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• W2056875662 abstract "Pathogenic and enteroinvasive bacteria have been shown to trigger the IκB/NF-κB transcriptional system and proinflammatory gene expression in epithelial cells. In this study, we investigated the molecular mechanism of the commensal Gram-negativeBacteroides vulgatus-induced NF-κB signal transduction in intestinal epithelial cells (IEC). We report that B. vulgatus induced interleukin-1 receptor-associated kinase-1 degradation, IκBα phosphorylation/degradation, RelA and Akt phosphorylation, as well as NF-κB DNA binding and NF-κB transcriptional activity in rat non-transformed IEC-6 cells. B. vulgatus- but not interleukin-1β-mediated NF-κB transcriptional activity was inhibited by dominant negative (dn) toll-like receptor 4. Of importance, B. vulgatus induced IκBα phosphorylation/degradation and IKKα/β and RelA phosphorylation in primary IEC derived from germ-free or mono-associated HLA-B27 transgenic and wild type rats, demonstrating the physiological relevance of non-pathogenic bacterial signaling in IEC. Adenoviral delivery of dn IKKβ or treatment with wortmannin inhibited B. vulgatus-induced endogenous RelA Ser-536 and GST-p65TAD (Ser-529/Ser-536) phosphorylation as well as NF-κB transcriptional activity in IEC-6 cells, suggesting a critical role of IKKβ and phosphatidylinositol 3-kinase/Akt in bacteria-induced RelA phosphorylation and NF-κB activation. Interestingly, B. vulgatus-induced IκBα degradation and NF-κB transcriptional activity in IEC transwell cultures were inhibited in the presence of lymphocytes. We propose that non-pathogenic B. vulgatusactivates the NF-κB signaling pathway through both IκB degradation and RelA phosphorylation but that immune cells mediate tolerance of IEC to this commensal bacteria. Pathogenic and enteroinvasive bacteria have been shown to trigger the IκB/NF-κB transcriptional system and proinflammatory gene expression in epithelial cells. In this study, we investigated the molecular mechanism of the commensal Gram-negativeBacteroides vulgatus-induced NF-κB signal transduction in intestinal epithelial cells (IEC). We report that B. vulgatus induced interleukin-1 receptor-associated kinase-1 degradation, IκBα phosphorylation/degradation, RelA and Akt phosphorylation, as well as NF-κB DNA binding and NF-κB transcriptional activity in rat non-transformed IEC-6 cells. B. vulgatus- but not interleukin-1β-mediated NF-κB transcriptional activity was inhibited by dominant negative (dn) toll-like receptor 4. Of importance, B. vulgatus induced IκBα phosphorylation/degradation and IKKα/β and RelA phosphorylation in primary IEC derived from germ-free or mono-associated HLA-B27 transgenic and wild type rats, demonstrating the physiological relevance of non-pathogenic bacterial signaling in IEC. Adenoviral delivery of dn IKKβ or treatment with wortmannin inhibited B. vulgatus-induced endogenous RelA Ser-536 and GST-p65TAD (Ser-529/Ser-536) phosphorylation as well as NF-κB transcriptional activity in IEC-6 cells, suggesting a critical role of IKKβ and phosphatidylinositol 3-kinase/Akt in bacteria-induced RelA phosphorylation and NF-κB activation. Interestingly, B. vulgatus-induced IκBα degradation and NF-κB transcriptional activity in IEC transwell cultures were inhibited in the presence of lymphocytes. We propose that non-pathogenic B. vulgatusactivates the NF-κB signaling pathway through both IκB degradation and RelA phosphorylation but that immune cells mediate tolerance of IEC to this commensal bacteria. intestinal epithelial cells nuclear transcription factor κB Toll-like