Histochem Cell Biol

DOI 10.1007/s00418-011-0902-3

ORIGINAL PAPER

Cigarette smoke and the terminal ileum: increased autophagy

in murine follicle-associated epithelium and Peyers patches
Stephanie Verschuere Liesbeth Allais Ken R. Bracke
Saskia Lippens Rebecca De Smet Peter Vandenabeele
Guy G. G. Brusselle Claude A. Cuvelier

Accepted: 12 December 2011

Springer-Verlag 2011

Abstract Cigarette smoke (CS) exposure is associated

with increased autophagy in several cell types, such as
bronchial epithelial cells. Smoking is also an environmental
risk factor in Crohns disease, in which impairment of the
autophagy-mediated anti-bacterial pathway has been implicated. So far, it is unknown whether CS induces autophagy
in the gut. Here, we examined the eVect of chronic CS
exposure on autophagy in the follicle-associated epithelium
(FAE) of murine Peyers patches. Transmission electron
microscopy revealed that the proportion of cell area occupied by autophagic vesicles signiWcantly increased in the
FAE after CS exposure. An increased number of autophagic vesicles was observed in the FAE, whereas the vesicle
size remained unaltered. Besides enterocytes, also M-cells
contain more autophagic vesicles upon CS exposure. In
addition, the mRNA level of the autophagy-related protein
Atg7 in the underlying Peyers patches is increased after
CS exposure, which indicates that the autophagy-inducing
eVect of CS is not limited to the FAE. In conclusion, our
results demonstrate that CS exposure induces autophagy in
murine FAE and in the underlying immune cells of Peyers
patches, suggesting that CS exposure increases the risk for
G. G. G. Brusselle, C. A. Cuvelier equally contributed to this paper.
S. Verschuere (&) L. Allais R. De Smet C. A. Cuvelier
Department of Pathology, University Hospital Ghent,
5 Blok A, De Pintelaan 185, 9000 Ghent, Belgium
e-mail: Stephanie.Verschuere@ugent.be
K. R. Bracke G. G. G. Brusselle
Laboratory for Translational Research in Obstructive
Pulmonary Diseases, Department of Respiratory Medicine,
Ghent University Hospital, Ghent, Belgium
S. Lippens P. Vandenabeele
Department for Molecular Biomedical Research,
VIB, University Ghent, Ghent, Belgium

Crohns disease by causing epithelial oxidative damage,

which needs to be repaired by autophagy.
Keywords Smoking Autophagy Follicle-associated
epithelium M-cells Peyers patches Crohns disease
Electron microscopy

Introduction
Smoking is the most important preventable cause of death
and disability worldwide. In developed countries, smoking
rates are estimated to be 37% for men and 21% for women,
whereas rates in developing countries are even higher
(van Zyl-Smit et al. 2010). Smoke-related morbidity and
mortality are not limited to respiratory and cardiovascular
diseases, but also involve other diseases, including several
malignancies and inXammatory disorders, like Crohns
disease (Sopori 2002).
Cigarette smoke (CS) exerts its detrimental eVects
through several mechanisms. Some CS compounds are carcinogens (such as benzopyrenes), toxins (e.g. carbon monoxide and nicotine), reactive particles (metal ions) or
oxidants (such as nitric oxide and superoxide anion) (Faux
et al. 2009). The latter induce the production of reactive
oxygen species. If these are not neutralized by anti-oxidants, this process leads to oxidative stress, which causes
damage to lipids, proteins, DNA and cell organelles
(StampXi and Anderson 2009). Eventually, oxidative cell
injury is repaired by autophagy or otherwise results in programed cell death (i.e., apoptosis) (Kim et al. 2008).
Autophagy in the airways is enhanced by CS exposure
(Brusselle et al. 2011). This homeostatic process plays an
important role in cell survival and energy metabolism in both
normal and stressful environments. Cellular constituents that

