Histochem Cell BiolDOI 10.1007/s00418-011-0902-3ORIGINAL 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 associatedwith increased autophagy in several cell types, such asbronchial epithelial cells. Smoking is also an environmentalrisk factor in Crohns disease, in which impairment of theautophagy-mediated anti-bacterial pathway has been impli-cated. So far, it is unknown whether CS induces autophagyin the gut. Here, we examined the eVect of chronic CSexposure on autophagy in the follicle-associated epithelium(FAE) of murine Peyers patches. Transmission electronmicroscopy revealed that the proportion of cell area occu-pied by autophagic vesicles signiWcantly increased in theFAE after CS exposure. An increased number of autopha-gic vesicles was observed in the FAE, whereas the vesiclesize remained unaltered. Besides enterocytes, also M-cellscontain more autophagic vesicles upon CS exposure. Inaddition, the mRNA level of the autophagy-related proteinAtg7 in the underlying Peyers patches is increased afterCS exposure, which indicates that the autophagy-inducingeVect of CS is not limited to the FAE. In conclusion, ourresults demonstrate that CS exposure induces autophagy inmurine FAE and in the underlying immune cells of Peyerspatches, suggesting that CS exposure increases the risk for

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 deathand disability worldwide. In developed countries, smokingrates 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 andmortality are not limited to respiratory and cardiovasculardiseases, but also involve other diseases, including severalmalignancies and inXammatory disorders, like Crohnsdisease (Sopori 2002).

Cigarette smoke (CS) exerts its detrimental eVectsthrough several mechanisms. Some CS compounds are car-cinogens (such as benzopyrenes), toxins (e.g. carbon mon-oxide and nicotine), reactive particles (metal ions) oroxidants (such as nitric oxide and superoxide anion) (Fauxet al. 2009). The latter induce the production of reactiveoxygen species. If these are not neutralized by anti-oxi-dants, this process leads to oxidative stress, which causesdamage to lipids, proteins, DNA and cell organelles(StampXi and Anderson 2009). Eventually, oxidative cellinjury is repaired by autophagy or otherwise results in pro-gramed 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 animportant role in cell survival and energy metabolism in bothnormal and stressful environments. Cellular constituents that

G. G. G. Brusselle, C. A. Cuvelier equally contributed to this paper.

S. Verschuere (&) L. Allais R. De Smet C. A. CuvelierDepartment of Pathology, University Hospital Ghent, 5 Blok A, De Pintelaan 185, 9000 Ghent, Belgiume-mail: Stephanie.Verschuere@ugent.be

K. R. Bracke G. G. G. BrusselleLaboratory for Translational Research in Obstructive Pulmonary Diseases, Department of Respiratory Medicine, Ghent University Hospital, Ghent, Belgium

S. Lippens P. VandenabeeleDepartment for Molecular Biomedical Research, VIB, University Ghent, Ghent, Belgium123

Histochem Cell Biolneed to be removed are sequestered in a double-membranedvacuole, the autophagosome, which then fuses with lyso-somes resulting in digestion of the entrapped material (Yangand Klionsky 2010). Autophagy has two main physiologicalfunctions: Wrstly, it recycles essential cell elements and sec-ondly, it eliminates unwanted cytoplasmatic elements, suchas damaged organelles (true autophagy) or foreign material(xenophagy). As a result, autophagy can oVer protectionagainst neurodegenerative disorders, Crohns disease andinfections by discarding damaged cell components or inva-sive bacteria (Rabinowitz and White 2010; Virgin andLevine 2009).

Crohns disease is a chronic relapsing inXammatorybowel disease mainly involving the terminal ileum and thecolon. The recent discovery of the involvement of the auto-phagic pathway in Crohns disease introduced a new era inthe research of inXammatory bowel disease. The Wrst evi-dence came from genome-wide association studies, under-scoring the association of functional polymorphisms in twoautophagy genes with Crohns disease (Brest et al. 2010).Since then, the role of (impaired) autophagy in the gut hasbeen intensively studied and autophagy has been demon-strated to be indispensable for innate immune responsesagainst invasive pathogens (Cadwell et al. 2008; Cooneyet al. 2010). Both smoking and autophagy polymorphismsare associated with an increased risk for especially ilealCrohns disease. However, the role of autophagy in Peyerspatches, which are considered as the onset site of ilealCrohns disease (Fujimura et al. 1996), has not been studied.

