nair, M. G. et al. Goblet cell-derived resistin-like
molecule β augments CD4+ T cell production of
IFN‑γ and infection-induced intestinal inflammation.
J. Immunol. 181, 4709–4715 (2008).
26. Bevins, C. L. & Salzman, N. H. Paneth cells,
antimicrobial peptides and maintenance of intestinal
homeostasis. Nature Rev. Microbiol. 9, 356–368
(2011).
27. Mukherjee, S. et al. Antibacterial membrane
attack by a pore-forming intestinal C‑type lectin.
Nature 505, 103–107 (2014).
28. Darmoul, D. & Ouellette, A. J. Positional specificity
of defensin gene expression reveals Paneth cell
heterogeneity in mouse small intestine.
Am. J. Physiol. 271, G68–G74 (1996).

150 | MARCH 2014 | VOLUME 14 www.nature.com/reviews/immunol

© 2014 Macmillan Publishers Limited. All rights reserved

 F O C U S O N H o m e os tat i c I m m un e R e sRpEons eS
29. Vaishnava, S. et al. The antibacterial lectin RegIIIγ
promotes the spatial segregation of microbiota and
host in the intestine. Science 334, 255–258 (2011).
In this study, the antimicrobial protein REGIIIγ
is identified as being necessary for the physical
separation of commensal bacteria from the surface
of the small intestinal epithelium, thus limiting the
activation of the intestinal immune response.
30. Meyer-Hoffert, U. et al. Secreted enteric antimicrobial
activity localises to the mucus surface layer. Gut 57,
764–771 (2008).
31. Hampe, J. et al. A genome-wide association scan of
nonsynonymous SNPs identifies a susceptibility
variant for Crohn disease in ATG16L1. Nature Genet.
39, 207–211 (2007).
32. Rioux, J. D. et al. Genome-wide association study
identifies new susceptibility loci for Crohn disease
and implicates autophagy in disease pathogenesis.
Nature Genet. 39, 596–604 (2007).
References 31 and 32 report the association
between genetic variants in the gene for the
autophagy protein ATG16L1 and Crohn’s
disease susceptibility, establishing a Crohn’s
disease-specific genetic link between IBD and
the autophagy pathway.
33. Cadwell, K. et al. A key role for autophagy and
the autophagy gene Atg16l1 in mouse and human
intestinal Paneth cells. Nature 456, 259–263
(2008).
34. Kaser, A. et al. XBP1 links ER stress to intestinal
inflammation and confers genetic risk for human
inflammatory bowel disease. Cell 134, 743–756
(2008).
35. Brandl, K. et al. Enhanced sensitivity to DSS colitis
caused by a hypomorphic Mbtps1 mutation disrupting
the ATF6‑driven unfolded protein response. Proc. Natl
Acad. Sci. USA 106, 3300–3305 (2009).
36. Cadwell, K. et al. Virus-plus-susceptibility gene
interaction determines Crohn’s disease gene Atg16L1
phenotypes in intestine. Cell 141, 1135–1145 (2010).
In this study, the interaction between
environmental exposures and genetic susceptibility
is shown to determine the penetrance of disease in
mouse models of intestinal inflammation.
37. Khor, B., Gardet, A. & Xavier, R. J. Genetics and
pathogenesis of inflammatory bowel disease. Nature
474, 307–317 (2011).
38. Benjamin, J. L., Sumpter, R. Jr, Levine, B. &
Hooper, L. V. Intestinal epithelial autophagy is
essential for host defense against invasive bacteria.
Cell Host Microbe 13, 723–734 (2013).
39. Adolph, T. E. et al. Paneth cells as a site of origin for
intestinal inflammation. Nature 503, 272–276 (2013).
This study demonstrates interactions between the
regulation of the UPR and the autophagy pathway
in Paneth cells and supports a model in which
alterations in these two responses regulate the
development of Crohn’s disease.
40. Johansen, F.‑E. & Kaetzel, C. S. Regulation of the
polymeric immunoglobulin receptor and IgA
transport: new advances in environmental factors
that stimulate pIgR expression and its role in
mucosal immunity. Mucosal Immunol. 4, 598–602
(2011).
41. Johansen, F.‑E. et al. Absence of epithelial
immunoglobulin a transport, with increased mucosal
leakiness, in polymeric immunoglobulin receptor/
secretory component–deficient mice. J. Exp. Med.
190, 915–922 (1999).
42. Shulzhenko, N. et al. Crosstalk between B
lymphocytes, microbiota and the intestinal epithelium
governs immunity versus metabolism in the gut.
Nature Med. 17, 1585–1593 (2011).
In this study, a compensatory response of IECs
in the absence of adaptive IgA directed against
commensal bacteria is characterized by the
engagement of immune pathways and
dysregulation of lipid metabolism.
