Immunity

Review

Barrier Epithelial Cells

and the Control of Type 2 Immunity
Hamida Hammad1,2,* and Bart N. Lambrecht1,2,3,*
1VIB Inflammation Research Center, Technologiepark 927, 9052 Ghent, Belgium
2Laboratory of Immunoregulation and Mucosal Immunology, Department of Respiratory Medicine, Ghent University, De Pintelaan 185, 9000
Ghent, Belgium
3Department of Pulmonary Medicine, ErasmusMC, Dr Molewaterplein 50, 3015 GE Rotterdam, the Netherlands

*Correspondence: hamida.hammad@irc.vib-ugent.be (H.H.), bart.lambrecht@irc.vib-ugent.be (B.N.L.)

http://dx.doi.org/10.1016/j.immuni.2015.07.007

Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4+ T helper 2 (Th2) cells, and
type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic
diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense
and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic
allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines
such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric
acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-medi-
ated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mech-
anisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads
to protection or disease.

Introduction class switching in B cells, resulting in the production of parasite-

More than 3 billion people suffer from helminth infection or
allergic diseases, like asthma, rhinitis, atopic dermatitis (AD),
food allergy, and anaphylaxis. Before the advent of sanitation
and access to modern medicine, infection with intestinal nema-
todes was ubiquitous, and it continues to be so in the absence of
sanitation. Probably the greatest evolutionary driving force for
type-2-cell-mediated immunity to exist is the ancient co-evolu-
tion of the mammalian mucosal immune system with these hel-
minths (Grencis et al., 2014). In some parts of the world, tick,
insect, and snake bites and stings are also a significant threat.
These infectious, toxic, and inflammatory disorders resemble
each other closely in that affected tissues, often the mucosal
barriers and the skin, mount a ‘‘type 2’’ cell-mediated immune
response, rich in mast cells, basophils, eosinophils, and alterna-
tively activated (or M2) macrophages that together release bene-
ficial pro-inflammatory mediators, toxin-neutralizing enzymes,
and helminth-killing toxins that, however, also cause significant
problems of itching, redness, swelling, and altered function of
the entire organ (Pulendran and Artis, 2012; Voehringer, 2013).
This type-2-cell-induced altered functional response can be
very appropriate; for example, in the case of helminth infection,
mediator-induced smooth muscle contraction and increased
mucus production and epithelial cell proliferation favor worm
expulsion (Maizels et al., 2012). Nevertheless, the type-2-cell-
mediated response can also be very detrimental; for example,
the smooth muscle contraction and mucus production after
inhalation of harmless allergens leads to chronic airway narrow-
ing in asthma and can also cause acute death in anaphylaxis
(Lambrecht and Hammad, 2015).
Integrated type-2-cell-mediated immune responses are en-
forced by the cytokines interleukin-4 (IL-4), IL-5, IL-9, and
IL-13, all known to be produced by CD4+ T helper 2 (Th2) lym-
phocytes of the adaptive immune system. IL-4 drives isotype
or allergen-specific IgE that arms effector mast cells and baso-
phils that carry the high-affinity IgE receptor FcεRI; IL-5 is
responsible for the generation and activation of eosinophils in
the bone marrow and tissues; IL-9 causes mast cell and ILC2
activation; IL-13 (and to a lesser extent IL-4) causes smooth
muscle hyperreactivity, goblet cell metaplasia, mucus hyperse-
cretion, and alternatively activated macrophages (AAM) differen-
tiation of monocytes; and Th2 cells also make amphiregulin
(Areg) that promotes epithelial repair from injury (Zaiss et al.,
2015). The cytokine IL-3 is made by naive T cells early after
T cell receptor TCR stimulation and enforces mast cell and baso-
phil functions but is not generally seen as a Th2-cell-associated
cytokine (Shen et al., 2008). Of note, not all Th2 cells make all Th2
cytokines, and unique production of IL-4 is more typical of Th2
cells in lymph nodes, where they boost IgE production; whereas
both IL-4- and IL-13-producing Th2 cells are found in tissues
(Liang et al., 2012). In addition to CD4+ Th2 cells, it is now also
clear that ILC2s that lack antigen-specific recognition receptors
are an important and much earlier source of the type-2-cell-
associated cytokines IL-5, IL-13, Areg, and (to a lesser extent)
IL-4 (Artis and Spits, 2015; Nussbaum et al., 2013). Even in the
complete absence of the adaptive immune system, ILC2s can
control a type-2-cell-mediated innate immune response, leading
to eosinophilic airway inflammation after protease allergen ex-
posure or to expulsion of helminths. In many cases, however,
ILC2s will have to conspire with CD4+ Th2 cells to sustain
type-2-cell-mediated immunity and acquire full-blown type 2
effector cell function such as parasite expulsion (Oliphant
et al., 2014). Here, we will review the pathways shared by barrier
epithelial cells (ECs) to recruit and activate various players of
the type-2-cell-mediated immune response. We will focus on
EC-derived factors (chemokines, cytokines, alarmins) induced
by diverse pattern recognition receptors and the importance of

Immunity 43, July 21, 2015 ª2015 Elsevier Inc. 29


Figure 1. Epithelial Cells and Dendritic Cell Crosstalk Control Th2
Cell Responses in the Lung
Upon allergen exposure, airway epithelial cells get activated to produce
chemokines and innate cytokines that will instruct immature dendritic cells
(iDCs) and activate ILC2s. ILC2-derived IL-5 contributes to airway eosino-
philia, whereas IL-13 drives goblet cell metaplasia (GCM) and cDC2 migration
to MLNs. In the MLN, cDC2, with the help of IL-4 derived from basophils, will
induce the differentiation of Th2 cells. IL-4 derived from Th2 cells promotes IgE
production by B cells. Th2 cells also produce IL-9, which causes ILC2 and
mast cell (MC) activation. Th2 cells also make amphiregulin (Areg) that pro-
motes epithelial repair from injury.
barrier integrity and mucus quality in murine models and in pa-
tients with Th2-cell-associated disorders.