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need to be removed are sequestered in a double-membraned

vacuole, the autophagosome, which then fuses with lysosomes resulting in digestion of the entrapped material (Yang
and Klionsky 2010). Autophagy has two main physiological
functions: Wrstly, it recycles essential cell elements and secondly, it eliminates unwanted cytoplasmatic elements, such
as damaged organelles (true autophagy) or foreign material
(xenophagy). As a result, autophagy can oVer protection
against neurodegenerative disorders, Crohns disease and
infections by discarding damaged cell components or invasive bacteria (Rabinowitz and White 2010; Virgin and
Levine 2009).
Crohns disease is a chronic relapsing inXammatory
bowel disease mainly involving the terminal ileum and the
colon. The recent discovery of the involvement of the autophagic pathway in Crohns disease introduced a new era in
the research of inXammatory bowel disease. The Wrst evidence came from genome-wide association studies, underscoring the association of functional polymorphisms in two
autophagy genes with Crohns disease (Brest et al. 2010).
Since then, the role of (impaired) autophagy in the gut has
been intensively studied and autophagy has been demonstrated to be indispensable for innate immune responses
against invasive pathogens (Cadwell et al. 2008; Cooney
et al. 2010). Both smoking and autophagy polymorphisms
are associated with an increased risk for especially ileal
Crohns disease. However, the role of autophagy in Peyers
patches, which are considered as the onset site of ileal
Crohns disease (Fujimura et al. 1996), has not been studied.
The aim of this study was to investigate whether CS
induces autophagy in follicle-associated epithelium (FAE)
and Peyers patches in the murine ileum. We recently demonstrated that chronic CS exposure induces apoptosis in the
FAE. We suggested that this is due to oxidative stress associated with smoking (Verschuere et al. 2011). In the present
study, we examined the presence of autophagic vesicles in
the FAE of air- and CS-exposed mice and we analyzed the
size and number of these vesicles by means of transmission
electron microscopy. Furthermore, we investigated the
expression of several autophagy-related genes in Peyers
patches of air- and smoke-exposed mice. Finally, autophagy
in M-cells was studied. We demonstrate that CS exposure is
associated with an increase in autophagy in both FAE and in
the Peyers patch. Our results contribute to a better understanding of the increased risk for Crohns disease in smokers.

the start of the CS exposure. The local Ethics Committee

for animal experimentation of the faculty of Medicine and
Health Sciences (Ghent, Belgium) approved all experiments (ECD 27/07).
Cigarette smoke exposure and sample collection
Mice were exposed to main stream CS, as described previously (Dhulst et al. 2005). BrieXy, mice were exposed to
the CS of Wve cigarettes (Reference Cigarette 3R4F without
Wlter; University of Kentucky, Lexington, KY, USA) four
times per day with a 30 min smoke-free interval, 5 days per
week for 24 weeks (chronic CS exposure). An optimal
smoke:air ratio of 1:6 was obtained. The control groups
were exposed to air. Carboxyhaemoglobin in serum of
smoke-exposed mice reached a non-toxic level of
8.70 0.31% (compared with 0.65 0.25% in airexposed mice), which is similar to carboxyhaemoglobin
blood concentrations of human smokers (Macdonald et al.
2004).
At 24 h after the last exposure, mice were killed with
an overdose of pentobarbital. Immediately after sacriWcing the animals, the abdominal cavity was opened and the
small intestine with Peyers patches was removed. Ileal
Peyers patches were sampled for transmission electron
microscopy (TEM), western blot or polymerase chain
reaction (PCR).
Transmission electron microscopy

Materials and methods

Samples of Peyers patches were Wxed in 0.1 M cacodylate

buVer containing 4% paraformaldehyde and 5% glutaraldehyde during 48 h. Then, samples were washed overnight
with 0.1 M Sodium cacodylate buVer. Following postWxation in 1% osmium tetroxide for 3 h, samples were dehydrated in a series of alcohol (15 50%, 15 70%, 15 90%,
3 30 100%) and embedded in Epon medium (Aurion,
Wageningen, The Nederlands). Semithin sections of 1 m
were cut and stained with Toluidine blue to select the most
appropriate area with follicle-associated epithelium. Ultrathin sections of 60 nm were cut and contrasted with uranyl
acetate and lead nitrate, followed by imaging with a Zeiss
TEM900 transmission electron microscope (Carl Zeiss,
Oberkochen, Germany) at 50 kV. Two diVerent regions of
FAE from six Peyers patches in each group were studied
(N = 6). Image analysis of autophagic vesicles in TEM photos was performed by ImageJ (NIH, Bethesda, MD, USA)
(Fig. 1a, b).

