Prostaglandin E2 Produced by Entamoeba histolytica Signals via EP4 Receptor and Alters Claudin-4 to Increase Ion Permeability of Tight Junctions
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.05.001
ISSN1525-2191
AutoresManigandan Lejeune, France Moreau, Kris Chadee,
Tópico(s)Barrier Structure and Function Studies
ResumoEntamoeba histolytica is a protozoan parasite that causes amebic dysentery characterized by severe watery diarrhea. Unfortunately, the parasitic factors involved in the pathogenesis of diarrhea are poorly defined. Prostaglandin E2 (PGE2) is a host lipid mediator associated with diarrheal diseases. Intriguingly, E. histolytica produces and secretes this inflammatory molecule. We investigated the mechanism whereby ameba-derived PGE2 induces the onset of diarrhea by altering ion permeability of paracellular tight junctions (TJs) in colonic epithelia. PGE2 decreased barrier integrity of TJs in a dose- and time-dependent manner, as measured by transepithelial resistance. PGE2 signals were selectively transduced via the EP4 receptor. Furthermore, PGE2 signaling decreased TJ integrity, as revealed by EP receptor-specific agonist and antagonist studies. Loss of mucosal barrier integrity corresponded with increased ion permeability across TJs. Subcellular fractionation and confocal microscopy studies highlighted a significant spatial alteration of an important TJ protein, claudin-4, that corresponded with increased sodium ion permeability through TJs toward the lumen. Moreover, PGE2-induced luminal chloride secretion was a prerequisite for alterations at TJs. Thus, the gradient of NaCl created across epithelia could serve as a trigger for osmotic water flow that leads to diarrhea. Our results highlight a pathological role for E. histolytica-derived PGE2 in the onset of diarrhea. Entamoeba histolytica is a protozoan parasite that causes amebic dysentery characterized by severe watery diarrhea. Unfortunately, the parasitic factors involved in the pathogenesis of diarrhea are poorly defined. Prostaglandin E2 (PGE2) is a host lipid mediator associated with diarrheal diseases. Intriguingly, E. histolytica produces and secretes this inflammatory molecule. We investigated the mechanism whereby ameba-derived PGE2 induces the onset of diarrhea by altering ion permeability of paracellular tight junctions (TJs) in colonic epithelia. PGE2 decreased barrier integrity of TJs in a dose- and time-dependent manner, as measured by transepithelial resistance. PGE2 signals were selectively transduced via the EP4 receptor. Furthermore, PGE2 signaling decreased TJ integrity, as revealed by EP receptor-specific agonist and antagonist studies. Loss of mucosal barrier integrity corresponded with increased ion permeability across TJs. Subcellular fractionation and confocal microscopy studies highlighted a significant spatial alteration of an important TJ protein, claudin-4, that corresponded with increased sodium ion permeability through TJs toward the lumen. Moreover, PGE2-induced luminal chloride secretion was a prerequisite for alterations at TJs. Thus, the gradient of NaCl created across epithelia could serve as a trigger for osmotic water flow that leads to diarrhea. Our results highlight a pathological role for E. histolytica-derived PGE2 in the onset of diarrhea. Entamoeba histolytica is an enteric-dwelling protozoan parasite that infects an estimated 500 million people worldwide.1Schuster F.L. Visvesvara G.S. Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease.Vet Parasitol. 2004; 126: 91-120Crossref PubMed Scopus (118) Google Scholar Alarmingly, E. histolytica accounts for more than 100,000 deaths annually.2Anane S. Khaled S. [Entamoeba histolytica and Entamoeba dispar: differentiation methods and implications].Ann Biol Clin (Paris). 