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The unusual suspects - Innate lymphoid cells as novel therapeutic targets in

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Article  in  Nature Reviews Gastroenterology &#38 Hepatology · May 2015

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 REVIEWS
The unusual suspects—innate lymphoid cells
as novel therapeutic targets in IBD
Rimma Goldberg, Natalie Prescott, Graham M. Lord, Thomas T. MacDonald and Nick Powell
Abstract | Innate lymphoid cells (ILCs) are a family of immune cells that selectively accumulate in mucosal
tissues serving as sentinels at the vanguard of host protective immunity. However, they are also implicated
as cellular mediators of immune-mediated diseases, most notably IBD. ILCs are subdivided into distinct
lineages based on the expression of effector cytokines and master transcription factors that programme their
differentiation and inflammatory behaviour. Strikingly, these subsets closely resemble CD4 + T‑cell lineages,
including T helper (TH)1, TH2 and TH17 cells that are similarly implicated in immune-mediated diseases.
However, ILCs that promote the maintenance of intestinal epithelial cells, mostly through production of IL‑22,
also exist. ILCs rapidly respond to environmental cues, including cytokines, metabolic signals and luminal
bacteria. They are potent and immediate producers of key cytokines linked to IBD pathogenesis, including
TNF, IL‑17, IL‑22 and IFN‑γ. Some subsets are implicated as mediators of chronic intestinal inflammation,
whereas others might provide protective functions. They are present in the gut of patients with IBD and,
intriguingly, closer scrutiny of IBD susceptibility loci shows that many of these genes are either expressed
by, or are intimately linked to, ILC function. Looking forward, targeting ILCs could represent a new IBD
treatment paradigm.
Goldberg, R. et al. Nat. Rev. Gastroenterol. Hepatol. 12, 271–283 (2015); published online 5 May 2015; doi:10.1038/nrgastro.2015.52

Introduction
IBD, chiefly comprising either Crohn’s disease or ulcera-
tive colitis, is a complex chronic immune-mediated disease
of unknown cause. Characteristically, mucosal tissues
become densely infiltrated with different immune cells,
which cooperate through complex networks of crosstalk
with stromal cells, epithelial cells, endothelial cells and the
contents of the gastrointestinal lumen, including bacte-
ria and food antigens. The roles and relative importance
of particular cellular components remains difficult to
define. As major producers of cytokines relevant to dis-
eases such as IBD, CD4+ T cells have dominated research
for some years. CD4+ T cells can be divided into distinct
lineages based on cytokines and master transcription
Department of
Experimental
factors expressed. Type 1 T helper (TH1) cells (express-
Immunobiology, ing IFN‑γ and the transcription factor T‑bet) and TH17
Division of cells (expressing IL‑17A, IL‑22 and the transcription factor
Transplantation
Immunology and RORγt) have been found to accumulate in inflammatory
Mucosal Biology (R.G., lesions of patients with Crohn’s disease and ulcerative
G.M.L., N.Powell),
Department of
colitis.1–4 TH2 cells (expressing IL‑5, IL‑13 and the tran-
Medical and Molecular scription factor GATA‑3) have been suggested to have a
Genetics (N.Prescott), role in ulcerative colitis by some groups,4,5 but this role
King’s College London,
London SE1 9RT, UK. has not been corroborated by others.6 Innate immune
Centre for Immunology cells also produce cytokines conventionally associated
and Infectious Disease,
Blizard Institute,
with effector T‑cell responses. Building on observations
Barts and the London of potent IL‑17A and IL‑22 responses in mice lacking
School of Medicine T cells and B cells,7 a landmark study by Cella et al.8 con-
and Dentistry, London
E1 2AT, UK (T.T.M.). firmed the presence of IL‑23-responsive innate mucosal
Correspondence to:
N.Powell Competing interests
nick.powell@kcl.ac.uk The authors declare no competing interests.
immune cells, which produce IL‑22, in human mucosa-
associated lymphoid tissue including Peyer’s patches.
Several mucosal-dwellin­g innate lymphocytes have since
been identified that produce IBD-relevant cytokines and
this novel family is termed innate lymphoid cells (ILCs).
A new population of effector lymphocytes
ILCs are effector cells defined by three main features:
the absence of recombined antigen-specific receptors,
such as those found on T cells and B cells; an absence
of phenotypic markers associated with ‘classic’ immune
cell lineages (with the exception of some natural killer
[NK] cell markers); and their lymphoid morphology.9
In mice, ILCs differentiate from a common precursor
in a process dependent on the transcriptional modula-
tor, DNA-binding protein inhibitor ID-2 (Id2).10,11 ILCs
were previously known by a variety of names, including
‘natural helper cells’ and ‘innate helper cells’. In an attempt
to standardize their nomenclature and acknowledge their
phenotypic and functional diversity, the ILC family is cur-
rently subdivided into ILC1, ILC2 and ILC3 subsets. These
subsets are based on the expression of lineage-defining
transcription factors, cytokine production profiles and
functional attributes.12 ILC subsets bear striking resem-
blance to recognized effector CD4+ T‑cell lineages, with
which they share phenotypic, morphological and func-
tional characteristics (Figure 1). ILC1 are defined by the
expression of the transcription factor T‑bet, which was
first described as the master transcriptional regulator of
TH1 cells13 and the production of canonical TH1 cytokines

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Key points cells, ILC3 participate in host resistance to extracellular

bacterial and fungal infections.15–18 ILC3 can be further
■■ Innate lymphoid cells (ILCs) are a novel population of innate lymphocytes that
subdivided according to whether they express natural
are selectively enriched at mucosal sites
■■ The ILC family comprises phenotypically and functionally distinct subsets that
cytotoxicity receptors (NCRs), such as those commonly
have been implicated in both maintenance and loss of mucosal homeostasis expressed by conventional NK cells. NCR+ ILC3 produce
■■ ILCs are early producers of pathogenic cytokines in the intestine, such as IL‑22 and NCR– ILC3 produce IL‑17 and IL‑22. RORγt-
interferon-γ and IL17, and have been implicated as important effector cells in dependent lymphoid tissue inducer (LTi) cells—which
preclinical models of inflammatory bowel disease (IBD) have a key role in the genesis of lymph nodes and mucosa-
■■ Other ILC subsets produce cytokines involved in promoting intestinal epithelial associated lymphoid tissue in the developing fetus through
homeostasis, such as IL-22, and might have protective roles in the gut expression of lymphotoxin—might also be considered
■■ The number and composition of ILC subsets accumulating in the intestine of
NCR– ILC3 cells. LTi cells express IL‑17 and IL‑22 and a
patients with IBD is dysregulated
■■ Selective therapeutic targeting of ILCs might represent a novel treatment proportion are CD4+.7
paradigm in IBD Conventional NK cells might be considered the
prototypical ILC and fit the paradigm of ILCs mirror-
ing T‑cell subsets. NK cells can be defined as cytotoxic
T cell ILC ILC1, akin to the relationship of CD8+ T cells to effec-
TH1 (LP) ILC1 (IE) ILC1 tor CD4+ lineages. However, important differences exist
* that distinguish conventional NK cells from more latterly
IFN-γ T-bet discovered ILCs lineages. First, NK cells develop from
T-bet T-bet
TNF Nfil3 different precursors via distinct developmental path-
ways to ILCs,10,11 which have been extensively reviewed
elsewhere.19 NK cells are also more commonly found in
TH17 NCR+ ILC3 NCR– ILC3 peripheral blood and solid organs, such as the spleen
and liver, and are fairly uncommon at mucosal sites. In
addition, NK cells are recognized for their cytolytic func-
IL-22
RORγt
IL-17
RORγt RORγt tions and are involved in host defence against viruses,
neoplastic change and cell senescence,20 which are not as
yet major roles assigned to mucosal ILCs. In this Review,
conventional NK cells will not be discussed.
TH2 ILC2

