Cysteine Protease–Dependent Mucous Disruptions and Differential Mucin Gene Expression in Giardia duodenalis Infection
2017; Elsevier BV; Volume: 187; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2017.07.009
ISSN1525-2191
AutoresChristina Amat, Jean‐Paul Motta, Elena Fekete, France Moreau, Kris Chadee, André G. Buret,
Tópico(s)Parasites and Host Interactions
ResumoThe intestinal mucous layer provides a critical host defense against pathogen exposure and epithelial injury, yet little is known about how enteropathogens may circumvent this physiologic barrier. Giardia duodenalis is a small intestinal parasite responsible for diarrheal disease and chronic postinfectious illness. This study reveals a complex interaction at the surface of epithelial cells, between G. duodenalis and the intestinal mucous layer. Here, we reveal mechanisms whereby G. duodenalis evades and disrupts the first line of host defense by degrading human mucin-2 (MUC2), depleting mucin stores and inducing differential gene expression in the mouse small and large intestines. Human colonic biopsy specimens exposed to G. duodenalis were depleted of mucus, and in vivo mice infected with G. duodenalis had a thinner mucous layer and demonstrated differential Muc2 and Muc5ac mucin gene expression. Infection in Muc2−/− mice elevated trophozoite colonization in the small intestine and impaired weight gain. In vitro, human LS174T goblet-like cells were depleted of mucus and had elevated levels of MUC2 mRNA expression after G. duodenalis exposure. Importantly, the cysteine protease inhibitor E64 prevented mucous degradation, mucin depletion, and the increase in MUC2 expression. This article describes a novel role for Giardia's cysteine proteases in pathogenesis and how Giardia's disruptions of the mucous barrier facilitate bacterial translocation that may contribute to the onset and propagation of disease. The intestinal mucous layer provides a critical host defense against pathogen exposure and epithelial injury, yet little is known about how enteropathogens may circumvent this physiologic barrier. Giardia duodenalis is a small intestinal parasite responsible for diarrheal disease and chronic postinfectious illness. This study reveals a complex interaction at the surface of epithelial cells, between G. duodenalis and the intestinal mucous layer. Here, we reveal mechanisms whereby G. duodenalis evades and disrupts the first line of host defense by degrading human mucin-2 (MUC2), depleting mucin stores and inducing differential gene expression in the mouse small and large intestines. Human colonic biopsy specimens exposed to G. duodenalis were depleted of mucus, and in vivo mice infected with G. duodenalis had a thinner mucous layer and demonstrated differential Muc2 and Muc5ac mucin gene expression. Infection in Muc2−/− mice elevated trophozoite colonization in the small intestine and impaired weight gain. In vitro, human LS174T goblet-like cells were depleted of mucus and had elevated levels of MUC2 mRNA expression after G. duodenalis exposure. Importantly, the cysteine protease inhibitor E64 prevented mucous degradation, mucin depletion, and the increase in MUC2 expression. This article describes a novel role for Giardia's cysteine proteases in pathogenesis and how Giardia's disruptions of the mucous barrier facilitate bacterial translocation that may contribute to the onset and propagation of disease. Giardia duodenalis (Giardia lamblia, Giardia intestinalis), the causative agent of giardiasis, is a protozoan parasite that infects animals and humans across the globe. Annually, it infects hundreds of millions of individuals worldwide, which led to its inclusion in the World Health Organization's Neglected Disease Initiative.1Savioli L. Smith H. Thompson A. Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’.Trends Parasitol. 2006; 22: 203-208Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar Moreover, giardiasis is a detriment to the agricultural economy because it causes weight loss in animals.2Olson M.E. McAllister T.A. Deselliers L. Morck D.W. Cheng K.J. Buret A.G. Ceri H. Effects of giardiasis on production in a domestic ruminant (lamb) model.Am J Vet Res. 1995; 56: 1470-1474PubMed Google Scholar, 3Geurden T. Vercruysse J. Claerebout E. Is Giardia a significant pathogen in production animals?.Exp Parasitol. 