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Intestinal epithelial tuft cells initiate type 2 mucosal
immunity to helminth parasites
François Gerbe, Emmanuelle Sidot, D. J. Smyth, M. Ohmoto, I. Matsumoto,
V. Dardalhon, Pierre Cesses, Laure Garnier, Marie Pouzolles, Bénédicte
Brulin, et al.
To cite this version:
François Gerbe, Emmanuelle Sidot, D. J. Smyth, M. Ohmoto, I. Matsumoto, et al.. Intestinal epithelial
tuft cells initiate type 2 mucosal immunity to helminth parasites. Nature, Nature Publishing Group,
2016, 529 (7585), pp.226–30. �10.1038/nature16527�. �hal-01940966�
226 | NATURE | VOL 529 | 14 JANUARY 2016
LETTER
doi:10.1038/nature16527
Intestinal epithelial tuft cells initiate type 2
mucosal immunity to helminth parasites
François Gerbe
1,2,3
, Emmanuelle Sidot
1,2,3
, Danielle J. Smyth
4
†, Makoto Ohmoto
5
, Ichiro Matsumoto
5
, Valérie Dardalhon
3,6
,
Pierre Cesses
1,2,3
, Laure Garnier
1,2,3
, Marie Pouzolles
3,6
, Bénédicte Brulin
1,2,3
, Marco Bruschi
1,2,3
, Yvonne Harcus
4
,
Valérie S. Zimmermann
3,6
, Naomi Taylor
3,6
, Rick M. Maizels
4
† & Philippe Jay
1,2,3
Helminth parasitic infections are a major global health and social
burden
1
. The host defence against helminths such as Nippostrongylus
brasiliensis is orchestrated by type 2 cell-mediated immunity
2
.
Induction of type 2 cytokines, including interleukins (IL) IL-4
and IL-13, induce goblet cell hyperplasia with mucus production,
ultimately resulting in worm expulsion
3,4
. However, the mechanisms
underlying the initiation of type 2 responses remain incompletely
understood. Here we show that tuft cells, a rare epithelial cell type in
the steady-state intestinal epithelium
5
, are responsible for initiating
type 2 responses to parasites by a cytokine-mediated cellular
relay. Tuft cells have a Th2-related gene expression signature
6
and we demonstrate that they undergo a rapid and extensive IL-
4Rα-dependent amplification following infection with helminth
parasites, owing to direct differentiation of epithelial crypt
progenitor cells. We find that the Pou2f3 gene is essential for tuft
cell specification. Pou2f3
−/−
mice lack intestinal tuft cells and have
defective mucosal type 2 responses to helminth infection; goblet
cell hyperplasia is abrogated and worm expulsion is compromised.
Notably, IL-4Rα signalling is sufficient to induce expansion of the
tuft cell lineage, and ectopic stimulation of this signalling cascade
obviates the need for tuft cells in the epithelial cell remodelling of the
intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2
immune responses. Our data reveal a novel function of intestinal
epithelial tuft cells and demonstrate a cellular relay required for
initiating mucosal type 2 immunity to helminth infection.
Experimental subcutaneous infection of mice with N. brasiliensis
(Nb) stage 3 larvae induces a typical type-2 response that involves a
remodelling of epithelial cell populations, with goblet cell hyperplasia
visible as soon as 5 days post-infection
3,4
. Nb L3 larvae first migrate
from their injection site to the lungs, where they moult to the L4 stage,
are coughed up, and swallowed to reach the intestines (day 2 post infec-
tion) where they mature and lay eggs (starting 5 days post-infection).
Nb induces a rapid and robust type 2 response, resulting in worm expul-
sion by 6–8 days post infection.