Intestinal ILCs
IL-5 ILC1, ILC2 and ILC3 cells have all been described in the
GATA3 IL-13 GATA3
mammalian gut under homeostatic conditions and roles
for these subsets have been described in host resistance to
Cytotoxic T cell NK cell intestinal pathogens. The first major population of ILCs
that were comprehensively described in human mucosal
IFN-γ
tissue was IL‑22 producing NCR+ (CD56+NKp44+) cells,8
T-bet Perforin T-bet
eomes Granzyme eomes which in later years was classified as NCR+ ILC3.12 These
Cytotxicity observations highlighted a new population of innate
lymphocytes present in mucosal barriers with distinct
functions from conventional NK cells and heralded a
new era in innate lymphocyte biology research.
CD4 CD94 CD127 IL-1R IL-15R IL-23R IL-33R
In the steady state, NCR+ cells comprise 5% of lym-
CD8 CD103 CCR6 IL-12R IL-18R IL-25R NCR phocytes in the human colon and small intestine.21,22 In
mice, fewer intestinal NCR+ ILCs exist; they make up 1%
Figure 1 | Human ILC subsets closely resemble
Nature T‑cell| Gastroenterology
Reviews lineages. Within the
& NCR –
ILC
Hepatology of immune cells in the colon, 2% in the small intestine
subset there are some CD4+ cells found in mice that are lymphoid tissue inducer and <0.5% of intraepithelial lymphocytes.15,23,24 NCR+
cells. *NCR (CD56, NKp44 and NKp46) expression is heterogeneous in ILC1. cells in the gut are heterogeneous and populations lacking
Abbreviations: GATA3, GATA binding protein 3; IE, intra-epithelial; LP, lamina propria;
NK function outnumber cells resembling conventional NK
NCR, natural cytotoxicity receptor; Nfil3, nuclear factor, interleukin 3 regulated; RORγt,
retinoic acid receptor related orphan receptor γt; T‑bet, T‑box expressed in T cells. cells.11,15,23,25 These novel mucosal NCR+ ILCs mostly lack
signature NK markers, such as perforin and granzyme B,
or molecules required to trigger apoptosis and kill target
such as IFN‑γ and TNF. As with their TH1 counterpart, cells, such as FasL. Unlike NK cells, they express inter­
ILC1 contribute to host resistance to intracellular infec- leukin 7 receptor (IL-7R, also known as CD127), produce
tion.11 ILC2 (previously known as nuocytes) are depend- different patterns of cytokines,8,15,23,26–28 have divergent
ent on the TH2 transcription factor GATA‑3 and produce transcriptional modules16 and originate from distinct
the TH2 cytokines IL‑5 and IL‑13; similar to CD4+ TH2 precursors and developmental pathways.11,15,23,25
cells they contribute to helminth immunity.14 ILC3 In mice, CD127+NCR+ ILCs are further subdivided into
express the transcription factor nuclear receptor RORγt three subsets, including: T‑bet-dependent ILC1, which
and produce IL‑17A and/or IL‑22. As with CD4+ TH17 produce IFN‑γ; RORγt-dependent NCR+ ILC3, which

