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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 hostmicrobial
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 hostcommensal 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 Crohns 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.

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.
emails: lancep@mail.med.
upenn.edu;
dartis@mail.med.upenn.edu
doi:10.1038/nri3608
1

Specialized epithelial cells constitute barrier surfaces that


separate mammalian hosts from the external environment. 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 carries with it the risk of infection and inflammation if epithelial 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 intestinal 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 immunecells.
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 pathology 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 infections, including infection with HIV and hepatitis virus2,3,
and metabolic disease4,5. Furthermore, hostmicrobial
interactions that occur at the IEC barrier contribute to a
broad range of extra-intestinal autoimmune and inflammatory diseases, including type1 diabetes, rheumatoid
arthritis and multiple sclerosis69. Hence, a comprehensive 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 hostviral
interactions in the intestine have been reviewed extensively elsewhere1014. Therefore, in this Review, we discuss 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, microbialsensing and immunoregulatory functions of IECs are
reviewed, with a particular focus on their relationship to
intestinal health and disease. We discuss the barrier function maintained by IEC-derived mucins and antimicrobial
proteins, the pathways through which IECs regulate
innate and adaptive immune cells present at the intestinal barrier and the contribution of IEC recognition of
microbial colonization to IEC function and homeostasis.

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Small intestine

Follicle-associated epithelium

Colon

Apoptotic
IECs

Commensal
bacteria

Mucus
Secondlayer
mucus

TFF3

sIgA

AMPs

M cell

Mucus

Enteroendocrine
cell

Goblet
cell

Enterocyte

B cell

Stromal
cell

Lymphoid
follicle
Paneth
cell

IESC

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
ofthe small intestine that
havean outer layer
consistingof mature,
absorptive enterocytes,
mucus-secreting goblet cells
and enteroendocrine cells.

Pluripotent intestinal
epithelial stem cells
(Pluripotent IESCs).
Tissue-resident stem cells
thatundergo continuous
self-renewal and are
responsible for regenerating
all lineages of mature
intestinal epithelial cells,
including enterocytes,
enteroendocrine cells,
gobletcells and Paneth cells.

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
epithelial
stem
Nature
Reviews
| 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 cryptvillus 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 (Mcells) 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


The intestinal epithelium is the largest of the bodys
mucosal surfaces, covering ~400m2 of surface area
with a single layer of cells organized into crypts and
villi (FIG.1) . This surface is continually renewed by
pluripotent intestinal epithelial stem cells (pluripotent
IESCs) that reside in the base of crypts, where the proliferation, differentiation and functional potential of
epithelial cell progenitors is regulated by the local stem
cell niche15,16 (BOX1). Although the majority of cells
bordering the intestinal lumen are absorptive enterocytes, which are adapted for metabolic and digestive
function, the diversity of functions that the intestinal
epithelium carries out is reflected by the presence of
additional specialized IEC lineages.
Secretory IECs, including enteroendocrine cells,
goblet cells and Paneth cells, are specialized for maintaining the digestive or barrier function of the epithelium. Enteroendocrine cells represent a link between the
central and enteric neuroendocrine systems through
the secretion of numerous hormone regulators of

digestive function. The luminal secretion of mucins


and antimicrobial proteins (AMPs) by goblet cells and
Paneth cells, respectively, establishes a physical and
biochemical barrier to microbial contact with the epithelial surface and underlying immune cells17,18 (FIG.1).
Collectively, the diverse functions of IECs result in
a dynamic barrier to the environment, which protects
the host from infection and continuous exposure to
potentially inflammatory stimuli.
Keeping the bugs at bayIEC secretory defences. The
secretion of highly glycosylated mucins into the intestinal lumen by goblet cells creates the first line of defence
against microbial encroachment. The most abundant
of these mucins, mucin2 (MUC2), plays an essential
part in the organization of the intestinal mucous layers
at the epithelial surface of the colon19. The importance
of mucin production by goblet cells is emphasized by
the spontaneous development of colitis and the predisposition to inflammation-induced colorectal cancers
observed in MUC2deficient mice20,21. Additional goblet

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Box 1 | The IESC niche
Along the cryptvillus 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 cryptvillus 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 differentiation16,183185.
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 WNTcatenin
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
WNTcatenin signalling by the pro-inflammatory cytokines interferon 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 hostcommensal microorganism interactions
into IEC homeostasis have been recently described188.