While the doublecortin-like kinase 1 (Dclk1)-expressing tuft cells
represent only 0.4% of intestinal epithelial cells in naive mice
5
, we found
that Nb infection resulted in a 8.5-fold expansion in tuft cells (Fig. 1a, b),
first detected by 5 days post-infection in intestinal crypts, where pro-
liferative epithelial progenitor cells reside, and also in the villi by 7 days
post infection (Fig. 1c, Extended Data Fig. 1a). The kinetics of tuft
cell expansion was equivalent to that of goblet cells (Fig. 1d, Extended
Data Fig. 1b). Neo-differentiated tuft cells were indistinguishable from
tuft cells present in naive mice, as evaluated by expression of estab-
lished tuft cell markers, including Dclk1, Sry-related transcription fac-
tor 9 (Sox9), and phospholipase C gamma 2 (Plcγ2) (Extended Data
Fig.1c)
6–8
. All tuft cells, characterized by Dclk1 and growth factor
independent 1b (Gfi1b)
8
expression also co-expressed the Pou domain,
class 2, transcription factor 3 (Pou2f3) (Fig. 2a). In addition, rare
(<3%, n = 400 cells counted) Pou2f3
+
;Dclk1
low
or Pou2f3
+
;Dclk1
−
cells
1
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, F-34094 Montpellier, France.
2
INSERM, U1191, F-34094 Montpellier, France.
3
Université de Montpellier, F-34000 Montpellier, France.
4
Institute of Immunology and Infection Research, School of Biological Sciences and Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh EH9 3JT, UK.
5
Monell
Chemical Senses Center, 3500 Market Street, Philadelphia, Pennsylvania 19104, USA.
6
Institut de Génétique Moléculaire de Montpellier, CNRS, UMR5535, F-34293 Montpellier, France.
†Present address: Wellcome Trust Centre for Molecular Parasitology, Institute for Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK.
ab
c
d
Naive Nb infected
Naive Nb infected
Naive
Nb infected
Tuft cells per crypt–villus axis
Naive
Nb
infected
0
5
10
15
20
25
P < 0.0001
Day 7 Day 7
Day 7 Day 7
Day 7 Day 7
Day 4
Day 4
Day 5
Day 5
Figure 1 | Rapid amplification of the tuft cell lineage following infection
with Nb. a, Presence of tuft cells in the intestinal epithelia of naive and
Nb-infected mice 7 days post infection, visualized by expression of the
Dclk1 marker. b, 8.7-fold increase of tuft cell numbers (1.8 ± 1.4 to
15.6 ± 4.8 per crypt–villus axis) in Nb-infected mice compared to naive
mice, 7 days post infection. (n = 50 crypt–villus units per mouse; 3 mice
per condition). Data are shown as means ± s.d. (P < 0.0001, two-tailed
Student’s t-test with Welch’s correction). c, Changes in the Dclk1-expressing
tuft cell population in intestinal crypts are presented at the indicated time
points post infection. Quantification is shown in Extended Data Fig. 1a.
d, Corresponding goblet cell hyperplasia associated with numerous and
larger mucus vacuoles, detected by periodic acid-Schiff (PAS) staining.
Dclk1 cells are also visualized in brown. Quantification is shown in
Extended Data Fig. 1b. Scale bars, 20 μm. All panels show representative
pictures of experiments replicated 3 times in 3 mice per condition.
© 2016 Macmillan Publishers Limited. All rights reserved
14 JANUARY 2016 | VOL 529 | NATURE | 227
Letter
reSeArCH
were found at the base of crypts, probably representing early differ-
entiating tuft cells since villus Pou2f3
+
cells always co-express Gfi1b
and Dclk1. Following infection, the percentage of proliferating tuft
cells in crypts increased from 13 ± 5.6% to 24 ± 14.9% (P = 0.035),
indicating that cell proliferation contributes to the amplifica
-
tion of the tuft lineage during type 2 responses (Extended Data
Fig. 1d, e). Examination of the location of tuft cells present in Nb-
infected mice revealed that some tuft cells differentiate close to
the stem cell zone (Extended Data Fig. 1d), suggesting that biased
differentiation from the recently described Lgr5
+
slowly cycling
early secretory progenitors
9
and Dll1
+
secretory progenitors
10
also contributes to tuft cell lineage amplification. The increase in
tuft cells was not due to a non-specific amplification of all secretory
cell lineages as the number of enteroendocrine cells expressing the
insulinoma-associated 1 (Insm1) marker
11
, another secretory line-
age of the intestinal epithelium, was significantly (P = 0.008) reduced
(Extended Data Fig. 1f, g).