43. Suzuki, K. et al. Aberrant expansion of segmented
filamentous bacteria in IgA-deficient gut. Proc. Natl
Acad. Sci. USA 101, 1981–1986 (2004).
44. Mabbott, N. A., Donaldson, D. S., Ohno, H.,
Williams, I. R. & Mahajan, A. Microfold (M) cells:
important immunosurveillance posts in the intestinal
epithelium. Mucosal Immunol. 6, 666–677 (2013).
45. Mowat, A. M. Anatomical basis of tolerance and
immunity to intestinal antigens. Nature Rev. Immunol.
3, 331–341 (2003).
46. Hase, K. et al. Uptake through glycoprotein 2
of FimH+ bacteria by M cells initiates mucosal
immune response. Nature 462, 226–230 (2009).
NATURE REVIEWS | IMMUNOLOGY VOLUME 14 | MARCH 2014 | 151
V IE W s
47. McDole, J. R. et al. Goblet cells deliver luminal antigen
to CD103+ dendritic cells in the small intestine.
Nature 483, 345–349 (2012).
This report shows that goblet cells can transport
soluble luminal antigens to lamina propria DCs,
implicating a pathway by which IECs directly
mediate antigen delivery to immune cells in
addition to M cell-mediated transport.
48. Rescigno, M. et al. Dendritic cells express tight
junction proteins and penetrate gut epithelial
monolayers to sample bacteria. Nature Immunol. 2,
361–367 (2001).
49. Chieppa, M., Rescigno, M., Huang, A. Y. C. &
Germain, R. N. Dynamic imaging of dendritic cell
extension into the small bowel lumen in response to
epithelial cell TLR engagement. J. Exp. Med. 203,
2841–2852 (2006).
This paper demonstrates a role for epithelial
cell TLR signalling in promoting DC sampling of
antigens from the intestinal lumen.
50. Shan, M. et al. Mucus enhances gut homeostasis and
oral tolerance by delivering immunoregulatory signals.
Science 342, 447–453 (2013).
51. Abreu, M. T. Toll-like receptor signalling in the
intestinal epithelium: how bacterial recognition
shapes intestinal function. Nature Rev. Immunol. 10,
131–144 (2010).
52. Chen, G. Y. & Núñez, G. Inflammasomes in intestinal
inflammation and cancer. Gastroenterology 141,
1986–1999 (2011).
53. Elinav, E., Henao-Mejia, J. & Flavell, R. A. Integrative
inflammasome activity in the regulation of intestinal
mucosal immune responses. Mucosal Immunol. 6,
4–13 (2013).
54. Li, X.‑D. et al. Mitochondrial antiviral signaling protein
(MAVS) monitors commensal bacteria and induces an
immune response that prevents experimental colitis.
Proc. Natl Acad. Sci. USA 108, 17390–17395
(2011).
55. Broquet, A. H., Hirata, Y., McAllister, C. S. &
Kagnoff, M. F. RIG‑I/MDA5/MAVS are required to
signal a protective IFN response in rotavirus-infected
intestinal epithelium. J. Immunol. 186, 1618–1626
(2011).
56. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F.,
Edberg, S. & Medzhitov, R. Recognition of commensal
microflora by Toll-like receptors is required for
intestinal homeostasis. Cell 118, 229–241 (2004).
In this study, mice deficient in TLR signalling or
greatly depleted of commensal bacteria exhibited
increased susceptibility to experimentally
induced intestinal inflammation, implicating
commensal microorganism-dependent signals
in the regulation of intestinal homeostasis and
response to injury.
57. Brandl, K. et al. MyD88 signaling in
nonhematopoietic cells protects mice against induced
colitis by regulating specific EGF receptor ligands.
Proc. Natl Acad. Sci. USA 107, 19967–19972
(2010).
58. Podolsky, D. K., Gerken, G., Eyking, A. & Cario, E.
Colitis-associated variant of TLR2 causes impaired
mucosal repair because of TFF3 deficiency.
Gastroenterology 137, 209–220 (2009).
59. Cario, E., Gerken, G. & Podolsky, D. K. Toll-like
receptor 2 enhances ZO‑1‑associated intestinal
epithelial barrier integrity via protein kinase C.
Gastroenterology 127, 224–238 (2004).
60. Greten, F. R. et al. IKKβ links inflammation and
tumorigenesis in a mouse model of colitis-associated
cancer. Cell 118, 285–296 (2004).
This study provides evidence that the NF‑κB
signalling pathway in IECs regulates the
development of inflammation-induced cancer
through the regulation of apoptosis.
61. Nenci, A. et al. Epithelial NEMO links innate
immunity to chronic intestinal inflammation.
Nature 446, 557–561 (2007).
62. Hugot, J.‑P. et al. Association of NOD2 leucine-rich
repeat variants with susceptibility to Crohn’s disease.
Nature 411, 599–603 (2001).