Initiation of Type-2-Cell-Mediated Immunity Requires
More than Just Dendritic Cells
Although we have a good idea of what constitutes a type-2-cell-
mediated immune response, the mechanisms that initiate,
perpetuate, and resolve it remain enigmatic. This is partly caused
by the fact that the triggers that cause type-2-cell-associated im-
munity are diverse, ranging from large extracellular multicellular
organisms (e.g., nematodes), venoms, tick saliva, excreta of
mites and cockroaches, mold and bacterial cell wall compo-
nents, and enzymes, food components, animal dander, plant
pollen, and even the inorganic alum and uric acid crystals
(Kool et al., 2008, 2011; Pulendran and Artis, 2012). In the labo-
ratory mouse, different models of parasite infection have been
used to study type-2-cell-mediated immunity, such as the hook-
worm Nippostrongylus brasiliensis, the roundworm Heligmoso-
moides polygyrus, the whipworm Trichuris muris, and Trichinella
spiralis, a nematode with an atypical life cycle. As experimental
allergens, most groups now use the complex house dust mite
(HDM) extracts or cockroach extracts (containing a multitude
of proteins, lipids, glycoconjugates, and microbiome-derived
molecules), the enzyme papain, or the particulate polysaccha-
ride polymer chitin, a constituent of the cell wall of fungi and
the exoskeleton of insects and crustaceans.
From these experimental studies, it has emerged that dendritic
cells (DCs), ILC2s, and basophils act very collaboratively and up-
stream in the type-2-cell-mediated immune response cascade
(Figure 1; Lambrecht and Hammad, 2014). Antigen-presenting
DCs that recognize an allergen or helminth present antigenic
30 Immunity 43, July 21, 2015 ª2015 Elsevier Inc.
Immunity
Review
peptides to T cells in the draining lymph nodes. Several studies
have reported that DCs are necessary and sufficient for inducing
adaptive Th2 cell responses to HDM allergen, papain, the hel-
minth Schistosoma mansoni, and proteins in inorganic crystals
(Hammad et al., 2010; Kool et al., 2008; Ohnmacht et al.,
2010). Although the exact DC subset initiating Th2 cell responses
to helminths has not been formally identified yet, in the lung and
in the skin, CD11b+CD172 (SIRP1a)+ conventional (c)DCs that
depend on the transcription factor IRF4 and KLF4 are necessary
and sufficient to induce adaptive Th2-cell-associated immune
responses (Akbari et al., 2014; Gao et al., 2013; Kumamoto
et al., 2013; Plantinga et al., 2013; Tussiwand et al., 2015; Wil-
liams et al., 2013). The mechanisms by which these DCs induce
Th2 cell immunity are poorly understood, compared with Th1,
Th17, and T regulatory (Treg) cell responses, but several studies
have shown the importance of the affinity of the major histocom-
patibility complex-T cell receptor (MHC-TCR) interaction, the
tumor necrosis factor (TNF) ligand family member OX40L, the
Notch ligand Jagged1, the cytokine IL-6, and the IRF4-induced
absence of IL-12 production (Akbari et al., 2014; Amsen et al.,
2004). Another reason why the initiation of Th2 cell immunity is
so obscure is that DCs do not produce the polarizing IL-4 cyto-
kine that drives Th2 cell immunity in many models. Basophils are
recruited to the lymph nodes very early in the course of a type-2-
cell-associated immune response and help DCs to polarize naive
T cells to the Th2 cell fate, by producing IL-4 (Figure 1; Hammad
et al., 2010; Sokol et al., 2008). After exposure to papain, DCs are
indeed not sufficient to polarize Th2 cell responses, but this also
depends on the experimental setting (Ohnmacht et al., 2010).
The basophils boost ILC2 effector function in a STAT-6 tran-
scription factor-dependent manner by producing IL-4 (Doherty
et al., 2012; Motomura et al., 2014), and basophils help eosino-
phil recruitment via IL-4-induced endothelial cell expression of
the integrin VCAM-1. Similarly, ILC2s are recruited early in a
type-2-cell-mediated immune response and might provide IL-
13 to boost DC function (mainly migration) and promote Th2
cell responses. For adaptive Th2-cell-mediated immunity to
papain, IL-13 (not IL-4) is the polarizing cytokine (Halim et al.,
2014). Eosinophils recruited into tissues and lymph nodes can
also be a source of IL-4 and can promote DC-driven Th2-cell-
mediated immunity via release of eosinophil peroxidase released
from granules (Chu et al., 2014). Under certain conditions, baso-
phils, eosinophils, and ILC2s can also directly function as anti-
gen-presenting cells (APCs), although they are clearly less
potent than DCs (Kambayashi and Laufer, 2014; Oliphant
et al., 2014).
Epithelial Cells Produce Chemokines that Recruit DCs,
Basophils, and ILC2s
Initiation of type-2-cell-mediated immunity in mucosal barriers
and skin is highly complex, requiring many cell types to respond
to type 2 immune stimuli in a rapid and coordinated manner, and
at the same time the barrier epithelial cells are very impermeable
due to the physical barrier of luminal mucus or epidermal kera-
tins, on account of the tight junction and adherens junction belt
connecting the ECs with one another. Many pathogens that
cause a type-2-cell-mediated immune response, like nema-
todes, have evolved mechanisms to penetrate through tissues,
via production of proteases that cleave mucus and tight
Immunity
Review
Figure 2. Epithelial Cells Are Upstream of the Th2 Cell Cascade
Exposure to type 2 cell immune stimuli (proteases, b-glucans, chitin, etc.)
favored by a barrier defect leads to the triggering of a wide range of pattern
recognition receptors on barrier epithelial cells. ECs have evolved receptors
like the protease-activated receptors (PARs), Toll-like receptors (TLRs), C-type
lectins (CLR), and inflammasomes to sense invasion and penetration. The
activation of these receptors will induce NF-kB activation, ROS production,
and the release of innate pro-Th2 cell cytokines like TSLP, GM-CSF, IL-25, and
IL-33. In addition, barrier ECs have the capacity to regulate type 2 immunity
through the production of endogenous danger-associated molecular patterns
(DAMPs) or alarmins like IL-1a, HMGB1, uric acid, ATP, or S100 proteins. The
release of DAMPs can amplify the production of TSLP, GM-CSF, IL-25, and IL-
33 by ECs. In response to these stimuli, immature DCs (iDCs) upregulate
OX40L and Notch ligand expression, downregulate IL-12 production, and
produce Th2-cell-attracting chemokines like CCL17 and CCL22. EC-derived
factors stimulate ILC2 proliferation and survival and their production of IL-5
and IL-13.