To determine whether the increase in the tuft cell population follow-
ing infection with Nb was specific to C57BL/6 mice, we infected BALB/c
mice and also observed a significant increase in tuft cell numbers
(14-fold, P < 0.0001; Extended Data Fig. 2a, b). Moreover, this response
seems to be a common adaptation to helminth infection in general, as
infection of C57BL/6 and BALB/c mice strains with Heligmosomoides
polygyrus
12
also resulted in a significant increase in tuft cell numbers
(6.1- and 8.3-fold, respectively, P < 0.0001; Extended Data Fig. 2c, d).
Tuft cell hyperplasia following Nb infection also occurred in Rag
−/−
mice (10-fold; P < 0.0001) and therefore does not require functional
adaptive immunity (Extended Data Fig. 2e, f).
Epithelial remodelling following helminth infection includes goblet
cell hyperplasia and changes in mucus composition, associated with
protective type 2 immunity
13,14
. To investigate the role of tuft cells
in this process, we identified and characterized a tuft-cell-deficient
mouse line. Mice deficient for the Pou2f3 transcription factor lack all
Pou2f3-expressing taste receptor cells including sweet, umami and
bitter taste cells
15
, as well as Trpm5-expressing chemosensory cells
in the nasal cavity
16
and olfactory epithelium
17
. Analysis of Pou2f3-
deficient mice revealed a unique phenotype in the intestinal epithe-
lium, with a complete absence of tuft cells as assessed by the absence
of Pou2f3, Dclk1 and Sox9 expression outside the crypt compartment
(Fig. 2b). The stem cell compartment, proliferation zone, and differ-
entiation of enterocytes, goblet, enteroendocrine and Paneth cells
were not affected (Fig. 2c and Extended Data Fig. 3). Furthermore,
the distribution of immune cells in lymph nodes, mesenteric lymph
nodes, spleen and lamina propria of Pou2f3
+/+
and Pou2f3
−/−
mice
was equivalent (Extended Data Fig. 4) and lymphocytes were capable
of responding to immune stimulation (Extended Data Fig. 5). Notably,
type 2 innate lymphoid cells (ILC2s), a lineage that plays a critical role
in secreting type 2 cytokines in response to helminth infection
18,19
,
were present in both the mesenteric lymph nodes and lamina propria
of Pou2f3
−/−
mice, at levels that were not significantly different from
wild-type mice. (Extended Data Fig. 6a–c). Therefore, the absence of
NS
Cell number per crypt–villus axis
0
5
10
15
20
Dclk1 Ki67
Olfm4 PAS UEA1 Insm1
a
b
c
Overlay
Pou2f3
G1b
Dclk1
Pou2f3
+/+
Pou2f3
+/+
Pou2f3
–/–
Pou2f3
–/–
NS
NS
NS
NS
P < 0.0001
Pou2f3 Pou2f3
Dclk1 Dclk1
Sox9 Sox9
Figure 2 | Absence of tuft cells in the intestinal epithelium of Pou2f3
−/−
mice. a, Pou2f3 is expressed specifically in tuft cells of the intestinal
epithelium as determined by co-staining for Pou2f3 and established
markers of tuft cells such as Dclk1 and Gfi1b. b, Pou2f3 deletion results
in the absence of tuft cells as monitored by staining intestinal epithelium
from Pou2f3
+/+
and Pou2f3
−/−
mice with Pou2f3-, Dclk1- and Sox9-
specific antibodies. a, b, Three mice per genotype were used for staining
experiments. Scale bars, 20 μm. c, Pou2f3 deficiency does not affect the
proliferation zone (P = 0.22), stem cell compartment (P = 0.66), enterocyte
(not counted), goblet (P = 0.83), Paneth (P = 0.60) or enteroendocrine
(P = 0.23) cell lineages as monitored by Ki67, Olfm4, alkaline phosphatase,
PAS staining, UEA1 lectin, and Insm1, respectively. (n = 50 crypt–villus
units per mouse; 3 Pou2f3
−/−
and 3 wild-type mice). Data are shown
as means ± s.d. A two-tailed Student’s t-test was used. Pictures show
representative experiments replicated 3 times.