2010; 124: 98-106Crossref PubMed Scopus (83) Google Scholar In humans, the pathogenic effects of G. duodenalis result in severe malnutrition and malabsorptive diarrhea and, in some cases, especially in young children and in those in developing countries, impaired weight gain and an ultimate failure to thrive.4Halliez M.C. Buret A.G. Extra-intestinal and long term consequences of Giardia duodenalis infections.World J Gastroenterol. 2013; 19: 8974-8985Crossref PubMed Scopus (248) Google Scholar, 5Platts-Mills J.A. Babji S. Bodhidatta L. Gratz J. Haque R. Havt A. et al.MAL-ED Network InvestigatorsPathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED).Lancet Glob Health. 2015; 3: e564-e575Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar Moreover, giardiasis has been implicated in long-term and extraintestinal consequences, including arthritis, skin allergies, myopathy, and diminished cognitive function.4Halliez M.C. Buret A.G. Extra-intestinal and long term consequences of Giardia duodenalis infections.World J Gastroenterol. 2013; 19: 8974-8985Crossref PubMed Scopus (248) Google Scholar Recent evidence has revealed that acute G. duodenalis infection may lead to chronic functional gastrointestinal disorders, including postinfectious irritable bowel syndrome (IBS).6Hanevik K. Dizdar V. Langeland N. Hausken T. Development of functional gastrointestinal disorders after Giardia lamblia infection.BMC Gastroenterol. 2009; 9: 27Crossref PubMed Scopus (121) Google Scholar Indeed, follow-up studies of a 2004 waterborne outbreak of giardiasis in Norway indicated that up to 80% of patients who experienced giardiasis developed IBS-like symptoms after the clearance of the parasite.6Hanevik K. Dizdar V. Langeland N. Hausken T. Development of functional gastrointestinal disorders after Giardia lamblia infection.BMC Gastroenterol. 2009; 9: 27Crossref PubMed Scopus (121) Google Scholar, 7Nygard K. Schimmer B. Sobstad O. Walde A. Tveit I. Langeland N. Hausken T. Aavitsland P. A large community outbreak of waterborne giardiasis-delayed detection in a non-endemic urban area.BMC Public Health. 2006; 6: 141Crossref PubMed Scopus (140) Google Scholar The mechanisms that underlie the transition of an acute, transient infection into chronic IBS symptoms remain unknown. Currently, no prophylaxis is available against giardiasis, but an established infection in humans is often treated with nitroimidazole drugs, including the common antiparasitic, metronidazole.8Pasupuleti V. Escobedo A.A. Deshpande A. Thota P. Roman Y. Hernandez A.V. Efficacy of 5-nitroimidazoles for the treatment of giardiasis: a systematic review of randomized controlled trials.PLoS Negl Trop Dis. 2014; 8: e2733Crossref PubMed Scopus (35) Google Scholar Unfortunately, increasing rates of resistance have been documented in the literature, underlining the need for novel therapeutic discovery.9Ansell B.R. McConville M.J. Ma'ayeh S.Y. Dagley M.J. Gasser R.B. Svard S.G. Jex A.R. Drug resistance in Giardia duodenalis.Biotechnol Adv. 2015; 33: 888-901Crossref PubMed Scopus (83) Google Scholar, 10Upcroft P. Upcroft J.A. Drug targets and mechanisms of resistance in the anaerobic protozoa.Clin Microbiol Rev. 2001; 14: 150-164Crossref PubMed Scopus (428) Google Scholar On ingestion, infectious G. duodenalis cysts undergo excystation in the small intestine to release the disease-causing trophozoites, which further divide and proliferate. Before attaching to epithelial cells via a ventral adhesive disk, trophozoites must penetrate the mucous layer, which is designed to protect against invading pathogens. The small intestinal mucous layer is irregular and loosely attached, whereas the colon is lined by a continuous, double mucous layer—an inner sterile layer firmly attached to the epithelium and a loose, porous layer that contains resident bacteria.11Johansson M.E. Phillipson M. Petersson J. Velcich A. Holm L. Hansson G.C. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria.Proc Natl Acad Sci U S A. 2008; 105: 15064-15069Crossref PubMed Scopus (1367) Google Scholar Goblet cells in the epithelium secrete mucins, of which 20 have been identified. Mucin-2 (MUC2), mucin-5AC (MUC5AC), mucin-5B (MUC5B), mucin-6 (MUC6), and mucin-19 (MUC19) are the oligomerizing secreted mucins. Human MUC5AC (mouse Muc5ac) is typically only found in the respiratory tract and stomach, whereas MUC2 (mouse Muc2) is the main component of mucus in the small and large intestines.12Johansson M.E. Sjovall H. Hansson G.C. The gastrointestinal mucus system in health and disease.Nat Rev Gastroenterol Hepatol. 2013; 10: 352-361Crossref PubMed Scopus (801) Google Scholar After oligomerization and glycosylation, mature mucins are packaged into secretory vesicles and stored in the apical cytoplasm of goblet cells.13Forstner G. Signal transduction, packaging and secretion of mucins.Annu Rev Physiol. 1995; 57: 585-605Crossref PubMed Scopus (146) Google Scholar, 14Birchenough G.M. Johansson M.E. Gustafsson J.K. Bergstrom J.H. Hansson G.C. New developments in goblet cell mucus secretion and function.Mucosal Immunol. 2015; 8: 712-719Crossref PubMed Scopus (378) Google Scholar Mucins are released constitutively via vesicle membrane fusion and released via exocytosis in response to external stimuli.13Forstner G. Signal transduction, packaging and secretion of mucins.Annu Rev Physiol. 1995; 57: 585-605Crossref PubMed Scopus (146) Google Scholar, 14Birchenough G.M. Johansson M.E. Gustafsson J.K. Bergstrom J.H. Hansson G.C. New developments in goblet cell mucus secretion and function.Mucosal Immunol. 2015; 8: 712-719Crossref PubMed Scopus (378) Google Scholar Once released, mucins expand in the luminal water to form a viscous protective barrier. Despite its critical importance to mucosal homeostasis, the regulation of mucin production and secretion in health and disease remains incompletely understood. The interactions between G. duodenalis and the intestinal mucous barrier remain obscure. Although isolated reports have described that some parasites may damage the mucous layer via secreted proteases,15Lidell M.E. Moncada D.M. Chadee K. Hansson G.C. Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel.Proc Natl Acad Sci U S A. 2006; 103: 9298-9303Crossref PubMed Scopus (209) Google Scholar, 16Hasnain S.Z. McGuckin M.A. Grencis R.K. Thornton D.J. Serine protease(s) secreted by the nematode Trichuris muris degrade the mucus barrier.PLoS Negl Trop Dis. 2012; 6: e1856Crossref PubMed Scopus (79) Google Scholar the role of Giardia's cysteine proteases, of which 27 with uncharacterized functions are found in the genome,17DuBois K.N. Abodeely M. Sakanari J. Craik C.S. Lee M. McKerrow J.H. Sajid M. Identification of the major cysteine protease of Giardia and its role in encystation.J Biol Chem. 2008; 283: 18024-18031Crossref PubMed Scopus (54) Google Scholar remains unclear. The results of this study describe how Giardia, via its cysteine proteases, degrades the intestinal mucous layer and depletes goblet cell mucins to impair mucous barrier function. Our findings also describe differential mucin gene expression in response to intestinal infection. Experiments involving animals were approved by the University of Calgary's Animal Care Committee that adheres to the governance of the Canadian Council on Animal Care (certificate AC13-0067). Studies involving human biopsy tissues were approved by the Conjoint Health Research Ethics Board at the University of Calgary and the Calgary Health Region (certificate REB14-2430 REN1). In accordance with Conjoint Health Research Ethics Board guidelines, subjects used in this study provided informed written or guardian-overseen consent. The irreversible broad-spectrum cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane (E64) was purchased from Sigma-Aldrich (Oakville, Ontario, Canada). The membrane-permeant, cathepsin B-specific inhibitor [2S,3S-3-propylcarbamoyloxirane-2-carbonyl]-l-isoleucyl-l-proline methyl ester (Ca074Me) was purchased from Peptide Institute, Inc. (Osaka, Japan). The selectivity of this inhibitor was recently validated.18Cotton J.A. Bhargava A. Ferraz J.G. Yates R.M. Beck P.L. Buret A.G. Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis.Infect Immun. 2014; 82: 2772-2787Crossref PubMed Scopus (73) Google Scholar, 19Bhargava A. Cotton J.A. Dixon B.R. Gedamu L. Yates R.M. Buret A.G. Giardia duodenalis surface cysteine proteases induce cleavage of the intestinal epithelial cytoskeletal protein villin via myosin light chain kinase.PLoS One. 2015; 10: e0136102Crossref PubMed Scopus (47) Google Scholar The selective phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) and the highly-selective cell-permeable protein kinase C (PKC) inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione [bisindolylmaleimide I (BIM1)] were purchased from Cayman Chemical (Ann Arbor, MI). The function-blocking antibody specific for αvβ3 integrin LM609 was purchased from EMD Millipore (Etobicoke, Ontario, Canada). 7-Hydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one, 7-hydroxy-3-(4-methoxyphenyl)chromone, and 7-hydroxy-4′-methoxyisoflavone (formononetin)20Lauwaet T. Andersen Y. Van de Ven L. Eckmann L. Gillin F.D. Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin.J Antimicrob Chemother. 2010; 65: 531-534Crossref PubMed Scopus (21) Google Scholar used to detach G. duodenalis trophozoites from cells were purchased from Sigma-Aldrich. G. duodenalis NF (Assemblage A) was originally isolated from contaminated water that caused a human epidemic of giardiasis in Newfoundland, Canada. G. duodenalis GS/M isolate (Assemblage B) was purchased from ATCC (Manassas, VA). Trophozoites were cultured axenically in polystyrene conical tubes with Keister's modified TY1-S-33 medium21Keister D.B. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile.Trans R Soc Trop Med Hyg. 1983; 77: 487-488Abstract Full Text PDF PubMed Scopus (790) Google Scholar supplemented with penicillin-streptomycin (Sigma-Aldrich) and used at culture peak density. Mucosal biopsy specimens from the descending colon of 5 healthy human donors were collected during routine screening for colon cancer (Intestinal Inflammation Tissue Bank, University of Calgary, Calgary, Alberta, Canada). On collection, samples were washed to remove loosely adherent mucus and bacteria, then incubated with 1.0 × 107/mL NF G. duodenalis trophozoites for 3 hours in prewarmed Hanks' balanced salt solution, as described previously.22Cotton J.A. Motta J.P. Schenck L.P. Hirota S.A. Beck P.L. Buret A.G. Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.PLoS One. 2014; 9: e109087Crossref PubMed Scopus (53) Google Scholar The human colonic epithelial cell line LS174T (ATCC CL-188) was grown in minimum essential medium Eagle solution (M7278; Sigma-Aldrich) supplemented with 100 μg/mL of streptomycin, 100 U/mL of penicillin, 200 mmol/L of l-glutamine, 5 mmol/L of sodium pyruvate (all purchased from Sigma-Aldrich), and 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Ottawa, Ontario, Canada). Cells were passaged at 80% confluence via detachment with 2× trypsin-EDTA and seeded onto 6-well plates (VWR, Mississauga, Ontario, Canada) for real-time quantitative PCR (qPCR) or 8-well chamber slides (Thermo Fisher Scientific) for staining. Cells were maintained at 37°C, 5% CO2, and 96% humidity and media replaced every 2 to 3 days. Cells on chamber slides were incubated with G. duodenalis NF or GS/M trophozoites at a multiplicity of infection of 20 trophozoites per cell (20:1), for various time points. G. duodenalis NF trophozoites were used in all in vitro studies with LS174T cells, unless otherwise stated. After incubation with G. duodenalis, cells were washed and exposed to formononetin in cell media to remove adherent trophozoites.20Lauwaet T. Andersen Y. Van de Ven L. Eckmann L. Gillin F.D. Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin.J Antimicrob Chemother. 