63. Ogura, Y. et al. A frameshift mutation in NOD2
associated with susceptibility to Crohn’s disease.
Nature 411, 603–606 (2001).
References 62 and 63 provide the first examples
of genetic variants linking bacterial recognition
with susceptibility to human IBD.
64. Zaki, M. H., Lamkanfi, M. & Kanneganti, T.‑D.
The Nlrp3 inflammasome: contributions to
intestinal homeostasis. Trends Immunol. 32,
171–179 (2011).
© 2014 Macmillan Publishers Limited. All rights reserved
65. Swanson, P. A. et al. Enteric commensal bacteria
potentiate epithelial restitution via reactive oxygen
species-mediated inactivation of focal adhesion
kinase phosphatases. Proc. Natl Acad. Sci. USA 108,
8803–8808 (2011).
66. Leoni, G. et al. Annexin A1, formyl peptide receptor,
and NOX1 orchestrate epithelial repair. J. Clin. Invest.
123, 443–454 (2012).
67. Lee, W.‑J. Bacterial-modulated host immunity and
stem cell activation for gut homeostasis. Genes Dev.
23, 2260–2265 (2009).
68. Hochmuth, C. E., Biteau, B., Bohmann, D. & Jasper, H.
Redox regulation by Keap1 and Nrf2 controls
intestinal stem cell proliferation in Drosophila. Cell
Stem Cell 8, 188–199 (2011).
69. Jones, R. M. et al. Symbiotic lactobacilli stimulate gut
epithelial proliferation via Nox-mediated generation of
reactive oxygen species. EMBO J. 32, 3017–3028
(2013).
70. Rakoff-Nahoum, S. & Medzhitov, R. Regulation of
spontaneous intestinal tumorigenesis through the
adaptor protein MyD88. Science 317, 124–127
(2007).
71. Lee, S. H. et al. ERK activation drives intestinal
tumorigenesis in Apcmin/+ mice. Nature Med. 16,
665–670 (2010).
72. Vlantis, K. et al. Constitutive IKK2 activation in
intestinal epithelial cells induces intestinal tumors
in mice. J. Clin. Invest. 121, 2781–2793 (2011).
73. Schwitalla, S. et al. Intestinal tumorigenesis initiated
by dedifferentiation and acquisition of stem-cell-like
properties. Cell 152, 25–38 (2013).
74. Fukata, M. et al. Toll-Like receptor‑4 promotes the
development of colitis-associated colorectal tumors.
Gastroenterology 133, 1869–1869 (2007).
75. Xiao, H. et al. The Toll–interleukin‑1 receptor
member SIGIRR regulates colonic epithelial
homeostasis, inflammation, and tumorigenesis.
Immunity 26, 461–475 (2007).
76. Dupaul-Chicoine, J. et al. Control of intestinal
homeostasis, colitis, and colitis-associated colorectal
cancer by the inflammatory caspases. Immunity 32,
367–378 (2010).
77. Hu, B. et al. Inflammation-induced tumorigenesis
in the colon is regulated by caspase‑1 and NLRC4.
PNAS 107, 21635–21640 (2010).
78. Zaki, M. H. et al. The NOD-like receptor NLRP12
attenuates colon inflammation and tumorigenesis.
Cancer Cell 20, 649–660 (2011).
79. Normand, S. et al. Nod-like receptor pyrin
domain‑containing protein 6 (NLRP6) controls
epithelial self-renewal and colorectal
carcinogenesis upon injury. Proc. Natl Acad. Sci. USA
108, 9601–9606 (2011).
80. Allen, I. C. et al. NLRP12 suppresses colon
inflammation and tumorigenesis through the
negative regulation of noncanonical NF‑κB signaling.
Immunity 36, 742–754 (2012).
81. Salcedo, R. et al. MyD88‑mediated signaling
prevents development of adenocarcinomas of the
colon: role of interleukin 18. J. Exp. Med. 207,
1625–1636 (2010).
82. Huber, S. et al. IL‑22BP is regulated by the
inflammasome and modulates tumorigenesis in
the intestine. Nature 491, 259–263 (2012).
83. Otte, J.‑M., Cario, E. & Podolsky, D. K. Mechanisms
of cross hyporesponsiveness to Toll-like receptor
bacterial ligands in intestinal epithelial cells.
Gastroenterology 126, 1054–1070 (2004).
84. Lotz, M. et al. Postnatal acquisition of endotoxin
tolerance in intestinal epithelial cells. J. Exp. Med.
203, 973–984 (2006).
This study demonstrates the inhibition of TLR
signalling in IECs and the acquisition of tolerance
to microbial colonization that occurs shortly after
birth to establish host–microbial homeostasis in
the intestine.
85. Vereecke, L. et al. Enterocyte-specific A20
deficiency sensitizes to tumor necrosis factor-induced
toxicity and experimental colitis. J. Exp. Med. 207,
1513–1523 (2010).