junctions. Also many allergens, like the HDM allergen Dermato-
phagoides pteronyssinus (Der p 1) are proteinases. Not surpris-
ingly, the barrier ECs have evolved receptors (like the prote-
ase-activated receptors or PARs) or pathways (reactive oxygen
species [ROS] sensing) to sense invasion and penetration and
have integrated this response to the initiation of type-2-cell-
mediated immunity to clear the insult and initiate the repair pro-
cess of the damaged barrier, e.g., via epithelial proliferation,
collagen deposition, or altered mucus production (Pulendran
and Artis, 2012). Type 2 cell stimuli on barrier ECs have in com-
mon that they recruit DCs, ILC2s, basophils, mast cell progeni-
tors, and eosinophils almost simultaneously. This happens
through an epithelial production of chemokines like CCL17 and
CCL22 (acting on CCR4+ ILC2s, basophils, Th2, and Treg cells)
and of eotaxins like CCL-11, -24, and -26 (acting on CCR3+ eo-
sinophils and Th2 cells). In support, transgenic overexpression of
CCL17 by keratinocytes is sufficient to induce an atopic derma-
titis (AD)-like disorder. In many type-2-cell-mediated disease
states like asthma, there is constitutive overexpression of
CCL17 in ECs (Sekiya et al., 2000). The barrier ECs also make
prostaglandin D2 that acts on the CRTH2 receptor to recruit
ILC2s, basophils, mast cells, and Th2 cells (Xue et al., 2014). A
striking feature is that many of the recruited innate type 2 cells
will start producing CCL17, CCL22, and prostaglandin D2
(PGD2) themselves, leading to a feed-forward loop that enforces
the response. As only one example, production of CCL17 and
CCL22 are defining characteristics of AAMs, and the alveolar
macrophages of human asthmatics are known to overproduce
PGD2 and CCL17 (Staples et al., 2012). Production of CCL11
(eotaxin), CCL17, and CCL22 by ECs is further enforced by IL-
4 and IL-13 in a STAT-6-dependent manner and inhibited by
the type 1 cytokine interferon-g (IFN-g). This observation ex-
plains the profound influence of STAT6 deficiency in ECs on in-
duction of type-2-cell-mediated immunity in the lung and gut
(Doherty et al., 2012).
Epithelial Cells Produce Cytokines that Activate DCs,
Basophils, and ILC2s
Barrier ECs can also produce several immunoregulatory and
instructive cytokines that will shape the immune response. In
many instances, antigens are cleared without the induction of in-
flammatory responses. In the gut and in the lung, EC-derived
transforming growth factor-b (TGF-b) production promotes the
differentiation of DCs with a tolerogenic potential, important for
the control of tolerance to food and environmental inhaled anti-
gens (Iliev et al., 2009; Wang et al., 2009a). However, in response
to most type-2-cell-mediated stimuli, barrier ECs produce a set
of prototypical cytokines (IL-33, thymic stromal lymphopoietin
[TSLP], and IL-25) that activate DCs to promote adaptive Th2
cell immunity, and license the innate type 2 cell response by acti-
vation of ILC2s, basophils, eosinophils, and mast cells (Figure 2;
Allakhverdi et al., 2007; Fallon et al., 2006; Halim et al., 2012;
Schmitz et al., 2005). This had led to the view that these cyto-
kines constitute an innate ‘‘pro-Th2’’ cell cluster of cytokines
that induce an almost stereotypical response, despite their
lack of structural homology and use of different receptor families
(Saenz et al., 2008). Indeed, DCs that recognize antigens in the
presence of TSLP, IL-25, or IL-33 express Ox40L and produce
CCL17 and CCL22, but in the absence of IL-12 production (Bes-
nard et al., 2011; Chu et al., 2013). The fact that ILC2s have been
discovered is also mainly due to their expansion in vivo after
administration of IL-25, IL-33, and TSLP (Fallon et al., 2006; Neill
et al., 2010; Saenz et al., 2010; Salimi et al., 2013). IL-25 and IL-
33 can induce different types of ILCs. IL-33 is found to induce
ILC2s whereas IL-25 preferentially elicits multipotent progenitor
(MPP) type 2 cells, a new population of innate cells promoting
type 2 cell immunity even in the absence of ILC2s (Saenz et al.,
2010, 2013). IL-25 has recently been shown to also drive inflam-
matory ILC2s, which can develop into natural IL-33-responsive
ILC2s and contribute to helminth expulsion (Huang et al.,
2015). The production of CCL17 and CCL22 chemokines is simi-
larly boosted when ECs are exposed to TSLP or IL-25, again
leading to a feed forward loop that enforces type 2 immunity.
Some effects on type-2-cell-mediated immunity are not shared
by these cytokines, and there are some subtle tissue-specific ef-
fects of the pro-Th2-cell-associated cytokines. TSLP, for
example, has the unique property of boosting the hematopoiesis
of basophils (Siracusa et al., 2011). IL-33 is not always pro-in-
flammatory and has also been shown to be involved in epithelial
repair from injury. In the gut, epithelial IL-33 drives the activation
of IL-33R-expressing Treg cells, converting them into fully sup-
pressive cells, an effect that could be overcome by IL-23 (Schier-
ing et al., 2014).
