Cigarette smoking alters epithelial apoptosis and immune composition in murine GALT

Cigarette smoking alters epithelial apoptosis and immune composition in murine GALT
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  Cigarette smoking alters epithelial apoptosis andimmune composition in murine GALT Stephanie Verschuere 1 , Ken R Bracke 2 , Tine Demoor 1 , Maud Plantinga 3 , Phebe Verbrugghe 4 , Liesbeth Ferdinande 1 ,Bart N Lambrecht 3 , Guy GG Brusselle 2, *  and Claude A Cuvelier 1, * Smokers have a twofold increased risk to develop Crohn’s disease (CD). However, little is known about the mechanismsthrough which smoking affects CD pathogenesis. Especially Crohn’s ileitis is negatively influenced by smoking. Inter-estingly, the ileum and, more in particular, the Peyer’s patches in the terminal ileum are also the sites where the first CDlesions are found. Several chemokines are implicated in the pathogenesis, among which is the CCL20-CCR6 pathway.Here, we studied the gut-associated lymphoid tissue in C57BL/6 wild-type mice and in CCR6-deficient mice after exposureto air or cigarette smoke for 24 weeks. Apoptotic index of the follicle-associated epithelium overlying the Peyer’spatches was evaluated. We found that chronic smoke exposure induced apoptosis in the follicle-associated epithelium.Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in Peyer’spatches. Important changes in immune cell composition were observed: total dendritic cells, CD4 þ  T cells (includingregulatory T cells) and CD8 þ  T cells increased significantly after smoke exposure. The CD11b þ  dendritic cell subsetalmost doubled. Interestingly, these changes were accompanied by an upregulated mRNA expression of the chemokinesCCL9 and CCL20. However, no differences in the increase of dendritic cells were observed between wild-type and CCR6-deficient mice. Our results show that cigarette smoke exposure increases apoptosis in the follicle-associated epitheliumand is associated with immune cell accumulation in Peyer’s patches. Laboratory Investigation  advance online publication, 2 May 2011; doi:10.1038/labinvest.2011.74 KEYWORDS:  apoptosis; cigarette smoke; Crohn’s disease; dendritic cell; intestinal epithelium; Peyer’s patch Crohn’s disease (CD) is an incapacitating inflammatory dis-ease of the gastrointestinal tract, mainly involving the term-inal ileum and colon. The social impact of CD is significant,as it is a chronic and relapsing disorder, which principall y affects young patients in the second and third decades of life. 1 Prevalence ranges from 174 to 210 per 100000 inhabitants inNorth America and Europe and is still increasing. 2,3 Despite years of intensive research, ample questions remain on theaetiology of CD. Defects in barrier function as well as al-terations in innate and adaptive immune system are involved,triggering an aberrant immune response against gut flora.Although the pathways leading to intestinal inflammation areincompletely understood, there is a wide consensus that bothgenetic and environmental factors are implicated. 4 Smoking is the most established environmental risk factorin CD. Moreover, smoking is one of the few reversible risk factors identified hitherto. Current smokers have a twofoldhigher lif etime risk to develop CD compared with never-smokers. 5 In addition, smoking is identified as an in-dependent risk factor for the development of clinical, surgicaland endoscopic recurrence after surgery. Former smokershave the same recurrence rates as never-smokers, supportingthe importance of smoking cessation for CD patients. 6 Fur-thermore, smoking is associated with more complicateddisease, a higher need for steroids and immunosuppressantsand poorer response rate to infliximab. 