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Table 1 | Role of intestinal ILCs in health and disease
Cell type Function in the gut
ILC1 Resistance to bacterial infection 11
ILC2 Eosinophil recruitment,37 immune response
to helminths14,38
ILC3 NCR+ Epithelial integrity,8,15,16 resistance to
mucosal infections15,16,19
ILC3 NCR– Currently unknown, possible role in
antifungal immunity17
Abbreviations: ILC, innate lymphoid cell; NCR, natural cytotoxicity receptor.
produce IL‑22; and a third population of ILC3, which
have previously expressed RORγt (as shown in elegant
fate mapping studies25), but have subsequently down-
regulated RORγt and differentiated into T‑bet-positive,
IFN‑γ-producing cells, probably under the influence of
IL‑12.11,15,23 An additional population of IFN‑γ-producing
NCR+ ILCs occupies the intraepithelial compartment.27
Phenotypically, intraepithelial ILC1 have overlapping fea-
tures with both conventional NK cells and lamina propria
ILC1. They are rare in health, but might have a role in
inflammation (as will be discussed later). They are T‑bet
dependent, do not express CD127, yet unlike conventional
NK cells their development is IL‑15 independent. They
also express signature NK molecules, such as perforin,
CD94 and CD160.27 The complexities of ILC heterogeneity
and lineage identity within and beyond the gut have been
reviewed elsewhere.29 Although most work on mucosal
ILCs in man has focussed on the tonsil, a broadly similar
picture is emerging in the human intestine. Early work
identified distinct populations of innate lymphocytes in
the gut that could be separated by differential expression
of NCRs and cytokine production profiles.22 A popula-
tion of CD3–CD56+IL-7R+ cells resembling murine NCR+
ILC3 were present, which expressed NKp44 (an NCR that
is not expressed by mice). NKp44+ innate lymphocytes
were enriched for IL-22 and RORC mRNA and lacked
IFN-γ mRNA.22 Although these initial studies would
have included mucosal NK cells (as innate lymphocytes
were defined as CD3–CD56+ cells), and subsequent studies
have used different ILC definitions and studied different
intestinal compartments (lamina propria versus intraepi-
thelial compartment), there is broad consensus that four
main ILC subsets exist in the human gut. These include
NKp44+CD103+ cells that are enriched in the intra­
epithelial compartment of the ileum and colon,27 lamina
propria NKp44+RORγt+ ILC3 that express IL-22 mRNA,26
lamina propria NKp44–c-kit– ILC1 that are enriched for
IFN-γ mRNA26 and NKp44–c-kit+RORγt+NCR– ILC3 that
have enriched expression of IL-17A mRNA.26
The role of NCR– ILC3 in the healthy gut is less clear.
They are defined by expression of RORγt and unlike
NCR+ ILC3 produce IL-17A sometimes in combination
with IL-22.30–32 A proportion of NCR– ILC3 also coexpress
T-bet, which licenses them to produce the canonical TH1
cytokine IFN-γ. NCR– ILC3 from mice genetically defi-
cient for T-bet are poor producers of IFN-γ, just like their
T-cell counterparts.30,31,33
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© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
Possible role in IBD
Increased in patients with Crohn’s disease,22,26,27 functionally
implicated in preclinical models of colitis25,27
Possible role in fibrosis in patients with Crohn’s disease53
Reduced numbers in patients with Crohn’s disease,22,26
possible role in protection from experimental colitis79,82
Increased innate IL-17 production patients with IBD,28
colitogenic in preclinical models30–32
In the germ-free intestine of embryos, ILC3 do not
express NCRs and instead they nearly all express CD4,
consistent with these ILCs having a similar phenotype to
LTi cells. However, with increasing age CD4+ ILC3 pro-
gressively diminish and are replaced by CD4– ILC3, includ-
ing both NCR– and NCR+ ILC3 populations.34 Although
the role of NCR– ILC3 is poorly understood, their capac-
ity to generate highly inflammatory cytokine profiles is
consistent with a protective function against intestinal
pathogens and, as discussed later, they might also be inap-
propriately mobilized during chronic intestinal inflam-
mation. Although some investigators have suggested a
dominant role for γδ T cells and natural TH17 cells in host
resistance to fungal infection,35 a role for IL‑17-producing
ILCs (a cytokine typically linked with NCR– ILC3) has
also been suggested.17 Consistent with an important role
for IL‑17 in antifungal immunity, increased oropharyn-
geal candidiasis was an observed complication in patients
with Crohn’s disease treated with IL‑17-neutralizing
­monoclonal antibodies.36
In mice, ILC2 account for 5% of small intestinal lym-
phocytes and under homeostatic conditions are respon-
sible for IL‑5-dependent recruitment and maintenance
of mucosal eosinophils.37 The function of ILC2 in the
human gut has yet to be verified, although it is tempting
to speculate that they might contribute to the early phase
of immunity to helminths, as they do in mice.14,38
ILCs and IBD
Intestinal ILCs have an important role in preclinical
models of intestinal inflammation (Table 1), which has
stimulated considerable enthusiasm for the possibility
that these cells could have a pathologically relevant role
in human IBD. Many animal studies have exploited mice
lacking T cells and B cells to avoid distractions or con-
founding contributions from adaptive immunity. To date,
ILC3, ILC1 and, to a lesser extent, ILC2 have all been
implicated in IBD.
Pathogenic ILC3
In a landmark study published in 2010, Powrie and col-
leagues32 were the first group to clearly identify a major role
for ILCs as mediators of chronic intestinal inflammation.
In this study CCR6+RORγt+CD127+NCR– ILC3 lacking
other lineage-specific markers, accumulated in the colon
following the onset of microbiota-dependent experimen-
tal colitis. This innate population of intestinal immune
REVIEWS
cells potently produced IL‑17A and IFN‑γ. Depletion
of intestinal ILCs or antagonism of their key cytokines
cured colitis, demonstrating an indispensable role for
ILCs, at least in this Helicobacter hepaticus model of IBD.32
Subsequent work in the Tbx21–/–Rag2–/– ulcerative colitis
(TRUC) mouse model confirmed and extended these
observations. TRUC mice develop microbiota-dependent
colitis resembling some aspects of human ulcerative colitis.
Histologically, inflammation is mostly superficial and,
although initially confined to the distal colon, is prone to
proximal extension with time and might be complicated
by the emergence of inflammation-dependent colonic epi-
thelial dysplasia and adenocarcinoma.30,39,40 In this innate
immune-mediated model of colitis, IL‑17A and IL‑22 pro-
ducing CCR6+RORγt+ CD127+NCR– ILC3 are expanded in
the diseased colon of TRUC mice.30 Colitis is abrogated
in mice lacking ILCs, or after their depletion with specific
monoclonal antibodies. However, it should be noted that
most depletion strategies have used anti-CD90 monoclonal
antibodies that are not specific for ILCs. In H. hepaticus and
TRUC mouse models of IBD, the ILC3 population pro-
duced IL‑17A and IL‑22 but did not express NCRs or CD4.
Crucially, innate immune cells isolated from inflamed
colon of patients with Crohn’s disease or ulcerative colitis
show increased gene expression of key ILC3 cytokines
(IL17A and IL22), transcription factors (RORC and the
AHR) and cytokine receptors (IL23R).28 These studies
support the view that intestinal NCR– ILC3 might have a
pathogenic role in chronic intestinal inflammation. Further
scrutiny of these cells in human IBD is now needed.
Pathogenic ILC1
In preclinical models of IBD IFN-γ producing intesti-
nal ILC1 have a functionally important role, including
IL-12 and IL-15 responsive T-bet dependent intra­­
epithelial NKp46+ ILC,27 and lamina propria NKp46+
ILC1. 25 Comparable populations of intra-epithelia­l
CD103 +NKp44 + ILC1 and lamina propria ILC1 are
similarly expanded in the ileum of patients with Crohn’s
disease in comparison with non-inflammatory control
patients,26,27 consistent with the possibility that ILC1
might have a pathogenic role in Crohn’s disease.
Protective IL-22 producing ILC3
In addition to expansion of ILC1 there also appears
to be a reciprocal reduction in IL-22 producing NCR+
ILC3 in patients with Crohn’s disease,22,26 which might
have detrimental consequences in the context of chronic
inflammation. NCR+ ILC3 are a major source of IL‑22,
a cytokine that conditions the intestinal epithelium and
promotes barrier integrity.15 The IL‑22 receptor is exclu-
sively expressed by the gut epithelium and IL‑22 ligation
triggers epithelial proliferation, repair and the produc-
tion of protective molecules, including antimicrobial
peptides, mucins, serum amyloid A, β‑defensins, Reg3γ
and lipocalin‑2. 8,42–44 IL‑22 blockade or depletion of
IL‑22-producing ILCs exacerbates intestinal inflamma-
tion resulting from acute Citrobacter rodentium infection
in mice, although it is important to acknowledge that this
model is for acute bacterial infection rather than IBD.15,18
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© 2015 Macmillan Publishers Limited. All rights reserved
Impaired tissue repair function resulting from the loss
of ILC3-producing IL‑22 cells, might be expected to be
detrimental in IBD, as patients without robust mucosal
healing have worse outcomes.45
IBD is associated with well-recognized extraintestinal
manifestations, including sacroiliitis and other arthropa-
thies.46 Similarly, ankylosing spondylitis (a seronegative
spondyloarthropathy) is associated with an intestinal
inflammation resembling Crohn’s disease, often affecting
the terminal ileum.47 As with IBD and other immune-
mediated diseases, chronic intestinal inflammation
associated with ankylosing spondylitis is genetically and
immunologically linked to the IL‑23–IL‑17 axis.48–50
A marked accumulation of IL‑22-producing NKp44+ ILCs
occurs in ileitis associated with ankylosing spondylitis.51,52
Whether these cells have a pathological role in ileitis associ-
ated with ankylosing spondylitis, or whether these cells are
serving to limit more severe disease emerging is unclear.
ILC2
As yet, no convincing data exists regarding a possible role
for intestinal lamina propria ILC2 as mediators of inflam-
mation in IBD. However, in Crohn’s disease, chronic intes-
tinal inflammation might be complicated by fibrostenotic
disease. A role for ILC2 as mediators of intestinal fibrosis
through the production of IL‑13 in the muscularis layer
of the gut has been proposed.53 Innate (CD3–) IL‑13-
producing KIR+ cells are expanded in the muscularis layer
of fibrotic intestinal strictures of patients with Crohn’s
disease undergoing operations for obstruction.53 Fibrotic
areas were characterized by increased deposition of col-
lagen, muscle hyperplasia and increased expression of
IL‑13 (and its receptor, IL‑13Rα2). Laser capture micros-
copy identified KIR+ cells as the chief source of IL‑13 in
the muscle layer and exposure of fibroblasts and muscle
cells to IL‑13 resulted in increased Signal Transducer and
Activator of Transcription (STAT)6 phosphorylation
and reduced activity of antifibrotic metalloproteinases
responsible for degrading fibrillar collagen.53 Further work
is needed to confirm whether these cells are ILC2 (or ILCs
at all) and whether ILC2 have mechanistically relevant
roles in intestinal fibrosis in animal models of disease.
Intestinal ILC activation
As well as providing important insights in to ILC biology,
recognition of key ILC activating pathways offers the
potential to identify therapeutically tractable molecu-
lar targets in IBD. Intestinal ILCs rapidly respond to
environmental cues, including dietary changes and
pathogen exposure.
Cytokines
The best known regulators of ILC behaviour are cytokines.
In the gut, IL‑23 is the canonical cytokine responsible
for triggering IL‑17 and/or IL‑22 secretion by ILC3.
Furthermore, cytokine production induced by IL‑23 is
synergistically enhanced by other cytokines, including
IL‑1α, IL‑1β, IL-2, IL‑6, IL‑7, IL‑15 and TL1A (also known
as TNFSF15).8,31,41,54,59 TNF, a key cytokine implicated
in IBD pathogenesis, also augments IL‑17 production

