Advertisement

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*

  • Dirk Haller
    Affiliations
    Department of Medicine and the Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599-7080
    Search for articles by this author
  • Maria P. Russo
    Affiliations
    Department of Medicine and the Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599-7080
    Search for articles by this author
  • R. Balfour Sartor
    Affiliations
    Department of Medicine and the Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599-7080
    Search for articles by this author
  • Christian Jobin
    Correspondence
    To whom correspondence should be addressed: Division of Digestive Disease and Nutrition, CB 7038, Glaxo Bldg., University of North Carolina, Chapel Hill, NC 27599-7080. Tel.: 919-966-7884; Fax: 919-966-7468
    Affiliations
    Department of Medicine and the Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599-7080
    Search for articles by this author
  • Author Footnotes
    * This work was supported by National Institutes of Health ROI Grant DK 47700, the Crohn's and Colitis Foundation of America (to C. J.), and by Deutsche Forschungsgemeinschaft Grant HA 3148/1-1 (to D. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:July 24, 2002DOI:https://doi.org/10.1074/jbc.M205737200
      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.
      IEC
      intestinal epithelial cells
      NF-κB
      nuclear transcription factor κB
      TLR4
      Toll-like receptor 4
      EMSA
      electrophoretic mobility shift assay
      RT
      reverse transcription
      IL-1β
      interleukin 1β
      TNF
      tumor necrosis factor
      IRAK
      interleukin 1 receptor-associated kinase
      ICAM-1
      intercellular adhesion molecule 1
      TAK1
      transforming growth factor-β-activated kinase 1
      IKK
      IκB kinase complex
      Ad
      adenoviral
      PI3K
      phosphatidylinositol 3-kinase
      cfu
      colony-forming units
      dn
      dominant negative
      mAb
      monoclonal antibody
      FACS
      fluorescence-activated cell sorter
      GST
      glutathioneS-transferase
      HA
      hemagglutinin
      FCS
      fetal calf serum
      m.o.i.
      multiplicity of infection
      PBMC
      peripheral blood mononuclear cells
      PBL
      peripheral blood lymphocytes
      LPS
      lipopolysaccharide
      TRAF
      TNF receptor-associated factor
      TAK
      transforming growth factor-β-activated kinase
      mAb
      monoclonal antibody
      MFI
      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 (
      • Sartor R.B.
      ,
      • Fiocchi C.
      ). 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 (
      • Blumberg R.S.
      • Saubermann L.J.
      • Strober W.
      ,
      • Bhan A.K.
      • Mizoguchi E.
      • Smith R.N.
      • Mizoguchi A.
      ,
      • Elson C.O.
      • Sartor R.B.
      • Tennyson G.S.
      • Riddell R.H.
      ). 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 (
      • Rath 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.
      ,
      • Taurog J.D.
      • Richardson J.A.
      • Croft J.T.
      • Simmons W.A.
      • Zhou M.
      • Fernandez-Sueiro J.L.
      • Balish E.
      • Hammer R.E.
      ). Reconstitution studies of gnotobiotic HLA-B27 transgenic rats (
      • Rath 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.
      ,
      • Rath H.C.
      • Wilson K.H.
      • Sartor R.B.
      ) and carrageenan-induced colitis in guinea pigs (
      • Onderdonk A.B.
      • Franklin M.L.
      • Cisneros R.L.
      ) 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 (
      • Rath H.C.
      • Wilson K.H.
      • Sartor R.B.
      ). 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 (
      • Kagnoff M.F.
      • Eckmann L.
      ). 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 (
      • Blumberg R.S.
      • Terhorst C.
      • Bleicher P.
      • McDermott F.V.
      • Allan C.H.
      • Landau S.B.
      • Trier J.S.
      • Balk S.P.
      ,
      • Panja A.
      • Barone A.
      • Mayer L.
      ,
      • Hershberg R.M.
      • Framson P.E.
      • Cho D.H.
      • Lee L.Y.
      • Kovats S.
      • Beitz J.
      • Blum J.S.
      • Nepom G.T.
      ). 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 (
      • McCormick B.A.
      • Colgan S.P.
      • Delp-Archer C.
      • Miller S.I.
      • Madara J.L.
      ,
      • Eckmann L.
      • Stenson W.F.
      • Savidge T.C.
      • Lowe D.C.
      • Barrett K.E.
      • Fierer J.
      • Smith J.R.
      • Kagnoff M.F.
      ,
      • Huang G.T.
      • Eckmann L.
      • Savidge T.C.
      • Kagnoff M.F.
      ,
      • Jung H.C.
      • Eckmann L.
      • Yang S.K.
      • Panja A.