5,7 Notwithstanding this substantial epidemiological evidence,little is known about the molecular and cellular mechanismsby which smoking affects the gut and interferes with CDpathogenesis. Hypotheses include modulation of humoraland cellular immune responses, changes in intestinal cytokinelevels, alterations in mucosal blood flow and modifications in Received 9 December 2010; revised 11 February 2011; accepted 11 March 2011; published online 2 May 2011 1 Department of Pathology, Ghent University, Ghent, Belgium;  2 Laboratory for Translational Research in Obstructive Pulmonary Diseases, Department of RespiratoryMedicine, Ghent University Hospital, Ghent, Belgium;  3 Laboratory of Immunoregulation and Mucosal Immunology, Department of Respiratory Medicine, GhentUniversity, Ghent, Belgium and  4 Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Perth,AustraliaCorrespondence: Dr S Verschuere, MD, Department of Pathology, 5 Blok A, University Hospital Ghent, De Pintelaan 185, Ghent 9000, Belgium.E-mail: *  These authors contributed equally to this work. Laboratory Investigation (2011),  1–12 &  2011 USCAP, Inc All rights reserved 0023-6837/11 $32.00  | Laboratory Investigation | Volume 00 00 2011  1  gut permeability or gut motility by smoke exposure. 8 How-ever, no single mechanism was able to explain the observedeffect of smoking on CD. Previously published humanstudies investigated distinct anatomical compartments (gutlavage fluid  versus  peripheral blood mononuclear cells  versus colonic mucosa), which makes it difficult to draw generalconclusions. 9–12 On the other hand, established animalmodels have several limitations to explore the effect of smoking on CD. First, most reports describe the effect of (sub)acute smoke exposure, whereas human smoking usually involves prolonged periods of smoke exposure. 13–15 Secondly,in several study  designs nicotine is administered orally orsubcutaneously. 16,17 Besides the fact that nicotine is not thesole active component in cigarette smoke, oral and sub-cutaneous administration stand far apart from smokeinhalation. A drawback of both human and animal experi-mental studies is that they generally focus on changes inthe—easier accessible—colon, whereas epidemiological datasuggest a smoke-induced increase in Crohn’s ileitis ratherthan colitis. 7,8 The purpose of this study was to investigate the influenceof cigarette smoke on the mucosal immune system of thesmall intestine in a murine model. Mice w ere exposed tomain stream cigarette smoke for 24 weeks. 18 Because of theknown relationship between smoking and Crohn’s ileitis,effects on the gut-associated lymphoid tissue (GALT) of thesmall intestine—and Peyer’s patches (PP) in particular—wereexamined. Smoking alone is insufficient to cause intestinalinflammation, as observed in both animal models andhuman individuals. However, in combination with other risk factors, like genetic factors or environmental triggers, it canlead to the development of CD and therefore we wereinterested in subclinical effects of smoke exposure in the gut,before full-blown inflammation. We hypothesized thatcigarette smoke alters the immune regulation in PP, predis-posing the gut to develop CD. Therefore, we first investigatedthe effect of smoke exposure on apoptosis in the follicle-associated epithelium (FAE) overlying PP. Secondly, weanalysed cellular composition and size of PP and mesentericlymph nodes. Furthermore, expression of chemokines wasexamined to elucidate how recruitment of immune cellstowards the PP is influenced by cigarette smoke. Finally, therole of the CCL20-CCR6 signalling pathway was furtherinvestigated by means of CCR6 knockout (KO) mice. Weshow that smoking alters apoptosis in the FAE and theimmune cell composition of PP, which may explain thehigher susceptibility of smokers to develop CD. MATERIALS AND METHODSAnimals Male C57BL/6 wild-type (WT) mice were purchased fromCharles River Laboratories. The CCR6 KO mice for this study were male inbred C57BL/6 mice, purchased from the JacksonLaboratory (Bar Harbor, ME, USA). All mice were 8–9 weeksold at the start of the smoke exposure. The local EthicsCommittee for animal experimentation of the faculty of Medicine and Health Sciences (Ghent, Belgium) approved allexperiments (ECD 27/07). Cigarette Smoke Exposure Mice were exposed to main stream cigarette smoke, asdescribed previously. 