86. Chassin, C. et al. miR‑146a mediates protective
innate immune tolerance in the neonate intestine.
Cell Host Microbe 8, 358–368 (2010).
87. Guma, M. et al. Constitutive intestinal NF‑κB does not
trigger destructive inflammation unless accompanied
by MAPK activation. J. Exp. Med. 208, 1889–1900
(2011).
88. Neish, A. S. et al. Prokaryotic regulation of epithelial
responses by inhibition of IκB‑α ubiquitination.
Science 289, 1560–1563 (2000).
R E V IE W S
89. Kumar, A. et al. The bacterial fermentation product
butyrate influences epithelial signaling via reactive
oxygen species-mediated changes in cullin‑1
neddylation. J. Immunol. 182, 538–546 (2009).
90. Kondo, T., Kawai, T. & Akira, S. Dissecting negative
regulation of Toll-like receptor signaling. Trends
Immunol. 33, 449–458 (2012).
91. Blander, J. M. & Sander, L. E. Beyond pattern
recognition: five immune checkpoints for scaling the
microbial threat. Nature Rev. Immunol. 12, 215–225
(2012).
92. Gewirtz, A. T., Navas, T. A., Lyons, S., Godowski, P. J. &
Madara, J. L. Cutting edge: bacterial flagellin activates
basolaterally expressed TLR5 to induce epithelial
proinflammatory gene expression. J. Immunol. 167,
1882–1885 (2001).
93. Rhee, S. H. et al. Pathophysiological role of Toll-like
receptor 5 engagement by bacterial flagellin in
colonic inflammation. Proc. Natl Acad. Sci. USA 102,
13610–13615 (2005).
94. Lee, J. et al. Maintenance of colonic homeostasis
by distinctive apical TLR9 signalling in intestinal
epithelial cells. Nature Cell Biol. 8, 1327–1336 (2006).
95. Barnich, N., Aguirre, J. E., Reinecker, H.‑C., Xavier, R.
& Podolsky, D. K. Membrane recruitment of NOD2
in intestinal epithelial cells is essential for nuclear
factor-κB activation in muramyl dipeptide recognition.
J. Cell Biol. 170, 21–26 (2005).
96. Lipinski, S. et al. RNAi screening identifies mediators
of NOD2 signaling: Implications for spatial specificity
of MDP recognition. Proc. Natl Acad. Sci. USA 109,
21426–21431 (2012).
97. Matzinger, P. The danger model: A renewed sense of
self. Science 296, 301–305 (2002).
98. Sander, L. E. et al. Detection of prokaryotic mRNA
signifies microbial viability and promotes immunity.
Nature 474, 385–389 (2011).
In this study, bacterial mRNA is identified as a
signal that enables the gauging of infectious risk
posed by live versus dead bacteria, exemplifying a
vita-PAMP.
99. Hooper, L. V., Stappenbeck, T. S., Hong, C. V. &
Gordon, J. I. Angiogenins: a new class of microbicidal
proteins involved in innate immunity. Nature Immunol.
4, 269–273 (2003).
100. Kobayashi, K. S. et al. Nod2‑dependent regulation of
innate and adaptive immunity in the intestinal tract.
Science 307, 731–734 (2005).
101. Vaishnava, S., Behrendt, C. L., Ismail, A. S.,
Eckmann, L. & Hooper, L. V. Paneth cells directly
sense gut commensals and maintain homeostasis at
the intestinal host-microbial interface. Proc. Natl
Acad. Sci. USA 105, 20858–20863 (2008).
102. Bruno, M. E. C., Frantz, A. L., Rogier, E. W.,
Johansen, F.‑E. & Kaetzel, C. S. Regulation of the
polymeric immunoglobulin receptor by the classical
and alternative NF‑κB pathways in intestinal
epithelial cells. Mucosal Immunol. 4, 468–478
(2011).
103. Rimoldi, M. et al. Intestinal immune homeostasis is
regulated by the crosstalk between epithelial cells and
dendritic cells. Nature Immunol. 6, 507–514 (2005).
This study describes the conditioning of mucosal
DCs towards a non-inflammatory phenotype by
interactions with IECs, representing a mechanism
for the indirect IEC-mediated regulation of
adaptive immune cell priming.
104. Zeuthen, L. H., Fink, L. N. & Frokiaer, H. Epithelial
cells prime the immune response to an array of gut-
derived commensals towards a tolerogenic phenotype
through distinct actions of thymic stromal
lymphopoietin and transforming growth factor‑β.
Immunology 123, 197–208 (2008).
105. Zaph, C. et al. Epithelial-cell-intrinsic IKKβ expression
regulates intestinal immune homeostasis. Nature
446, 552–556 (2007).