induced by IL‑23 in ILC3 of mice with colitis; although,
this association might be an indirect effect mediated
through the induction of the IL‑1 family of cytokines.30,31
Cytokine production by ILC1 is stimulated by IL‑12 in
synergy with IL‑15 and IL‑18.26,27 TL1A also potentiates
IL-22 production induced by IL-1 and IL-23,41 mirror-
ing its effects on TH17 cells.55 The complexity of proxi-
mal cytokine inter­actions in driving mucosal ILC effector
function has been demonstrated in experiments con-
ducted in highly purified intestinal ILCs. These studies
indicate that even canonical proximal cytokine signals
such as IL‑23 have little cell-intrinsic capacity to trigger
cytokine production by ILCs unless they are present
together with other combinations of cytokines, including
IL‑1α, IL‑1β and IL‑7.59,31
Cytokines have key roles orchestrating ILC plasticity.
Conversion of one ILC subset into another is an emerging
concept in ILC biology with implications likely to be rele­
vant in chronic inflammatory states. Under homeostatic
conditions the most effective resource-conserving option
would be to favour the accumulation of ILCs responsible
for overseeing basal epithelial maintenance and limiting
the expansion of inflammatory subsets in the absence of
a threat from pathogens. Once exposed to a pathogen,
rapid promotion of the differentiation of resident homeo-
static ILCs towards more inflammatory subsets ready for
combat might afford some survival advantages. In Crohn’s
disease, it seems that control over this dynamic balance
has been lost with ongoing disproportionate skewing
towards inflammatory, IFN‑γ producing NKp44– ILC1
at the expense of IL‑22-producing NKp44+ ILC3.22,26 The
cytokines IL‑12 and IL‑23 probably contribute to the regu-
lation of this process. Human ILC3 cultured with IL‑12
differentiate into ILC1. Although it is possible that ILC1
might also differentiate towards ILC3 under the influence
of IL‑23, these experiments were less convincing and were
limited by difficulties in maintaining ILC1 in the absence
of IL‑12.26
Proximal cytokine triggers of ILC2 activation include
IL‑25, IL‑33, TSLP and TL1A. A clear role for ILC2 or
their proximal stimulating cytokines in IBD has yet to
be defined.
Activating receptors
Evidence of direct stimulation of ILCs through microbial
pattern-recognition receptors, such as Toll-like receptors
(TLRs) is reasonably scarce.56,57 NCR+ ILC3 express TLRs
(especially TLR2), albeit at lower levels than classic TLR-
bearing cells, such as monocytes.56 Exposure of ILC3 to
TLR agonists triggers cellular activation (upregulation of
CD69), increased cytokine production and proliferation,
however, prior exposure to cytokines, such as IL-23 or
IL-1β might be required to optimally sensitise ILCs to
TLR agonist activation.56,57
Cytokine production by ILCs might also be increased
by the engagement of other activating cell-surface recep-
tors, a phenomenon that is well recognized in conven-
tional NK cells.58 Ligation of NKp44 triggers cytokine
production and augments the activation of NCR+ ILC3 by
IL‑23 and IL‑1β.59 Furthermore, NKp44 ligand binding
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REVIEWS
alters the profile of the cytokines secreted. Stimulation
of ILC3 with IL‑23 and IL‑1 leads to production of IL‑22
and granulocyte-macrophage colony-stimulating factor
(GM-CSF). However, engagement of NKp44 resulted
in reduced IL‑22 expression and selective induction of
TNF.59 The ligands for NKp44 are poorly characterized,
but epithelial cellular stress and pathogen exposure trig-
gers NKp44-dependent activation. Studies investigating
other NCRs, including NK1.1 and NKp46 have shown
that unlike splenic NK cells, intestinal NCR+ ILC3 are
not activated to produce cytokines by crosslinking of
these NCRs.60 However, the ligation of unknown acti-
vation receptors probably does occur, because optimal
activation of ILCs by macrophages requires cell contacts
as well as secreted cytokines. These data indicate that
ILC3 cytokine production is context dependent, thus
enabling these cells to sense and differentially respond
to ­important changes in the local microenvironment.
Interaction between ILCs and mucosal cells
Crosstalk between ILCs and other immune and non­
immune cells in the gut coordinates host responses to
environ­mental cues and the outcome of these interactions
has important consequences for the host with respect
to maintenance or indeed loss of intestinal homeostasis
(Figure 2).
ILCs and intestinal mononuclear phagocytes
ILC and mononuclear phagocyte interactions have a
critical role in fine tuning the responsiveness of the
mucosal immune system that influences the balance
between homeostatic maintenance or the emergence of
IBD. Intestinal mononuclear phagocytes are function-
ally diverse and include dendritic cell populations and
tissue-resident macrophages that differentiate from
emigrated inflammatory monocytes and dendritic cell
populations.61–63 Mononuclear phagocytes have a crucial
role in the activation of intestinal ILCs. In the intestine
of patients with Crohn’s disease, CD14+CX3CR1+ mono-
nuclear phagocytes are enriched in diseased mucosa and
are major producers of key cytokines involved in the
activation of ILCs, including IL‑23, IL‑1β, IL‑6, TNF and
TL1A.41,64,65 CD14+CX3CR1+ mononuclear phagocytes are
better than other macrophage or dendritic cell populations
at promoting ILC activation, which is potently augmented
after stimulation with TLR agonists or heat-killed intes-
tinal bacteria—providing one possible explanation for
how ILCs might respond to the intestinal microbiota.22,41
Confocal microscopy demonstrates that CX3CR1+ mono-
nuclear phagocytes form intimate contacts with ILC3 in
the gut.41 Furthermore, optimal stimulation of intestinal
ILC3 by CD14+ mononuclear phagocytes also requires
direct cell contact demonstrating that mononuclear-
phagocyte-mediated activation of ILCs requires signals
beyond secreted soluble cytokines.66
Dialogue between mononuclear phagocytes and ILCs
also affects regulatory T (TREG) cells in the gut, which
have a critical role in limiting or reversing intestinal
inflammation. Intestinal colonization with commensal
bacteria is sensed by intestinal mononuclear phagocytes,
REVIEWS

Homeostasis Inflammation

β-defensins
Antimicrobial peptides
Intraepithelial Epithelial
Epithelial proliferation Fucosylation ILC1 apoptosis or damage

Lamina propria NCR ligand

IL-22R IL-22
TNF
DC
IFN-γ 02–
elastase
NCR
IL-12 IL-12
ILC3 ILC1
IL-23
IL-10 IL-17
MHCII IL-23,
IL-1β, TL1A NCR–
IL-23, IL1-β, TNF, IL-6 ILC3
T cell
CD14+
CX3CR1+ MP Stromal
cell IL-13 Neutrophil
Intestinal fibrosis Collagen KIR
Muscularis

ILC2?