Animals

RNA preparation and RT-PCR

Male C57BL/6 wild-type mice were purchased from

Charles River Laboratories. All mice were 89 weeks old at

RNA from Peyers patches was extracted using the

Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany).

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Histochem Cell Biol

expression of the autophagy-related genes Atg5, Atg7,

Atg16l1, Beclin-1 and reference genes Gapdh (glyceraldehyde-3-phosphate dehydrogenase), Hprt1 (hypoxanthine
phosphoribosyltransferase 1) and Tfrc (transferrin receptor) was analyzed with the TaqMan Gene Expression
Assays (Applied Biosystems). Real-time PCR reactions
were performed in duplicate using diluted cDNA template
and the LightCycler480 Probes Master (Roche). AmpliWcations were performed on a LightCycler480 detection
system (Roche) with the following cycling conditions:
10 min incubation at 95C and 50 cycles of 95C for 10 s
and 60C for 15 s. Expression of target genes was corrected by a normalization factor that was calculated based
on the expression of the three reference genes (Gapdh,
Hprt1, Tfrc), using the geNorm applet according to the
guidelines and theoretical framework previously described
(Vandesompele et al. 2002).
Western blotting

Fig. 1 Transmission electron microscopy of follicle-associated epithelium, illustrating the applied quantiWcation method. a Overview of
follicle-associated epithelium (FAE). 2 diVerent regions of FAE of
Peyers patches, each containing at least 10 epithelial cells, were selected
and all cells in this area were examined. The number of cells scored per
Peyers patch was between 25 and 50 cells, with no diVerence in cell
numbers examined between the 2 groups. b Cell area (full line) and the
area occupied by autophagic vesicles (dotted line) per epithelial cell
were calculated with ImageJ image analysis. N = 6 mice per group

Subsequently, cDNA was obtained by reverse transcription of RNA with the Transcriptor First Strand cDNA synthesis kit (Roche) following manufacturers instructions
and using a 2:1 ratio of hexa:oligodT primers. mRNA

Peyers patches were lysed in ice-cold 0.1% triton X-100/

0.1% tween-20/PBS and sonicated. As positive controls,
lysates of the IL-3-dependent mouse pro-B-cell line Ba/F3
were used, with and without IL-3 deprivation as described
previously (Wirawan et al. 2010). Samples were heated to
95 for 10 min in Laemmli loading buVer before loading.
Then, lysates were separated in SDS-PAGE gels and transferred to nitrocellulose membranes by wet blotting. After
blocking in PBS supplemented with 0.2% Tween-20 (v/v)
and 3% (w/v) non-fat dry milk, the membrane was incubated with primary antibodies overnight at 4 and washed.
The primary antibodies used were anti-LC3B (Cell Signaling, Danvers, MA, USA) and anti-actin (MP Biomedicals,
Irvine, CA, USA). Following incubation with HRP-conjugated secondary antibody against rabbit immunoglobulin
(Amersham Biosciences, Uppsala, Sweden) and against
mouse immunoglobulin (GE Healthcare, Buckinghamshire,
UK), immunoreactive proteins were visualized by
enhanced chemiluminescence (Perkin Elmer, Boston, MA,
USA). QuantiWcation of the LC3-II/LC3-I ratio was performed by ImageJ software.
Statistical analysis
Reported values are expressed as mean SEM (standard
error of the mean) and error bars were marked as the
SEM. Statistical analysis was performed by SPSS 16
Software (SPSS 16 Inc., Chicago, IL, USA) using Student
t test for normally distributed populations and Mann
Whitney U test for populations where normal distribution
was not accomplished. A P value of <0.05 was considered
signiWcant.