This study demonstrates a crucial role for
IEC-intrinsic NF-κB signalling and the production
of the cytokine TSLPthymic stromal lymphopoietin
in regulating intestinal immune responses and
coordinating anti-helminth immunity.
106. Atarashi, K. et al. Induction of colonic regulatory T
cells by indigenous Clostridium species. Science 331,
337–341 (2011).
107. Zaph, C. et al. Commensal-dependent expression of
IL‑25 regulates the IL‑23–IL‑17 axis in the intestine.
J. Exp. Med. 205, 2191–2198 (2008).
108. He, B. et al. Intestinal bacteria trigger T cell-
independent immunoglobulin A2 class switching by
inducing epithelial-cell secretion of the cytokine
APRIL. Immunity 26, 812–826 (2007).
152 | MARCH 2014 | VOLUME 14 www.nature.com/reviews/immunol
109. Xu, W. et al. Epithelial cells trigger frontline
immunoglobulin class switching through a pathway
regulated by the inhibitor SLPI. Nature Immunol. 8,
294–303 (2007).
References 108 and 109 identify IEC production
of APRIL and BAFF in response to microbial
stimulation as important for the regulation of
B cell CSR and the mucosal IgA response.
110. Taylor, B. C. et al. TSLP regulates intestinal immunity
and inflammation in mouse models of helminth
infection and colitis. J. Exp. Med. 206, 655–667
(2009).
111. Pabst, O. & Bernhardt, G. The puzzle of intestinal
lamina propria dendritic cells and macrophages.
Eur. J. Immunol. 40, 2107–2111 (2010).
112. Bogunovic, M. et al. Origin of the lamina propria
dendritic cell network. Immunity 31, 513–525
(2009).
113. Varol, C. et al. Intestinal lamina propria dendritic cell
subsets have different origin and functions. Immunity
31, 502–512 (2009).
114. Rivollier, A., He, J., Kole, A., Valatas, V. & Kelsall, B. L.
Inflammation switches the differentiation program
of Ly6Chi monocytes from antiinflammatory
macrophages to inflammatory dendritic cells in the
colon. J. Exp. Med. 209, 139–155 (2012).
115. Zigmond, E. & Jung, S. Intestinal macrophages: well
educated exceptions from the rule. Trends Immunol.
34, 162–168 (2013).
116. Schulz, O. et al. Intestinal CD103+, but not CX3CR1+,
antigen sampling cells migrate in lymph and serve
classical dendritic cell functions. J. Exp. Med. 206,
3101–3114 (2009).
117. Macpherson, A. J. & Uhr, T. Induction of protective
IgA by intestinal dendritic cells carrying commensal
bacteria. Science 303, 1662–1665 (2004).
118. Coombes, J. L. et al. A functionally specialized
population of mucosal CD103+ DCs induces Foxp3+
regulatory T cells via a TGF‑β– and retinoic acid–
dependent mechanism. J. Exp. Med. 204,
1757–1764 (2007).
119. Sun, C.‑M. et al. Small intestine lamina propria
dendritic cells promote de novo generation of Foxp3
T reg cells via retinoic acid. J. Exp. Med. 204,
1775–1785 (2007).
120. Mora, J. R. et al. Selective imprinting of gut-homing
T cells by Peyer’s patch dendritic cells. Nature 424,
88–93 (2003).
121. Johansson-Lindbom, B. et al. Functional specialization
of gut CD103+ dendritic cells in the regulation of
tissue-selective T cell homing. J. Exp. Med. 202,
1063–1073 (2005).
122. Iwata, M. et al. Retinoic acid imprints gut-homing
specificity on T cells. Immunity 21, 527–538 (2004).
123. Jaensson, E. et al. Small intestinal CD103+ dendritic
cells display unique functional properties that are
conserved between mice and humans. J. Exp. Med.
205, 2139–2149 (2008).
124. Mora, J. R. & von Andrian, U. H. Differentiation and
homing of IgA-secreting cells. Mucosal Immunol. 1,
96–109 (2008).
125. Niess, J. H. et al. CX3CR1‑mediated dendritic cell
access to the intestinal lumen and bacterial clearance.
Science 307, 254–258 (2005).
126. Hadis, U. et al. Intestinal tolerance requires
gut homing and expansion of FoxP3+ regulatory
T cells in the lamina propria. Immunity 34, 237–246
(2011).
127. Murai, M. et al. Interleukin 10 acts on regulatory
T cells to maintain expression of the transcription
factor Foxp3 and suppressive function in mice with
colitis. Nature Immunol. 10, 1178–1184 (2009).
128. Kayama, H. et al. Intestinal CX3C chemokine receptor
1high (CX3CR1high) myeloid cells prevent T‑cell-
dependent colitis. Proc. Natl Acad. Sci. USA 109,
5010–5015 (2012).