© 2016 Macmillan Publishers Limited. All rights reserved
228 | NATURE | VOL 529 | 14 JANUARY 2016
Letter
reSeArCH
Pou2f3 does not affect global immunity or intestinal epithelium for-
mation. Rather, Pou2f3 represents the first identified transcription factor
that is specifically required to specify the tuft cell lineage in the intestinal
epithelium, analogously to Sox9 for Paneth
20,21
and Ngn3 for enteroen-
docrine
22
cell lineages. Thus, Pou2f3
−/−
mice represent a powerful model
to study the function of tuft cells.
Pou2f3
+/+
and Pou2f3
−/−
mice were infected with Nb and ana-
lysed at several time points. In Pou2f3
+/+
mice, only few worms were
found after 9 days and expulsion was nearly complete after 13 days. In
sharp contrast, numerous worms were found in Pou2f3
−/−
mice up to
42 days post infection (Fig. 3a, b), not only in the proximal part of
the small intestine, their normal site of attachment
23
, but also in more
distal locations. Together, these data strongly suggest that a compro-
mised type-2 response is responsible for prolonged worm survival in
Pou2f3
−/−
tuft-cell-deficient mice.
To understand the mechanisms underlying the delayed worm
expulsion in Pou2f3-deficient mice, we analysed the type-2 response-
dependent remodelling of the intestinal epithelium 7 days after infec-
tion, a time point at which adult worms were detected in all infected
animals. In Pou2f3
+/+
mice, the intestinal epithelium displayed exten-
sive and generalized goblet cell hyperplasia, with large mucus vacu-
oles, and tuft cell hyperplasia (Fig. 3c). Expectedly, Pou2f3
−/−
mice
completely lacked tuft cells and, in contrast to Pou2f3
+/+
mice, were
devoid of overt goblet cell hyperplasia, with focal and moderate hyper-
plasia limited to the most proximal small intestine, and lower goblet
cell numbers than wild-type mice (Fig. 3c, Extended Data Fig. 7a, and
Supplementary Information 1 and 2). Therefore, tuft-cell-deficient
mice have a delayed type 2 response, with deficient mucosal goblet cell
hyperplasia and delayed control of Nb infection.
The goblet cell-produced Resistin-like beta (Retnlβ) molecule,
strongly induced by type 2 cytokines, has direct anti-helminth activ-
ity that facilitates expulsion
3,24
. We compared expression of Retnlβ in
wild-type and Pou2f3
−/−
mice 7 days after Nb infection, when worm
expulsion had started in wild-type mice. Retnlβ was strongly expressed
in hyperplastic goblet cells in Pou2f3
+/+
mice, but was only weakly
expressed in Pou2f3
−/−
mice (Fig. 3c, d and Extended Data Fig. 7a).
Moreover, while IL-4 levels were equivalent in mucosal tissue of
Nb-infected Pou2f3
+/+
and Pou2f3
−/−
mice, IL-13 levels were markedly
decreased in the latter (Fig. 3d). As both IL-4 and IL-13 type 2 cytokines
are known to regulate Retnlβ expression
3
, and IL-4 is dispensable dur-
ing type 2 responses to Nb
25
, our data strongly suggest that defective
IL-13 production is responsible for the decreased Retnlβ expression
in Nb-infected Pou2f3
−/−
mice. Thus, we identify a defective IL-13/
Retnlβ axis in tuft-cell-deficient mice with impaired worm expulsion.
We next studied the link between tuft cells and type-2-mediated
mucosal adaptation following Nb infection. IL-4Rα signalling is
essential for both goblet cell hyperplasia and type 2 immune responses
occurring upon helminth infection, and deletion of the Il4rα gene abro-
gates Nb expulsion
23,26
. Importantly, the Nb-induced tuft cell hyper-
plasia occurring in wild-type mice 7 days post infection was absent in
Il4rα
−/−
mice, as was goblet cell hyperplasia (Fig. 3e, Extended Data
Fig. 7b). This demonstrates the critical role of IL-4Rα signalling in
the expansion of the tuft cell population following helminth infection.