19 Briefly, groups of 10–12 mice wereexposed to the tobacco smoke of five cigarettes (ReferenceCigarette 3R4F without filter; University of Kentucky,Lexington, KY, USA) four times per day with a 30-minsmoke-free interval, 5 days per week for 24 weeks (chronicsmoke exposure). An optimal smoke:air ratio of 1:6 wasobtained. The control groups were exposed to air. Carbox- yhaemoglobin in serum of smoke-exposed mice reached anon-toxic level of 8.7 ± 0.31% (compared with 0.65 ± 0.25%in air-exposed mice), which is similar to carbox- yhaemoglobin blood concentrations of human smokers. 20 IgA Quantification in Luminal Samples At 24h after the last exposure, mice were weighed and killedwith an overdose of pentobarbital. The abdominal cavity wasopened and the small intestine was removed. Next, intestinalwashes were collected by flushing the small intestine with2ml of ice-cold PBS with 0.1% BSA and 1mg/ml proteaseinhibitors (Protease Inhibitor Cocktail, Complete Mini,Roche, Basel, Switzerland). The collected samples were cen-trifuged for 10min at 4 1 C and the supernatant was stored at  80 1 C for IgA analysis. A commercially available ELISA kit(Alpha Diagnostic International, San Antonio, TX, USA) wasused to determine IgA titre in intestinal washes. Single-Cell Suspensions of PP and MesentericLymph Nodes After flushing, PP and mesenteric lymph nodes were removedfrom the small intestine. Samples were minced by scissorsand put in digestion medium in a humidified incubator at37 1 C and 5% CO 2  for 30min. Fragments were resuspended,fresh digestion medium was added and incubation at 37 1 Cwas continued for 15min. Then, samples were centrifugedand resuspended in PBS containing 10mM EDTA for 5minat room temperature on a shaker. After red blood cell lysis,the cells were washed, passed through a 50- m m strainer andkept on ice until labelling. Cell counting was performed witha Z2 Beckman Coulter particle counter (Beckman Coulter,Ghent, Belgium). Flow Cytometry of PP and Mesenteric Lymph Nodes Single-cell populations of mouse PP and mesenteric lymphnodes were stained with MHCII-PE-Cy5 (eBioscience Inc.,San Diego, CA, USA), CD11c-PE-TexRed (InvitrogenCorp., Carlsbad, CA, USA), CD11b-Horizon V450 (BDPharmingen, San Diego, CA, USA), CD19-AlexaFluor 700(eBioscience), CD3-AlexaFluor 700 (eBioscience), CD4-APC-Cy7 (BD Pharmingen) and CD8-PE-Cy7 (eBioscience). AnAqua Live/Death marker (Invitrogen) was used to identify  Smoking alters intestinal apoptosis and GALT S Verschuere  et al  2  Laboratory Investigation | Volume 00 00 2011 |  live cells. For analysis of regulatory T cells (Treg), use wasmade of anti-CD4-PE-Cy5, anti-CD25-PE-Cy7 and anti-FoxP3-APC (all purchased from eBioscience). Acquisitionwas performed on an LSR cytometer equipped with FACS-Diva software (both BD Pharmingen). The FlowJo softwarewas used for data analysis (TreeStar Inc., Ashland, OR, USA). Quantification of Apoptosis Samples of PP and ileum were fixed in 4% buffered formalin,processed for paraffin embedding and eventually cut into2- m m-thick sections. After antigen retrieval and blocking,primary antibody (polyclonal rabbit anti-active capsase-3;R&D systems, Minneapolis, MN, USA) or rabbit IgG isotypecontrol (Abcam, Cambridge, UK) was applied for 1h, fol-lowed by biotin-labelled goat anti-rabbit secondary antibody (DAKO, Carpinteria, CA, USA) for 30min and HRP-con- jugated streptavidin (DAKO) for 30min. AEC (3-amino-9-ethyl-carbazole) was used as enzyme substrate before coun-terstaining with haematoxylin.For TUNEL (terminal deoxynucleotidyl