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Histochem Cell Biol

Fig. 2 Autophagy in follicle-associated epithelium increases after

smoke exposure. a In FAE cells of air-exposed mice, only a limited
percentage of the cell area is occupied by autophagic vesicles
(arrows). Most epithelial cells do not contain vesicles larger than

2 m2. b Detail of autophagic vesicles in air-exposed FAE. c In

smoke-exposed FAE cells, a greater proportion of the cell area is occupied by autophagic vesicles (arrows). d Detail of autophagic vesicles
in smoke-exposed FAE

Results

The area occupied by autophagic vesicles (both autophagosomes and autolysosomes) in FAE cells increased
signiWcantly in the smoke-exposed group compared to airexposed mice (Fig. 3a). The mean vesicle area per epithelial cell doubled from 1.1 0.4 m2 in the air group to
2.4 0.4 m2 in the CS group (P < 0.05). Also when
expressed as cell area percentage occupied by autophagic
vesicles, the diVerence was statistically signiWcant.
To further investigate what accounts for the increase in
autophagic vesicle area in FAE of smoke-exposed mice,
number and size of the autophagic vesicles were determined. To exclude lysosomes, only vesicles with an area of
minimum 0.2 m2 were taken into account (Levine et al.
2011). The size of autophagic vesicles did not diVer
between both groups (Fig. 3b), but FAE cells contained a
signiWcantly higher number of vesicles after CS exposure
(Fig. 3c). This implies that the higher autophagic vesicle
area in epithelial cells of Peyers patches is caused by an
increased number of vesicles, rather than by an enlargement
of the vesicles. No diVerences in maturation stage of the
autophagic vesicles were observed between both groups.

Chronic smoke exposure increases the amount

of autophagic vesicles in the follicle-associated epithelium
We previously demonstrated that CS induced apoptosis in
follicle-associated epithelium (FAE) of Peyers patches
(Verschuere et al. 2011). To investigate whether chronic
CS exposure also inXuenced autophagy in the FAE, we
performed transmission electron microscopy (TEM),
which is the gold-standard method for detection of
autophagy (Fig. 1). FAE of Peyers patches from smokeexposed mice was compared to Peyers patches from airexposed animals (Fig. 2ad). The mean number of FAE
cells analyzed per Peyers patch was 37.5 in air-exposed
mice and 32.0 in smoke-exposed mice, and this was not
statistically diVerent. No diVerence in mean cell area of
FAE cells was observed between both groups. CS exposure did not cause ultrastructural damage to cells or
organelles, such as mitochondria or the endoplasmatic
reticulum.

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Histochem Cell Biol

Fig. 3 Cigarette smoke increases autophagic vesicle number but not

size in the follicle-associated epithelium. a The proportion of the FAE
cell area occupied by autophagic vesicles increase from 0.85 0.27%
in air-exposed mice to 1.91 0.28% in smoke-exposed mice.
b Vesicle size does not diVer after air and smoke exposure

(2.2 0.9 m2 vs. 2.5 0.3 m2). c The number of autophagic vesicles is 0.5 0.1 vesicles per cell in air-exposed FAE versus 1.1 0.1
vesicles per cell in smoke-exposed FAE. Data are represented as
mean SEM. N = 6 mice per group. NS non-signiWcant; *P < 0.05

Fig. 4 Expression of autophagy

genes in Peyers patches following smoke exposure mRNA
expression of autophagy-related
genes in Peyers patches as
determined by RT-PCR, relative
to expression of 3 reference
genes (Hprt, Gapdh and Tfrc).
a Expression of Beclin-1 tends
to increase after smoke exposure
(1.11 0.15 vs. 1.47 0.09).
b Expression of Atg5 tends to
increase from 1.62 0.29 in airexposed mice to 2.30 0.19 in
smoke-exposed mice.
c Expression of Atg7 increases
signiWcantly from 1.27 0.15 in
air-exposed mice to 2.00 0.13
in smoke-exposed mice.
d Expression of Atg16l1 is not
altered by smoke exposure. Data
are represented as mean SEM.
N = 6 mice per group. NS nonsigniWcant; *P < 0.05

Cigarette smoke-induced autophagy is present

in the underlying Peyers patches
Next, we investigated whether smoke-induced autophagy
was also observed in the entire Peyers patch. mRNA
expression of four autophagy-related genes in Peyers
patches was measured by RT-PCR. mRNA expression of
Beclin-1, a protein involved in the nucleation of autophagosomes, and of Atg5, which plays a role in the autophagosome vesicle elongation and completion, showed a
tendency towards increase after CS exposure (Fig. 4a, b).
mRNA expression of the autophagy protein Atg7, which
also assists in autophagosome vesicle maturation, is signiWcantly higher following CS (Fig. 4c). In contrast, another
protein involved in autophagosome formation further in the
autophagy cascade, Atg16l1, did not show increased
expression after CS exposure (Fig. 4d).