We then examined whether IL-4Rα signalling is sufficient to trig-
ger tuft cell lineage hyperplasia by injecting naive C57BL/6 mice with
recombinant murine IL-4 and/or IL-13 (rIL-4/rIL-13) for 5 days and
assessing the histology of the intestinal epithelium. rIL-4/rIL-13 injec-
tion induced goblet cell hyperplasia together with tuft cell expansion
(Extended Data Fig. 7c). Importantly, treatment of Pou2f3
−/−
mice with
rIL-4/rIL-13 also resulted in goblet as well as Paneth cell hyperplasia,
indicating a function of tuft cells upstream of IL-4/IL-13 (Extended Data
Fig. 7c, d). Moreover, ectopic IL-4/IL-13 induced Retnlβ expression in
goblet cells, independently of the Pou2f3 genotype. Retnlβ expression
was found predominantly in crypts and was therefore delayed com-
pared to the onset of goblet cell hyperplasia (Extended Data Fig. 7c), and
quantitatively lower than in an infectious context (Fig. 3c). Thus, IL-4Rα
signalling is sufficient to induce an expansion of the tuft cell lineage.
Furthermore, ectopic stimulation of this signalling cascade obviates
the need for tuft cells in the epithelial cell remodelling of the intestine,
including induction of Retnlβ expression by hyperplastic goblet cells.
To determine whether the IL-4/IL-13-induced goblet cell hyperplasia
was epithelial-cell-autonomous, we used an ex vivo organoid culture
0
2
4
6
8
ab
Worm burden
Eggs per gram faeces (10
3
)
0
20
40
60
80
91342
56710121520 26 33 40
Pou2f3
+/+
Pou2f3
–/–
Pou2f3
+/+
Pou2f3
–/–
Dclk1
PAS-Prox
PAS-Dist Retnlβ-Prox
Retnlβ-Dist
Dclk1
PAS-Prox
PAS-Dist Retnlβ-Prox
Retnlβ-Dist
c
e
Pou2f3
–/–
Pou2f3
+/+
Dclk1
PAS
Il4RD
+/+
Il4RD
–/–
Il4RD
+/+
Il4RD
–/–
Il4RD
+/+
Il4RD
–/–
Il4RD
+/+
Il4RD
–/–
Naive
Naive
Naive
Naive
Nb infected
Nb infected
Nb infected
Nb infected
d
RetnlE
Il13
Gapdh
WT
KO
Naive
H
2
O
Figure 3 | Impaired type 2 responses in tuft cell-deficient mice.
a, Live adult worm counts in the small intestines of wild-type and Pou2f3
−/−
mice at days 9, 13 and 42 post infection with Nb (n = 3 wild-type
mice and 4 Pou2f3
−/−
mice for each time point except for day 9 where
n = 3 Pou2f3
−/−
mice). b, Kinetic of Nb infection in 3 wild-type and 4
Pou2f3
−/−
mice, assessed by faecal eggs count. a, b, Each circle represents
an individual mouse. The x axis indicates time (days) post-infection.
Average values ± s.d. are shown. c, Immunohistochemistry illustrating the
proximal and distal small intestinal epithelium of infected wild-type and
Pou2f3
−/−
mice 7 days after infection (n = 3 mice per genotype).
Dclk1 and PAS stainings, respectively, reveal tuft and goblet cells, as well as
Retnlβ production. d, Quantification of IL-13 and Retnlβ in the intestinal
mucosa of naive, and Nb-infected Pou2f3
+/+
and Pou2f3
−/−
mice by
RT–PCR, 7 days after infection. Representative gels are shown with relative
Gapdh expression presented as an internal control. e, Histological analysis
showing tuft (Dclk1 staining) and goblet (PAS staining) cells in naive and
Nb-infected Il4Rα
+/+
and Il4Rα
−/−
mice 7 days post infection (n = 3 mice
per genotype). Scale bars, 20 μm. All panels show representative pictures of
experiments replicated 3 times.