The aim of this study was to investigate whether CSinduces autophagy in follicle-associated epithelium (FAE)and Peyers patches in the murine ileum. We recently dem-onstrated that chronic CS exposure induces apoptosis in theFAE. We suggested that this is due to oxidative stress associ-ated with smoking (Verschuere et al. 2011). In the presentstudy, we examined the presence of autophagic vesicles inthe FAE of air- and CS-exposed mice and we analyzed thesize and number of these vesicles by means of transmissionelectron microscopy. Furthermore, we investigated theexpression of several autophagy-related genes in Peyerspatches of air- and smoke-exposed mice. Finally, autophagyin M-cells was studied. We demonstrate that CS exposure isassociated with an increase in autophagy in both FAE and inthe Peyers patch. Our results contribute to a better under-standing of the increased risk for Crohns disease in smokers.

Materials and methods

Animals

Male C57BL/6 wild-type mice were purchased fromCharles River Laboratories. All mice were 89 weeks old at

the start of the CS exposure. The local Ethics Committeefor animal experimentation of the faculty of Medicine andHealth Sciences (Ghent, Belgium) approved all experi-ments (ECD 27/07).

Cigarette smoke exposure and sample collection

Mice were exposed to main stream CS, as described previ-ously (Dhulst et al. 2005). BrieXy, mice were exposed tothe CS of Wve cigarettes (Reference Cigarette 3R4F withoutWlter; University of Kentucky, Lexington, KY, USA) fourtimes per day with a 30 min smoke-free interval, 5 days perweek for 24 weeks (chronic CS exposure). An optimalsmoke:air ratio of 1:6 was obtained. The control groupswere exposed to air. Carboxyhaemoglobin in serum ofsmoke-exposed mice reached a non-toxic level of8.70 0.31% (compared with 0.65 0.25% in air-exposed mice), which is similar to carboxyhaemoglobinblood concentrations of human smokers (Macdonald et al.2004).

At 24 h after the last exposure, mice were killed withan overdose of pentobarbital. Immediately after sacriWc-ing the animals, the abdominal cavity was opened and thesmall intestine with Peyers patches was removed. IlealPeyers patches were sampled for transmission electronmicroscopy (TEM), western blot or polymerase chainreaction (PCR).

Transmission electron microscopy

Samples of Peyers patches were Wxed in 0.1 M cacodylatebuVer containing 4% paraformaldehyde and 5% glutaralde-hyde during 48 h. Then, samples were washed overnightwith 0.1 M Sodium cacodylate buVer. Following postWx-ation in 1% osmium tetroxide for 3 h, samples were dehy-drated 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 mwere cut and stained with Toluidine blue to select the mostappropriate area with follicle-associated epithelium. Ultra-thin sections of 60 nm were cut and contrasted with uranylacetate and lead nitrate, followed by imaging with a ZeissTEM900 transmission electron microscope (Carl Zeiss,Oberkochen, Germany) at 50 kV. Two diVerent regions ofFAE from six Peyers patches in each group were studied(N = 6). Image analysis of autophagic vesicles in TEM pho-tos was performed by ImageJ (NIH, Bethesda, MD, USA)(Fig. 1a, b).

RNA preparation and RT-PCR

RNA from Peyers patches was extracted using theQiagen RNeasy Mini Kit (Qiagen, Hilden, Germany).123

Histochem Cell BiolSubsequently, cDNA was obtained by reverse transcrip-tion of RNA with the Transcriptor First Strand cDNA syn-thesis kit (Roche) following manufacturers instructionsand using a 2:1 ratio of hexa:oligodT primers. mRNA

expression of the autophagy-related genes Atg5, Atg7,Atg16l1, Beclin-1 and reference genes Gapdh (glyceralde-hyde-3-phosphate dehydrogenase), Hprt1 (hypoxanthinephosphoribosyltransferase 1) and Tfrc (transferrin recep-tor) was analyzed with the TaqMan Gene ExpressionAssays (Applied Biosystems). Real-time PCR reactionswere performed in duplicate using diluted cDNA templateand the LightCycler480 Probes Master (Roche). AmpliW-cations were performed on a LightCycler480 detectionsystem (Roche) with the following cycling conditions:10 min incubation at 95C and 50 cycles of 95C for 10 sand 60C for 15 s. Expression of target genes was cor-rected by a normalization factor that was calculated basedon the expression of the three reference genes (Gapdh,Hprt1, Tfrc), using the geNorm applet according to theguidelines and theoretical framework previously described(Vandesompele et al. 2002).