129. Kang, S. et al. Intestinal epithelial cell-derived
semaphorin 7A negatively regulates development of
colitis via αvβ1 integrin. J. Immunol. 188, 1108–1116
(2012).
130. Saenz, S. A. et al. IL25 elicits a multipotent progenitor
cell population that promotes TH2 cytokine responses.
Nature 464, 1362–1366 (2010).
131. Siracusa, M. C. et al. TSLP promotes
interleukin‑3‑independent basophil haematopoiesis
and type 2 inflammation. Nature 477, 229–233
(2011).
132. Saenz, S. A. et al. IL‑25 simultaneously elicits distinct
populations of innate lymphoid cells and multipotent
progenitor type 2 (MPPtype2) cells. J. Exp. Med. 210,
1823–1837 (2013).
© 2014 Macmillan Publishers Limited. All rights reserved
133. Siracusa, M. C. et al. Thymic stromal lymphopoietin-
mediated extramedullary hematopoiesis promotes
allergic inflammation. Immunity 39, 1158–1170
(2013).
134. Spits, H. & Cupedo, T. Innate lymphoid cells:
emerging insights in development, lineage
relationships, and function. Annu. Rev. Immunol. 30,
647–675 (2012).
135. Monticelli, L. A., Sonnenberg, G. F. & Artis, D.
Innate lymphoid cells: critical regulators of allergic
inflammation and tissue repair in the lung.
Curr. Opin. Immunol. 24, 284–289 (2012).
136. Kim, B. S. et al. TSLP elicits IL‑33‑independent innate
lymphoid cell responses to promote skin
inflammation. Sci. Transl. Med. 5, 170ra16 (2013).
137. Tait Wojno, E. D. & Artis, D. Innate lymphoid cells:
balancing immunity, inflammation, and tissue repair
in the intestine. Cell Host Microbe 12, 445–457
(2012).
138. Walker, J. A., Barlow, J. L. & McKenzie, A. N. J.
Innate lymphoid cells — how did we miss them?
Nature Rev. Immunol. 13, 75–87 (2013).
139. Cherrier, M., Ohnmacht, C., Cording, S. & Eberl, G.
Development and function of intestinal innate
lymphoid cells. Curr. Opin. Immunol. 24, 277–283
(2012).
140. Spits, H. et al. Innate lymphoid cells — a proposal for
uniform nomenclature. Nature Rev. Immunol. 13,
145–149 (2013).
141. Bernink, J. H. et al. Human type 1 innate lymphoid
cells accumulate in inflamed mucosal tissues.
Nature Immunol. 14, 221–229 (2013).
142. Fuchs, A. et al. Intraepithelial type 1 innate
lymphoid cells are a unique subset of IL‑12- and
IL‑15‑responsive IFN‑γ‑producing cells. Immunity 38,
769–781 (2013).
143. Moro, K. et al. Innate production of TH2 cytokines by
adipose tissue-associated c‑Kit+Sca‑1+ lymphoid cells.
Nature 463, 540–544 (2009).
144. Neill, D. R. et al. Nuocytes represent a new innate
effector leukocyte that mediates type‑2 immunity.
Nature 464, 1367–1370 (2010).
References 143 and 144 identify a population of
innate lymphoid cells that mediate early TH2
cytokine production in response to the cytokines
IL-25 and IL-33 and during intestinal helminth
infection, that promotes goblet cell hyperplasia
and worm expulsion.
145. Price, A. E. et al. Systemically dispersed innate
IL‑13‑expressing cells in type 2 immunity. Proc. Natl
Acad. Sci. USA 107, 11489–11494 (2010).
146. Monticelli, L. A. et al. Innate lymphoid cells promote
lung-tissue homeostasis after infection with influenza
virus. Nature Immunol. 12, 1045–1054 (2011).
147. Chang, Y.‑J. et al. Innate lymphoid cells mediate
influenza-induced airway hyper-reactivity
independently of adaptive immunity. Nature Immunol.
12, 631–638 (2011).
148. Wilhelm, C. et al. An IL‑9 fate reporter demonstrates
the induction of an innate IL‑9 response in lung
inflammation. Nature Immunol. 12, 1071–1077
(2011).
149. Halim, T. Y. F., Krauß, R. H., Sun, A. C. & Takei, F.
Lung natural helper cells are a critical source of Th2
cell-type cytokines in protease allergen-induced
airway inflammation. Immunity 36, 451–463
(2012).
150. Mjösberg, J. et al. The transcription factor GATA3 Is
essential for the function of human type 2 innate
lymphoid cells. Immunity 37, 649–659 (2012).
151. Mebius, R. E., Rennert, P. & Weissman, I. L.
Developing lymph nodes collect, CD4+CD3− LTβ+ cells
that can differentiate to APC, NK cells, and follicular
cells but not T or B cells. Immunity 7, 493–504
(1997).