Furthermore, western blot analysis was performed for

detection of processed microtubule-associated protein 1
light chain 3 (LC3). The modiWcation of the soluble LC3-I
into the lipidated form LC3-II, which attaches to the autophagosomal membrane, is considered to be a marker for
autophagy (Barth et al. 2010). However, no diVerences
were detected between both groups. The levels of LC3BII were comparable in Peyers patches of mice exposed to
air and CS (Fig. 5a). Moreover, the LC3B-II/LC3B-I ratio
did not diVer between air- and smoke-exposed mice
(Fig. 5b).
M-cells are also aVected by smoke-induced autophagy
Finally, we compared the amount of autophagic vesicles in
the diVerent cell types of the FAE. The most striking diVerence between FAE and absorptive intestinal epithelium is

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Histochem Cell Biol

Fig. 5 Protein expression of LC3 in Peyers patches is not altered by

cigarette smoke exposure. a Western blot analysis of LC3 on Peyers
patches. Control 1 Ba/F3 cells without IL-3 deprivation, showing moderate levels of autophagy. Control 2 Ba/F3 cells with deprivation of
IL-3, inducing a marked increase in autophagy as evidenced by

increased LC3-II levels. Both the air- and smoke-exposed group

showed very low levels of LC3-II. b LC3-II/LC3-I ratio was not
altered by smoke exposure. Data are represented as mean SEM.
N = 6 mice per group. NS non-signiWcant

the presence of M-cells, which are specialized gateways

transporting intact molecules and microorganisms through
the epithelium for antigen presentation to the underlying
immune cells of the Peyers patch. This plays a crucial role
in the induction of innate immunity through generation of
IgA responses (Huett and Xavier 2010). Therefore, we
were interested whether CS exposure did induce autophagic
changes in M-cells as well.
M-cells can be easily recognized on TEM images due to
the presence of apical microfolds and an intraepithelial
pocket (Fig. 6a). The number of M-cells was comparable in
both groups. In air-exposed mice, a mean number of 4.5 Mcells was observed on a mean total cell number of 37.5
FAE cells, whereas the FAE of smoke-exposed mice contained a mean of 4.2 M-cells on 32.0 cells (13.6% of FAE
cells in air-exposed mice, vs. 12.0% in smoke-exposed
mice). However, the number of autophagic vesicles in airexposed M-cells was lower than in the overall FAE
(0.39 0.22% of the cell area in M-cells compared with
0.85 0.27% in all epithelial cells), although this diVerence was not statistically signiWcant.
In contrast to the low baseline level of autophagy in
M-cells, the area occupied by autophagic vesicles per
M-cell increased signiWcantly following CS exposure
(Fig. 6b, c). Consistent with the Wndings in the FAE in general, the number of vesicles per M-cell increased signiWcantly from 0.37 0.17 per cell in air-exposed mice
towards 1.22 0.24 in smoke-exposed mice (P < 0.05).
The mean autophagic vesicle size in M-cells remained
unaltered following CS exposure.