Western blotting

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/F3were used, with and without IL-3 deprivation as describedpreviously (Wirawan et al. 2010). Samples were heated to95 for 10 min in Laemmli loading buVer before loading.Then, lysates were separated in SDS-PAGE gels and trans-ferred to nitrocellulose membranes by wet blotting. Afterblocking in PBS supplemented with 0.2% Tween-20 (v/v)and 3% (w/v) non-fat dry milk, the membrane was incu-bated with primary antibodies overnight at 4 and washed.The primary antibodies used were anti-LC3B (Cell Signal-ing, Danvers, MA, USA) and anti-actin (MP Biomedicals,Irvine, CA, USA). Following incubation with HRP-conju-gated secondary antibody against rabbit immunoglobulin(Amersham Biosciences, Uppsala, Sweden) and againstmouse immunoglobulin (GE Healthcare, Buckinghamshire,UK), immunoreactive proteins were visualized byenhanced chemiluminescence (Perkin Elmer, Boston, MA,USA). QuantiWcation of the LC3-II/LC3-I ratio was per-formed by ImageJ software.

Statistical analysis

Reported values are expressed as mean SEM (standarderror of the mean) and error bars were marked as theSEM. Statistical analysis was performed by SPSS 16Software (SPSS 16 Inc., Chicago, IL, USA) using Studentt test for normally distributed populations and MannWhitney U test for populations where normal distributionwas not accomplished. A P value of

Histochem Cell BiolResults

Chronic smoke exposure increases the amount of autophagic vesicles in the follicle-associated epithelium

We previously demonstrated that CS induced apoptosis infollicle-associated epithelium (FAE) of Peyers patches(Verschuere et al. 2011). To investigate whether chronicCS exposure also inXuenced autophagy in the FAE, weperformed transmission electron microscopy (TEM),which is the gold-standard method for detection ofautophagy (Fig. 1). FAE of Peyers patches from smoke-exposed mice was compared to Peyers patches from air-exposed animals (Fig. 2ad). The mean number of FAEcells analyzed per Peyers patch was 37.5 in air-exposedmice and 32.0 in smoke-exposed mice, and this was notstatistically diVerent. No diVerence in mean cell area ofFAE cells was observed between both groups. CS expo-sure did not cause ultrastructural damage to cells ororganelles, such as mitochondria or the endoplasmaticreticulum.

The area occupied by autophagic vesicles (both auto-phagosomes and autolysosomes) in FAE cells increasedsigniWcantly in the smoke-exposed group compared to air-exposed mice (Fig. 3a). The mean vesicle area per epithe-lial cell doubled from 1.1 0.4 m2 in the air group to2.4 0.4 m2 in the CS group (P < 0.05). Also whenexpressed as cell area percentage occupied by autophagicvesicles, the diVerence was statistically signiWcant.

To further investigate what accounts for the increase inautophagic vesicle area in FAE of smoke-exposed mice,number and size of the autophagic vesicles were deter-mined. To exclude lysosomes, only vesicles with an area ofminimum 0.2 m2 were taken into account (Levine et al.2011). The size of autophagic vesicles did not diVerbetween both groups (Fig. 3b), but FAE cells contained asigniWcantly higher number of vesicles after CS exposure(Fig. 3c). This implies that the higher autophagic vesiclearea in epithelial cells of Peyers patches is caused by anincreased number of vesicles, rather than by an enlargementof the vesicles. No diVerences in maturation stage of theautophagic vesicles were observed between both groups.

Fig. 2 Autophagy in follicle-associated epithelium increases aftersmoke exposure. a In FAE cells of air-exposed mice, only a limitedpercentage 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 Insmoke-exposed FAE cells, a greater proportion of the cell area is occu-pied by autophagic vesicles (arrows). d Detail of autophagic vesiclesin smoke-exposed FAE123

Histochem Cell BiolCigarette smoke-induced autophagy is present in the underlying Peyers patches

Next, we investigated whether smoke-induced autophagywas also observed in the entire Peyers patch. mRNAexpression of four autophagy-related genes in Peyerspatches was measured by RT-PCR. mRNA expression ofBeclin-1, a protein involved in the nucleation of autophago-somes, and of Atg5, which plays a role in the autophago-some vesicle elongation and completion, showed atendency towards increase after CS exposure (Fig. 4a, b).mRNA expression of the autophagy protein Atg7, whichalso assists in autophagosome vesicle maturation, is signiW-cantly higher following CS (Fig. 4c). In contrast, anotherprotein involved in autophagosome formation further in theautophagy cascade, Atg16l1, did not show increasedexpression after CS exposure (Fig. 4d).