Figure 2 | ILCs interact with other key mucosal cells in IBD. In the steady state, ILC3
Nature help to| Gastroenterology
Reviews maintain homeostasis. ILC3
& Hepatology
secrete IL‑22, which binds to its receptor on gut epithelial cells, thereby promoting the production of IL‑10 and antimicrobial
peptides, increasing basal epithelial cell proliferation and regulating glycosylation patterns (fucosylation) of epithelial cell-
surface molecules. MHC II bearing ILC3 suppress T‑cell activation, as they do not express co-stimulatory molecules. Under
inflammatory conditions, ILC3 differentiate towards IFN‑γ producing ILC1, under the influence of IL‑12. IFN‑γ drives
activation of macrophages and other mononuclear phagocytes. Epithelial stress induces ligands that crosslink NKp44,
switching the profile of cytokines produced by ILC3 from IL‑22 to TNF, which instigates widespread proinflammatory action,
including direct epithelial apoptosis and neutrophil recruitment. NCR– ILC3 produce proinflammatory cytokines such as
IL‑17 (and sometimes IFN‑γ) that activate and recruit neutrophils. CD3–KIR+ cells that possibly represent ILC2 reside in the
muscularis layer in fibrotic and/or strictured areas of intestine in patients with Crohn’s disease. ILC2 produce the
fibrogenic cytokine IL‑13. Abbreviations: DC, dendritic cell; ILC, innate lymphoid cell; KIR, killer-cell immunoglobulin-like
receptor; MHC, major histocompatability complex; MP, mononuclear phagocyte; NCR, natural cytotoxicity receptor.

which respond by producing IL‑1β and in turn stimulates
GM‑CSF production by ILC3—the major producer of
this cytokine in the gut.67 ILC3 derived GM‑CSF is a key
growth and survival factor for intestinal mononuclear
phagocytes and neutrophils. GM‑CSF also drives retinoic
acid and TGF‑β production to augment TREG-cell genera-
tion. Deletion of the gene encoding GM‑CSF is associ-
ated with reduced activity of the retinoic-acid-generating
enzyme aldehyde dehydro­genase and reduced production
of TGF‑β and IL‑10 in r­elevant intestinal mononuclear
phagocyte subsets.
ILC crosstalk with adaptive immune cells
ILC2 and ILC3, but not ILC1, express major histo­
compatibility complex (MHC) class II and interact directly
with CD4+ T cells. 38,57,68 ILC2 express MHC II, the co-
stimulatory molecules CD80 and CD86 and trigger anti-
gen-specific CD4+ T‑cell proliferation and TH2 cytokine
production. Ablation of ILC2 results in diminished CD4+
TH2-cell generation. Within this cognate interaction,
T cells produce IL‑2 to promote ILC growth and ILCs
reciprocally induce antigen-specific T‑cell proliferation
and cytokine production in an MHC-dependent manner.
The outcome of ILC3 interactions with CD4+ T cells
depends on the presence of danger signals and other tissue
specific factors. ILC3 from the intestine of humans and
mice express MHC II and can take up, process and present
antigen in the context of MHC II to CD4+ T cells.57,69 In
the gut, ILC3 lack expression of co-stimulator­y molecules,
including CD80, CD86 and CD40, which suppresses T‑cell
proliferation and cytokine production. Mice with ILC3
intrinsic deletion of MHC II have dysregulated intesti-
nal CD4+ T‑cell responses directed against the commen-
sal microbiota; this dysregulated response culminates in
exaggerated IL‑17 production and spontaneous colitis,68–70
consistent with an immunoregulatory role for ILC3 by lim-
iting microbiota-reactive responses of T cells in the gut.
However, IL‑1β-activated ILC3 in the spleen, upregulate
MHC together with co-stimulatory molecules resulting
in effective CD4+ T-cell priming and potent proliferative
responses. Furthermore, this interaction was bi­directional
with activated T cells providing activating signals to ILCs;57
therefore, it is likely that under some conditions ILCs might
promote T‑cell activation. Other data shows that memory
CD4+ T‑cell persistence and survival in vivo (in the absence
of antigen) is dependent on RORγt+ LTi cells.71 As such,

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© 2015 Macmillan Publishers Limited. All rights reserved