      • Fierer J.
      • Morzycka- Wroblewska E.
      • Kagnoff M.F.
      ,
      • Savkovic S.D.
      • Koutsouris A.
      • Hecht G.
      ,
      • Schulte R.
      • Wattiau P.
      • Hartland E.L.
      • Robins-Browne R.M.
      • Cornelis G.R.
      ). 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 (
      • Tak P.P.
      • Firestein G.S.
      ).
      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 (
      • Jobin C.
      • Sartor R.B.
      ), 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 (
      • Zhang G.
      • Ghosh S.
      ,
      • Tapping R.I.
      • Akashi S.
      • Miyake K.
      • Godowski P.J.
      • Tobias P.S.
      ,
      • Faure E.
      • Equils O.
      • Sieling P.A.
      • Thomas L.
      • Zhang F.X.
      • Kirschning C.J.
      • Polentarutti N.
      • Muzio M.
      • Arditi M.
      ). 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 (
      • Beutler B.
      ,
      • Hatada E.N.
      • Krappmann D.
      • Scheidereit C.
      ). 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 (
      • Sizemore N.
      • Lerner N.
      • Dombrowski N.
      • Sakurai H.
      • Stark G.R.
      ,
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Wang D.
      • Baldwin A.S.J.
      ,
      • Bird T.A.
      • Schooley K.
      • Dower S.K.
      • Hagen H.
      • Virca G.D.
      ,
      • Zhong H.
      • SuYang H.
      • Erdjument-Bromage H.
      • Tempst P.
      • Ghosh S.
      ,
      • Wang D.
      • Westerheide S.D.
      • Hanson J.L.
      • Baldwin A.S.J.
      ). 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 (
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Madrid L.V.
      • Mayo M.W.
      • Reuther J.Y.
      • Baldwin A.S.J.
      ,
      • Madrid L.V.
      • Wang C.Y.
      • Guttridge D.C.
      • Schottelius A.J.
      • Baldwin A.S.J.
      • Mayo M.W.
      ). Potential kinases involved in signal-induced RelA phosphorylation are the casein kinase II, Akt, and IKK (
      • Sizemore N.
      • Lerner N.
      • Dombrowski N.
      • Sakurai H.
      • Stark G.R.
      ,
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ,
      • Madrid L.V.
      • Mayo M.W.
      • Reuther J.Y.
      • Baldwin A.S.J.
      ,
      • Schmitz M.L.
      • Bacher S.
      • Kracht M.
      ). 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 (
      • Cario E.
      • Podolsky D.K.
      ,
      • Cario E.
      • Rosenberg I.M.
      • Brandwein S.L.
      • Beck P.L.
      • Reinecker H.C.
      • Podolsky D.K.
      ,
      • Cario E.
      • Brown D.
      • McKee M.
      • Lynch-Devaney K.
      • Gerken G.
      • Podolsky D.K.
      ), 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.

      DISCUSSION

      In this study, we demonstrate that a non-pathogenic enteric Gram-negative B. vulgatus bacteria strain has the ability to signal to the NF-κB system in IEC lines by inducing IRAK-1 degradation, IκBα phosphorylation and degradation, NF-κB DNA binding activity, and NF-κB transcriptional activity. Interestingly,B. vulgatus also stimulates endogenous RelA serine 536 and GST-p65TAD phosphorylation (Ser-529/Ser-536). Although previous studies using pathogenic strains of invasive Salmonella species (
      • Elewaut D.
      • DiDonato J.A.
      • Kim J.M.
      • Truong F.
      • Eckmann L.
      • Kagnoff M.F.
      ), enteroadherent E. coli (
      • Savkovic S.D.
      • Koutsouris A.
      • Hecht G.
      ), andCryptosporidium (
      • Chen X.M.
      • Levine S.A.
      • Splinter P.L.
      • Tietz P.S.
      • Ganong A.L.
      • Jobin C.
      • Gores G.J.
      • Paya C.V.
      • LaRusso N.F.