transferase dUTPnick-end labelling), the  in situ  cell death detection kit alkalinephosphatase (AP) of Roche was used. Staining was per-formed as recommended by Roche. Briefly, after pre-treat-ment using citrate, sections were incubated with TUNELreaction mixture for 60min at 37 1 C in a humidified dark chamber. After rinsing, Converter-AP was applied to thesections for 30min in the dark. Finally, sections were in-cubated with substrate solution for 15min in the dark atroom temperature and counterstained with haematoxylin.The apoptotic index in the FAE was determined as thepercentage of apoptotic cells per 100 epithelial cells en-umerated, as described by Heczko  et al  . 21 Quantification wasperformed at six different levels of the PP. Quantification of apoptosis in the subepithelial dome was accomplished by means of a Zeiss KS400 image analyser platform. Quantifi-cation was performed at five different levels of the PP; thenumber of apoptotic cells per area of the subepithelial domewas calculated. Immunofluorescent Double Staining for UEA-1 andCaspase-3 Fluorescent double-labelling started with antigen retrieval,followed by blocking with 1% BSA and 0.1% FSG in PBS andincubation with the rabbit anti-caspase-3 antibody (R&Dsystems) and the secondary antibody (goat anti-rabbit Alexa596, Invitrogen). Subsequently, FITC-conjugated UEA-1(Sigma Aldrich, St Louis, MO, USA) was applied, followed by DAPI nuclear staining. For immunofluorescent staining forUEA-1 alone, antigen retrieval and blocking was followed by incubation with FITC-conjugated UEA-1 and DAPI staining. Immunohistochemistry for CCL9 and CCL20 Cryosections of PP were air-dried and fixed with ice-coldacetone. Endogenous peroxidase activity was quenched with1% H 2 O 2 , followed by blocking of nonspecific binding siteswith 2% rabbit serum and 1% BSA in PBS. Subsequently,slides were incubated with the primary antibody (polyclonalgoat anti-CCL9 and polyclonal goat anti-CCL20, both R&DSystems) or goat IgG isotype control (Santa Cruz Bio-technology, Santa Cruz, CA, USA) for 90min at 37 1 C andwith the biotinylated rabbit anti-goat secondary antibody (DAKO) for 30min at room temperature. Then, HRP-con- jugated streptavidin (DAKO) was applied for 30min. AECwas used as enzyme substrate before counterstaining withhaematoxylin. RNA Preparation and RT-PCR RNA from PP was extracted using the Qiagen RNeasy MiniKit (Qiagen, Hilden, Germany). Subsequently, cDNA wasobtained by reverse transcription of RNA with the Tran-scriptor First Strand cDNA synthesis kit (Roche) followingmanufacturer’s instructions and using a 2:1 ratio of hex-a:oligodT primers. Expression of target genes  CCL2, CCL9,CCL19, CCL20, CCR1, CCR6, tumour necrosis factor- a  (TNF- a ), IL-1 b  , IFN- g  , IL-6, IL-10 and TGF- b  and reference genes Gapdh  (glyceraldehyde-3-phosphate dehydrogenase),  Hprt1 (hypoxanthine phosphoribosyltransferase 1) and  Tfrc  (transferrin receptor) mRNA was analysed with the TaqManGene Expression Assays (Applied Biosystems). Real-timePCR reactions were performed in duplicate using dilutedcDNA template and the LightCycler480 Probes Master(Roche). Amplifications were performed on a LightCycler480detection system (Roche) with the following cycling condi-tions: 10min incubation at 95 1 C and 50 cycles of 95 1 C for10s and 60 1 C for 15s. Expression of target genes was cor-rected by a normalization factor that was calculated based onthe expression of three reference genes ( Gapdh ,  Hprt1 ,  Tfrc  ),using the geNorm applet according to the guidelines andtheoretical framework described previously. 