Furthermore, western blot analysis was performed fordetection of processed microtubule-associated protein 1light chain 3 (LC3). The modiWcation of the soluble LC3-Iinto the lipidated form LC3-II, which attaches to the auto-phagosomal membrane, is considered to be a marker forautophagy (Barth et al. 2010). However, no diVerenceswere detected between both groups. The levels of LC3B-II were comparable in Peyers patches of mice exposed toair and CS (Fig. 5a). Moreover, the LC3B-II/LC3B-I ratiodid 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 inthe diVerent cell types of the FAE. The most striking diVer-ence between FAE and absorptive intestinal epithelium is

Fig. 3 Cigarette smoke increases autophagic vesicle number but notsize in the follicle-associated epithelium. a The proportion of the FAEcell 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 vesi-cles is 0.5 0.1 vesicles per cell in air-exposed FAE versus 1.1 0.1vesicles per cell in smoke-exposed FAE. Data are represented asmean SEM. N = 6 mice per group. NS non-signiWcant; *P < 0.05

Fig. 4 Expression of autophagy genes in Peyers patches follow-ing 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 air-exposed 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 non-signiWcant; *P < 0.05123

Histochem Cell Biolthe presence of M-cells, which are specialized gatewaystransporting intact molecules and microorganisms throughthe epithelium for antigen presentation to the underlyingimmune cells of the Peyers patch. This plays a crucial rolein the induction of innate immunity through generation ofIgA responses (Huett and Xavier 2010). Therefore, wewere interested whether CS exposure did induce autophagicchanges in M-cells as well.

M-cells can be easily recognized on TEM images due tothe presence of apical microfolds and an intraepithelialpocket (Fig. 6a). The number of M-cells was comparable inboth groups. In air-exposed mice, a mean number of 4.5 M-cells was observed on a mean total cell number of 37.5FAE cells, whereas the FAE of smoke-exposed mice con-tained a mean of 4.2 M-cells on 32.0 cells (13.6% of FAEcells in air-exposed mice, vs. 12.0% in smoke-exposedmice). However, the number of autophagic vesicles in air-exposed M-cells was lower than in the overall FAE(0.39 0.22% of the cell area in M-cells compared with0.85 0.27% in all epithelial cells), although this diVer-ence was not statistically signiWcant.

In contrast to the low baseline level of autophagy inM-cells, the area occupied by autophagic vesicles perM-cell increased signiWcantly following CS exposure(Fig. 6b, c). Consistent with the Wndings in the FAE in gen-eral, the number of vesicles per M-cell increased signiW-cantly from 0.37 0.17 per cell in air-exposed micetowards 1.22 0.24 in smoke-exposed mice (P < 0.05).The mean autophagic vesicle size in M-cells remainedunaltered following CS exposure.

Discussion

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

Peyers patches. Electron microscopic study after 24 weeksof CS exposure revealed that FAE of smoke-exposed micecontains a signiWcantly higher amount of autophagic vesi-cles than FAE of air-exposed mice. The higher number ofautophagic vesicles is not only present in enterocytes, butalso in M-cells. Furthermore, several autophagy-relatedgenes have upregulated mRNA expression in Peyerspatches following CS exposure.

A Wrst important Wnding was the signiWcantly increasedproportion of FAE cell area occupied by autophagic vesi-cles after CS exposure. This might indicate an induction ofthe autophagic pathway (leading to an increased productionof vesicles) or a blockade in the terminal vesicle degrada-tion and fragmentation step (causing an accumulation oflarge mature autolysosomes). Further analysis of the vesi-cles revealed that the increased proportion was due to ahigher number of autophagic vesicles, rather than to a big-ger vesicle size. It was not feasible to make a reproducibledistinction between early autophagic compartments (auto-phagosomes) and late autophagic structures (autolyso-somes), and therefore we preferred to quantify allautophagic vesicles instead of making arbitrary sub-classi-Wcations.