      ) show NF-κB activation and chemokine expression in IEC lines, this is the first demonstration that non-pathogenic bacteria induce gene expression through NF-κB translocation and RelA phosphorylation. B. vulgatus-induced RelA phosphorylation is IKKβ- and PI3K/Akt-dependent. The physiological relevance of B. vulgatus-induced NF-κB signal transduction was demonstrated in primary rat IEC. First, we showed that the potential of primary naive IEC to initiate IκBα phosphorylation/degradation and IKKα/β and RelA phosphorylation in response to B. vulgatus stimulation. Second, and most importantly, we showed that RelA phosphorylation and ICAM-1 protein expression are induced in primary IEC derived fromB. vulgatus mono-associated wild type rats shortly after bacterial colonization (3 days). The pivotal role of the IκB/NF-κB transcriptional system in B. vulgatus signaling to IEC was clearly demonstrated using adenoviral delivery of Ad5IκBαAA and Ad5dnIKKβ, which completely blocked bacteria-induced NF-κB transcriptional activity and ICAM-1 mRNA and protein expression in IEC-6 cells. Bacterial activation of IEC was specific for Gram-negative bacteria but was not dependent on bacterial adherence or invasion, as demonstrated by the use of Gram-negative bacterial lysates and LPS for IEC stimulation. This shows that non-pathogenic as well as pathogenic bacteria utilize the NF-κB signaling cassette to induce gene expression in IEC.
      It has been shown that LPS signals through the pattern recognition receptor TLR4 to activate multiple signaling cascades including the IκB/NF-κB system (
      • Beutler B.
      ,
      • Wright S.D.
      ). TLR4 protein was detected in tissue sections from patients with ulcerative colitis and Crohn's disease, suggesting the potential ability of intestinal epithelial cells to respond to luminal Gram-negative bacteria or LPS (
      • Cario E.
      • Podolsky D.K.
      ). Cario et al. (
      • Cario E.
      • Podolsky D.K.
      ,
      • Cario E.
      • Rosenberg I.M.
      • Brandwein S.L.
      • Beck P.L.
      • Reinecker H.C.
      • Podolsky D.K.
      ) showed that human IEC lines express TLR4 and CD14 and respond to high doses (5–10 μg/ml) of LPS by inducing NF-κB DNA binding activity. However, the exact molecular mechanisms of Gram-negative bacteria or LPS signal transduction in IEC are still unclear.
      We provide evidence that TLR4 and several IL-1 receptor/TLR4 signaling proteins participate in B. vulgatus-mediated NF-κB activation. First, we showed that the mRNA of TLR4 and its co-receptor MD-2 were expressed in CaCO-2 and dnTLR4 blocked B. vulgatus but not IL-1β-stimulated NF-κB transcriptional activity, demonstrating a selective role of TLR4 in B. vulgatus signaling to NF-κB in IEC. The relatively low expression of TLR4 and MD-2 may explain the weak induction of NF-κB transcriptional activity following B. vulgatus stimulation in CaCO-2 cells. Interestingly, similar findings were reported for the stimulation of CaCO-2 cells with LPS (
      • Abreu M.T.
      • Vora P.
      • Faure E.
      • Thomas L.S.
      • Arnold E.T.
      • Arditi M.
      ). Of importance, these authors were able to enhance colonic IEC responsiveness to LPS by transfecting expression vectors encoding for MD-2 and TLR4, suggesting that both receptors are important in conveying LPS signaling to NF-κB. In addition, we showed that IRAK-1 protein, a signaling molecule involved in the IL-1R/TLR pathway, is degraded in B. vulgatus-stimulated IEC-6 cells. Previous studies have shown that LPS-induced NF-κB activation is partially impaired in macrophages isolated from IRAK-1−/− mice (
      • Swantek J.L.
      • Tsen M.F.
      • Cobb M.H.
      • Thomas J.A.
      ), whereas macrophages isolated from IRAK-4−/− are strongly unresponsive (
      • Suzuki N.
      • Suzuki S.
      • Duncan G.S.
      • Millar D.G.
      • Wada T.
      • Mirtsos C.
      • Takada H.
      • Wakeham A.
      • Itie A., Li, S.
      • Penninger J.M.
      • Wesche H.
      • Ohashi P.S.
      • Mak T.W.
      • Yeh W.C.
      ). It remains to be seen whether B. vulgatus utilizes IRAK-4 to signal to NF-κB in IEC.
      Although TAK1 has been linked to LPS-mediated NF-κB signaling in macrophages (
      • Irie T.
      • Muta T.
      • Takeshige K.
      ), Ad5dnTAK1 only partially inhibited B. vulgatus-induced RelA nuclear translocation (data not shown) and transcriptional activity. Interestingly, B. vulgatus-induced NF-κB transcriptional activity and ICAM-1 gene expression is completely blocked by Ad5dnIKKβ or Ad5IκBAA. This suggests that the signaling proteins IKKβ and IκBα constitute critical control points of Gram-negative bacterial signaling to NF-κB and gene expression in IEC. Together, these results suggest that NF-κB activation by B. vulgatus likely requires TLR4, IRAK-1, IKK, and IκBα in IEC.
      An emerging control point in NF-κB activation is signal-induced RelA serine phosphorylation, which enhances its transcriptional activity (
      • Schmitz M.L.
      • Bacher S.