Autophagy
A cellular process by which
cytoplasmic organelles and
macromolecular complexes
are engulfed by double
membrane-bound vesicles
for delivery to lysosomes
and subsequent degradation.
This process is involved in
constitutive turnover of
proteins and organelles and is
central to cellular activities that
maintain a balance between
the synthesis and breakdown
of various proteins.

Unfolded protein response


(UPR). A response that
increases the ability of the
endoplasmic reticulum to
fold and translocate proteins,
decreases the synthesis of
proteins, causes the arrest of
the cell cycle and promotes
apoptosis.

Plasma cells
Terminally differentiated cells
of the Bcell lineage that
secrete large amounts of
antibodies.

Lamina propria
Connective tissue that
underlies the epithelium of the
mucosa and contains stromal
and haematopoietic cells.

cell-derived products, such as trefoil factor 3 (TFF3) and


resistin-like molecule (RELM), further contribute to
the regulation of a physical barrier in the intestine. TFF3
provides structural integrity to mucus through mucin
crosslinking and acts as a signal that promotes epithelial repair, migration of IECs and resistance to apoptosis22,23. RELM functions to promote MUC2 secretion,
regulate macrophage and adaptive Tcell responses
during inflammation and, in the setting of nematode
infection, directly inhibit parasite chemotaxis24,25.
Intestinal barrier function is further reinforced by
the secretion of AMPs by IECs. Enterocytes are capable of producing some AMPs, such as the Ctype lectin regenerating islet-derived protein III (REGIII),
throughout the small intestine and colon. By contrast,
Paneth cells are uniquely adapted for the secretion of
many additional AMPs, including defensins (cryptdins
in mice), cathelicidins and lysozyme, in the crypts of
the small intestine18,26. These AMPs disrupt highly conserved and essential features of bacterial biology, such as
surface membranes, which are targeted by pore-forming
defensins and cathelicidins, and Gram-positive cell wall
peptidoglycans, which are targeted by Ctype lectins18,27.
This strategy enables the broad regulation of both commensal and pathogenic bacteria and limits resistance
of bacteria to antimicrobial responses. Regional variation in AMP production exists along the longitudinal
axis of the intestinal tract 28. Although further analysis
is required, this distribution may reflect anatomically
restricted hostcommensal bacteria interactions that
drive the differential regulation of IEC responses or
serve to shape heterogeneity in the composition and
localization of microbial communities.

Paneth cell- and enterocyte-derived REGIII has


recently been described as a mediator of hostmicrobial
segregation in the gut 29. Similar to the function and
regulation of MUC2 in the colon, REGIII acts to
exclude bacteria from the epithelial surface of the
small intestine, and its production is dependent on
IEC-intrinsic recognition of commensal microbial signals29. Interactions between AMPs, including REGIII,
and mucins lead to concentrated antimicrobial activity
at the epithelial surface30. Thus, the combined functions of secretory IECs seem to limit the quantity and
diversity of live bacteria that can reach the epithelial surface or interact with the underlying mucosal
immunesystem.
The importance of maintaining the health of secretory 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 3137. 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 susceptible 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 environmental interactions that contribute to disease pathogenesis36. 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 engagement of these pathways by Paneth cells is required for
maintaining intestinal homeostasis in mice, and their
combined absence leads to the development of a spontaneous disease resembling human Crohns 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 important link between the disruption of Paneth cell function
and the potential origins of intestinal inflammation.
Finally, IECs directly transport secretory immunoglobulins 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 Bcells and IECs provides an adaptive
immune component to the epithelial barrier that regulates commensal bacterial populations to maintain
IEC and immune cell homeostasis4143. Future studies
to better understand how mucus, AMP and secretory
immunoglobulin dynamics can be regulated to support barrier function will enable the development of
therapeutic interventions for preserving intestinal
homeostasis.