Another important actor of barrier type 2 immunity is granu-
locyte macrophage colony stimulating factor (GM-CSF), which
is released by ECs and stimulates the proliferation, maturation,
and function of APCs like DCs and macrophages and also
Immunity 43, July 21, 2015 ª2015 Elsevier Inc. 31

promotes eosinophil survival (Figure 2). Transgenic GM-CSF
overexpression in the lungs of mice induces spontaneous
Th2 cell sensitization to the inhaled innocuous protein oval-
bumin (OVA), via activation of IL-33 and Ox40L+ DCs (Llop-
Guevara et al., 2014). In contrast, the neutralization of GM-
CSF abolishes sensitization to HDM allergen and attenuates
the adjuvant effects of diesel particles on allergic sensitization
(Bleck et al., 2006; Willart et al., 2012). More recent data show
that GM-CSF lowers the threshold for allergen to induce fea-
tures of allergic asthma (Llop-Guevara et al., 2014). Moreover,
ECs from asthmatic patients overproduce GM-CSF, suggest-
ing that, in such patients, its production could be epigeneti-
cally regulated (Hackett et al., 2011). Whether GM-CSF plays
an equally important role in other barrier epithelia remains to
be shown.
Finally, the TNF family member cytokine TL1A (TNFSF15) is
made under conditions of type-2-cell-mediated immunity like
helminth infection and HDM exposure. ILC2s express high
amounts of DR3 (TNFRSF15), the receptor for TL1A, and ligation
of DR3 leads to activation and proliferation of ILC2s, necessary
for worm expulsion and airway allergy, and to differentiation of
Th9 cells (Meylan et al., 2014; Richard et al., 2015; Yu et al.,
2014).
Epithelial-Derived DAMPs and Alarmins Promote
Th2-Cell-Mediated Immunity
In addition to the polarizing cytokines IL-25, IL-33, and TSLP,
barrier ECs have the capacity to regulate type-2-cell-mediated
immunity through the production of endogenous danger-associ-
ated molecular patterns (DAMPs) or alarmins. We have previ-
ously shown that exposure of asthmatic patients to a single
HDM administration via the airways leads to the release of ATP
and uric acid in the bronchoalveolar lavage (Idzko et al., 2007;
Kool et al., 2011). Although the exact cellular source of these
DAMPs has not been formally identified in these patients, several
reports show that ECs directly release ATP when stimulated
by allergens like grass pollen or Alternaria alternata (O’Grady
et al., 2013). In mice, a single exposure to HDM is sufficient to
trigger uric acid (UA) production by airway ECs (Kool et al.,
2011), which is crucial in boosting the production of HDM-
induced TSLP, GM-CSF, IL-25, and IL-33. The degradation of
uric acid by uricase completely prevents allergic sensitization
(Kool et al., 2011). The protease allergens bromelain and papain
similarly induce UA production in the lung, leading to IL-33 and
TSLP induction (Hara et al., 2014).
The alarmins High Mobility Group Box 1 (HMGB1), S100 family
proteins, IL-33, and IL-1a are normally localized in the nucleus.
These can, however, be released by non-programmed cell
death, or they can be actively secreted in a process not requiring
the classical cellular secretion pathway involving vesicular trans-
port from the Golgi complex. IL-1a and HMGB1 are expressed
by lung ECs (Figure 2), and exposure to HDM has been shown
to induce release in the lung fluid. Neutralization of IL-1a or
HMGB1 is able to block production of the pro-Th2-cell-derived
cytokines IL-33, IL-25, and GM-CSF in the lung after HDM
administration, showing that the epithelial alarmins act very up-
stream in the allergic type 2 cascade (Hara et al., 2014; Ullah
et al., 2014; Willart et al., 2012). Adoptive transfer of mesen-
chymal stem cells has been shown to suppress features of
32 Immunity 43, July 21, 2015 ª2015 Elsevier Inc.
Immunity
Review
asthma, via interleukin-1 receptor antagonist (IL-1RA)-mediated
suppression of IL-1a and HMGB1 (Duong et al., 2015). Increased
amounts of HMGB1 are found in the skin of mice with AD lesions
(Karuppagounder et al., 2014, 2015). Similarly, induction of type-
2-cell-mediated immunity to nematodes in the gut and to aller-
gens in the skin requires IL-1. The IL-1R can also be triggered
by IL-1b, whose release often depends on the Nlrp3 inflamma-
some. In keratinocytes, HDM has been shown to lead to Nlrp3-
dependent production of IL-1b (Dai et al., 2011). In the lung,
type-2-cell-mediated immunity to HDM is, however, not affected
by loss of caspase-1, Nlrp3, or Asc-1 (Kool et al., 2011).
Psoriasin (S100A7) is a keratinocyte-derived anti-microbial
protein that is highly expressed in the skin of patients with
atopic dermatitis. More recently, the expression of other mem-
bers of the S100 protein family, S100A8 and S100A9, has also
been found to be increased in lesional AD skin (Gittler et al.,
2012) and by keratinocytes after exposure to HDM (Jin et al.,
2014). These proteins function as intracellular differentiation
markers, and upon secretion, they act as amplifiers of inflam-
matory responses in several inflammatory diseases via two re-
ceptors, Toll-like receptor 4 (TLR4) and receptor of advanced
glycation endproducts (RAGE), that also binds to HMGB1.
S100A9-treated keratinocytes show a dramatic RAGE-depen-
dent increase in the amounts of IL-33 mRNA, whereas no sig-
nificant changes in TSLP or TNF-a mRNA expression are
observed (Jin et al., 2014). The role of these S100 proteins
in vivo in relevant type-2-mediated diseases still needs to be
fully addressed.
Many of the alarmins and DAMPs are released by necrotic cell
death. However, there is little evidence for massive cell death of
ECs after exposure to type-2-cell-mediated immune stimuli in
the lungs. Lung ECs have been shown, however, to recognize
and internalize apoptotic cells in a pathway requiring the actin
binding protein Rac1. Recognition of apoptotic cells leads to a
dampened IL-33 response in ECs, and epithelial-cell-specific
loss of Rac1 exacerbates type-2-cell-mediated immune inflam-
mation in an asthma model (Juncadella et al., 2013).
Pattern Recognition Receptors Prime Type-2-Cell-
Mediated Immunity in Epithelial Cells
Pattern recognition receptor (PRR) triggering on ECs induces the
release of cytokines and chemokines that attract and activate
immune cells. As an example, HDM allergen extract leads to a
chemokine response in isolated lung ECs in a process requiring
b-glucans, suggesting involvement of the dectin receptor (Na-
than et al., 2009). We and others have shown a few years ago,
by using radiation-induced bone marrow chimeric mice, that
TLR4 triggering on lung structural cells is required for the recog-
nition of HDM (and cockroach) and for the development of Th2-
cell-associated asthma features (Hammad et al., 2009; Ullah
et al., 2014). More recently, via the Cre-Lox technology, it has
been confirmed that Th2-cell-mediated immunity to HDM de-
velops only when TLR4 is triggered on Clara cell secretory pro-
tein (CCSP)+ airway epithelial cells (McAlees et al., 2015).