2005; 63 (French): 7-13PubMed Google Scholar The disease amebiasis, which manifests as amebic colitis in 10% to 20% of infected subjects, is characterized by acute watery and/or bloody diarrhea.1Schuster F.L. Visvesvara G.S. Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease.Vet Parasitol. 2004; 126: 91-120Crossref PubMed Scopus (118) Google Scholar, 3Haque R. Mondal D. Duggal P. Kabir M. Roy S. Farr B.M. Sack R.B. Petri Jr, W.A. Entamoeba histolytica infection in children and protection from subsequent amebiasis.Infect Immun. 2006; 74: 904-909Crossref PubMed Scopus (131) Google Scholar, 4Haque R. Mondal D. Kirkpatrick B.D. Akther S. Farr B.M. Sack R.B. Petri Jr, W.A. Epidemiologic and clinical characteristics of acute diarrhea with emphasis on Entamoeba histolytica infections in preschool children in an urban slum of Dhaka, Bangladesh.Am J Trop Med Hyg. 2003; 69: 398-405PubMed Google Scholar, 5Niyogi S.K. Saha M.R. De S.P. Enteropathogens associated with acute diarrhoeal diseases.Indian J Public Health. 1994; 38: 29-32PubMed Google Scholar The mechanism by which E. histolytica causes acute diarrhea is not clearly understood. E. histolytica in contact with colonic epithelial cells induces a robust inflammatory response involving proinflammatory cytokines and other inflammatory mediators that causes what is known as invasive or inflammatory diarrhea.6Navaneethan U. Giannella R.A. Mechanisms of infectious diarrhea.Nat Clin Pract Gastroenterol Hepatol. 2008; 5: 637-647Crossref PubMed Scopus (120) Google Scholar However, the acute nature of the disease indicates the possibility of pathogenesis even in the absence of parasite-epithelial contact. We speculate that E. histolytica can cause diarrhea in an epithelial contact-independent manner; however, the parasite factor or factors responsible for this effect is not clearly understood. Prostaglandin E2 (PGE2) is an important host inflammatory mediator that is associated with various diarrheal pathologies, including cholera, entero-invasive bacterial diseases, and inflammatory bowel diseases.7Beubler E. Schuligoi R. Mechanisms of cholera toxin-induced diarrhea.Ann N Y Acad Sci. 2000; 915: 339-346Crossref PubMed Scopus (12) Google Scholar, 8Resta-Lenert S. Barrett K.E. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium: role of iNOS and COX-2.Gastroenterology. 2002; 122: 1070-1087Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 9Alcantara C. Stenson W.F. Steiner T.S. Guerrant R.L. Role of inducible cyclooxygenase and prostaglandins in Clostridium difficile toxin A-induced secretion and inflammation in an animal model.J Infect Dis. 2001; 184: 648-652Crossref PubMed Scopus (45) Google Scholar, 10Ahrenstedt O. Hällgren R. Knutson L. Jejunal release of prostaglandin E2 in Crohn's disease: relation to disease activity and first-degree relatives.J Gastroenterol Hepatol. 1994; 9: 539-543Crossref PubMed Scopus (23) Google Scholar PGE2 levels are increased by 10-fold in the colonic mucosa during E. histolytica infection.11Stenson W.F. Zhang Z. Riehl T. Stanley Jr, S.L. Amebic infection in the human colon induces cyclooxygenase-2.Infect Immun. 2001; 69: 3382-3388Crossref PubMed Scopus (46) Google Scholar Amebic trophozoites induce host cells such as macrophages, polymorphonuclear cells, and colonic epithelial cells to synthesize PGE2.11Stenson W.F. Zhang Z. Riehl T. Stanley Jr, S.L. Amebic infection in the human colon induces cyclooxygenase-2.Infect Immun. 2001; 69: 3382-3388Crossref PubMed Scopus (46) Google Scholar, 12Sánchez-Ramírez B. Ramírez-Gil M. Vázquez-Moctezuma I. Ramos-Martínez E. Talamás-Rohana P. Entamoeba histolytica: induction of cyclooxygenase-2 expression during amoebic liver abscess formation in hamsters (Mesocricetus auratus).Exp Parasitol. 2004; 106: 119-125Crossref PubMed Scopus (14) Google Scholar Remarkably, E. histolytica trophozoites also constitutively produce and secrete PGE2.13Belley A. Chadee K. Eicosanoid production by parasites: from pathogenesis to immunomodulation?.Parasitol Today. 