ILC3 could profoundly affect chronic diseases linked to
chronic activation of memory T-cell populations, such as
IBD. Studies into the functional outcomes of T‑cell cross-
talk with different ILC subsets, in the context of chronic
intestinal inflammation in humans, are now needed.
ILC interaction with adaptive immunity is not limited
to T cells. Communication between ILCs and B cells
augments immunoglobulin formation in a T‑cell-
independent manner. ILCs and B cells co-localize at
splenic marginal zones and interact through B‑cell acti-
vating factor (BAFF), CD40L, Notch ligand Delta-like
1 (DLL1) and lymphotoxin-dependent mechanisms to
augment antibody production by B1 cells.72
ILCs interact with the intestinal epithelium
IL‑22-producing ILC3 have a critical role maintaining
epithelial homeostasis and providing functional barrier
protection from luminal microbes. ILCs implicated in IBD
pathogenesis are in intimate contact with and adhere to
epithelial cells. Indeed, some ILC1 subsets express CD103
and occupy the intraepithelial compartment. 27 ILC3
express CCR6, which is the receptor for the chemokine
CCL20 that is produced by and encourages trafficking to
Peyer’s patches and the gastrointestinal epithelium. ILC3
in contact with the epithelium trigger STAT1 and STAT3
phosphorylation within epithelial cells and stimulate their
proliferation and production of IL‑10.8 Many of these
properties are dependent on IL‑22 production by ILC3.
In the gut, IL‑22 receptor is exclusively found on epithelial
cells.73,74 As well as inducing antimicrobial peptides and
promoting mucin production, IL‑22 conditions intestinal
epithelial cells by shaping glycosylation patterns. IL‑22 sig-
nalling in intestinal epithelium triggers induction of the
enzyme fucosyltransferase 2 that regulates fucosylation of
intestinal epithelial cells.75,76 Disruption of epithelial fuco-
sylation dependent on IL‑22 or the IL‑22 receptor results
in loss of epithelial colonization with bacteria that support
mutualism; instead, bacteria with pathogenic properties
expand unchecked, such as Enterococcus faecalis, which
favours the development of colitis and augments the
virulenc­e of other intestinal pathogens.75,76
ILC interaction with intestinal luminal contents
Microbiota
Mucosal immune handling of commensal microbes is
probably a critical event in the maintenance of homeo­
stasis or the emergence of IBD. Unlike intestinal mono-
nuclear phagocytes that reach dendrites through epithelial
tight junctions and contact luminal bacteria, no evidence
exists that demonstrates ILCs interact directly with
luminal bacteria. Although intestinal ILC subsets might
express TLRs, TLR expression in ILCs from patients with
IBD has not been verified. Whether ILCs interact directly
with conserved microbial molecules in vivo has yet to be
established. However, ILCs probably respond to luminal
microbiota through crosstalk with intermediary cells,
including the epithelial cells and mononuclear phagocytes.
Nevertheless, experimental ablation of intestinal ILCs
severely disrupts mucosal-associated microbial commu-
nities, which profoundly affects the homeostatic control
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© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
mechanisms that regulate host–microbe interactions.
For instance, containment of intestinal bacteria is lost
when intestinal ILC3 are depleted.77 Interestingly, the dis-
seminated bacteria were not random mixtures of diverse
luminal species, but were from the Alcaligenes genus,
which constitutively colonize gut-associated lymphoid
tissue and Peyer’s patches of healthy mammals; there-
fore, ILCs serve to contain bacteria in lymphoid tissues.
Patients with Crohn’s disease have high serum titres of
Alcaligenes-specific IgG, consistent with ILC dysfunction
and increased systemic exposure to these bacteria.77 ILCs
also regulate the interaction of mucosal-associated bac-
terial species known to direct the differentiation of host
immune cells. Intestinal colonization with segmented fila-
mentous bacteria has a central role in the differentiation of
intestinal TH17 cells in mice.78 In the absence of ILC3 the
attendant reduction in mucosal IL‑22 production results
in outgrowth of intestinal segmented filamentous bacteria,
which in turn drives expansion of intestinal CD4+ TH17
cells and heightens susceptibility to colitis.79
Evidence that the luminal microbiota influence ILCs in
IBD comes from human studies examining patients with
Crohn’s disease and surgical diversion of the faecal stream.
Although ILC3 could be detected in both the afferent limb
of the gut (still in contact with the intestinal microbiota)
and the efferent limb of the gut (no longer exposed to the
faecal stream), ILCs isolated from the efferent limb pro-
duced less cytokine.41 Whether this phenomena results
from direct exposure of ILCs to microbial products or
whether it is dependent on intermediary cells is unclear;
however, in the same study ILCs were highly activated by
CXC3R1highCD14high mononuclear phagocytes that had
been primed with microbial products, which favours the
latter explanation. Importantly, NKp44+ ILC3 are present
in the fetal gut by the third trimester of gestation, inde-
pendent of microbial colonization. However, these cells
have reduced IL22 expression than phenotypically similar
ILCs from the adult gut,80 implying that IL-22-induction
by NKp44+ ILCs is driven by the microbiota.
Diet
Dietary factors also profoundly influence intestinal ILC
homeostasis. Vitamin A deficiency, which globally is one
of the most common micronutrient deficiencies, is associ-
ated with profound changes in the relative composition of
intestinal ILC subsets.81 Strikingly, IL‑22-producing ILC3
are severely diminished and ILC2 reciprocally expanded in
vitamin-A-deficient mice.81 Vitamin-A-deficient animals
exhibit increased susceptibility to intestinal bacterial infec-
tions and enhanced protection from intestinal worms.81
Conversely, administration of excess vitamin A expands
IL‑22-producing ILC3 and protects mice from experi-
mental colitis.82 In addition, excess vitamin A halts ILC2
growth and survival.81 A gradient of dietary vitamin A
dynamically regulates the composition of intestinal ILCs
and orientates particular host protective mechanisms.
Evolutionary selection pressures might favour the regula-
tion of host immunity by exogenous factors such as diet.
Indeed, food scarcity, starvation and micronutrient defi-
ciencies often go hand-in-hand with endemic parasitic
REVIEWS
worm infestation.83 Improving protection from helminths
that would otherwise compete for resources might be
expected to provide a survival advantage to the host.
ILCs express the aryl hydrocarbon receptor, which has
a central role in the development of numerous cell types,
including ILCs. Intracellular aryl hydrocarbon receptor
is responsive to dietary molecules and ligands including
flavonoids, polyphenolics and other aromatic hydrocar-
bons. Aryl hydrocarbon receptor deficiency results in
diminished numbers of IL‑22-producing ILCs in the gut,
impaired small intestinal lymphoid follicle formation and
increased susceptibility to gastrointestinal infection.84,85
Induction of arylhydrocarbon receptor activity in ILCs
by dietary ligands might provide an elegant link between
dynamic environmental changes, such as food intake,
and functional innate immune responses, which could
in part depend on the induction of Notch signalling.86
How these dietary agonists might affect intestinal ILC
phenotype through activation of the aryl hydrocarbon
receptor, especially in the context of chronic intestinal
inflammation is not yet known.
IBD genetics and intestinal ILC phenotype
A meta-analysis published in 2011 of several international
IBD GWAS followed by extensive replication in >75,000
individuals has now set the number of IBD-associated
genetic loci to 163.87 By looking across all loci for patterns
of genes whose protein products co-occur in similar cel-
lular pathways or interact directly, it is possible to make
some informed choices about which genes are more likely
to be involved in IBD pathogenesis. Jostins et al.87 used
this approach to prioritize 300 of over 1,000 genes at the
163 IBD loci. Genes involved in the regulation of cytokine
production or in sensing and responding to bacteria were
especially enriched.87 Gene expression data in mouse
immune cells87,88 show expression of IBD genes enriched
predominantly in innate immune cells (in particular in
dendritic cells) rather than adaptive immune cells; these
experiments did not analyse ILC populations.
One of the most important outcomes of GWAS in IBD
was highlighting the role of the TH17 pathway, through
associations with the genes encoding the IL‑23 receptor
and its intracellular signalling pathway components STAT3,
JAK2 and TYK2.87,89,90 As well as promoting differentiation
and maintenance of TH17 cells,91 IL‑23 also activates ILC3.
Low frequency and rare loss-of-function variants in IL23R
(p.Arg86Gln, p.Gly149Arg, p.Val362Ile)90,92,93 that are pre-
dicted to result in partial loss-of-function, have reduced
frequency in IBD. Carriage of these variants would lead
to an attenuated TH17–ILC3 response in healthy indi-
viduals in whom they are more common, corroborating
the hypothesis that IBD might result from dysregulated
TH17–ILC3 responses. Genetic variation at loci encod-
ing other genes that affect TH17–ILC3 responses include
RORC, IL22, IL1R, IL6ST, TL1A and CCR6.87 Likewise,
genes conventionally associated with the TH1 response, are
also expressed by ILC1; therefore, alterations in the expres-
sion of these genes or the function of their protein products
might similarly affect ILC1 phenotype including: STAT1;
STAT4; IL12RB2; and IFNG. SNPs at the IL12B locus,
278  |  MAY 2015  |  VOLUME 12  www.nature.com/nrgastro
© 2015 Macmillan Publishers Limited. All rights reserved
which encodes the IL12p40 subunit common to both IL‑12
and IL‑23, are also robustly associated with IBD.87 Indeed,
at least 29 genes of the currently detected 163 IBD loci are
crucially implicated in ILC biology and thus could be con-
sidered an ‘ILC gene’ (Table 2). These genes encode key
effector cytokines, cell-surface receptor components that
enable ILC responsiveness to key stimulatory cytokines,
intracellular signalling pathways, transcription factors
and ILC-stimulating cytokines themselves. Although the
fine mapping data that is expected to improve elucidation
of true causal variants at these loci is yet to emerge, it is
intriguing that at more than half of these loci the best-
associated genetic variants are closest to an ‘ILC gene’ and
in most cases that gene is implicated by a combination of
multiple gene prioritization strategies including pathway
and functional analyses.87
ILCs as therapeutic targets in IBD
In view of the prominent role of ILCs in preclinical models
of IBD and their expansion in mucosal lesions of patients
with IBD, it is appealing to speculate that selectively target-
ing ILCs could be a useful treatment strategy. ILCs produce
or respond to key cytokines implicated in IBD pathogenesis
and other potentially drug-sensitive pathways to target also
exist in these cells. To date, few data exists regarding the
effects that established IBD treatments (such as aminosal-
icylates or immunomodulators) have on the phenotype of
mucosal ILCs. However, data shows that IL‑22 and TNF
production by NCR+ ILC3 is suppressed by ciclosporin A,59
which is sometimes used to treat steroid refractory ulcera-
tive colitis. The increasing use of biologic agents to target
specific aspects of host immunity in inflammatory dis-
eases might be an especially useful strategy to limit ILC-
mediated intestinal pathology. Therapeutic strategies might
include prevention of ILC activation by blockade of activat-
ing and/or survival signals, interference with intracellular
signalling pathways, neutralization of the effector cytokines
that they produce, or perturbation of ILC trafficking to
target organs (Figure 3). Some of these strategies might
be additionally advantageous by simultaneously inhibit-
ing other immune cells implicated in IBD pathogenesis,
such as T cells or some myeloid lineages. With the promise
of personalized medicine on the horizon it is exciting to
speculate that specific biologic therapies might be selected
based on dominant immune response pathways present in
individual patients.94
Targeting ILC activation
Targeting proximal cytokines (or their receptors) or other
activating signals is a theoretically attractive proposition in
IBD and offers the advantage of limiting multiple down-
stream effector functions. Selectively targeting of IL‑23 is
an obvious strategy to follow to limit pathogenic ILC acti-
vation, especially in the gut. As mentioned earlier, the IL‑23
heterodimer is comprised of an IL‑12p40 subunit (which
is also a subunit of the IL‑12 heterodimer) with a novel
IL‑23p19 subunit. In the gut, IL‑23 potently stimulates
IL‑17A and IL‑22 production by pathogenic ILC328,30,32
and might also stimulate IFN‑γ production by ILC1.22
Therapeutic neutralization of IL‑23p19 or blockade of its
 REVIEWS