2010; 65: 531-534Crossref PubMed Scopus (21) Google Scholar Cells were fixed in 4% paraformaldehyde (Santa Cruz, Mississauga, Ontario, Canada), stained with fluorescein-coupled wheat germ agglutinin (WGA) (Thermo Fisher Scientific), and mounted with DAPI (Sigma-Aldrich). Images were acquired using a Leica DMR Microscope (epifluorescence) and Leica DM IRE2 (confocal). A ratio of WGA-positive surface area to DAPI-positive surface area was performed on ImageJ software version 1.51 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). For inhibition studies, cells were pretreated for 30 minutes with 20 μmol/L PI3K inhibitor LY294002, 10 μmol/L PKC inhibitor BIM1, or 5 μg/mL antiintegrin αvβ3 antibody LM609 for 5 minutes before incubation with G. duodenalis trophozoites. Male and female Muc2−/− mice of C57BL/6 genetic origin, aged 5 to 9 weeks, originally sourced from Velcich et al,23Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295: 1726-1729Crossref PubMed Scopus (716) Google Scholar were bred in-house as described in Cobo et al.24Cobo E.R. Kissoon-Singh V. Moreau F. Chadee K. Colonic MUC2 mucin regulates the expression and antimicrobial activity of beta-defensin 2.Mucosal Immunol. 2015; 8: 1360-1372Crossref PubMed Scopus (65) Google Scholar Male and female C57BL/6 mice, aged 5 to 9 weeks were ordered from Charles River Laboratories (St-Constant, Quebec, Canada). Mice were orally gavaged with 1.0 × 107 GS/M G. duodenalis trophozoites in 0.1 mL of 70% TY1-S-33, 30% NaHCO3 medium or with 0.1 mL of 70% TY1-S-33, 30% NaHCO3 medium as the vehicle control. Mice were weighed daily and euthanized by cervical dislocation. Trophozoite counts were performed by excising 3-cm sections from the jejunum and colon of euthanized mice. The sections were cut longitudinally, placed in tubes with 1 mL of phosphate-buffered saline (PBS; Sigma-Aldrich), and placed on ice for 15 minutes to aid in trophozoite detachment from the tissue. Before counting, the tubes were thoroughly vortexed to detach remaining trophozoites, and 10 μL of solution was pipetted into a hemocytometer for counting with a light microscope. For bacterial translocation assays, mouse liver and spleen were collected aseptically, weighed, homogenized, and plated on 5% Columbia blood agar plates (VWR). Samples were incubated aerobically and anaerobically for 48 hours. Bacterial colony-forming units were enumerated and normalized to grams of tissue. For cytokine assays, mouse jejunal tissue was homogenized in 1 mL of lysis buffer [20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5% Tween-20, and a Complete Minitab protease inhibitor cocktail (Sigma-Aldrich)]. Cytokine expression levels were assessed via a Luminex Discovery Assay (Eve Technologies, Calgary, Alberta, Canada) according to the manufacturer's protocol. Sections 1 cm thick of proximal jejunum and proximal colon were fixed in Carnoy's fixative (60% ethanol-100%, 30% chloroform, 10% glacial acetic acid) or 4% paraformaldehyde. Visualization of intestinal microbiota was performed using fluorescent in situ hybridization (FISH) according to a previously published protocol.25Motta J.P. Flannigan K.L. Agbor T.A. Beatty J.K. Blackler R.W. Workentine M.L. Da Silva G.J. Wang R. Buret A.G. Wallace J.L. Hydrogen sulfide protects from colitis and restores intestinal microbiota biofilm and mucus production.Inflamm Bowel Dis. 2015; 21: 1006-1017Crossref PubMed Scopus (112) Google Scholar The mucous layer was stained in these sections using fluorescein-WGA to stain sialic acid and N-acetylglucosamine residues, and slides were mounted with DAPI. Quantifications of mucous layer thickness and WGA-positive staining were determined using ImageJ software. Human biopsy specimens were fixed in 4% paraformaldehyde, and mucin content was stained using periodic acid–Schiff/Alcian blue (PAS/AB) as per manufacturer instructions (Newcomer Supply, Middleton, WI). Mucin surface area was quantified using ImageJ software. Secreted mucin was purified from LS174T human goblet-like cells as previously described.26Belley A. Keller K. Grove J. Chadee K. Interaction of LS174T human colon cancer cell mucins with Entamoeba histolytica: an in vitro model for colonic disease.Gastroenterology. 