Indeed, HDM-induced ECs produce the chemokines CCL2 and
CCL20, as well as IL-1a, HMGB1, uric acid, TSLP, GM-CSF,
IL-25, and IL-33, in a TLR4-dependent manner (Hammad et al.,
2009; Kool et al., 2011; Willart et al., 2012). How exactly TLR4
gets activated on ECs is a matter of intense study. Some
Immunity
Review
allergens like Der p 2 bind to TLR4 by mimicking the MD2
molecule (Trompette et al., 2009). Cat and dog allergens facili-
tate TLR4 signaling by binding to LPS directly (Herre et al.,
2013). The protease allergens from Aspergillus or endogenously
released proteases lead to cleavage of fibrinogen into smaller
fragments that can directly trigger TLR4 on lung ECs to boost
type-2-cell-mediated immunity (Millien et al., 2013).
Just like ECs in the lung, keratinocytes express a wide range of
PRRs and can therefore also be activated by numerous PAMPs
or DAMPs. A recent study reported that papain, a cysteine pro-
tease used as a model allergen, or Der p 2, a major allergen of
HDM, are able to induce Th2-cell-mediated responses when
applied epicutaneously, in a TLR4-independent manner (Strem-
nitzer et al., 2014, 2015), despite earlier reports of TLR4 depen-
dence when the antigen was injected subcutaneously. Nickel
allergy is one of the most frequent forms of contact hypersensi-
tivity (CHS). Only the human TLR4 molecule is able to bind to
nickel, so mice do not develop CHS. However, mice carrying a
transgenic human TLR4 became fully sensitive to CHS to nickel
(Schmidt et al., 2010). The exposure of human keratinocytes to
double-stranded RNA (TLR3 ligand), flagellin (TLR5 ligand),
and diacylated lipopeptide (TLR2-TLR6 ligand) stimulates these
cells to express TSLP (Takai et al., 2014). Of note, Staphylo-
coccus aureus, a Gram-positive opportunistic bacterium that is
known to colonize the skin of patients with atopic dermatitis, is
also able to stimulate TSLP release by keratinocytes. This could,
for instance, explain how the environment or infections can
contribute to the initiation and/or amplification of Th2-cell-type
skin inflammation including atopic dermatitis (AD) and the
‘‘atopic march,’’ a process describing the progression from
AD to other allergic disorders such as asthma, rhinitis, or food
allergy.
Many allergens are proteases and many nematodes release
proteases. Protease-activated receptors (PAR) are character-
ized by a mechanism of self-activation after specific proteolytic
cleavage of their extracellular domain. Four PARs have been
identified, and PAR2 is the only member activated by serine pro-
teases. Almost all skin cell types express PAR2, including kera-
tinocytes. Lesional skin of AD patients express high amounts of
PAR2, and the activation of PAR2 is thought to be involved in the
pruritus observed in AD (Lee et al., 2010) through the induction of
the innate pro-Th2-cell-associated cytokine TSLP by keratino-
cytes (Moniaga et al., 2013), suggesting that PAR-2 antagonists
might represent a potential therapeutic for treating skin allergic
disorders. Although in the lung, PAR2 activation by the proteo-
lytic activities of some allergens like mold (Chiu et al., 2007),
cockroach (Page et al., 2010), or fungi (Boitano et al., 2011)
seems crucial for generating type responses, in the skin, this
phenomenon has not been well studied.
Even if C-type lectin receptors, TLRs, and PARs are crucial for
type-2-cell-mediated immunity to develop, we urgently need to
understand better how these receptors promote type-2-cell-
associated immune responses when triggered on ECs, whereas
they tend to stimulate Th1 and/or Th17 cell immunity when they
are triggered on hematopoietic cells. It could be that down-
stream signaling from these receptors is fundamentally different.
It has also been insufficiently studied what impact binding to LPS
(e.g., by allergen components) or the presence of a concomitant
ROS response has on PRR signaling in barrier ECs.
The Threshold of EC Activation Influences the Induction
of Type-2-Cell-Mediated Immunity
The activation threshold of ECs and their capacity to activate
immune responses can be profoundly influenced by prior ex-
posure to PAMPs or DAMPs, and this is certainly relevant for
barrier ECs, exposed to both the outside world and organismal
microbiome. This could lead to either increased PRR expression
or modulation of signaling molecules downstream of PRR. As
such, exposure of ECs to respiratory syncitial virus (RSV) or to
cigarette smoke (which causes damage to cells and the release
of reactive oxygen species [ROS]) can sensitize the airway
epithelium to a subsequent environmental exposure (LPS) by
altered expression and membrane localization of TLR4 (Pace
et al., 2008). These data could explain why early exposure to
RSV or cigarette smoke predisposes some individuals to
develop allergic inflammation and asthma. The activation
threshold of ECs can also be regulated at the level of signaling
molecules downstream of PRRs. Triggering of most PRRs leads
to the activation of the transcription factor NF-kB, and many of
the innate pro-Th2-cell-associated cytokines have an NF-kB
binding site in the promotor region (Swamy et al., 2010). The acti-
vation of the classical NF-kB pathway within the lung epithelium
has been shown to play a crucial role in allergic airway inflamma-
tion. The constitutive activation of NF-kB in airway ECs has been
found to be sufficient to activate DCs, breach inhalational toler-
ance, and promote sensitization to the harmless inhaled allergen
ovalbumin (OVA) (Ather et al., 2011), whereas inhibition of epithe-
lial NF-kB reduced Th2 cell recruitment and airway remodeling
(Broide et al., 2005). A more recent study confirmed the impor-
tance of this signaling pathway in ECs after exposure to HDM
(Tully et al., 2013). An early gene induced after NF-kB activation
is the ubiquitin-editing enzyme A20 (also known as TNFAIP3),
which acts as a negative regulator of inflammatory gene induc-
tion in the epithelium, by deubiquitinating K63-linked ubiquitin
chains on TRAF6, a protein mediating signal transduction from
TNF/Toll/IL-1 family members (Kelly et al., 2011). Delivery of
the A20 binding protein ABIN-1 to lung ECs is associated with
a considerable reduction of all features of allergic asthma (El
Bakkouri et al., 2005). The expression of A20 in the gut epithe-
lium is strongly correlated with the appearance of the gut micro-
biome (Wang et al., 2009b). In the lung, induction of A20 might be
induced in the early postnatal period as well, requiring instruction
from the microbial world. Future studies are required to fully un-
derstand how NF-kB is activated or de-activated in ECs. Also,
the existence of epigenetic modifications in this pathway,
caused by previous environmental triggers (viruses, pollutants),
which would alter the threshold of allergen recognition, need to
be further investigated.