© 2016 Macmillan Publishers Limited. All rights reserved
14 JANUARY 2016 | VOL 529 | NATURE | 229
Letter
reSeArCH
system
27
that allows physiological responses of an isolated intestinal
epithelium to be analysed in the absence of stromal cues. As expected,
tuft cells were absent in Pou2f3
−/−
organoid cultures (Extended Data
Fig. 8a). Moreover, in wild-type organoids, the tuft cell population
increased as early as 48 h following addition of rIL-4/rIL-13 (Extended
Data Fig. 8a, b). Treatment with rIL-4 or rIL-13 alone yielded identical
results to the rIL-4/rIL-13 mixture (Extended Data Fig. 8c). Treatment
of Pou2f3
−/−
organoids with rIL-4/rIL-13 also triggered goblet cell
hyperplasia equivalent to that detected in Pou2f3
+/+
organoids, as
indicated by Retnlβ expression (Extended Data Fig. 8d), revealing
the critical role of type 2 cytokine signalling downstream of the tuft
cell lineage. Furthermore, these data demonstrate that the intestinal
epithelial response to IL-4/IL-13 is epithelium-autonomous and does
not require additional stromal signals. Together, our data identify a
novel function of tuft cells in initiating the mucosal type 2 responses
with a positive feedback loop through IL-13-producing immune cells
that, in turn, amplify the tuft cell lineage.
Finally, we investigated the physiological function of the tuft cell
hyperplasia, fully established by 7 days post-infection when worm
expulsion starts. IL-25 is an alarmin molecule produced by an as yet
unidentified intestinal epithelial cell type, capable of initiating type 2
responses by stimulating ILC2s to produce IL-4 and IL-13
18,19
. We thus
analysed Il25 messenger RNA expression in Pou2f3
+/+
and Pou2f3
−/−
mice infected with Nb. Nine days after infection, Il25 expression was
higher in the intestinal mucosa of Pou2f3
+/+
mice than in tuft-cell-
deficient Pou2f3
−/−
mice (Fig. 4a). Moreover, IL-25 protein expression
was restricted to tuft cells in naive mice (Fig. 4b and Extended Data
Fig. 9a) and consistent with these data, Il25 mRNA was only detected
in the FACS-enriched tuft cell fraction of the intestinal epithelium
(Fig. 4c and Extended Data Fig. 9b). Following Nb infection, IL-25
expression remained restricted to tuft cells (Fig. 4b). Concomitant
with tuft cell hyperplasia, epithelial IL-25 expression peaks 9 days
after infection with Nb, at the time of worm expulsion, for which
it is required
28
. In accord with a critical role for IL-25-secreting
tuft cells in the expansion of ILC2s, we found that the percentage
of Lin
−
CD127
+
Gata3
+
KLRG1
+
ILC2s was not significantly aug-
mented by Nb infection of Pou2f3
−/−
mice, but was significantly
augmented in wild-type mice. Indeed, tuft cells were required for
the global induction of an adaptive immune response as helminth
infection induced an approximately 2.5-fold expansion of both ILC2
and Th2 subsets in mesenteric lymph nodes, whereas these sub-
sets remained unchanged in the infected Pou2f3
−/−
mice (P = 0.02,
P = 0.0005, respectively; Extended Data Fig. 6d–f). It is likely that
these immune defects are directly due to the paucity of IL-25 as
treatment of Nb-infected Pou2f3
−/−
mice with rIL-25 almost com-
pletely compensated for the absence of tuft cells, promoting an effi-
cient worm expulsion (Fig. 4d). IL-25 thus provides a mechanistic
link between tuft cells, promotion of type 2 responses and worm
expulsion.
Taken together, our data reveal a critical function of tuft cells in ini
-
tiating mucosal type 2 responses following infection with helminths
through IL-25 secretion. In the absence of tuft cells, IL-25 and IL-13
expression remain low, and type 2 mucosal responses and worm expul-
sion are delayed. Our study demonstrates a requirement for tuft cells
upstream of IL-4/IL-13, with these cytokines driving tuft cell hyperpla-
sia, thereby amplifying a feed-forward loop to orchestrate a rapid and
effective anti-helminth immunity (Fig. 4e).
Online Content Methods, along with any additional Extended Data display items and
Source Data, are available in the online version of the paper; references unique to
these sections appear only in the online paper.