1996; 111: 1484-1492Abstract Full Text PDF PubMed Scopus (47) Google Scholar In brief, supernatant collected from LS174T cells was dialyzed, lyophilized, and resuspended in Tris-HCl buffer that contained sodium azide, followed by fractionation on a Sepharose 4B gel filtration column. High-molecular-weight mucin fractions in the void volume were dialyzed again and concentrated using centrifugal filter units. G. duodenalis NF trophozoites secreted product was obtained from centrifugation of trophozoites incubated in PBS for 2 hours and concentrated using 3-kDa centrifugal filter units (EMD Millipore). Secreted products were incubated with or without 20 μg/mL of E64 for 30 minutes before the addition of purified mucin. Purified mucin (3 μg) was incubated with Giardia's secreted products in PBS at 37°C for 3 hours. Solutions were resuspended in Laemlli buffer; proteins were separated by electrophoresis (4% stacking and 7% resolving gel) and transferred onto nitrocellulose membranes (GE Healthcare, Mississauga, Ontario, Canada). Membranes were incubated with affinity-purified rabbit IgG mucin antibody,26Belley A. Keller K. Grove J. Chadee K. Interaction of LS174T human colon cancer cell mucins with Entamoeba histolytica: an in vitro model for colonic disease.Gastroenterology. 1996; 111: 1484-1492Abstract Full Text PDF PubMed Scopus (47) Google Scholar and bands were visualized with ECL-plus chemiluminescence detection (GE Healthcare). Mucin-specific band intensity (540 kDa) was quantified using ImageJ software. Total RNA was extracted from 1 cm of mouse jejunum tissue or LS174T cells using an RNeasy Mini Kit (Qiagen, Montreal, Quebec, Canada) as per manufacturer's instructions. cDNA was synthesized with a QuantiTect Reverse Transcription Kit (Qiagen). RT-PCR was performed using a Rotor Gene-Q (Qiagen). For qPCR with LS174T cells, human MUC2 was normalized to β2-microglobulin (B2M NM_004048, PPH01094E, RT2 qPCR Primer Assay; Qiagen) expression. For qPCR with mouse tissue, murine Muc2 and Muc5ac were normalized to β-actin expression. All primer sets are listed in Table 1,27Iwashita J. Sato Y. Sugaya H. Takahashi N. Sasaki H. Abe T. mRNA of MUC2 is stimulated by IL-4, IL-13 or TNF-alpha through a mitogen-activated protein kinase pathway in human colon cancer cells.Immunol Cell Biol. 2003; 81: 275-282Crossref PubMed Scopus (109) Google Scholar, 28Wallace J.L. Vong L. Dharmani P. Srivastava V. Chadee K. Muc-2-deficient mice display a sex-specific, COX-2-related impairment of gastric mucosal repair.Am J Pathol. 2011; 178: 1126-1133Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 29Osada T. Uehara H. Kim H. Ikai A. mRNA analysis of single living cells.J Nanobiotechnology. 2003; 1: 2Crossref PubMed Scopus (56) Google Scholar, 30Morampudi V. Dalwadi U. Bhinder G. Sham H.P. Gill S.K. Chan J. Bergstrom K.S. Huang T. Ma C. Jacobson K. Gibson D.L. Vallance B.A. The goblet cell-derived mediator RELM-beta drives spontaneous colitis in Muc2-deficient mice by promoting commensal microbial dysbiosis.Mucosal Immunol. 2016; 9: 1218-1233Crossref PubMed Scopus (66) Google Scholar, 31Png C.W. Linden S.K. Gilshenan K.S. Zoetendal E.G. McSweeney C.S. Sly L.I. McGuckin M.A. Florin T.H. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.Am J Gastroenterol. 2010; 105: 2420-2428Crossref PubMed Scopus (859) Google Scholar (University Core DNA Services at the University of Calgary, Calgary, Alberta, Canada). Data were analyzed using a 2−ΔΔCT method and expressed as a fold change compared with wild-type (WT) noninfected mice or LS174T cells not exposed to G. duodenalis.Table 1Targets and Primer Sets for RT-PCRTarget mRNASource referenceHuman MUC2Iwashita et al27Iwashita J. Sato Y. Sugaya H. Takahashi N. Sasaki H. Abe T. mRNA of MUC2 is stimulated by IL-4, IL-13 or TNF-alpha through a mitogen-activated protein kinase pathway in human colon cancer cells.Immunol Cell Biol. 2003; 81: 275-282Crossref PubMed Scopus (109) Google ScholarMouse Muc2Fwd: 5′-GAAGCCAGATCCCGAAACCA-3′Rev: 5′-CCAGCTTGTGGGTGAGGTAG-3′Mouse Muc5acWallace et al28Wallace J.L. Vong L. Dharmani P. Srivastava V. Chadee K. Muc-2-deficient mice display a sex-specific, COX-2-related impairment of gastric mucosal repair.Am J Pathol. 2011; 178: 1126-1133Abstract Full Text Full Text PDF PubMed Scopus (20) Google ScholarMouse B-actinOsada et al29Osada T. Uehara H. Kim H. Ikai A. mRNA analysis of single living cells.J Nanobiotechnology. 