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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 moleculeA, 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.

Peyers patches
Groups of lymphoid aggregates
located in the submucosa of
the small intestine that contain
many immune cells, including
Bcells, Tcells and dendritic
cells. They have a luminal
barrier consisting of specialized
epithelial cells, called microfold
cells, which sample the lumen
and transport antigens.

Pattern-recognition
receptors
(PRRs). Receptors that
recognize structures shared
by foreign microorganisms
or endogenous molecules
associated with pathogenesis.
Signalling through these
receptors promotes
tissue-specific innate immune
responses including the
production of cytokines.

Toll-like receptor
(TLR). An evolutionarily
conserved pattern-recognition
receptor located at the cell
surface or at intracellular
membranes. The natural
ligands of TLRs are conserved
molecular structures found in
bacteria, viruses and fungi.

Sampling of luminal contents by IECs. Despite the barrier 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 (Mcells), mediate the sampling
of luminal antigens and intact microorganisms for presentation to the underlying mucosal immune system44.
These specialized IECs are concentrated in the follicleassociated epithelium overlaying the luminal surface of
intestinal lymphoid structures, including Peyers patches
and isolated lymphoid follicles44,45.
Although nonspecific uptake and transcytosis of antigens represents a well-established mechanism of sampling
by Mcells, it has recently been demonstrated that more
efficient mechanisms of receptor-mediated transport also
exist. The surface glycoprotein GP2 acts as a receptor for
the bacterial pilus protein FimH, and the Mcell-mediated
transport of the pathogen Salmonella enterica across the
epithelial barrier depends on GP2FimH interaction46.
This suggests that Mcells are capable of both specific
receptor-mediated microbial uptake and nonspecific
antigen uptake from the intestinal lumen. Although the
active transport of luminal contents across the epithelial
barrier was thought to be a unique function of Mcells, it
was recently shown that small-intestinal goblet cells also
contribute to this process through the delivery of soluble
luminal antigens to subepithelial dendritic cells (DCs)47.
Although both Mcells and goblet cells seem to be capable
of antigen delivery to the lamina propria, the functional
importance and contribution of these two pathways to
the development of anti-pathogen responses or to the
maintenance of immune tolerance remains incompletely
understood.

In addition to luminal antigen sampling by IECs,


subepithelial mononuclear phagocytes, through interactions 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, transport through these pathways may alter bacteria and
antigens to enable controlled transport of antigens to
be differentiated from dysregulated bacterial translocation50. 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 environment and as active participants in the directing of
mucosal immune cell responses (see Supplementary
information S1 (table)). Members of the Toll-like receptor (TLR)51, NOD-like receptor (NLR)52,53 and RIGIlike
receptor (RLR) families54,55 provide distinct pathways for
the recognition of microbial ligands or endogenous signals 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 damage 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
MYD88deficient and broad-spectrum antibiotic-treated
mice, that commensal bacteria-derived signals contribute 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

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a

Commensal bacteria

Mucin

AMPs

TFF3

TLR9
(apical)

ROS
Tight
junction

TLR3,
TLR7,
TLR8

Endosome

MYD88
Ub
Ub
Ub
I B
NLRP3,
NLRP6,
NLRC4

Heat-shock
proteins
IB
p50 p65

NF-B

NF-B

p50 p65

p50 p65

NOD1,
NOD2

Inammasome

RIP2
FRMPD2

IL-1
and IL-18

(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.

RIGIlike receptor
(RLR). A pattern-recognition
receptor located in the cytosol
that responds to viral RNA.