In the skin, barrier homeostasis is also maintained by Notch
signaling. Keratinocyte-specific loss of the metalloproteinase
ADAM17 leads to reduced ligand-independent Notch-1 and
-2 signaling, known to suppress production of TSLP, and these
mice develop a severe form of atopic dermatitis, driven by
TSLP, as well as skin barrier defects (Murthy et al., 2012).
The amounts of Notch target genes are severely reduced in
skin of patients with AD. How exactly Notch gets activated
on keratinocytes is a matter of intense study, but the skin
microbiome has a suppressive effect on TSLP expression
(Yockey et al., 2013).
Immunity 43, July 21, 2015 ª2015 Elsevier Inc. 33

Whatever the mechanism by which ECs can downregulate
responses to type 2 immune stimuli, it will be important to
further investigate the therapeutic potential of dampening EC re-
sponses. The evolutionary driving pressure to dampen type-2-
cell-mediated immunity might stem from the chronic relationship
between host, its microbiome, parasitic nematodes (which often
occupy the host for life), and the ubiquitous nature of nematode
infection before the advent of modern medicine.
Altered Epithelial Barrier Function Contributes to
Type-2-Cell-Mediated Immunity
The relationship between exposure to some inhaled antigens
and barrier dysfunction is complex because some proteolytically
active allergens like HDM or fungal proteases could be at the
origin of barrier dysfunction but, at the same time, barrier
dysfunction might facilitate allergens to gain access to immune
cells. In lung biopsies of asthmatic patients, tight junctions
(mostly E-cadherin) are defective, and when ECs from asth-
matics are cultured ex vivo, they also show defective tight junc-
tion development (Xiao et al., 2011). Allergen extracts can also
induce a rapid and transient destabilization of tight junctions
and a loss of E-cadherin expression (Figure 3, left). This effect
has been shown to be dependent on the enzymatic activity of
HDM, cockroach, pollen, and fungi. It is important to note that
DCs receive a tonic inhibitory signal through homotypic E-cad-
herin interactions between DCs and ECs (Nawijn et al., 2011).
A defective E-cadherin expression could activate DCs, and
therefore facilitate allergic sensitization. Moreover, a loss of E-
cadherin in cultured ECs results in the increased production of
TSLP (Heijink et al., 2007), and this event could constitute an
early step in the break of inhalational tolerance leading to Th2
cell sensitization and asthma development. E-cadherin is also
a ligand of KLRG-1 expressed by human and mouse ILC2s. Liga-
tion of KLRG-1 by E-cadherin suppresses the production of IL-5
and IL-13 by ILC2s, and in this way, a loss of E-cadherin might
promote type-2-cell-mediated innate immunity (Salimi et al.,
2013). This is relevant in the skin, because the expression of E-
cadherin is reduced in patients with AD.
34 Immunity 43, July 21, 2015 ª2015 Elsevier Inc.
Immunity
Review
Figure 3. Altered Epithelial Barrier Function
Contributes to Type 2 Immunity
In the gut or in the lung (left), exposure to allergen-
or helminth-derived proteases induces a rapid and
transient destabilization of tight junction proteins
(claudin, occludin) and a loss of E-cadherin
expression by epithelial cells. In the skin and the
esophagus (right), genetic polymorphisms have
been described in several genes involved in
epithelial barrier integrity (filaggrin [FLG], SPINK5,
CAPN14). All these processes lead to a facilitated
penetration of antigens, an increased access to
DCs, and Th2 cell sensitization.
Normal mucosal barriers (skin, GI
tract, lung) can be viewed as very tight
barriers, whose function is to retain mois-
ture and to prevent the penetration of
allergens and microbes. Increasing evi-
dence from genome-wide association
studies (GWASs) and from experimental
models have suggested that a defective
barrier function along with an exaggerated or abnormal immune
response might contribute to the pathophysiology of common
allergic disorders like AD and to less common disorders of
type-2-cell-mediated immunity like eosinophilic esophagitis
(Figure 3, right). As such, filaggrin (FLG) protein is crucial for
the terminal differentiation of keratinocytes. In case of FLG mu-
tations, the epidermal barrier is disrupted due to decreased tight
junction protein expression, and this might lead to the develop-
ment of percutaneous allergen sensitization and Th2 cell re-
sponses (Mócsai et al., 2014). FLG mutations are correlated
with a higher risk of developing allergic diseases like AD, allergic
rhinitis, food allergies, and atopic asthma even though FLG is not
expressed in airway ECs, illustrating that asthma development in
individuals with AD might be secondary to allergic sensitization
that occurs after the breakdown of the epidermal skin barrier
(Ring et al., 2012). Tight junction (TJ) proteins like claudins are
located on keratinocytes of the stratum granulosum, situated
just below the stratum corneum (Kuo et al., 2013). Reduced
expression of the TJ proteins claudin-1 and claudin-23 is
observed only in patients with AD (De Benedetto et al., 2011).