receptor 4 electrophoretic mobility shift assay reverse transcription interleukin 1β tumor necrosis factor interleukin 1 receptor-associated kinase intercellular adhesion molecule 1 transforming growth factor-β-activated kinase 1 IκB kinase complex adenoviral phosphatidylinositol 3-kinase colony-forming units dominant negative monoclonal antibody fluorescence-activated cell sorter glutathioneS-transferase hemagglutinin fetal calf serum multiplicity of infection peripheral blood mononuclear cells peripheral blood lymphocytes lipopolysaccharide TNF receptor-associated factor transforming growth factor-β-activated kinase monoclonal antibody mean fluorescent intensity Inflammatory bowel diseases, including human ulcerative colitis and Crohn's disease, are chronic immune-mediated diseases of the distal intestinal tract with unknown etiologies (1Sartor R.B. Am. J. Gastroenterol. 1997; 92: 5-11PubMed Google Scholar, 2Fiocchi C. Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1865) Google Scholar). Various pathogenic mechanisms have been proposed, including inflammatory responses to a persistent luminal pathogen or abnormal luminal constituent, autoimmunity, or an overly aggressive immune response to normal luminal constituents such as commensal enteric bacteria. The hypothesis that aberrant immune responses to non-pathogenic commensal luminal bacteria can cause colitis in genetically predisposed individuals is supported by several rodent models of experimental colitis (3Blumberg R.S. Saubermann L.J. Strober W. Curr. Opin. Immunol. 1999; 11: 648-656Crossref PubMed Scopus (407) Google Scholar, 4Bhan A.K. Mizoguchi E. Smith R.N. Mizoguchi A. Immunol. Rev. 1999; 169: 195-207Crossref PubMed Scopus (137) Google Scholar, 5Elson C.O. Sartor R.B. Tennyson G.S. Riddell R.H. Gastroenterology. 1995; 109: 1344-1367Abstract Full Text PDF PubMed Scopus (1097) Google Scholar). For example, HLA-B27/β2-microglobulin transgenic rats raised under specific pathogen-free conditions spontaneously develop colitis, whereas germ-free (sterile) conditions prevent the development of chronic inflammation (6Rath H.C. Herfarth H.H. Ikeda J.S. Grenther W.B. Hamm T.E.J. Balish E. Taurog J.D. Hammer R.E. Wilson K.H. Sartor R.B. J. Clin. Invest. 1996; 98: 945-953Crossref PubMed Scopus (693) Google Scholar, 7Taurog J.D. Richardson J.A. Croft J.T. Simmons W.A. Zhou M. Fernandez-Sueiro J.L. Balish E. Hammer R.E. J. Exp. Med. 1994; 180: 2359-2364Crossref PubMed Scopus (947) Google Scholar). Reconstitution studies of gnotobiotic HLA-B27 transgenic rats (6Rath H.C. Herfarth H.H. Ikeda J.S. Grenther W.B. Hamm T.E.J. Balish E. Taurog J.D. Hammer R.E. Wilson K.H. Sartor R.B. J. Clin. Invest. 1996; 98: 945-953Crossref PubMed Scopus (693) Google Scholar, 8Rath H.C. Wilson K.H. Sartor R.B. Infect. Immun. 1999; 67: 2969-2974Crossref PubMed Google Scholar) and carrageenan-induced colitis in guinea pigs (9Onderdonk A.B. Franklin M.L. Cisneros R.L. Infect. Immun. 1981; 32: 225-231Crossref PubMed Google Scholar) implicateBacteroides vulgatus as particularly important to the induction of colitis in these models. Of note, this B. vulgatus strain induced no colitis in wild type rats, documenting its wild type nature (8Rath H.C. Wilson K.H. Sartor R.B. Infect. Immun. 1999; 67: 2969-2974Crossref PubMed Google Scholar). Despite these observations suggesting a central role of non-pathogenic resident luminal bacteria in the regulation of intestinal inflammation, the mechanisms by which bacteria influence the mucosal immune response responsible for inducing and perpetuating chronic colitis remain unclear. A single layer of intestinal epithelial cells (IEC)1 isolate the host from the gut luminal environment. These cells are considered to be an integral and essential component of the innate mucosal immune system of the host (10Kagnoff M.F. Eckmann L. J. Clin. Invest. 