Secondly, CS was shown to increase the mRNA expres-sion of the autophagy protein Atg7, and two other autoph-agy-related proteins showed a trend towards increasedmRNA expression. Although autophagy proteins are alsoinvolved in other cellular processes, a tendency towardsincreased expression in three diVerent components of theautophagic pathway suggests that it is the autophagic path-way itself that is induced by CS. The increases in expres-sion are modest, but all three genes show a similar trend.In contrast, another marker for autophagy, the rate of con-version of LC3-I to LC3-II, showed no increase after CSexposure. We speculate that this is because the smallchanges in mRNA expression do not result in changes in

Fig. 5 Protein expression of LC3 in Peyers patches is not altered bycigarette smoke exposure. a Western blot analysis of LC3 on Peyerspatches. Control 1 Ba/F3 cells without IL-3 deprivation, showing mod-erate levels of autophagy. Control 2 Ba/F3 cells with deprivation ofIL-3, inducing a marked increase in autophagy as evidenced by

increased LC3-II levels. Both the air- and smoke-exposed groupshowed very low levels of LC3-II. b LC3-II/LC3-I ratio was notaltered by smoke exposure. Data are represented as mean SEM.N = 6 mice per group. NS non-signiWcant123

Histochem Cell Biolprotein expression large enough to be picked up by a rela-tively rough technique as western blotting.

In addition, we demonstrated that also M-cells areinvolved in CS-induced autophagy. Whereas basal autoph-agy in M-cells was very low, CS signiWcantly increased theamount of autophagic vesicles to levels comparable withthat of neighbouring enterocytes. Up till now, autophagy inM-cells has never been described. Previous studies onendosomal and lysosomal compartments in M-cellsreported conXicting results (Allan et al. 1993; Owen et al.1986). However, as the main function of M-cells is totransport intact antigens and microorganisms throughthe epithelial layer, it could be expected that basal autoph-agy is minimal. After CS exposure, however, autophagy inM-cells almost equalized autophagy in enterocytes. Wehave no indication whether this is due to a changed han-dling of exogenous antigens (resulting in increased xeno-phagy) or to a higher need for disposal of damaged cellorganelles (true autophagy).

The identiWcation of murine M-cells relies on histo-chemical staining (Ulex Europeaus Agglutinin 1 orAnnexin V) or on TEM (Verbrugghe et al. 2006). TEM isalso one of the most sensitive techniques to detect autopha-gic vesicles, and is considered as the gold standard forautophagy detection (Barth et al. 2010). Therefore, wechose for TEM to evaluate autophagy, as we are experi-enced with this technique for examination of FAE andM-cells (Cuvelier et al. 1994; Verbrugghe et al. 2008). ThediYculties associated with the investigation of M-cells andFAE and the subsequent choice for TEM implicate that thisstudy is descriptive, rather than mechanistical. Notwith-standing this limitation, we believe that it extends the cur-rent knowledge on FAE and M-cell function, as this is theWrst report describing autophagy in FAE cells in normaland pathological conditions.

Our Wndings are consistent with the previous reports,describing CS-induced autophagy both in vitro and in vivo inlung tissue and in pulmonary epithelial cells (Brusselle et al.2011). Activation of autophagy by CS was also observed inhuman umbilical vein endothelial cells (HUVECs) (Csordaset al. 2011). However, no data have been published yet onthe eVect of smoking on intestinal autophagy. The exactmechanisms through which CS triggers the autophagic path-way are not elucidated. Autophagy is induced by environ-mental stress conditions such as genotoxic agents andoxidative stress, both occurring in the context of CS exposure(Levine and Yuan 2005). These stimuli might act on genesactivating or downregulating the autophagy pathway, includ-ing 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 autoph-agy and apoptosis in the context of CS exposure. The con-nections between these two pathways are complex and

Fig. 6 Smoke-induced autophagy is also present in M-cells. a TEMimage of an M-cell after air exposure, showing the hallmarks for rec-ognizing these cells: the presence of microfolds instead of the micro-villi of the neighbouring enterocytes and an intra-epithelial pocket,Wlled with immune cells. No autophagic vesicles are present. b M-cellof smoke-exposed mouse showing the presence of autophagic vesicles.c The percentage of M-cell area occupied by autophagic vesiclesincreases from 0.39 0.22% in air-exposed mice to 1.79 0.77% insmoke-exposed mice. Data are represented as mean SEM. N = 6mice per group. *P < 0.05123