Peyers patches. Electron microscopic study after 24 weeks

of CS exposure revealed that FAE of smoke-exposed mice
contains a signiWcantly higher amount of autophagic vesicles than FAE of air-exposed mice. The higher number of
autophagic vesicles is not only present in enterocytes, but
also in M-cells. Furthermore, several autophagy-related
genes have upregulated mRNA expression in Peyers
patches following CS exposure.
A Wrst important Wnding was the signiWcantly increased
proportion of FAE cell area occupied by autophagic vesicles after CS exposure. This might indicate an induction of
the autophagic pathway (leading to an increased production
of vesicles) or a blockade in the terminal vesicle degradation and fragmentation step (causing an accumulation of
large mature autolysosomes). Further analysis of the vesicles revealed that the increased proportion was due to a
higher number of autophagic vesicles, rather than to a bigger vesicle size. It was not feasible to make a reproducible
distinction between early autophagic compartments (autophagosomes) and late autophagic structures (autolysosomes), and therefore we preferred to quantify all
autophagic vesicles instead of making arbitrary sub-classiWcations.
Secondly, CS was shown to increase the mRNA expression of the autophagy protein Atg7, and two other autophagy-related proteins showed a trend towards increased
mRNA expression. Although autophagy proteins are also
involved in other cellular processes, a tendency towards
increased expression in three diVerent components of the
autophagic pathway suggests that it is the autophagic pathway itself that is induced by CS. The increases in expression are modest, but all three genes show a similar trend.
In contrast, another marker for autophagy, the rate of conversion of LC3-I to LC3-II, showed no increase after CS
exposure. We speculate that this is because the small
changes in mRNA expression do not result in changes in

Discussion
In this study, we provide the Wrst evidence that chronic
exposure to cigarette smoke (CS) can induce autophagy in

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Histochem Cell Biol

Fig. 6 Smoke-induced autophagy is also present in M-cells. a TEM

image of an M-cell after air exposure, showing the hallmarks for recognizing these cells: the presence of microfolds instead of the microvilli of the neighbouring enterocytes and an intra-epithelial pocket,
Wlled with immune cells. No autophagic vesicles are present. b M-cell
of smoke-exposed mouse showing the presence of autophagic vesicles.
c The percentage of M-cell area occupied by autophagic vesicles
increases from 0.39 0.22% in air-exposed mice to 1.79 0.77% in
smoke-exposed mice. Data are represented as mean SEM. N = 6
mice per group. *P < 0.05

protein expression large enough to be picked up by a relatively rough technique as western blotting.
In addition, we demonstrated that also M-cells are
involved in CS-induced autophagy. Whereas basal autophagy in M-cells was very low, CS signiWcantly increased the
amount of autophagic vesicles to levels comparable with
that of neighbouring enterocytes. Up till now, autophagy in
M-cells has never been described. Previous studies on
endosomal and lysosomal compartments in M-cells
reported conXicting results (Allan et al. 1993; Owen et al.
1986). However, as the main function of M-cells is to
transport intact antigens and microorganisms through
the epithelial layer, it could be expected that basal autophagy is minimal. After CS exposure, however, autophagy in
M-cells almost equalized autophagy in enterocytes. We
have no indication whether this is due to a changed handling of exogenous antigens (resulting in increased xenophagy) or to a higher need for disposal of damaged cell
organelles (true autophagy).
The identiWcation of murine M-cells relies on histochemical staining (Ulex Europeaus Agglutinin 1 or
Annexin V) or on TEM (Verbrugghe et al. 2006). TEM is
also one of the most sensitive techniques to detect autophagic vesicles, and is considered as the gold standard for
autophagy detection (Barth et al. 2010). Therefore, we
chose for TEM to evaluate autophagy, as we are experienced with this technique for examination of FAE and
M-cells (Cuvelier et al. 1994; Verbrugghe et al. 2008). The
diYculties associated with the investigation of M-cells and
FAE and the subsequent choice for TEM implicate that this
study is descriptive, rather than mechanistical. Notwithstanding this limitation, we believe that it extends the current knowledge on FAE and M-cell function, as this is the
Wrst report describing autophagy in FAE cells in normal
and pathological conditions.
Our Wndings are consistent with the previous reports,
describing CS-induced autophagy both in vitro and in vivo in
lung tissue and in pulmonary epithelial cells (Brusselle et al.
2011). Activation of autophagy by CS was also observed in
human umbilical vein endothelial cells (HUVECs) (Csordas
et al. 2011). However, no data have been published yet on
the eVect of smoking on intestinal autophagy. The exact
mechanisms through which CS triggers the autophagic pathway are not elucidated. Autophagy is induced by environmental stress conditions such as genotoxic agents and
oxidative stress, both occurring in the context of CS exposure
(Levine and Yuan 2005). These stimuli might act on genes
activating or downregulating the autophagy pathway, including early growth response-1 (Egr-1) and heme oxygenase-1
(HO-1) (Chen et al. 2008; Kim et al. 2008).
Another unsolved issue is the relation between autophagy and apoptosis in the context of CS exposure. The connections between these two pathways are complex and