2003; 1: 2Crossref PubMed Scopus (56) Google ScholarBacteroidetesMorampudi et al30Morampudi V. Dalwadi U. Bhinder G. Sham H.P. Gill S.K. Chan J. Bergstrom K.S. Huang T. Ma C. Jacobson K. Gibson D.L. Vallance B.A. The goblet cell-derived mediator RELM-beta drives spontaneous colitis in Muc2-deficient mice by promoting commensal microbial dysbiosis.Mucosal Immunol. 2016; 9: 1218-1233Crossref PubMed Scopus (66) Google ScholarFirmicutesMorampudi et al30Morampudi V. Dalwadi U. Bhinder G. Sham H.P. Gill S.K. Chan J. Bergstrom K.S. Huang T. Ma C. Jacobson K. Gibson D.L. Vallance B.A. The goblet cell-derived mediator RELM-beta drives spontaneous colitis in Muc2-deficient mice by promoting commensal microbial dysbiosis.Mucosal Immunol. 2016; 9: 1218-1233Crossref PubMed Scopus (66) Google ScholarProteobacteriaMorampudi et al30Morampudi V. Dalwadi U. Bhinder G. Sham H.P. Gill S.K. Chan J. Bergstrom K.S. Huang T. Ma C. Jacobson K. Gibson D.L. Vallance B.A. The goblet cell-derived mediator RELM-beta drives spontaneous colitis in Muc2-deficient mice by promoting commensal microbial dysbiosis.Mucosal Immunol. 2016; 9: 1218-1233Crossref PubMed Scopus (66) Google ScholarAkkermansia muciniphilaPng et al31Png C.W. Linden S.K. Gilshenan K.S. Zoetendal E.G. McSweeney C.S. Sly L.I. McGuckin M.A. Florin T.H. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.Am J Gastroenterol. 2010; 105: 2420-2428Crossref PubMed Scopus (859) Google ScholarRuminococcus gnavusPng et al31Png C.W. Linden S.K. Gilshenan K.S. Zoetendal E.G. McSweeney C.S. Sly L.I. McGuckin M.A. Florin T.H. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.Am J Gastroenterol. 2010; 105: 2420-2428Crossref PubMed Scopus (859) Google Scholar16s rRNAPng et al31Png C.W. Linden S.K. Gilshenan K.S. Zoetendal E.G. McSweeney C.S. Sly L.I. McGuckin M.A. Florin T.H. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.Am J Gastroenterol. 2010; 105: 2420-2428Crossref PubMed Scopus (859) Google Scholar Open table in a new tab Total DNA from frozen feces samples were extracted using a DNA Mini Stool Kit (Qiagen) and following a previously published protocol,30Morampudi V. Dalwadi U. Bhinder G. Sham H.P. Gill S.K. Chan J. Bergstrom K.S. Huang T. Ma C. Jacobson K. Gibson D.L. Vallance B.A. The goblet cell-derived mediator RELM-beta drives spontaneous colitis in Muc2-deficient mice by promoting commensal microbial dysbiosis.Mucosal Immunol. 2016; 9: 1218-1233Crossref PubMed Scopus (66) Google Scholar with slight modifications. Quantitative RT-PCR was performed with a QuantiFast SYBR green PCR kit (Qiagen) on a Rotor Gene-Q (Qiagen). Primer sets for all bacteria are listed in Table 1 (University Core DNA Services at the University of Calgary). Percent composition of each group was normalized to total 16S rRNA quantity (total bacteria). All values are expressed as means ± SEM, unless otherwise noted. Statistical analysis was performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc., San Diego, CA). The normality of the data was assessed before statistical analysis. Differences between groups were assessed using one-way analysis of variance with Tukey or Bonferroni multiple comparison's test or two-way analysis of variance with Bonferroni posttests for comparison of three or more groups. For differences between two groups, a U-test for comparison of two nonparametric groups or a t-test for parametric groups were used. To determine the effects of G. duodenalis in the human gut, colonic biopsy specimens from healthy donors were exposed to live trophozoites. Human colonic biopsies exposed to G. duodenalis trophozoites for 3 hours had reduced intracellular mucin compared with vehicle (PBS)–treated controls (Figure 1, A and B). PAS/AB staining revealed that biopsy specimens exposed to G. duodenalis contained a mean of 13.25% ± 1.26% mucous-positive area per crypt, which was significantly lower than the control biopsy specimens (17.39% ± 1.26%) (Figure 1B). These results demonstrate that G. duodenalis reduces goblet cell mucin stores in human intestinal tissues. To assess the mechanisms of goblet cell depletion, human LS174T goblet-like cells were exposed to G. duodenalis trophozoites over increasing time points at a multiplicity of infectio
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