Once these two physical barriers (filaggrin and tight junctions)
are breached, there is an increased risk for AD patients to get
colonized by Staphylococcus aureus. Because S. aureus can
produce high amounts of serine proteases that can degrade
the skin barrier further (Schlievert et al., 2010), this can contribute
to exacerbation of Th2-cell-associated symptoms of the dis-
ease. The balance between proteases and protease inhibitors
therefore seems to be important in maintaining homeostasis in
the skin. Lymphoepithelial Kazal-type-related inhibitor (LEKTI)
is an inhibitor of several serine proteases in the skin, thereby con-
trolling epidermal barrier function (Ovaere et al., 2009). A prema-
ture stop codon mutation in SPINK5, which encodes LEKTI, is
associated with Netherton syndrome, a rare autosomal-reces-
sive skin disease with severe skin inflammation and constant
allergic manifestations (Di et al., 2009). In addition, several
studies have reported that the polymorphisms in SPINK5 are
associated with AD (Walley et al., 2001). In eosinophilic esopha-
gitis, recent studies have also shown evidence for defective
Immunity
Review
epithelial barrier function, with decreased expression of LEKTI
and filaggrin, and SNPs in the CAPN14 gene that codes for an
intracellular cysteine protease belonging to the calpain family
(Simon et al., 2015; Sleiman et al., 2014). Calpains have been
associated with asthma development and the blockade of these
proteins in an asthma model results in a marked improvement
of asthma features (Aich et al., 2012). Altogether, these data
suggest that a very fine balance between proteases, protease in-
hibitors, and junctions contributing to optimal barrier function
therefore seems to be crucial in maintaining immune homeosta-
sis at barrier sites.
Production of Mucus and Mucus-Associated Bioactive
Molecules Promote Type-2-Cell-Mediated Immunity
The airways and the intestinal mucosa are covered with a thick
layer of mucous, consisting of a highly ordered well-hydrated
gel composed of high-molecular-weight glycosylated proteins,
produced by goblet cells, and containing antimicrobial peptides
and metabolites derived from commensal microbiota, and form-
ing yet another barrier to the entry of type 2 cell stimuli. One of the
features of allergic asthma, and one of the major defense mech-
anisms against intestinal nematodes, is IL-13-driven goblet cell
metaplasia (GCM), an increase in goblet cell numbers mostly
due to the transdifferentiation of airway ciliated and Clara cells
or enterocytes to goblet cells, resulting in more mucus produc-
tion, of a different quality. In a healthy gut, expression of the
mucins Muc2 and Muc3 is predominant (Buisine et al., 2001).
Exposure of enterocytes to IL-13 and GCM leads to the produc-
tion of more highly charged glycoproteins and of Mucin 5AC, an
important factor for nematode expulsion in the gut (Hasnain
et al., 2011a). Several transcription factors have been involved
in this process (Hasnain et al., 2011c; Le Cras et al., 2011). A
recent paper has described that SPDEF and Foxa3 overexpres-
sion in ECs is sufficient to induce airway goblet cell differentiation
in neonatal and adult lungs, but strikingly, this also leads to
eosinophilic airway inflammation and the recruitment of group
2 ILCs and DCs (Rajavelu et al., 2015). This is caused by
the SPDEF- and Foxa3-induced release of EC-derived IL-33,
TSLP, and GM-CSF. SPDEF- and Foxa3-induced GCM might
therefore play a role in the establishment of Th2 cell polarization,
influencing subsequent responses to different environmental
exposures. Similarly, Muc5AC-deficient mice infected with the
nematode T. muris had increased concentrations of IFN-g (Has-
nain et al., 2011a). Clearly more research is needed before
we understand the full impact of altered secretory capacity of
ECs on immune polarization in the mucosa. One possibility is
that secretion of mucins interferes with the unfolded protein
response ([UPR], a cellular response to stress), that can intersect
with inflammatory response pathways, including NF-kB activa-
tion (Janssens et al., 2014). In airway epithelial cells of human
asthmatics, IL-13-induced GCM leads to induction of Xbp1
splicing (Martino et al., 2013). This process is very similar to
mucus production in the gut and depends on the mucosal-
restricted endoplasmic reticulum (ER) stress sensor IRE1b, lead-
ing to induction of the protein disulfide isomerase Agr2. Genetic
polymorphisms in XBP1 and AGR2 are associated with IBD and
could act far upstream in the immunological cascade.
RELMb is produced by goblet cells in the intestine and in the
lung and is secreted into the lumen at high concentration as a ho-
modimer. In the gut and in the lung, RELMb is induced by the Th2
cell cytokines IL-4 and IL-13 produced during a helminth infec-
tion or after exposure to fungal allergens (Herbert et al., 2009).
RELMb has been shown to have very distinct effects in the gut
and in the lung. In the gut, RELMb is crucial in promoting protec-
tion against gastrointestinal worm infection and against colitis
development by regulating mucous secretion, thus contributing
to barrier integrity (Krimi et al., 2008). In the lung, RELMb favors
leukocyte recruitment, collagen deposition, and lung remodeling
(Fang et al., 2015).
Alterations in EC-Repairing Factors Might Promote
Type-2-Cell-Mediated Immunity
Given the complex composition of mucus and the metabolites
and antimicrobial peptides contained in it, it will be important
to study whether type 2 immune cells sense mucus composition
directly, and whether mucus composition is altered by type 2 im-
mune cells. In this regard, IL-22-producing ILC3s have already
been shown to alter the glycosylation state of gut epithelial cells
in a microbiome-dependent manner, leading to protection from
invasive pathogens (Goto et al., 2014). IL-22 is also found in
the airways of asthmatic humans and mice, but the exact source
(Th22 cells or NKp46+ ILCs) and function requires further study.
Whereas Il22 / mice have severely reduced type 2 immunity in
an OVA-alum model of asthma (Besnard et al., 2011), recombi-
nant IL-22 was also shown to reduce airway inflammation by
reducing the epithelial production of IL-25 (Takahashi et al.,
2011). It is unclear at present whether alterations in mucus con-
tent or glycosylation would contribute to the suppression of IL-
25. During nematode infection, IL-22 has been found responsible
for the induction of GCM and worm expulsion (Turner et al.,
2013). Amphiregulin (made by Th2, ILC2, and mast cells) has
also been shown to alter the mucin composition in the lung
and gut (Okumura et al., 2005). Amphiregulin also causes a hy-
perproliferative state in the gut epithelium, which eventually
leads to parasite expulsion. Undoubtedly, this hyperproliferative
response will also alter cytokine production by ECs.