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Histochem Cell Biol

remain poorly understood. Autophagy can protect against

apoptosis by cleaning up damaged organelles and restoring
cell function, but excessive autophagy may cause uncontrolled degradation of cellular constituents, resulting in cell
death. Apoptosis in turn can induce autophagy, which subsequently eliminates apoptotic bodies and by this prevents
inXammation (Levine et al. 2011; Ravikumar et al. 2010)
but it may also prevent autophagy when acting as a survival
mechanism by caspase-dependent cleavage of Atg proteins
(Wirawan et al. 2011). In a previous report, we described
the presence of CS-induced apoptosis in the FAE of murine
Peyers patches (Verschuere et al. 2011), so also in the
intestine smoke-induced apoptosis and autophagy occur in
the same cell type. We speculate that CS exerts an injurious
eVect on the FAE and Peyers patches through oxidative
stress. If autophagy is unable to clean up the damage, this
insurmountable injury will result in apoptosis.
Currently, autophagy in the gut epithelium attracts a lot
of attention, because of the recently discovered link
between Crohns disease and autophagy genes. Several
genome-wide association studies identiWed polymorphisms
in the autophagy genes ATG16L1 and IRGM as risk factors
for Crohns disease. The ATG16L1 variant associated with
Crohns disease, T300A, results in a deWcient bacteriainduced autophagy (Kuballa et al. 2008). Furthermore, also
NOD2, another known risk factor for Crohns disease, can
activate the autophagic pathway. Genetic variants in NOD2
or in autophagy genes which result in reduced autophagy,
may diminish intestinal defence mechanisms against invasive bacteria, subsequently increasing the susceptibility to
Crohns disease (Homer et al. 2010; Netea and Joosten
2010).
Interestingly, active smoking and a homozygous genotype for the T300A variant were found to have a synergistic
eVect in the pathogenesis of Crohns disease, with an odds
ratio of 7.65 to develop the disease (Fowler et al. 2008).
This implies that the negative eVect of cigarette smoke on
CD is reinforced by a deWcient autophagy. As smoking
itself induces autophagy, one can hypothesize that autophagy is a protective mechanism, reversing the cellular injury
caused by smoking. The impairment of this repair process
can cause an accumulation of damaged organelles in cells,
increasing apoptosis and tissue injury. Further research will
need to elucidate the exact role of CS-induced autophagy
in homeostatic conditions and in intestinal inXammation,
and focus on the eVect of CS in a context of impaired
autophagy.
In conclusion, this study is the Wrst to describe smokeinduced autophagy in the FAE covering murine Peyers
patches. Both in enterocytes and in M-cells, TEM demonstrated an increase in autophagic vesicle number following
CS exposure. Our Wndings may point to an important role
for autophagy in protecting the FAE against smoke-induced

123

oxidative damage and can help to understand the role of

smoking in the pathogenesis of Crohns disease, in which
autophagy is impaired.
Acknowledgments We are grateful to Barbara Gilbert for providing
help with the Western blot technique for LC3. We thank Dorothea van
Limbergen and Ran Rumes for the processing of the samples for electron microscopy, and Katrien De Visschere, Isabelle Rottiers, Eliane
Castrique, Christelle Snauwaert, Marie-Rose Mouton, Katleen De
Saedeleer, Anouk Goethals, Ann Neesen, Indra De Borle, Greet Barbier en Evelien Spruyt for the excellent technical support. Funding was
provided by the Special Research Fund of Ghent University
(01J17507) and Concerted Research Action of Ghent University (BOF
10/GOA/021). Stephanie Verschuere is supported by a doctoral Grant
from the Special Research Fund of Ghent University (01D21009). Ken
Bracke is a postdoctoral researcher of the Fund for ScientiWc Research
(FWO) in Flanders.

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