Table 2 | IBD risk-associated loci determined by genetic studies that contain known ILC genes
Chromosome Best SNP marker Known ILC genes in locus Associated with P value for
(no. of additional genes) association
5 rs10065637 IL6ST (2) Reduced risk for CD 3.68 × 10–12
4 rs3774959 NFKB1* (3) Increased risk for UC 3.66 × 10–12
1 rs11209026 IL23R*, IL12RB2 (4) Reduced risk for IBD 8.12 × 10–161
1 rs4845604 RORC* (1) Reduced risk for IBD 3.52 × 10–16
2 rs917997 IL1R2, IL1R1, IL1RL1, IL1RL2 (5) Increased risk for IBD 3.12 × 10–20
2 rs1517352 STAT1, STAT4* (2) Reduced risk for IBD 3.28 × 10–11
4 rs2472649 IL8 (10) Reduced risk for IBD 2.57 × 10–08
4 rs7657746 IL2 (3) Reduced risk for IBD 2.76 × 10–13
5 rs2188962 IL13, CSF2 (16) Increased risk for IBD 1.35 × 10–52
5 rs6871626 IL12B* (3) Increased risk for IBD 1.43 × 10–42
6 rs1819333 CCR6 (5) Reduced risk for IBD 6.76 × 10–21
9 rs10758669 JAK2* (4) Increased risk for IBD 7.88 × 10–45
9 rs4743820 NFIL3 (2) Reduced risk for IBD 3.60 × 10–09
9 rs4246905 TNFSF15* (4) Reduced risk for IBD 2.80 × 10–32
10 rs12722515 IL15RA (7) Reduced risk for IBD 3.76 × 10–10
12 rs7134599 IFNG*, IL22 (2) Increased risk for IBD 8.51 × 10–32
17 rs12942547 STAT3* (3) Reduced risk for IBD 5.51 × 10–22
19 rs11879191 TYK2 (4) Reduced risk for IBD 2.04 × 10–18
*Genes closest to the major disease-associated SNP. At least 18 out of the 163 chromosomal loci identified so far to be highly significantly associated with UC,
CD or the combined phenotype of IBD, harbour genes encoding proteins essential in ILC biology. Abbreviations: CCR6, chemokine (C-C motif) receptor 6; CD,
Crohn’s disease; ILC, innate lymphoid cell; IL6ST, interleukin 6 signal transducer; JAK2, janus kinase 2; NFIL3, nuclear factor interleukin 3 regulated; NFKB1,
nuclear factor κβ 1; RORC, RAR-related orphan receptor C; SNP, single nucleotide polymorphism; STAT, signal transducer and activator of transcription; TNFSF15,
tumour necrosis factor (ligand) superfamily, member 15; TYK2, tyrosine kinase 2; UC, ulcerative colitis. Permission obtained from NPG © Jostins, L. Nature 491,
119–124 (2012).

receptor (IL‑23R) attenuates ILC-mediated colitis in mouse
disease models.30,32 Drugs targeting the IL‑23 specific p19
subunit, including BI655066 and AMG139 are currently
being evaluated in clinical trials in patients with Crohn’s
disease.95 As well as suppressing ILCs, IL‑23 antagonism
probably limits other IL‑23-responsive cells, includ-
ing TH17 cells, γδ T cells and neutrophils, which are an
additional source of innate IL‑17 and IL‑22.96 Targeting
IL‑12p40 offers the advantage of simultaneously neu-
tralizing two key pathways involved in ILC activation
(IL‑12-induced ILC1 activation and IL‑23-induced ILC3
activation). IL‑12p40 neutralization with monoclonal
antibodies (ustekinumab and briakinumab) or inhibition
of its synthesis with oral agents (STA‑5,326) are promis-
ing treatments for Crohn’s disease and await evaluation in
ulcerative colitis.97,98
Other cytokines responsible for triggering ILCs, or aug-
menting IL‑23-induced cytokine production include IL‑1,
IL‑6, IL‑15, IL‑18 and TL1A.28,31,41,54,59 Notably, levels of
these cytokines are increased in the gut of patients with
IBD99–103 and are therefore biologically plausible thera-
peutic targets. IL‑6 augments IL‑17 and IL‑22 production
by ILC3 in mouse and human ILC3, and experimental
blockade of this cytokine reduces effector cytokine pro-
duction and attenuates ILC dependent colitis in TRUC
mice (Powell, unpublished data). Initial studies evaluating
tocilizumab (an IL‑6-receptor-blocking monoclonal anti-
body) in patients with Crohn’s disease are very encourag-
ing 104 and other IL‑6-neutralizing or IL‑6R-blocking drugs
(PF04236921, C326) are in early-phase clinical trials.105,106
Therapeutic blockade of IL‑1β is therapeutic in ILC-
dependent preclinical models of IBD.107 IL‑1R antagonism
(for example, with anakinra, canakinumab, or rilonacept)
is efficacious in other inflammatory diseases108–110 and
trials in patients with IBD are awaited.
Targeting intracellular signalling
Engagement of cytokines with their specific receptors
triggers intracellular signalling pathways, which initiate
downstream production of effector cytokines by ILCs
typically through induction of selective transcriptional
modules. Many cytokine receptors signal through Janus
kinase (JAK) and STAT pathways. For instance, IL‑23
triggers phosphorylation of STAT3 (and to a lesser extent
STAT5), whereas IL‑7 is a potent stimulator of STAT5
phosphorylation in human mucosal ILCs.59 Specific
inhibition of JAK/STAT signalling suppresses effector
cytokine production by ILCs.57,59 Small-molecule inhibi-
tors of JAK/STAT signalling are in advanced development
or are even currently being trialed in patients with IBD,
including pimozide (an oral STAT5 inhibitor) and tofaci-
tinib (a JAK3 inhibitor). The efficacy and safety of oral
STAT3 inhibitors are being evaluated in clinical trials in
malignant disease.111,112 In IBD, STAT3 inhibition would
be expected to suppress the activation of ILCs mediated by
IL‑23 and IL‑6, limiting the pathogenic potential of ILCs
in the gut. Other cytokines responsible for stimulating
ILCs, such as IL‑1 family cytokines or TL1A signalling

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© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS

ILC survival or depletion

p19
Prevent activation CD90 Block effector
IL-23 IL-23R IL-7 cytokines
BI655066 p40
AMG139 IL-7R IFN-γ Fontolizumab
p40 IL-12R STAT3 STAT5
Ustekinumab ILC
Briakinumab IL-12 Infliximab
STAT1 TNF Adalimumab
p35 Golimumab
Tocilizumab
Olokizumab IL-6R Signal inhibition
BMS945429 Pimozide Secukinumab
PF04236921 IL-6 STAT3 Tofacitinib Brodalimumab
C326 STAT inhibitors IL-17

Anakinra Anrukinzumab
Rilonacept IL-1R Tralokinzumab
Canakinumab NFκB QAX576
IL-22
IL-1α/β

IL-13
TL1A DR3
α4β7 CCR6 Vedolizumab
Etrolizumab
Natulizumab Inhibit migration

MAdCAM-1 PF-547659

Endothelial cell

Figure 3 | Current and emerging therapeutic strategies in IBD and how theyNature
might Reviews
affect ILC function. Abbreviations:
| Gastroenterology CCR6,
& Hepatology
C-C chemokine receptor type 6; DR3, death receptor 3; ILC, innate lymphoid cell; MAdCAM-1, mucosal vascular addressin
cell adhesion molecule 1; STAT, signal transducer and activator of transcription; TL1A, Tumor necrosis factor ligand
superfamily member 15.