22 Statistical analysis Reported values are expressed as mean ± s.e.m. (standarderror of the mean) and error bars were marked as the s.e.m.Statistical analysis was performed by SPSS 16 Software (SPSS16 Inc., Chicago, IL, USA) using Student’s  t- test for normally distributed populations and Mann–Whitney   U  -test for po-pulations where normal distribution was not accomplished.For comparison of more than two groups, use was made of two-way analysis of variance (ANOVA), followed by   post hoc  least significant difference test, or non-parametric Kruskal–Wallis test if conditions for ANOVA were not met. A  P  -valueof less than 0.05 was considered significant. RESULTSCigarette Smoke Exposure Induces Apoptosis inFollicle-Associated Epithelium of PP To investigate whether cigarette smoke affects the FAE of PP, the apoptotic index was determined by means of immunohistochemistry for active caspase-3, a marker forapoptosis. Following smoke exposure, significantly more FAE Smoking alters intestinal apoptosis and GALT S Verschuere  et al  | Laboratory Investigation | Volume 00 00 2011  3  cells stained positive for caspase-3 compared to air-exposedanimals (Figure 1a and b). This difference in apoptotic index was confirmed using TUNEL, an alternative detection methodfor apoptosis (data not shown). To establish whether cigarettesmoke-induced apoptosis was limited to the FAE, apoptosis inthe subepithelial dome was quantified as well. The number of apoptotic cells in the subepithelial dome was similar in bothgroups (Figure 1c and d). In contrast to FAE, the villousepithelium of the ileum did not show increased apoptosisafter smoke exposure (Supplementary Figure 1A–G).An important feature of FAE is the presence of M cells,epithelial cells whose main function is transcytosis of antigens and macromolecules through the epithelial barrierinto the PP, allowing generation of mucosal immuneresponses. First, we quantified M-cell numbers in the FAE by means of staining for UEA-1, a murine M-cell marker.Interestingly, smoke-exposed mice had a significantly lower proportion of M cells than air-exposed animals(Figure 2a and b). Secondly, to investigate whether smoke-induced apoptosis also affects M cells, we performed afluorescent double staining for active-caspase-3 and UEA-1.In both groups, very few M cells showed signs of apoptosis(Figure 2c). Moreover, M-cell apoptosis did not differbetween air- and smoke-exposed mice. Figure 1  Smoke exposure induces apoptosis in follicle-associated epithelium (FAE) of Peyer’s patches (PP). ( a ) Immunohistochemistry for active caspase-3 ina PP of a smoke-exposed mouse, with apoptotic cells in the FAE. Inset: details of an apoptotic cell. ( b ) Apoptotic index, defined as the percentage of apoptotic cells in FAE (determined by active caspase-3 immunohistochemistry), after smoke exposure (1.76 ± 0.14%) compared with air exposure(0.98 ± 0.09%). Each group consists of 20 PP from four different mice. ( c ) The subepithelial dome was defined as the region of the PP between the FAE andthe germinal follicles. Image analysis software calculated the number of apoptotic cells (determined by active caspase-3 immunohistochemistry) in this area.( d ) Apoptotic cell number per area in the subepithelial dome. Data are from one representative of two independent experiments. Data are represented asmean ± s.e.m. NS: nonsignificant;  *** P  o 0.001. Smoking alters intestinal apoptosis and GALT S Verschuere  et al  4  Laboratory Investigation | Volume 00 00 2011 |  Immune Cell Composition of PP is Influenced bySmoke Exposure To further investigate the influence of cigarette smoke on theGALT in the small intestine, PPs were quantified. Similarnumbers were found in air- and smoke-exposed animals(5–9PP per mouse). Furthermore, size of ileal PP did notdiffer between both groups (Supplementary Figure 2A).Next, the different cell populations in PP were examinedby means of flow cytometry. We observed an increase indendritic cells (DCs) in smoke-exposed animals (Figure 3aand b). The rise was even more obvious in the CD11b þ  DCsubset, which practically doubled (Figure 3c).Although the total lymphocyte number, defined as the sumof CD3 þ  and