IKK
IKK IKK

NF-B

Pro-caspase 1

NOD-like receptor

TRIF

TLR2, TLR4,
TLR5 or TLR9

APRIL, BAFF, IL-25,


retinoic acid, TGF
and TSLP

EGFR
EGFR ligands

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
Nature
| Immunology
conserved microbial-associated molecular motifs and pathogen-specific virulence properties.
TLRsReviews
recruit the
signalling
adaptors MYD88 and TIR-domain-containing adaptor protein inducing interferon (TRIF) on ligation to signal molecules
via nuclear factorB (NFB), 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 NFB and MAPKs, whereas other IEC-expressed NLRs, including NOD-, LRR- and pyrin
domain-containing3 (NLRP3), NLRP6 and NOD-, LRR- and CARD-containing4 (NLRC4), form inflammasome complexes
with pro-caspase1 for the cleavage and activation of interleukin1 (IL1) and IL18. 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-B signalling, whereas TLR signalling from the basolateral pole promotes NF-B 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 (TGF) and thymic
stromal lymphopoietin (TSLP)), FRMPD2, FERM and PDZ domain-containing 2; IB, inhibitor of NFB; IKK, IB kinase;
ROS, reactive oxygen species; Ub, ubiquitin.

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.

enhanced integrity of apical tight-junction complexes59


(FIG.2). Furthermore, IEC-specific deletion of elements
necessary for the activation of the transcription factor
complex nuclear factor-B (NFB) downstream of TLR
signalling in mice, including the inhibitor of NFB

(IB) kinase (IKK) complex or NFB essential modulator (NEMO), results in enhanced DSS-induced or spontaneous colitis60,61. These studies establish an essential
role for TLRs, in addition to other NFB signalling
pathways, in epithelial homeostasis andrepair.

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Nuclear factor-B
(NFB). A family of
transcription factors important
for pro-inflammatory and
anti-apoptotic responses that
are activated by the ubiquitindependent degradation of
their respective inhibitors,
members of the inhibitor of
NFB (IB) family. This process
is mediated by the kinases, IB
kinase1 (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
interleukin1 (IL1) and
IL18.

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.

As additional families of TLRs, such as the NLRs and


RLRs, have been ascribed roles in regulating inflammatory immune cell responses, they have also been shown
to be important in IECs for the regulation of intestinal
homeostasis5255. The identificationof nucleotide-binding oligomerization domain 2 (NOD2), an NLR family
member that recognizes bacterial muramyl dipeptide
(MDP), as the first genetic susceptibility locus for
Crohns 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 caspase1 and NLRs, including IEC-expressed NOD-, LRR- and pyrin domaincontaining3 (NLRP3), NLRP6, NLRP12 and NOD-,
LRR- and CARD-containing4 (NLRC4), have a complex
influence over inflammation and epithelial repair, as demonstrated by both pathological and protective roles in constitutive knockout mouse models52,53,64 (see Supplementary
informationS1 (table)). As NLRs are expressed by several cell populations in the intestine, conditional knockout 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 redoxsensitive 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
responses6769.
The protective effects of microbial recognition by
IECs may come at a cost. Although commensal microbial 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 NFB signalling have all
been implicated in promoting tumour development and
progression in multiple genetic7073 and inflammationinduced60,72,74,75 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, NFB
can enhance WNT signalling in terminally differentiated
IECs to promote their dedifferentiation into stem celllike
tumour initiators73.
Paradoxically, some NLR signalling pathways protect
against tumorigenesis, partly through the regulation of
cell death and proliferation in damaged or transformed
IECs7680 and through the regulation of tissue repair
responses mediated by interleukin18 (IL18) signalling 53,81,82. The complexity of the multiple roles of microbial recognition by IECs serves to further highlight the