1997; 100: 6-10Crossref PubMed Scopus (267) Google Scholar). IEC constitutively express, or can be induced to express, co-stimulatory molecules and components of the human major histocompatibility complex including class II and classical I and non-classical class Ib human major histocompatibility complex molecules (11Blumberg R.S. Terhorst C. Bleicher P. McDermott F.V. Allan C.H. Landau S.B. Trier J.S. Balk S.P. J. Immunol. 1991; 147: 2518-2524PubMed Google Scholar, 12Panja A. Barone A. Mayer L. J. Exp. Med. 1994; 179: 943-950Crossref PubMed Scopus (63) Google Scholar, 13Hershberg R.M. Framson P.E. Cho D.H. Lee L.Y. Kovats S. Beitz J. Blum J.S. Nepom G.T. J. Clin. Invest. 1997; 100: 204-215Crossref PubMed Scopus (190) Google Scholar). Moreover, proinflammatory stimuli (e.g. TNF and IL-1) as well as certain enteric pathogens (e.g. Salmonella species, Yersinia enterocolitica, and enteropathogenic Escherichia coli) induce the expression and secretion of a wide range of inflammatory and chemoattractive cytokines in IEC including TNF, IL-8, MCP-1, IP-10, GROα, inducible nitric-oxide synthase, and COX-2 as well as the adhesion molecule ICAM-1 (14McCormick B.A. Colgan S.P. Delp-Archer C. Miller S.I. Madara J.L. J. Cell Biol. 1993; 123: 895-907Crossref PubMed Scopus (382) Google Scholar, 15Eckmann L. Stenson W.F. Savidge T.C. Lowe D.C. Barrett K.E. Fierer J. Smith J.R. Kagnoff M.F. J. Clin. Invest. 1997; 100: 296-309Crossref PubMed Scopus (174) Google Scholar, 16Huang G.T. Eckmann L. Savidge T.C. Kagnoff M.F. J. Clin. Invest. 1996; 98: 572-583Crossref PubMed Scopus (151) Google Scholar, 17Jung H.C. Eckmann L. Yang S.K. Panja A. Fierer J. Morzycka- Wroblewska E. Kagnoff M.F. J. Clin. Invest. 1995; 95: 55-65Crossref PubMed Google Scholar, 18Savkovic S.D. Koutsouris A. Hecht G. Am. J. Physiol. 1997; 273: C1160-C1167Crossref PubMed Google Scholar, 19Schulte R. Wattiau P. Hartland E.L. Robins-Browne R.M. Cornelis G.R. Infect. Immun. 1996; 64: 2106-2113Crossref PubMed Google Scholar). As shown in multiple cell systems including IEC, most of these proinflammatory molecules are in part regulated at the transcriptional level by the transcription factor NF-κB (20Tak P.P. Firestein G.S. J. Clin. Invest. 2001; 107: 7-11Crossref PubMed Scopus (3287) Google Scholar). Activation of the IκB/NF-κB system is a complex process that involves the participation of multiple adapter proteins and kinases acting in a coordinated fashion to give specificity to the cell surface stimuli. Although IL-1 and TNF signaling events leading to NF-κB activation has been well studied (21Jobin C. Sartor R.B. Am. J. Physiol. 2000; 278: C451-C462Crossref PubMed Google Scholar), the molecular mechanisms of bacterial signaling to the IκB/NF-κB transcriptional system in IEC are still largely unknown. The major molecular determinant of Gram-negative bacteria responsible for NF-κB activation is the glycolipid lipopolysaccharide (LPS). This bacterial product signals to the IκB/NF-κB system by using the cell surface toll-like receptor (TLR) 4 and its co-receptor MD-2 which then utilize downstream components of the IL-1 signaling cascade (22Zhang G. Ghosh S. J. Clin. Invest. 2001; 107: 13-19Crossref PubMed Scopus (614) Google Scholar, 23Tapping R.I. Akashi S. Miyake K. Godowski P.J. Tobias P.S. J. Immunol. 