Received 19 August; accepted 10 December 2015.
1. Hotez, P. J. et al. Helminth infections: the great neglected tropical diseases.
J. Clin. Invest. 118, 1311–1321 (2008).
2. Allen, J. E. & Maizels, R. M. Diversity and dialogue in immunity to helminths.
Nature Rev. Immunol. 11, 375–388 (2011).
3. Herbert, D. R. et al. Intestinal epithelial cell secretion of RELM-beta
protects against gastrointestinal worm infection. J. Exp. Med. 206, 2947–2957
(2009).
4. McKenzie, G. J., Bancroft, A., Grencis, R. K. & McKenzie, A. N. A distinct role
for interleukin-13 in Th2-cell-mediated immune responses. Curr. Biol. 8,
339–342 (1998).
5. Gerbe, F. et al. Distinct ATOH1 and Neurog3 requirements dene tuft cells as
a new secretory cell type in the intestinal epithelium. J. Cell Biol. 192,
767–780 (2011).
6. Bezençon, C. et al. Murine intestinal cells expressing Trpm5 are mostly brush
cells and express markers of neuronal and inammatory cells. J. Comp. Neurol.
509, 514–525 (2008).
7. Gerbe, F., Brulin, B., Makrini, L., Legraverend, C. & Jay, P. DCAMKL-1 expression
identies tuft cells rather than stem cells in the adult mouse intestinal
epithelium. Gastroenterology 137, 2179–2180 (2009).
Helminth
Villus
Crypt
IL-25
IL-25
IL-25
IL-25
IL-25
ILC2
Th2
IL-4
IL-13
Goblet cells
hyperplasia
Tuft cell lineage
amplication
Tuft cells
Goblet cells
Paneth cells
Enterocytes
CBC cells
Nb
day 6
Nb
day 7
Nb
day 8
Nb
day 9
0
10
20
30
40
Pou2f3
+/+
Pou2f3
–/–
Pou2f3
–/–
+rIL-25
Eggs per gram faeces (10
3
)
WT
WT
KO
H2O
KO
H2O
a b
c
d
e
Il25
Gapdh
Naive Nb day 7
IL-25 Pou2f3
Siglec-F
+
–
+
–
+
–
Il25
Pou2f3
Gapdh
NS
NS
NS
NS
NS
NS
P = 0.006
P = 0.005
P = 0.001
P = 0.0006
P = 0.034
P = 0.011
Nb
day 5
Figure 4 | Tuft cells express IL-25, and rIL-25 is sufficient to initiate
type 2 mucosal responses in the absence of tuft cells. a, Analysis of Il25
mRNA expression in Pou2f3
+/+
and Pou2f3
−/−
mice infected with Nb,
9 days post-infection, by RT–PCR. Gapdh expression is presented as an
internal control. b, Immunohistochemistry showing IL-25 expression
in naive and Nb–infected wild type mice. Blue staining, nuclear Pou2f3
expression revealed with NBT/BCIP. Brown staining, IL-25 expression
revealed with DAB (n = 3 naive and 3 infected mice). Scale bars, 20 μm.
c, PCR with reverse transcription (RT–PCR) showing predominant Il25
and Pou2f3 mRNA expression in the FACS-enriched tuft cells fractions
(+) and the other epithelial cells (−), obtained from 3 independent
mice. Gapdh is shown as an internal control. d, Rescue of the Pou2f3
deficiency by treatment with exogenous rIL-25, as assessed by egg counts
during a time course of infection with Nb (n = 7 mice for the wild-type
and Pou2f3
−/−
mice, and n = 6 for the rIL-25-treated Pou2f3
−/−
mice).
Average ± s.d. are presented, as well as exact P values when < 0.05 (two-tailed
Mann–Whitney U-test). e, Scheme illustrating the function of tuft cells in
initiating type 2 responses following infection with intestinal helminths.
Left, normal epithelium undergoing infection with a helminth. Right, tuft
cell-dependent epithelial remodelling during type 2 responses. All panels
show representative pictures of experiments replicated 3 times.
© 2016 Macmillan Publishers Limited. All rights reserved