delicate nature of the balance that exists between homeostasis and inflammation and its importance in maintaining healthy hostmicroorganism symbiosis.
Specialized regulation of PRR pathways in IECs. The
proximity of IECs to an abundance of luminal microbial
signals necessitates specialized mechanisms for maintaining 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
informationS1 (table)). The disruption of these regulatory pathways or constitutive activation of NFB predispose 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 NFB, broadly tolerizing IECs to microbial stimulation through PRR signalling 88,89. Although additional
mechanisms exist for the negative regulation of PRR signalling 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 pathogenic microorganisms for the scaling of an appropriate
inflammatory response91. The polarized nature of the
intestinal epithelium allows for the anatomical segregation of PRRs (FIG.2). Invitro and invivo models demonstrate differential responsiveness of IECs to apical versus
basolateral stimulation with multiple TLR ligands9294.
For example, although basolateral exposure of IECs to
TLR9 ligands results in canonical activation and nuclear
translocation of NFB, apical exposure results in a
net inhibitory effect through the stabilization of IB94.
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 regulation of additional PRR pathways95,96. Through a series
of elegant genetic screens, FERM and PDZ domaincontaining2 (FRMPD2) which is a positive regulator of NOD2mediated NFB 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 Crohns diseaseassociated variants of NOD2 contain mutations in this
LRR domain. These NOD2-mutant proteins were shown
invitro 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.

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Finally, mechanisms by which IECs may break their
relative tolerance to microbial signals in settings of pathogen infection are poorly defined. In contrast to sterile
sites in the body, control of inflammation in the intestine 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 mediated 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 bacterial secretion systems and toxins that penetrate into the
cellular cytosol91,98. Although these mechanisms for scaling 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 secretory IgA contribute to epithelial barrier function. Reduced
mucous layer thickness in germ-free mice can be reversed
by treatment with TLR ligands, indicating that commensal bacteria-dependent signals regulate mucus production by goblet cells17. Similarly, the expression of many
epithelial cell-derived AMPs is enhanced by, or dependent
on, the presence of commensal microbial signals18,29,99101.
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 (REFS100,101).
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 NFBdependent signalling in response
to commensal microbial signals40,102. Finally, the integrity of tight junctions and transepithelial permeability
are regulated by commensal microbial signals, including
TLR2dependent redistribution of the tight-junction proteins to apical cellcell contacts59. Thus, the ability of IECs
to sense their microbial surroundings has an integral role
in regulating their barrier function.
Viability-associated PAMPs
(Vita-PAMPs). Members of a
special class of pathogenassociated 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.

Regulation of immune cells by IECs


IECs produce numerous immunoregulatory signals that
are necessary for tolerizing immune cells, limiting steadystate 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 signals by IECs to mucosal immune cells. The production of the cytokines thymic stromal lymphopoietin
(TSLP)103105, transforming growth factor (TGF)104,106
and IL25 (REF.107) and the Bcell-stimulating factors

a proliferation-inducing ligand (APRIL; also known


as TNFSF13) and Bcell-activating factor (BAFF; also
known as TNFSF13B)108,109 by IECs is promoted by commensal bacteria via PRR signalling (FIG.2). We discuss
below the immunoregulatory functions of IECs, describing 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 phagocytes (FIG.3). IEC-derived TSLP, TGF and retinoic acid,
produced in response to commensal bacteria-derived signals, promote the development of DCs and macrophages
with tolerogenic properties, including the production
of IL10 and retinoic acid103,104,110. Considerable hetero
geneity exists among intestinal mononuclear phagocytes,
the classification of which has been previously complicated by conflicting nomenclature, as well as phenotypical
and functional plasticity in settings of inflammation111.
However, two distinct populations that have been
characterized are the preDCderived CD11c+CD103+
DCs and monocyte-derived CD11clowF4/80+CX3CR1hi
intestineresident macrophages112115.
CD103+ DCs act as migratory antigen-presenting cells
and upon activation traffic to secondary lymphoid tissues, including the mesenteric lymph nodes and Peyers
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 boxP3
(FOXP3+) regulatory Tcells by a TGF- and retinoic
aciddependent mechanism116,118,119. Furthermore, the
production of retinoic acid by IEC-conditioned CD103+
DCs is responsible for the imprinting of gut-homing
properties on Tcells, allowing for the targeting of recirculating mature cells to the original site of antigen encounter in the intestinal lamina propria120124. Thus, in addition
to promoting naive Tcell 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 intestineresident macrophages lack migratory properties in the
steady state and instead persist in close physical contact
with IECs, where they act as avid phagocytes to mediate clearance of pathogens and commensal bacteria
that traverse the epithelial barrier 116,125. Their expression 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 implicated in the maintenance of mucosal tolerance, as they
have been shown to promote the survival and local