2000; 165: 5780-5787Crossref PubMed Scopus (303) Google Scholar, 24Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). For example, TLR4 engagement by LPS leads to the sequential recruitment/activation of the myeloid differentiation protein (MyD88), the IL-1 receptor-associated kinase (IRAK), the TNF receptor-associated factor (TRAF) 6, and the transforming growth factor-β-activated kinase (TAK) 1. Although controversial, the signal may converge on the NF-κB-inducing kinase, which then activates the IκB kinase (IKK) complex. The activated IKK complex phosphorylates IκBα at serine residues 32 and 36, which triggers its ubiquination/degradation and subsequent release of NF-κB, which then translocates to the nucleus and activates the transcription of κB-dependent genes (25Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar, 26Hatada E.N. Krappmann D. Scheidereit C. Curr. Opin. Immunol. 2000; 12: 52-58Crossref PubMed Scopus (314) Google Scholar). In addition to nuclear translocation, modification of NF-κB transcriptional activity by phosphorylation of the RelA subunit has been shown to be an important regulatory element of the pathway (27Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 28Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 29Wang D. Baldwin A.S.J. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 30Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 31Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 32Wang D. Westerheide S.D. Hanson J.L. Baldwin A.S.J. J. Biol. Chem. 2000; 275: 32592-32597Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). TNF and IL-1β induce phosphorylation of serine 529 and/or 536 of the RelA transactivating domain 1 (TAD1), which increased NF-κB transcriptional activity (28Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 33Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin A.S.J. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 34Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin A.S.J. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (587) Google Scholar). Potential kinases involved in signal-induced RelA phosphorylation are the casein kinase II, Akt, and IKK (27Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 28Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 33Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin A.S.J. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 35Schmitz M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The precise mechanism for increased transcriptional activity is not clear but may involve enhanced recruitment of transcriptional co-activator such as cAMP-response element-binding protein-binding protein (CBP/p300) to the promoter site of some specific genes. Although IEC lines were shown to express TLR4 (36Cario E. Podolsky D.K. Infect. Immun. 2000; 68: 7010-7017Crossref PubMed Scopus (1062) Google Scholar, 37Cario E. Rosenberg I.M. Brandwein S.L. Beck P.L. Reinecker H.C. Podolsky D.K. J. Immunol. 2000; 164: 966-972Crossref PubMed Scopus (643) Google Scholar, 38Cario E. Brown D. McKee M. Lynch-Devaney K. Gerken G. Podolsky D.K. Am. J. Pathol. 2002; 160: 165-173Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), the functional role of this receptor and the downstream signal transduction, including RelA phosphorylation triggered by commensal non-pathogenic bacteria in IEC, is still unknown. In this study, we characterized the molecular mechanisms of NF-κB activation triggered by the Gram-negative, commensal bacterial strainB. vulgatus in the rat intestinal epithelial cell line IEC-6, the human colonic cell line CaCO-2, and primary IEC derived from germ-free or B. vulgatus mono-associated HLA-B27/β2-microglobulin