1995; 11: 327-334Abstract Full Text PDF PubMed Scopus (69) Google Scholar The identity/similarity of the parasite PGE2 with that of the host has been confirmed by gas chromatography/mass spectrometry analysis.14Belley A. Chadee K. Production of prostaglandin E(2) by Entamoeba histolytica via a novel cyclooxygenase.Arch Med Res. 2000; 31: S74-S75Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Nevertheless, PGE2 is the only prostanoid present in the parasite SPs. Parasitic production of PGE2 was significantly increased in the presence of arachidonic acid (AA),13Belley A. Chadee K. Eicosanoid production by parasites: from pathogenesis to immunomodulation?.Parasitol Today. 1995; 11: 327-334Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 15Dey I. Keller K. Belley A. Chadee K. Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.Proc Natl Acad Sci USA. 2003; 100: 13561-13566Crossref PubMed Scopus (56) Google Scholar the precursor for PGE2, which is usually present in high concentrations in the gut.16Meyer B.J. Mann N.J. Lewis J.L. Milligan G.C. Sinclair A.J. Howe P.R. Dietary intakes and food sources of omega-6 and omega-3 polyunsaturated fatty acids.Lipids. 2003; 38: 391-398Crossref PubMed Scopus (417) Google Scholar, 17Sioen I.A. Pynaert I. Matthys C. De Backer G. Van Camp J. De Henauw S. Dietary intakes and food sources of fatty acids for Belgian women, focused on n-6 and n-3 polyunsaturated fatty acids.Lipids. 2006; 41: 415-422Crossref PubMed Scopus (106) Google Scholar More importantly, E. histolytica has a COX-like gene that encodes a functional cyclooxygenase enzyme for the biosynthesis of PGE2; however, E. histolytica COX is primitive, and is unique in its pharmacological sensitivity to nonsteroidal anti-inflammatory drugs in being sensitive only to aspirin (ASA).15Dey I. Keller K. Belley A. Chadee K. Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.Proc Natl Acad Sci USA. 2003; 100: 13561-13566Crossref PubMed Scopus (56) Google Scholar Ameba COX has little homology to COX-1/2 enzymes from different species, in that the AA-binding domain and heme-coordinating and catalytic sites are absent.15Dey I. Keller K. Belley A. Chadee K. Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.Proc Natl Acad Sci USA. 2003; 100: 13561-13566Crossref PubMed Scopus (56) Google Scholar At present, we do not know the exact functional/biological significance of ameba-derived PGE2 in the pathogenesis of amebiasis. We have recently shown that E. histolytica PGE2 signals via the host epithelial EP4 receptors to induce robust IL-8 secretion that could augment inflammation.18Dey I. Chadee K. Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells.Infect Immun. 2008; 76: 5158-5163Crossref PubMed Scopus (35) Google Scholar Because PGE2 is strongly associated with various diarrheal diseases, however, we reasoned that ameba-derived PGE2 might play a major role in the onset of diarrhea during acute amebic colitis. In general, secretory diarrhea is characterized by an alteration of the ion barrier of intestinal epithelium19Berkes J. Viswanathan V.K. Savkovic S.D. Hecht G. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation.Gut. 2003; 52: 439-451Crossref PubMed Scopus (459) Google Scholar through increased luminal secretion of chloride ions (anions), a phenomenon that is observed with cholera toxin in the ileal mucosa.20Field M. Fromm D. al-Awqati Q. Greenough 3rd, W.B. Effect of cholera enterotoxin on ion transport across isolated ileal mucosa.J Clin Invest. 1972; 51: 796-804Crossref PubMed Scopus (217) Google Scholar To maintain charge balance in the lumen, sodium ions (cations) are pulled across the epithelium, also drawing water molecules and thus causing diarrhea.19Berkes J. Viswanathan V.K. Savkovic S.D. Hecht G. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation.Gut. 2003; 52: 439-451Crossref PubMed Scopus (459) Google Scholar PGE2 is a potent colonic epithelial chloride ion secretagogue21Keeler R. Wong N.L. Evidence that prostaglandin E2 stimulates chloride secretion in cultured A6 renal epithelial cells.Am J Physiol. 1986; 250: F511-F515PubMed Google Scholar, 22Bunce K.T. Spraggs C.F. Stimulation of electrogenic chloride secretion by prostaglandin E2 in guinea-pig isolated gastric mucosa.J Physiol. 1988; 400: 381-394PubMed Google Scholar, 23Deachapunya C. O'Grady S.M. Regulation of chloride secretion across porcine endometrial epithelial cells by prostaglandin E2.J Physiol. 1998; 508: 31-47Crossref PubMed Scopus (37) Google Scholar; however, it is not known whether it induces sodium ion flux across the epithelium. Colonic epithelial tight junctions (TJs) act as an important barrier for paracellular movement of macromolecules and ions across the epithelium.24Schneeberger E.E. Lynch R.D. The tight junction: a multifunctional complex.Am J Physiol Cell Physiol. 2004; 286: C1213-C1228Crossref PubMed Scopus (1109) Google Scholar, 25Harhaj N.S. Antonetti D.A. Regulation of tight junctions and loss of barrier function in pathophysiology.Int J Biochem Cell Biol. 2004; 36: 1206-1237Crossref PubMed Scopus (449) Google Scholar, 26González-Mariscal L. Betanzos A. Nava P. Jaramillo B.E. Tight junction proteins.Prog Biophys Mol Biol. 2003; 81: 1-44Crossref PubMed Scopus (920) Google Scholar Among the 40 different proteins that constitute the TJ complex, the claudin family is responsible for ion barrier maintenance of the paracellular space.27Turksen K. Troy T.C. Barriers built on claudins [Erratum appeared in J Cell Sci 2004, 117:4341].J Cell Sci. 2004; 117: 2435-2447Crossref PubMed Scopus (345) Google Scholar, 28Balkovetz D.F. Claudins at the gate: determinants of renal epithelial tight junction paracellular permeability.Am J Physiol Renal Physiol. 2006; 290: F572-F579Crossref PubMed Scopus (79) Google Scholar We speculate that PGE2 secreted by E. histolytica breaks the TJ ion barrier by selectively altering claudin family members, allowing free paracellular permeability to sodium ions. Any alteration in the TJ ion barrier will markedly affect epithelial transport mechanisms associated with electrolyte and water imbalance. Here, we present a novel mechanism by which E. histolytica disrupts colonic TJ barrier function and offer important insight into the cellular and molecular basis of epithelial barrier/ion transport function in intestinal amebiasis. To date, there has been no report suggesting a contact-independent disruption of TJs by E. histolytica. The focus of our study was therefore to identify a role for E. histolytica-derived PGE2 in altering the colonic epithelial TJ ion barrier and to define the mechanism by which PGE2 causes diarrhea. Secreted products (SP) of E. histolytica were prepared and collected as described previously.29Yu Y. Chadee K. Entamoeba histolytica stimulates interleukin 8 from human colonic epithelial cells without parasite-enterocyte contact.Gastroenterology. 1997; 112: 1536-1547Abstract Full Text PDF PubMed Scopus (80) Google Scholar Briefly, trophozoites of HM1-IMSS, a highly virulent strain of E. histolytica, were cultured until log phase in TYI-S-33 medium containing 20% serum. Trophozoites were washed three times in ice-cold Hanks' balanced salt solution (HBSS), adjusted to a final concentration of 1 × 107/mL HBSS, and incubated for 2 hours at 37°C under periodic swirling. The resulting SP were collected by centrifugation (1000 × g for 5 minutes) and assayed for protein amount using Bradford's reagent, normalized (0.1 μg/μL of SP), aliquoted, and stored at −80°C until further use. Trophozoite viability after incubation was >95% as determined by trypan blue exclusion assay. In some experiments, E. histolytica trophozoites were incubated with a nonspecific COX-1/2 inhibitor (1 mmol/L ASA) or with a specific COX-1/2 inhibitor (50 μmol/L indomethacin) for 16 hours. COX inhibitors were removed by centrifugation, followed by 2 hours of incubation in fresh HBSS for collecting SP as described previously.18Dey I. Chadee K. Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells.Infect Immun. 2008; 76: 5158-5163Crossref PubMed Scopus (35) Google Scholar In another experiment, 100 μmol/L AA was added to trophozoites in HBSS at the start of the 2-hour incubation and SP was collected. PGE2 in SP was assayed using an Assay Designs Correlate-CLIA kit (no. 910–001; Enzo Life Sciences, Plymouth Meeting, PA). This highly sensitive PGE2 chemiluminescence enzyme immunoassay is designed for quantitative determination of human PGE2 in biological samples with 2000 Ω·cm2 was reached, the medium from either the apical or basal compartment was replaced with medium containing the desired stimulant and TER was measured. Changes in TER in response to stimuli were normalized to that of the baseline resistance (resistance at the starting time point) and represented as percentage of the control over a desired time course. For experiments involving EP receptor agonists/antagonists, the concentration and the conditions for treatment/pretreatment were determined based on previous reports.30Tang C.H. Yang R.S. Fu W.M. Prostaglandin E2 stimulates fibronectin expression through EP1 receptor, phospholipase C, protein kinase Calpha, and c-Src pathway in primary cultured rat osteoblasts.J Biol Chem. 2005; 280: 22907-22916Crossref PubMed Scopus (89) Google Scholar, 31Chen B.C. Liao C.C. Hsu M.J. Liao Y.T. Lin C.C. Sheu J.R. Lin C.H. Peptidoglycan-induced IL-6 production in RAW 264.7 macrophages is mediated by cyclooxygenase-2, PGE2/PGE4 receptors, protein kinase A, I kappa B kinase, and NF-kappa B.J Immunol. 2006; 177: 681-693PubMed Google Scholar, 32Shao J. Sheng H. Prostaglandin E2 induces the expression of IL-1alpha in colon cancer cells.J Immunol. 2007; 178: 4097-4103PubMed Google Scholar, 33Dey I. Giembycz M.A. Chadee K. Prostaglandin E(2) couples through EP(4) prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines.Br J Pharmacol. 2009; 156: 475-485Crossref PubMed Scopus (27) Google Scholar, 34Cherukuri D.P. Chen X.B. Goulet A.C. Young R.N. Han Y. Heimark R.L. Regan J.W. Meuillet E. Nelson M.A. The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells.Exp Cell Res. 2007; 313: 2969-2979Crossref PubMed Scopus (65) Google Scholar The permeability of TJs to macromolecules/solute in response to PGE2 was measured by studying the apical to basal translocation of 14C-labeled ethanolamine (a small paracellular tracer, ∼4.9 Å in diameter), as described previously.35Tang V.W. Goodenough D.A. Paracellular ion channel at the tight junction.Biophys J. 2003; 84: 1660-1673Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar The tracer was used at a concentration of 0.33 μCi/well. After incubation with tracer for the desired time intervals, 40-μL samples from both chambers were collected in separate scintillation vials containing 10 mL of scintillation fluid. Radioactivity was measured using a Beckman liquid scintillation counter (Beckman Coulter, Brea, CA). Permeability to [14C]ethanolamine was measured as the apparent permeability coefficient Papp and was expressed as the transport enhancement ratio R, as described previously.36Grobovac V. Bernkop-Schnürch A. Improvement of the intestinal membrane permeability of low molecular weight heparin by complexation with stem bromelain.Int J Pharm. 2006; 326: 153-159Crossref PubMed Scopus (22) Google Scholar A 20% NaCl dilution potential assay was performed as described previously.37Rao R.K. Li L. Baker R.D. Baker S.S. Gupta A. Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G332-G340PubMed Google Scholar Briefly, medium was discarded from a Transwell plate with a confluent T84 monolayer and cells were bathed in PBS + bovine serum albumin (BSA) (140 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4, and 0.6% BSA), with 0.5 and 1.5 mL in the apical and basal chambers, respectively. After apical stimulation with 1 μmol/L PGE2 in PBS + BSA, 20% dilution in either the apical or the basal chamber was developed by replacing 100 or 300 μL of apical or basal solution with a solution of equally osmolar mannitol + BSA (140 mmol/L mannitol, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4 and 0.6% BSA), respectively. The electrical potential was recorded before and after 20% dilution and at 5-minute intervals from the start until 30 minutes. The 20% dilution potential difference was calculated as the difference between the initial potential and the potential after dilution. For measuring sodium potential, T84 monolayer on a Transwell plate was washed with PBS + BSA and stimulated apically with 1 μmol/L PGE2 in mannitol + BSA. At various time points, 25-μL samples were collected from the apical chamber and sodium potential was measured using a highly sensitive sodium electrode (Thermo Orion, Beverly, MA), as described previously.35Tang V.W. Goodenough D.A. Paracellular ion channel at the tight junction.Biophys J. 2003; 84: 1660-1673Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar For experiments involving pharmacological inhibition of membrane sodium channel/transporters or cystic fibrosis transmembrane regulator (CFTR), inhibitor concentration and conditions for pretreatment were determined based on previous reports.38de Jong J.C. Willems P.H. Mooren F.J. van den Heuvel L.P. Knoers N.V. Bindels R.J. The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer.J Biol Chem. 2003; 278: 24302-24307Crossref PubMed Scopus (78) Google Scholar, 39D'Andrea L. Lytle C. Matthews J.B. Hofman P. Forbush 3rd, B. Madara J.L. Na: K:2Cl cotransporter (NKCC) of intestinal epithelial cells Surface expression in response to cAMP.J Biol Chem. 1996; 271: 28969-28976Crossref PubMed Scopus (91) Google Scholar, 40Kim J.A. Kang Y.S. Lee S.H. Lee E.H. Yoo B.H. Lee Y.S. Glibenclamide induces apoptosis through inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(−) channels and intracellular Ca(2+) release in HepG2 human hepatoblastoma cells.Biochem Biophys Res Commun. 1999; 261: 682-688Crossref PubMed Scopus (51) Google Scholar T84 cells grown to confluence on regular 6-well plates were fractionated after appropriate treatment, based on a previously described protocol41Sheth P. Basuroy S. Li C. Naren A.P. Rao R.K. Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions.J Biol Chem. 2003; 278: 49239-49245Crossref PubMed Scopus (152) Google Scholar with slight modifications. The cell monolayer was washed twice with ice-cold PBS, scraped using a rubber policeman, and collected in an Eppendorf tube. Cells were centrifuged at 1250 × g for 5 minutes at 4°C. The pellet was resuspended in 400 μL of ice cold cell lysis buffer (50 mmol/L Tris-HCl, 140 mmol/L EDTA, 30 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride and protease inhibitor cocktail tablet) containing 1% Triton X-100, incubated for 30 minutes on ice and centrifuged at 20,000 × g for 30 minutes at 4°C. The supernatant was labeled as Triton X-100 soluble fraction (SF) or noncytoskeletal fraction. The pellet was again resuspended in 200 μL of cell lysis buffer containing 1% SDS, and then was sonicated and centrifuged at 20,000 × g for 30 minutes at 4°C. The resulting supernatant was labeled as Triton X-100 insoluble fraction (IF) or cytoskeletal fraction. Protein content of subcellular fractions was estimated using the Bradford method and was adjusted for a final concentration of 0.5 μg/μL. Equal volume of 1× sample buffer was added to the samples, boiled at 100°C