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R E V IE W S
Innate immune regulation

IL-25,
IL-33,
TSLP
IL-13,
amphiregulin

Adaptive immune regulation


SEMA7A
TLA

Commensal
bacterium

IL-25

IFN,
TNF

IL-1,
IL-23

IL-17,
IL-22

TSLP,
TGF,
RA

IEL

sIgA

TSLP
APRIL,
BAFF

IL-7,
IL-15

IL-12
IL-10

ILC2

IL-25

IgA+ plasma cell


ILC3

ILC1

TSLP

TReg cell

DC

Lamina propria

TCR
MHC
Macrophage Monocyte
Basophil
progenitor

Peyers patch
or mesenteric
lymph node

Naive T cell

Type 2
MPP
Mast cell

Basophil

RA,
TGF

B cell

Basophil

IL-10,
RA,
TGF

TReg cell
Direct IEC eect
Indirect IEC eect
Immune response
Dierentiation

Figure 3 | IECs regulate innate and adaptive immunity. Intestinal epithelial cell (IEC)-derived cytokines interleukin25
Nature progenitors
Reviews | Immunology
(IL25) and thymic stromal lymphopoietin (TSLP) elicit the expansion and differentiation of basophil
and
multipotent progenitor type2 (type2MPP) cells, respectively. IL25, IL33 and TSLP stimulate group 2 innate lymphoid
cells (ILC2s), whereas IL25 suppresses innate lymphoid cell subset 1 (ILC1) and ILC3 function by limiting macrophage
production of pro-inflammatory cytokines IL1, IL12 and IL23. IECs condition dendritic cells (DCs) and macrophages
towards a tolerogenic phenotype through the production of TSLP, transforming growth factor (TGF) and retinoic acid
(RA). TheseDCs promote the differentiation of naive CD4+ Tcells into regulatory T (TReg) cells and the maturation of Bcells
into IgA-secreting plasma cells. Mucosal cell-derived DCs also imprint a gut-homing phenotype on primed Bcells and
Tcells through the production of RA. After trafficking to the intestine, TReg cells are expanded in number by macrophages
that are conditioned to produce IL10 by TSLP-mediated stimulation and through contact-dependent interactions with
IEC-expressed semaphorin 7A (SEMA7A). The production of a proliferation-inducing ligand (APRIL) and Bcell-activating
factor (BAFF) by IECs and by TSLP-stimulated macrophages and DCs promotes class-switch recombination and
the production of IgA by Bcells in the intestinal lamina propria. IEL, intra-epithelial lymphocyte; IFN, interferon;
sIgA, secretory IgA; TCR, Tcell 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 Tcells 126.


CX3CR1hi macrophages promote tolerance in the intestinal lamina propria through the production of IL10,
which leads to suppression of inflammatory cytokine
production by colitogenic Tcells and promotion of
regulatory Tcell function127,128. IECs maintain this
tolerogenic function through their production of soluble factors, such as TSLP, TGF and retinoic acid103,104,110,
as well as through contact-dependent interactions
involving IEC expression of the integrin ligand semaphorin 7A, which induces IL10 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 infection. In this setting, the IEC-derived cytokines TSLP
and IL25 promote the expansion and differentiation of
haematopoietic progenitor cells towards mononuclear

and myeloid cell phenotypes that promote the development of type2 cytokine responses at mucosal sites130133.
These cells include a distinct population of basophil progenitors and a population of multipotent progenitor cells,
which undergo extramedullary haematopoiesis and represent 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 homeostasis. 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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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.