CD19 þ  cells, did not change after cigarettesmoke exposure (data not shown), shifts were seen in totalB-cell percentage (Supplementary Figure 2B). The T-cellpercentage increased significantly after smoke exposure(Supplementary Figure 2C).T-cell subsets were further analysed. The CD8 þ  T-cellsubset increased significantly after smoke exposure (Supple-mentary Figure 2D), as well as the CD4 þ  T-cell subset andTreg (Figure 4a–c).To examine the influence of smoke exposure on otherlymphoid structures related to the small intestine, mesentericlymph nodes were analysed. Size, determined as total cellcount of three lymph nodes, did not differ between bothgroups. Furthermore, no difference in immune cell compo-sition of mesenteric lymph nodes was observed between air-and smoke-exposed mice (data not shown). Cigarette Smoke Exposure Suppresses IL-10 Expressionin PP To examine whether the observed changes in immune cellcomposition in PP were associated with inflammation,mRNA of pro-inflammatory and anti-inflammatory cyto-kines were studied. No changes in expression of TNF- a ,IL-1 b , IFN- g  or IL-6 were detected, indicating that smokeexposure did not induce alterations in pro-inflammatory cytokine levels. Also TGF- b 1, an anti-inflammatory cytokine,did not undergo any changes in its mRNA expression.However, IL-10 expression showed a significant decrease aftersmoke exposure (Supplementary Figure 3A–F).Furthermore, the lower B-cell percentage in PP promptedus to investigate the influence of cigarette smoke inhalationon mucosal IgA antibody production in the small intestine.PP are important inductive sites for IgA responses in the gutand IgA þ  B cells, which are precursors for IgA-producingplasma cells, are mainly generated in the PP. 23 Total luminalIgA immunoglobulin concentration was measured by ELISA. However, no significant differences were seen betweenair- and smoke-exposed mice after 24 weeks of exposure(data not shown). Expression of DC-Attracting Chemokines Increases onCigarette Smoke Exposure To unravel the mechanisms leading to an increase in DC andthe CD11b þ  DC subset in PP, mRNA expression of che-mokines involved in recruitment of DC was studied. Ex-pression of CCL9 and CCL20 increased after smoke exposure(Figure 5a and b). These chemokines are known to be ex-pressed by the FAE and attract CD11b þ  DC to the sub-epithelial dome through interaction with their respectivereceptors CCR1 and CCR6, both present on DC. 24 In con-trast, expression of CCL19, a chemokine involved in at-tracting DC to the interfollicular region of PP, was not alteredafter cigarette smoke exposure (Figure 5c). Also CCL2, achemokine that has a role in migration and maturation of monocyte-derived DC, was not expressed differentially in air-and smoke-exposed mice (Figure 5d). Furthermore, mRNAexpression of the chemokine receptors CCR1 and CCR6 wasdetermined. CCR1 expression was not influenced by cigarettesmoke exposure (Figure 5e). Expression of CCR6 decreasedin the smoke-exposed group compared with air-exposedmice (Figure 5f ). Figure 2  M-cell percentage decreases after exposure to cigarette smoke. ( a ) M-cell percentage, defined as the percentage of Ulex europaeus agglutinin-1(UEA-1)-positive cells in the follicle-associated epithelium (FAE), in smoke- (7.67 ± 0.59%) and air-exposed animals (9.99 ± 0.62%). Each group consists of 20 PP from four different mice. ( b ) Immunofluorescent staining with UEA-1 (green) for quantification of M cells in the FAE. ( c ) Confocal microscopyshows absence of colocalization of active caspase-3 (red) and the M-cell marker UEA-1 (green). Data are from one representative of two independentexperiments. Data are represented as mean ± s.e.m.  ** P  o 0.01. Smoking alters intestinal apoptosis and GALT S Verschuere  et al  | Laboratory Investigation | Volume 00 00 2011  5
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