152. Zenewicz, L. A. et al. Innate and adaptive
interleukin‑22 protects mice from inflammatory
bowel disease. Immunity 29, 947–957 (2008).
153. Sonnenberg, G. F., Monticelli, L. A., Elloso, M. M.,
Fouser, L. A. & Artis, D. CD4+ lymphoid tissue-inducer
cells promote innate immunity in the gut. Immunity
34, 122–134 (2011).
154. Sonnenberg, G. F. et al. Innate lymphoid cells
promote anatomical containment of lymphoid-resident
commensal bacteria. Science 336, 1321–1325
(2012).
In this study, innate lymphoid cell-derived IL-22 is
demonstrated to mediate the containment of
specialized intestinal lymphoid tissue-resident
commensal bacteria to prevent systemic immune
activation.
 F O C U S O N ho m e os tat i c i m m un e r e sRpEons eS
155. Hanash, A. M. et al. Interleukin‑22 protects intestinal
stem cells from immune-mediated tissue damage and
regulates sensitivity to graft versus host disease.
Immunity 37, 339–350 (2012).
156. Kirchberger, S. et al. Innate lymphoid cells sustain
colon cancer through production of interleukin‑22 in
a mouse model. J. Exp. Med. 210, 917–931 (2013).
157. Muñoz, M. et al. Interleukin (IL)-23 mediates Toxoplasma
gondii–induced immunopathology in the gut via
matrixmetalloproteinase‑2 and IL‑22 but independent
of IL‑17. J. Exp. Med. 206, 3047–3059 (2009).
158. Buonocore, S. et al. Innate lymphoid cells drive
interleukin‑23‑dependent innate intestinal pathology.
Nature 464, 1371–1375 (2010).
159. Geremia, A. et al. IL‑23–responsive innate lymphoid
cells are increased in inflammatory bowel disease.
J. Exp. Med. 208, 1127–1133 (2011).
160. Coccia, M. et al. IL‑1β mediates chronic intestinal
inflammation by promoting the accumulation of
IL‑17A secreting innate lymphoid cells and CD4+
Th17 cells. J. Exp. Med. 209, 1595–1609 (2012).
161. Sawa, S. et al. RORγt+ innate lymphoid cells regulate
intestinal homeostasis by integrating negative signals
from the symbiotic microbiota. Nature Immunol. 12,
320–326 (2011).
162. Vonarbourg, C. et al. Regulated expression of nuclear
receptor RORγt confers distinct functional fates to NK
cell receptor-expressing RORγt+ innate lymphocytes.
Immunity 33, 736–751 (2010).
163. Satoh-Takayama, N. et al. Microbial flora drives
interleukin 22 production in intestinal NKp46+ cells
that provide innate mucosal immune defense.
Immunity 29, 958–970 (2008).
164. Yu, Q. et al. MyD88‑dependent signaling for IL‑15
production plays an important role in maintenance
of CD8αα TCRαβ and TCRγδ intestinal intraepithelial
lymphocytes. J. Immunol. 176, 6180–6185 (2006).
165. Cheroutre, H., Lambolez, F. & Mucida, D. The light and
dark sides of intestinal intraepithelial lymphocytes.
Nature Rev. Immunol. 11, 445–456 (2011).
166. Edelblum, K. L. et al. Dynamic migration of γδ
intraepithelial lymphocytes requires occludin.
Proc. Natl Acad. Sci. USA 109, 7097–7102 (2012).
167. Ismail, A. S. et al. γδ intraepithelial lymphocytes are
essential mediators of host-microbial homeostasis at
the intestinal mucosal surface. Proc. Natl Acad. Sci.
USA 108, 8743–8748 (2011).
168. Mucida, D. et al. Transcriptional reprogramming
of mature CD4+ helper T cells generates distinct
MHC class II‑restricted cytotoxic T lymphocytes.
Nature Immunol. 14, 281–289 (2013).
169. Gebhardt, T., Mueller, S. N., Heath, W. R. &
Carbone, F. R. Peripheral tissue surveillance and
residency by memory T cells. Trends Immunol. 34,
27–32 (2013).
NATURE REVIEWS | IMMUNOLOGY VOLUME 14 | MARCH 2014 | 153
V IE W s
170. Sathaliyawala, T. et al. Distribution and
compartmentalization of human circulating and
tissue-resident memory T cell subsets. Immunity 38,
187–197 (2013).
171. Schön, M. P. et al. Mucosal T lymphocyte numbers are
selectively reduced in integrin αE (CD103)-deficient
mice. J. Immunol. 162, 6641–6649 (1999).
172. El‑Asady, R. et al. TGF‑β-dependent CD103
expression by CD8+ T cells promotes selective
destruction of the host intestinal epithelium
during graft-versus-host disease. J. Exp. Med. 201,
1647–1657 (2005).