for 10 minutes, and used for Western blotting. Approximately 2.5 to 5.0 μg of total protein was loaded per well of 7.5% to 15% SDS-PAGE (depending on the molecular weight of the TJ protein being probed) and electrophoresis was performed. Proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) followed by blocking in 5% skim milk powder in TBS-T (20 mmol/L Tris-HCl, pH 7.5, 500 mmol/L NaCl, 0.1% Tween 20) for 1 hour at room temperature. Membranes were incubated with appropriate primary antibodies in 1% skim milk-TBS-T at 4°C, overnight. Blots were washed three times with TBS-T and then incubated in horseradish peroxidase-conjugated secondary antibodies in 1% skim milk-TBS-T for 2 hours at room temperature. The blots were washed with TBS-T and were developed using Immobilon Western chemiluminescent HRP substrate (Millipore, Billerica, MA) according to the manufacturer's instructions. T84 monolayers grown on glass coverslips in regular six-well culture plates were used. After appropriate treatment, monolayer was washed with ice cold PBS and fixed with 3.75% paraformaldehyde at 4°C for 30 minutes. Fixed cells were permeabilized using 0.5% Triton X-100 in PBS for 15 minutes and blocked with 5% BSA in PBS-0.5% Tween 20 (PBS-T) for 1 hour at room temperature. Monolayers were incubated overnight in 1:100 dilution of appropriate primary antibodies at 4°C. After two washes in PBS-T, monolayers were coincubated with fluorescein isothiocyanate or phycoerythrin-conjugated secondary antibodies (1:200 dilution) for 1 hour at room temperature. Coverslip was mounted on clean glass slide using Vectashield (Vector Laboratories, Burlingame, CA). Slides were examined using a FluoView FV1000 confocal immunofluorescence microscope (Olympus). Immunofluorescence signals from the tags for respective proteins were determined in individual en face (xy axes) planes throughout the cellular z axis at 0.35-μm intervals. Z-planes obtained were stacked and analyzed using Volocity software version 5.4 (PerkinElmer, Waltham, MA) to obtain PXZ and three-dimensional (3D) reconstructions of the Z-stacks. Data were analyzed by two-way analysis of variance followed by a Bonferroni post hoc test for comparison between groups using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA). In the figures, data are reported as means ± SD of three independent experiments. To test the hypothesis that secreted components derived from highly virulent wild-type (Wt) E. histolytica alter the integrity of the colonic epithelial monolayer during pathogenesis of intestinal amebiasis, the effect of SP on TER of T84 colonic epithelial monolayer was analyzed. When the monolayer was exposed on the apical/luminal side to SP, there was a decrease in the integrity of TER that occurred in a dose- and time-dependent manner, compared with the nonstimulated controls (Figure 1). With high doses of SP (48 μg), there was a 55% decrease in TER as early as 5 minutes, followed by a gradual recovery but with a 28% decrease (P < 0.001) remaining even at 60 minutes. This is in contrast to the response observed with low doses of SP (8 μg), which caused a modest 19% decrease in TER at 5 minutes (P < 0.05), followed by recovery to control values as early as 30 minutes. An intermediate dose of SP (24 μg) caused 34% (P < 0.001) and 19% (P < 0.05) decreases at 5 and 30 minutes, respectively, with recovery to control values at 60 minutes. Thus, the effects of E. histolytica SP on TER follow a dose-response relationship. More importantly, these results show that alteration in colonic epithelial barrier function occurred in the absence of parasite-epithelial cell contact. We have previously shown that trophozoites of E. histolytica constitutively produce and secrete PGE2 and have confirmed its identity with mammalian PGE2 by gas chromatography/mass spectrometry analysis.13Belley
Referência(s)