transgenic as well as wild type rats. We report that B. vulgatus induces NF-κB activation in IEC through components of the IL-1R/TLR4 pathway and the induction of RelA phosphorylation. The presence of lymphocytes negatively regulates bacteria-induced NF-κB activity, suggesting a potential role of immune cells in controlling IEC responsiveness to commensal bacteria. B. vulgatusderived from a guinea pig with carrageenan-induced colitis (a gift from A. B. Onderdonk, Harvard University, Cambridge, MA) was anaerobically grown at 37 °C in brain heart infusion broth supplemented with cysteine (0.05%), hemin (5 mg/liter), and resazurin.E. coli derived from a patient with active Crohn's disease (provided by the Clinical Microbiology Laboratory of the University of North Carolina Hospitals, Chapel Hill) and the human oral isolateEnterococcus faecalis (a gift from M. Huycki, University of Oklahoma State), which has been shown to induce experimental colitis in mono-associated IL-10−/− mice (39Kim S.C. Tonkonogy S.L. Balish E. Warner T. Sartor R.B. Gastroenterology. 2001; 20: A82Crossref Google Scholar), were aerobically grown in Luria broth containing tryptone (1%), yeast extract (0.5%), and NaCl (0.5%). The probiotic strain Lactobacillus paracasei strain GG of human intestinal origin (ConAgra, Lincoln, NE) was anaerobically grown in MRS broth 37 °C. All bacteria were harvested by centrifugation (3,000 × g, 15 min) at stationary growth phase, washed three times with phosphate-buffered saline (1×, pH 7.2, Invitrogen), and subsequently diluted to obtain final cell densities of 5 × 107 cfu/ml in Dulbecco's modified Eagle's medium (Invitrogen). Bacterial lysates were prepared as described previously (40Cong Y. Brandwein S.L. McCabe R.P. Lazenby A. Birkenmeier E.H. Sundberg J.P. Elson C.O. J. Exp. Med. 1998; 187: 855-864Crossref PubMed Scopus (333) Google Scholar). The rat nontransformed small intestinal epithelial cell line IEC-6 (passage 5–0) (ATCC CRL 1592, American Type Culture Collection (ATCC), Manassas, VA) and the human adenocarcinoma intestinal epithelial cell line CaCO-2 (passage 30–50) (ATCC HTB 38) were grown to confluency in 12- or 6-well plates (Nunc) as described previously (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar). CaCO-2 cells were used in liposome-based transfections, because IEC-6 cells are not permissive to transfections. Confluent cell monolayers were stimulated with 5 × 107 cfu/ml bacteria, bacterial lysate (200 or 50 μg of protein/ml), LPS (5 or 1 μg/ml; from E. coli serotype O111:B4, Sigma), TNF (5 ng/ml), and IL-1β (10 ng/ml) (both from R & D Systems, Minneapolis, MN) for various times. To prevent bacterial growth, gentamicin (100 μg/ml) was added to the cultures after 2 h. Where indicated, cells were pretreated with cycloheximide (50 μg/ml, Sigma), triptolide (100 ng/ml, Biomol, Plymouth Meeting, PA), or wortmannin (100 nm, Sigma). Transwell leukocyte co-cultures with confluent CaCO-2 monolayers were established as described previously (42Haller D. Bode C. Hammes W.P. Pfeifer A.M.A. Schiffrin E.J. Blum S. Gut. 2000; 47: 79-87Crossref PubMed Scopus (386) Google Scholar, 43Haller D. Serrant P. Peruisseau G. Hammes W.P. Bode C. Schiffrin E.J. Blum S. Microbiol. Immunol. 