through non-JAK/STAT signalling cascades (such as the
MyD88–IRAK–NFκB and MAP kinase pathways), might
also represent therapeutically tractable targets in IBD.
Targeting ILC effector cytokines
Selective neutralization of key ILC effector cytokines,
such as IL‑17, IL‑22 and IFN‑γ is an attractive alterna-
tive strategy. Levels of these cytokines are all elevated in
diseased mucosa of patients with IBD.3,28 IL‑17 is one of
the major effector cytokines produced by disease-causing
intestinal ILCs and neutralization of this cytokine attenu-
ates colitis in preclinical models of disease.30,32 The results
of therapies targeting IL‑17 in Crohn’s disease were hotly
anticipated and appropriately powered studies assessing
the role of neutralizing anti-IL‑17A monoclonal antibody
(secukinumab), or blockade of its receptor IL‑17RA (bro-
dalimumab) have now been reported (the latter only in
abstract form so far).36,113 Both drugs lacked overall benefit
and on the contrary were associated with clinically signifi-
cant disease deterioration leading to premature termina-
tion of both trials. Interestingly, subgroup analysis in the
secukinumab trial—which set out to evaluate the role of
genetic polymorphisms in the IL‑23–IL‑17 axis in the
context of anti-IL‑17 therapy—found that patients with
a specific polymorphism at the TL1A locus (rs4263839)
responded markedly better to anti-IL‑17A therapy than
patients lacking the polymorphism.36 In this subgroup
analysis all nine patients carrying the TL1A rs4263839
minor allele improved following secukinumab therapy,
whereas six of seven patients lacking the minor allele
deteriorated. Intriguingly, as well as promoting effector-
T‑cell responses, TL1A also augments IL‑23-induced
cytokine production by intestinal ILCs,22,41 consistent
with the possibility that some subgroups of patients with
IBD might be responsive to anti-IL‑17 treatment, but that
thera­peutic sensitivity hinges on genetic factors r­esponsible
for regulating host TH17 and ILC3 cells.
The major effector cytokine of ILC1 is IFN‑γ, which
might also be coexpressed by some ILC3.32 Fontolizumab
(a monoclonal antibody targeting IFN‑γ) has been evalu-
ated in patients with Crohn’s disease, including an appro-
priately powered phase II clinical trial.114 Although this
study did not meet its primary end point (remission at
4 weeks), a clear and statistically significant efficacy signal
was seen beyond 4 weeks and clinical response or remis-
sion rates were especially prominent in patients with sig-
nificant inflammation (as determined by raised C-reactive
protein levels).114 ILC1 (and ILC3 under some conditions)
also produce TNF, antagonism of which is now a main-
stay of treatment in IBD,115,116 although TNF is produced
by other intestinal immune cells including mononuclear
phagocytes and T cells.
Overall, with the exception of anti-TNF agents,115,116
strategies targeting single effector cytokines have been dis-
appointing in clinical trials in IBD. As ILCs produce several
important effector cytokines, often simultaneously, it might
be necessary to target multiple effector cytokines simul-
taneously to effectively suppress ILC-mediated intestinal
pathology. Concomitant blockade of IL‑17 and IFN‑γ is
more effective than anti-IL‑17 or anti-IFN‑γ monotherapy

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© 2015 Macmillan Publishers Limited. All rights reserved
 REVIEWS

in preclinical models of ILC-mediated colitis.32 Dual block-
ade approaches have yet to be evaluated in patients with
IBD. However, ABT‑122, a bi-specific antibody construct
that simultaneously neutralizes TNF and IL‑17, is currently
being evaluated in patients with rheumatoid arthritis.117
Targeting ILC survival and trafficking
An alternative strategy to combat IBD pathogenesis
mediated by ILCs is to reduce intestinal ILC numbers by
starving them of survival signals, depleting them in situ,
or preventing them from trafficking to the gut. ILC
survival and maintenance is dependent on IL‑7; IL‑7R
blockade markedly reduces intestinal ILC numbers and
attenuates colitis severity in TRUC mice.30 IL‑7 also aug-
ments pathogenic cytokine production by ILC359 and
blockade might offer the additional benefit of reducing
ILC cytokine production.
ILC depletion strategies (for example, anti-CD90 mono-
clonal antibodies) have been highly successful in preclinical
models of IBD,30,32 although these strategies have yet to be
tried in human disease. Expression of CD90 in human ILCs
is unknown, neither is it known whether alternative surface
markers could be targeted. Drugs targeting inflammatory
cell trafficking to the gut are currently in clinical practice
for patients with IBD and newer agents continue to emerge.
Many of these treatments target interactions between inte-
grins expressed by leucocytes (such as α4β7) and its specific
ligand MAdCAM‑1, an adhesion molecule located on high
endothelial venules supplying the gut. Interactions between
α4β7 and MAdCAM‑1 govern trafficking and extravasa-
tion of some leucocyte populations into the intestinal
lamina propria and Peyer’s patches. ILC precursors and
some ILC subsets express the integrin α4β7, which enables
their recruitment to the gut.11,118 Drugs specifically target-
ing adhesion interactions between α4β7 and MAdCAM‑1
are effective in patients with IBD and additional agents are
in the pipeline.119–124 To anticipate that the blockade of this
trafficking pathway would prevent the recruitment of ILC
precursors to the gut for therapeutic benefit is logical.
A definite role for ILC2 in IBD has yet to be established,
although therapeutic approaches to negate the pathogenic
potential of ILC2 might include neutralization of proximal
activating signals (IL‑25, IL‑33, TSLP and TL1A) and neu-
tralization of ILC2 effector cytokines (IL‑5 and IL‑13).
Early-phase trials of IL‑13 blockade (tralokinumab,
anrukinzumab and QAX576) are underway in IBD.125–127
If a convincing role for IL‑13-producing ILC2 in intestinal
fibrosis holds up, then anti-IL‑13 therapies might be best
deployed in patients with Crohn’s disease at high risk of
developing fibrostenotic complications.
Conclusions
ILCs are newly described innate immune cells that accu-
mulate in inflammatory lesions of patients with IBD. At
the mucosal surfaces these cells react to environmental
or inflammatory cues and rapidly respond to new threats
or signals. They are a potent early source of IBD-relevant
effector cytokines and mechanistic studies show that they
are functionally important in preclinical models of disease.
Insights into how therapeutic modulation of ILCs might
affect clinical outcomes in IBD are eagerly awaited. The
exciting possibility that these cells might be important
drivers of mucosal inflammation in Crohn’s disease and/
or ulcerative colitis could herald the emergence of new
therapeutic strategies to combat these diseases. Targeting
conserved cytokine pathways used by ILCs and T cells to
simultaneously antagonize potentially harmful pathways
shared by these effector cells might be advantageous.
However, the complexity of mucosal immune response
networks means that challenges remain. IBD is hetero-
geneous and dominant immune pathways operational in
one patient might have less significant roles in others. This
heterogeneity is reflected in the complex genetic architec-
ture of IBD. Studies looking at ILC populations in patients
with IBD demonstrate marked variation in ILC abundance
and responsiveness to activating cytokine signals. Smarter
ways of stratifying patients with IBD according to their
particular mucosal immune phenotype to appropriately
select personalized patient-specific immune based thera-
pies is needed. Defining which ILC subsets are present,
what signals they respond to and what effector responses
they generate, could hold the key to deciding which bio-
logical therapy will have the greatest chance of therapeutic
success in individual patients.

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Author contributions
N.Powell, R.G. and N.Prescott contributed equally
to researching data for the article, contributing to
discussion of content and writing. All authors
contributed equally to reviewing and editing the
article before submission.