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
elsewhere134,138140. ILCs are characterized by their developmental requirements and differential cytokine expression 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 cellassociated cytokines interferon- (IFN) and
tumour necrosis factor (TNF) in response to IL12
and/or IL15 (REF.140). Although NK cells can directly
kill target cells through cytotoxic activity, other ILC1s are
limited to cytokine production in response to stimulation. 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 cellassociated cytokines IL5 and IL13 (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 secretion143145. Furthermore, ILC2s present in the lung promote airway hyperresponsiveness or
tissue repair in mouse models of allergy and influenza
virus infection146149. 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 IL25, IL33 and TSLP143145,150.
The contribution of microbial stimulation to these signals 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 cellassociated cytokines, including IL17A and IL22, in
response to stimulation by IL23 (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.
IL22 has an important role in protecting the intestinal
epithelium following injury or infection by bacterial
pathogens152,153. In addition, ILC3derived IL22 supports the anatomical containment of gut-associated
lymphoid tissue-resident commensal bacteria and the
protection of IESCs in models of graft-versus-host disease154156. These tissue-protective functions of IL22 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 IL22 function. By contrast, ILC3derived IL17 is thought to have a primarily
pro-inflammatory effect in the intestine and has been
implicated in both mouse colitis and human IBD158160.

IECs play an indirect part in the regulation of ILC3s


in response to commensal bacteria-derived signals. For
example, IEC-derived IL25 leads to the suppression of
IL23 production by macrophages and decreased IL22
production by ILC3s161. By contrast, commensal bacteriadependent signals have also been shown to stimulate the
production of IL7 by IECs162, which supports the production of IL22 by ILC3s through the stabilization of the
transcription factor retinoid-related orphan receptor-t
(RORt; encoded by RORC)162,163. These seemingly conflicting 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 contributions to human disease and develop means of clinically
targeting their protective or detrimental functions.
Tissue-resident Tcells. Following priming by intestinederived antigen-presenting cells in secondary lymphoid
tissues, conventional effector Tcells 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 Tcells are subject to
the direct influence of IECs for their functional maintenance 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 164166. IELs display an
activated phenotype and include conventional Tcells, as
well as subsets of cells expressing a restricted repertoire
of Tcell receptor specificities and specialized properties,
including Tcells and NKTcells165,167. 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+ Tcells can undergo transcriptional
reprogramming when they become IELs to develop a
distinct phenotype resembling that of CD8+ cytotoxic
Tcells168. Although the influence of the local environment 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 Tcells 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 challenge. This is thought to be particularly important
in CD8+Tcell-dependent memory responses169. As such,
CD8+ Tcells with a tissue-resident memory phenotype
are uniquely enriched among Tcells present in the
intestinal IEL compartment of mice and humans169,170.

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Class-switch recombination
(CSR). The process by which
proliferating Bcells 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 E7 integrin), 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 contribute 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+ Tcell populations expressing homodimers of the
co-receptor subunit CD8 (known as CD8+ IELs),
which interact with the IEC-expressed MHC classIlike
molecule, thymus leukaemia antigen (TLA) 173 .
Understanding how such memory cell populations are
maintained is of particular interest in the design of efficient 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 preventing infections with pathogens such as HIV and enteric
viruses176.
IgA-secreting plasma cells. The maturation of naive
Bcells 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 Tcell 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), IL10 and retinoic
acid, in conjunction with TGF signalling 117,124,178 (FIG.3).

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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
Crohns and Colitis Foundation of America (D.A.).

Competing interests statement

The authors declare no competing interests.

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