173. Huang, Y. et al. Mucosal memory CD8+ T cells are
selected in the periphery by an MHC class I molecule.
Nature Immunol. 12, 1086–1095 (2011).
174. Mackay, L. K. et al. Long-lived epithelial immunity by
tissue-resident memory T (TRM) cells in the absence of
persisting local antigen presentation. Proc. Natl Acad.
Sci. USA 109, 7037–7042 (2012).
175. Shin, H. & Iwasaki, A. A vaccine strategy that protects
against genital herpes by establishing local memory
T cells. Nature 491, 463–467 (2012).
176. Hansen, S. G. et al. Profound early control of highly
pathogenic SIV by an effector memory T‑cell vaccine.
Nature 473, 523–527 (2011).
177. Cerutti, A. The regulation of IgA class switching.
Nature Rev. Immunol. 8, 421–434 (2008).
178. Mora, J. R. et al. Generation of gut-homing IgA-
secreting B cells by intestinal dendritic cells.
Science 314, 1157–1160 (2006).
179. Litinskiy, M. B. et al. DCs induce CD40‑independent
immunoglobulin class switching through BLyS and
APRIL. Nature Immunol. 3, 822–829 (2002).
180. Castigli, E. et al. TACI and BAFF‑R mediate isotype
switching in B cells. J. Exp. Med. 201, 35–39 (2005).
181. Salzer, U. et al. Mutations in TNFRSF13B encoding
TACI are associated with common variable
immunodeficiency in humans. Nature Genet. 37,
820–828 (2005).
182. Castigli, E. et al. TACI is mutant in common variable
immunodeficiency and IgA deficiency. Nature Genet.
37, 829–834 (2005).
183. Barker, N. et al. Identification of stem cells in small
intestine and colon by marker gene Lgr5. Nature 449,
1003–1007 (2007).
184. Takeda, N. et al. Interconversion between intestinal
stem cell populations in distinct niches. Science 334,
1420–1424 (2011).
185. Yan, K. S. et al. The intestinal stem cell markers Bmi1
and Lgr5 identify two functionally distinct populations.
Proc. Natl Acad. Sci. USA 109, 466–471 (2012).
186. Capaldo, C. T. et al. IFN‑γ and TNF‑α‑induced
GBP‑1 inhibits epithelial cell proliferation through
suppression of β‑catenin/TCF signaling.
Mucosal Immunol. 5, 681–690 (2012).
© 2014 Macmillan Publishers Limited. All rights reserved
187. Nava, P. et al. Interferon‑γ regulates intestinal
epithelial homeostasis through converging β‑catenin
signaling pathways. Immunity 32, 392–402 (2010).
188. Alenghat, T. et al. Histone deacetylase 3 coordinates
commensal-bacteria-dependent intestinal
homeostasis. Nature 504, 153–157 (2013).
189. Blair, S. A., Kane, S. V., Clayburgh, D. R. & Turner, J. R.
Epithelial myosin light chain kinase expression and
activity are upregulated in inflammatory bowel
disease. Lab. Invest. 86, 191–201 (2006).
190. Marchiando, A. M. et al. Caveolin‑1–dependent
occludin endocytosis is required for TNF-induced tight
junction regulation in vivo. J. Cell Biol. 189, 111–126
(2010).
191. Wang, F. et al. Active deformation of apoptotic
intestinal epithelial cells with adhesion-restricted
polarity contributes to apoptotic clearance.
Lab. Invest. 91, 462–471 (2011).
192. Eisenhoffer, G. T. et al. Crowding induces live cell
extrusion to maintain homeostatic cell numbers
in epithelia. Nature 484, 546–549 (2012).
193. D’Incà, R. et al. Intestinal permeability test as a
predictor of clinical course in Crohn’s disease.
Am. J. Gastroenterol. 94, 2956–2960 (1999).
194. Wyatt, J. & Vogelsang, H. Intestinal permeability
and the prediction of relapse in Crohn’s disease.
Lancet 341, 1437 (1993).
195. Khounlotham, M. et al. Compromised intestinal
epithelial barrier induces adaptive immune
compensation that protects from colitis. Immunity 37,
563–573 (2012).
196. Su, L. et al. Targeted epithelial tight junction
dysfunction causes immune activation and
contributes to development of experimental colitis.
Gastroenterology 136, 551–563 (2009).
Acknowledgements
The authors thank all members of the Artis laboratory for
discussions and critical reading of the manuscript. This work
is supported by US National Institutes of Health grants
(AI061570, AI095608, AI087990, AI074878, AI095466,
AI106697, AI102942 and AI097333 to D.A.; T32AI00744
to L.W.P.), the Burroughs Wellcome Fund Investigator in
Pathogenesis of Infectious Disease Award (D.A.) and the
Crohn’s and Colitis Foundation of America (D.A.).
Competing interests statement
The authors declare no competing interests.
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