Trefoil factors are epithelial-derived cytokines that promote
epithelial healing, mucosal secretion, and intestinal integrity.
Allergens like HDM, Aspergillus, and OVA lead to increased pro-
duction of TFF2 in lung ECs, in a STAT6-dependent manner (Ni-
kolaidis et al., 2003). Although TFF-2 is required for repair of lung
injury after N. brasiliensis passage through the lungs, TFF2 is also
a potent inducer of IL-33. TFF2 is thus able to promote allergen-
driven airway type-2-cell-mediated immunity.
Epithelial Cytokines in Human Type-2-Cell-Associated
Chronic Inflammatory Diseases
Several recent reviews deal with epithelial pathways in chronic
type-2-cell-mediated immune diseases, like asthma, atopic
dermatitis, and chronic helminth infections, so we will only briefly
illustrate some studies on the importance of ECs in controlling
these diseases (Grencis et al., 2014; Hallstrand et al., 2014; Lam-
brecht and Hammad, 2012). The evidence for increased produc-
tion of pro-Th2 cell cytokines by ECs is overwhelming in asthma,
and many genetic association studies point to an important role
for ECs in this disease. In mice, the overexpression of TSLP in the
lung epithelium leads to the spontaneous development of an
asthma-like disease (Zhou et al., 2005). A wide variety of stimuli,
Immunity 43, July 21, 2015 ª2015 Elsevier Inc. 35

including proteolytic allergens, diesel-exhaust particles, ciga-
rette smoke, and respiratory viruses can induce the epithelial
release of TSLP (Bleck et al., 2010). The lung epithelium of asth-
matics contains a higher number of TSLP+ cells, and the bron-
choalveolar lavage of these patients had higher concentrations
of TSLP protein compared to healthy individuals (Bleck et al.,
2015). Genetic polymorphisms in the promoter region of TSLP
and IL33R (IL1RL1) are associated with increased risk of asthma
(Harada et al., 2011). Of note, airway ECs from asthmatic pa-
tients secrete more TSLP when exposed to dsRNA (viral analog)
(Brandelius et al., 2011). This might partly explain why patients
with asthma get an exacerbation of their disease after being
exposed to such triggers (Jackson and Johnston, 2010). It is
therefore very likely that an aberrant production of TSLP might
influence both the susceptibility of certain individuals to develop
asthma and the clinical complications developing after exposure
to some environmental triggers. Although IL-25 is produced by
ECs of patients with Th2-cell-high corticosteroid-responsive
asthma (Cheng et al., 2014), the precise role of this cytokine is
still unclear. Recent studies have linked IL-25 and IL-33 to vi-
rus-induced asthma exacerbations (Bousquet et al., 2014).
Although there are no reports showing virus-induced increased
IL-33 production by ECs in asthmatic patients, it is very plausible
that the injury caused by respiratory viruses to ECs might lead to
enhanced IL-25 and IL-33 production, thereby driving an acute
exacerbation of allergic asthma via DC and/or ILC2 activation
(Kumar et al., 2014). Also, primary ECs from allergic children
exposed to RSV are found to produce low amounts of type I
IFN and had higher viral shedding compared to healthy ECs
(Spann et al., 2014). Of note, these features are absent when
adult ECs were used (Patel et al., 2014).
TSLP has also emerged as a key cytokine in the pathogenesis
of Th2-cell-associated AD (Ziegler and Artis, 2010). Early studies
have shown that TSLP overexpression in keratinocytes leads to
the spontaneous development of AD-like disease in mice (Li
et al., 2005). Moreover, TSLP overexpression in keratinocytes
during epicutaneous OVA or HDM sensitization has the potential
to aggravate features of asthma upon re-exposure to the allergen
via the airways. Of note, epicutaneous sensitization with peanut
promotes intestinal food allergy through a mechanism involving
TSLP production by keratinocytes (Noti et al., 2014). These
data strongly suggest that TSLP (over)produced in the skin could
be a risk factor for the development of Th2-cell-mediated dis-
eases like allergic airway inflammation or food allergy. Increased
expression of TSLP has been demonstrated in skin keratinocytes
from AD patients, and elevated serum TSLP concentrations have
been reported in AD children. The association of AD with a poly-
morphism of the IL1RL1 gene (Shimizu et al., 2005) suggests that
the IL-33-ST2 system might contribute to the development or
worsening of the disease. IL-33 expression is upregulated in ker-
atinocytes and endothelial cells in AD (Oboki et al., 2010; Savinko
et al., 2012), as well as in the serum of AD patients (Tamagawa-
Mineoka et al., 2014). The specific overexpression of IL-33 by
keratinocytes leads to the spontaneous development of derma-
titis with eosinophilic infiltration and activation of IL-5-producing
ILC2s (Imai et al., 2013). Moreover, IL-33 release by keratinocytes
has been shown to drive the pro-Th2 cell capacities of skin DCs in
a food allergy model based on the epicutaneous application of
peanut extracts (Tordesillas et al., 2014). The blockade of ST2
36 Immunity 43, July 21, 2015 ª2015 Elsevier Inc.
Immunity
Review
at the time of epicutaneous sensitization strongly reduces the
ability of skin-derived DCs to induce the proliferation and Th2-
cell-associated cytokine production by allergen-specific CD4+
T cells (Tordesillas et al., 2014).
Conclusions
There has been great progress in the study of type-2-cell-medi-
ated immune responses driven by allergens and nematodes in
barrier sites of the body. From these studies, a picture emerges
whereby epithelial cells constitute a first physical, chemical, and
immunological barrier that can be seen almost as an integral
part of the innate defense mechanism. The ECs express pattern
recognition receptors, integrate information from many receptors
simultaneously, and talk to the innate and adaptive immune sys-
tem cells by releasing chemokines, cytokines, and alarmins. This
recognition event is tightly regulated and influenced by prior expo-
sure history and is terminated appropriately when the type-2-cell-
mediated immune stimulus disappears. In certain chronic inflam-
matory diseases like asthma and AD, dysregulated EC responses
or barrier function could be the driver of the disease process. In the
future, the challenge will be to translate this new knowledge to
novel preventive or curative strategies for these disorders.
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