2002; 46: 195-205Crossref PubMed Scopus (42) Google Scholar). Briefly, human peripheral blood mononuclear cells (PBMC) derived from healthy volunteers were isolated using Ficoll-Paque 1077 (Amersham Biosciences) gradient centrifugation (500 × g, 30 min). To purify leukocyte subpopulations, PBMC were incubated for 2 h at 37 °C and 5% CO2 on 225-cm2 tissue culture plates (Costar) to allow adherence. Non-adherent peripheral blood lymphocytes (PBL) were separated from adherent cells by aspiration. Adherent peripheral blood monocytes were washed 3 times with phosphate-buffered saline (1 time) and harvested by cell scraping. PBMC, PBL, or monocytes were added to the basolateral compartment of 6-well transwell inserts (0.4-mm pore size) at cell densities of 2 × 106/ml. IEC/leukocyte co-cultures were stimulated with bacteria by adding 5 × 107cfu/ml B. vulgatus to the apical surface of IEC monolayers and incubated for 4 h at 5% CO2 and 37 °C. Germ-free (sterile) HLA-B27 transgenic and wild type Fisher F344 rats were euthanized, and the entire small intestine as well as the large intestine were removed and placed in calcium/magnesium-free Hanks' buffered saline solution (Invitrogen) containing 5% FCS. The small intestine was cut longitudinally, washed 3 times in calcium/magnesium-free Hanks' buffered saline solution (Invitrogen), cut into pieces 0.5 cm long, and incubated at 37 °C in 40 ml of RPMI 1640 containing 5% FCS and 1 mmdithiothreitol for 30 min in an orbital shaker. The supernatant was filtered and centrifuged for 5 min at 400 × g, and the cell pellet was resuspended in RPMI 1640 containing 5% FCS. The remaining tissue was incubated in 40 ml of phosphate-buffered saline (1×) containing 0.5 mm dithiothreitol and 1.5 mm EDTA for an additional 15 min. The supernatant was filtered and centrifuged for 5 min at 400 × g, and the cell pellet was resuspended in RPMI 1640 containing 5% FCS. Finally, primary IEC were collected by centrifugation through a 25–40% discontinuous Percoll gradient at 600 × g for 20 min. Cell viability and purity was assessed by trypan blue exclusion and FACS analysis using rat anti-CD3 mAb (BD Biosciences, clone G4.18). Cells were >85% viable and >90% pure. Primary rat IEC at a concentration of 2 × 106 cells/ml were incubated for 2 h in 5% CO2 at 37 °C and then stimulated with 5 × 107 cfu/ml B. vulgatus, L. paracasei, or medium alone for 4 h. Fisher F344 rats raised under germ-free conditions were transferred to B. vulgatus isolators at the age of 10 weeks. The animals were removed from the gnotobiotic isolator after 3 days and euthanized by CO2 asphyxiation within the next 3 h. Bacterial colonization was documented by fecal culture. Primary IEC were isolated from the small intestine, cecum, and colon as described above. Cells were prepared for Western blot as well as FACS analysis. IEC-6 cells were infected overnight with adenoviral dominant negative (dn) IKKβ (Ad5dnIKKβ), dnTAK1 (Ad5dnTAK1), dnTRAF-2 (Ad5dnTRAF-2), and Ad5IκBαAA in serum-free media (Opti-MEM, Invitrogen) at different multiplicity of infection (m.o.i., 0, 25, 50, and 100). The Ad5IκBαAA, Ad5TRAF-2, Ad5dnIKKβ, and Ad5TAK1 were described previously (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar, 44Kotani K. Ogawa W. Hino Y. Kitamura T. Ueno H. Sano W. Sutherland C. Granner D.K. Kasuga M. J. Biol. Chem. 1999; 274: 21305-21312Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 45Jobin C. Panja A. Hellerbrand C. Iimuro Y. Didonato J. Brenner D.A. Sartor R.B. J. Immunol. 1998; 160: 410-418PubMed Google Scholar, 46Jobin C. Bradham C.A. Russo M.P. Juma B. Narula A.S. Brenner D.A. Sartor R.B. J. Immunol. 1999; 163: 3474-3483PubMed Google Scholar, 47Jobin C. Holt L. Bradham C.A. Streetz K. Brenner D.A. Sartor R.B. J. Immunol. 1999; 162: 4447-4454PubMed Google Scholar, 48Bradham C.A. Hatano E. Brenner D.A. Am. J. Physiol. 