Histochem Cell Biolremain poorly understood. Autophagy can protect againstapoptosis by cleaning up damaged organelles and restoringcell function, but excessive autophagy may cause uncon-trolled degradation of cellular constituents, resulting in celldeath. Apoptosis in turn can induce autophagy, which sub-sequently eliminates apoptotic bodies and by this preventsinXammation (Levine et al. 2011; Ravikumar et al. 2010)but it may also prevent autophagy when acting as a survivalmechanism by caspase-dependent cleavage of Atg proteins(Wirawan et al. 2011). In a previous report, we describedthe presence of CS-induced apoptosis in the FAE of murinePeyers patches (Verschuere et al. 2011), so also in theintestine smoke-induced apoptosis and autophagy occur inthe same cell type. We speculate that CS exerts an injuriouseVect on the FAE and Peyers patches through oxidativestress. If autophagy is unable to clean up the damage, thisinsurmountable injury will result in apoptosis.

Currently, autophagy in the gut epithelium attracts a lotof attention, because of the recently discovered linkbetween Crohns disease and autophagy genes. Severalgenome-wide association studies identiWed polymorphismsin the autophagy genes ATG16L1 and IRGM as risk factorsfor Crohns disease. The ATG16L1 variant associated withCrohns disease, T300A, results in a deWcient bacteria-induced autophagy (Kuballa et al. 2008). Furthermore, alsoNOD2, another known risk factor for Crohns disease, canactivate the autophagic pathway. Genetic variants in NOD2or in autophagy genes which result in reduced autophagy,may diminish intestinal defence mechanisms against inva-sive bacteria, subsequently increasing the susceptibility toCrohns disease (Homer et al. 2010; Netea and Joosten2010).

Interestingly, active smoking and a homozygous geno-type for the T300A variant were found to have a synergisticeVect in the pathogenesis of Crohns disease, with an oddsratio of 7.65 to develop the disease (Fowler et al. 2008).This implies that the negative eVect of cigarette smoke onCD is reinforced by a deWcient autophagy. As smokingitself induces autophagy, one can hypothesize that autoph-agy is a protective mechanism, reversing the cellular injurycaused by smoking. The impairment of this repair processcan cause an accumulation of damaged organelles in cells,increasing apoptosis and tissue injury. Further research willneed to elucidate the exact role of CS-induced autophagyin homeostatic conditions and in intestinal inXammation,and focus on the eVect of CS in a context of impairedautophagy.

In conclusion, this study is the Wrst to describe smoke-induced autophagy in the FAE covering murine Peyerspatches. Both in enterocytes and in M-cells, TEM demon-strated an increase in autophagic vesicle number followingCS exposure. Our Wndings may point to an important rolefor autophagy in protecting the FAE against smoke-induced

oxidative damage and can help to understand the role ofsmoking in the pathogenesis of Crohns disease, in whichautophagy is impaired.

Acknowledgments We are grateful to Barbara Gilbert for providinghelp with the Western blot technique for LC3. We thank Dorothea vanLimbergen and Ran Rumes for the processing of the samples for elec-tron microscopy, and Katrien De Visschere, Isabelle Rottiers, ElianeCastrique, Christelle Snauwaert, Marie-Rose Mouton, Katleen DeSaedeleer, Anouk Goethals, Ann Neesen, Indra De Borle, Greet Barbi-er en Evelien Spruyt for the excellent technical support. Funding wasprovided by the Special Research Fund of Ghent University(01J17507) and Concerted Research Action of Ghent University (BOF10/GOA/021). Stephanie Verschuere is supported by a doctoral Grantfrom the Special Research Fund of Ghent University (01D21009). KenBracke is a postdoctoral researcher of the Fund for ScientiWc Research(FWO) in Flanders.

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Cigarette smoke and the terminal ileum: increased autophagy in murine follicle-associated epithelium and Peyer's patchesAbstractIntroductionMaterials and methodsAnimalsCigarette smoke exposure and sample collectionTransmission electron microscopyRNA preparation and RT-PCRWestern blottingStatistical analysis

ResultsChronic smoke exposure increases the amount of autophagic vesicles in the follicle-associated epitheliumCigarette smoke-induced autophagy is present in the underlying Peyer's patchesM-cells are also affected by smoke-induced autophagy

DiscussionAcknowledgmentsReferences