2001; 281: G1279-G1289PubMed Google Scholar, 49Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar). Ad5κB-LUC consisting of three consensus NF-κB-binding sites was linked to luciferase. Ad5GFP containing green fluorescent protein and Ad5LacZ containing the E. coli β-galactosidase were used as viral negative control (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar). The adenoviruses were washed off, and fresh medium containing serum without antibiotics was added. Cells were stimulated at various time points with B. vulgatus (5 × 107 cfu/ml), TNF (5 ng/ml), and IL-1β (10 ng/ml). Dominant negative TAK1 and the super-repressor IκBαAA contained an extra 27-bp DNA nucleotides coding for a peptide derived from hemagglutinin (HA) gene (YPYDVPDYA). Dominant negative IKKβ and dnTRAF-2 contained an extra 24-bp DNA nucleotides coding for the FLAG peptide (DYLDDDDL). Expression of HA- and FLAG-tagged mutant molecules in IEC-6 cells was controlled by immunofluorescent microscopy and Western blot analysis using mouse anti-HA (Roche Molecular Biochemicals) and mouse anti-FLAG M2 (Eastman Kodak Co.) mAb. CaCO-2 cells were transfected using LipofectAMINE Reagent (Invitrogen) as described previously (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar). The (κB)3-luciferase motif consists of three consensus NF-κB sites linked to luciferase (47Jobin C. Holt L. Bradham C.A. Streetz K. Brenner D.A. Sartor R.B. J. Immunol. 1999; 162: 4447-4454PubMed Google Scholar). Plasmids expressing dominant negative TLR4 (0.8 μg; generous gift of Dr. Bruce Beutler, Scripps Research Institute) or (κB)3-luciferase (1 μg) were transfected in combination or alone as described as under “Results,” and the total amount of DNA was equalized with empty vector. Transfected cells were incubated overnight after which the DNA/LipofectAMINE was replaced with serum-containing media. Cells were then stimulated with B. vulgatus (5 × 107cfu/ml), TNF (5 ng/ml), IL-1β (10 ng/ml), or medium for 12 h. Cell extracts were prepared using enhanced luciferase assay reagents (Analytical Luminescence, San Diego, CA). Luciferase assay were performed on a Monolight 2010 luminometer for 20 s (Analytical Luminescence, San Diego, CA), and results were normalized for extract protein concentrations measured with the Bio-Rad protein assay kit. RNA was isolated using Trizol (Invitrogen), and 1 μg of total RNA was reverse-transcribed and amplified (RT-PCR) using specific primers for rat ICAM-1, COX-2, and β-actin as described previously (50Jobin C. Hellerbrand C. Licato L.L. Brenner D.A. Sartor R.B. Gut. 1998; 42: 779-787Crossref PubMed Scopus (108) Google Scholar). The oligonucleotide TLR4 primers used are as follows: TLR4-A (5′), 5-TGTCCCTGAACCCTATGAAC-3 (positions 795–812); TLR4-B (3′), 5-ACTCAAATCTCTCAAAAGGC-3 (positions 1211–1230). The oligonucleotide MD-2 primers used are as follows: MD-2A (5′), 5-GAAGCTCAGAAGCAGTATTGGGTC-3 (positions 174–197); MD-2B (3′), 5-GGAGTTTGTCATCCTACACCAACC-3 (positions 572–596). The length of the amplified product was 438 and 422 bp, respectively. The PCR products (5 μl) were subjected to electrophoresis on 2% agarose gels containing GelStar fluorescent dye (FMC, Philadelphia, PA). Fluorescent staining was captured using an AlphaImager 2000 (AlphaInnotech, San Leandro, CA). IEC were stimulated for various times (0–4 h) with bacteria, bacterial products, or cytokines. The cells were lysed in 1× Laemmli buffer, and 20 μg of protein was subjected to electrophoresis on 10% SDS-polyacrylamide gels. Where indicated IEC-6 cells were pretreated for 1 h with 20 μm of the proteasome inhibitor MG132 (Peptide Institute, Japan). Anti-phosphoserine IκBα (Cell Signaling, Beverly, MA), anti-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA), anti-IRAK-1 (a generous gift from D. K. Miller, Merck), anti-phosphoserine IKKα/β (Cell Signaling, Beverly, MA), anti-phosphoserine RelA (Ser-536, Cell Signaling, Beverly, MA), anti-phosphoserine Akt (Ser-473, Cell Signaling, Beverly, MA), and anti-β-actin (ICN, Costa Mesa, CA) were used to detect immunoreactive phospho-IκBα, total IκBα, IRAK-1, phospho-IKKα/β, phospho-RelA, and β-actin, respectively, using enhanced chemiluminescence light-detecting kit (Amersham Biosciences) as described previously (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar). IEC-6 cells were stimulated for various times (0–4 h) with B. vulgatus (5 × 107 cfu/ml), and nuclear extracts were prepared as described previously (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar). Extracts (5 μg) were incubated with radiolabeled double-stranded class I major histocompatibility complex κB sites (GGCTGGGGATTCCCCATCT), separated by nondenaturing electrophoresis, and analyzed by autoradiography as described previously (41Jobin C. Haskill S. Mayer L. Panja A. Sartor R.B. J. Immunol. 1997; 158: 226-234PubMed Google Scholar). IKK activity on B. vulgatus-induced serine RelA phosphorylation was determined by immunocomplex kinase assay as described previously (51Schwabe R.F. Brenner D.A. Am. J. Physiol. 2002; 283: G204-G211Crossref PubMed Scopus (120) Google Scholar). Briefly, IEC-6 cells were lysed in Triton lysis buffer containing protease and phosphatase inhibitors after stimulation with B. vulgatus at various times. Where indicated IEC-6 cells were pretreated for 45 min with triptolide (100 ng/ml) or wortmannin (100 nm) or infected for 16 h with Ad5dnIKKβ, Ad5IκBαAA, or Ad5LacZ. 300 μg was immunoprecipitated with 2 μl of anti-IKKγ (Cell Signaling, Beverly, MA), and the kinase reaction was performed by incubating 25 ml of kinase buffer containing 20 mm Tris (pH 7.5), 10 mm MgCl2, 5 mm dithiothreitol, 50 mm ATP, and 0.5 mCi of [32P]ATP with either glutathione S-transferase (GST)-p65-(1–305) or GST-p65-(354–551) (a generous gift of Dr. Hiroaki Sakurai, Tanabe Seiyaku, Osaka, Japan) as substrate for 30 min at 30 °C. Substrate protein was resolved by gel electrophoresis, and phosphate incorporation was assessed by autoradiography and PhosphorImager analysis (Amersham Biosciences). IEC were stimulated with bacteria, bacterial products, or cytokines. ICAM-1 cell surface expression was analyzed after 24 h. Cell staining was performed for 20 min at 4 °C with saturating concentrations of the fluorescein isothiocyanate-conjugated mouse anti-rat ICAM-1 mAb (IgG1, 1A29, Pharmingen). Fluorescein isothiocyanate-conjugated mouse IgG1 (G155–228, MOPC-21, G155–178, Pharmingen) was used as isotype control. The samples were analyzed using a FACSCalibur® (BD Biosciences). Data are expressed as means ± S.D. of triplicates. Statistical significance was performed by the two-tail Student's t test for paired data and was considered significant if p values were <0.05 and <0.01. The commensal enteric Gram-negative species B. vulgatus and E. coli as well as the Gram-positive E. faecalis andL. paracasei were used at a concentration of 5 × 107 cfu/ml to stimulate the nontransformed rat intestinal cell line IEC-6. As shown in Fig.1 A, the Gram-negative bacteriaB. vulgatus an" @default.
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• W2056875662 title "IKKβ and Phosphatidylinositol 3-Kinase/Akt Participate in Non-pathogenic Gram-negative Enteric Bacteria-induced RelA Phosphorylation and NF-κB Activation in Both Primary and Intestinal Epithelial Cell Lines" @default.
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