      • Kracht M.
      ). Interestingly, we found that B. vulgatus stimulation induced endogenous RelA 536 phosphorylation in both primary and IEC lines. In addition, immunoprecipitated IKKγ from B. vulgatus-stimulated cells phosphorylates GST-p65TAD-(354–551), which contains the important Ser-529 and Ser-536 residues. It has been shown that the PI3K/Akt and IKK are critical kinases for cytokine-induced RelA phosphorylation and NF-κB transcriptional activity (
      • Sizemore N.
      • Lerner N.
      • Dombrowski N.
      • Sakurai H.
      • Stark G.R.
      ,
      • Madrid L.V.
      • Mayo M.W.
      • Reuther J.Y.
      • Baldwin A.S.J.
      ,
      • Madrid L.V.
      • Wang C.Y.
      • Guttridge D.C.
      • Schottelius A.J.
      • Baldwin A.S.J.
      • Mayo M.W.
      ,
      • Mayo M.W.
      • Madrid L.V.
      • Westerheide S.D.
      • Jones D.R.
      • Yuan X.J.
      • Baldwin A.S.J.
      • Whang Y.E.
      ). The functional consequence of RelA phosphorylation is highlighted by the blockade of B. vulgatus-induced NF-κB transcriptional activity by Ad5dnIKKβ and by the pharmacological inhibitor wortmannin, both of which prevent RelA phosphorylation. The kinase as well as the signaling pathway responsible for B. vulgatus-induced RelA phosphorylation is still unknown. Our data demonstrate for the first time that both IKK and PI3K/Akt participate in B. vulgatus-mediated NF-κB transcriptional activity. Further studies are required to define the exact signaling cascade involved in bacteria-induced RelA phosphorylation and NF-κB activity.
      Interestingly, although the Chinese herb derivative triptolide has been shown to inhibit NF-κB transcriptional activity (
      • Lee K.Y.
      • Chang W.
      • Qiu D.
      • Kao P.N.
      • Rosen G.D.
      ), it failed to prevent bacteria-induced Ser-536 and Ser-529 phosphorylation. RelA is phosphorylated at various serine residues (Ser-276, Ser-529, and Ser-536) in response to either IL-1 or TNF stimulation (
      • Schmitz M.L.
      • Bacher S.
      • Kracht M.
      ,
      • Ghosh S.
      • Karin M.
      ). Therefore, the possibility remains that triptolide interferes with the phosphorylation of serine residue 276. However, B. vulgatusfailed to induce phosphorylation of GST-p65TAD-(1–305) which includes the Ser-276 phosphorylation site. Therefore, although triptolide blocksB. vulgatus-induced NF-κB transcriptional activity, its mechanism of action is unlikely to involve inhibition of RelA phosphorylation. Recruitment of transcriptional co-activator/co-repressor played an important role in the regulation of NF-κB transcriptional activity (
      • Ghosh S.
      • Karin M.
      ,
      • Zhong H.
      • May M.J.
      • Jimi E.
      • Gosh S.
      ). In addition, chromatin remodeling through modulation of histone acetylation defines access of transcription factors to gene promoter elements and therefore transcriptional activity (
      • Ghosh S.
      • Karin M.
      ,
      • Saccani S.
      • Pantano S.
      • Natoli G.
      ,
      • Zhong H.
      • May M.J.
      • Jimi E.
      • Gosh S.
      ,
      • Johnstone R.W.
      ). A possible mechanism of triptolide interference with NF-κB transcriptional activity may include blockade of transcriptional co-activator or modulation of the histone acetylation status.
      The finding that commensal bacteria normally present in high concentration in the distal intestine are able to trigger NF-κB activation in IEC in vitro contrasts with the absence of chronic inflammation seen in the normal host. Multiple mechanisms ensure in vivo hyporesponsiveness of native IEC to bacteria including low expression of TLR4 and MD-2 in colonic epithelial cell lines (
      • Abreu M.T.
      • Vora P.
      • Faure E.
      • Thomas L.S.
      • Arnold E.T.
      • Arditi M.
      ) and down-regulatory signals from adjacent lamina propria immune cells. Previous results have shown that bacterial mediated activation of IEC is modulated by the presence of immunocompetent cells using transwell co-cultures (
      • Haller D.
      • Bode C.
      • Hammes W.P.
      • Pfeifer A.M.A.
      • Schiffrin E.J.
      • Blum S.
      ,
      • Haller D.
      • Serrant P.
      • Peruisseau G.
      • Hammes W.P.
      • Bode C.
      • Schiffrin E.J.
      • Blum S.
      ). Interestingly, the presence of human peripheral blood lymphocytes inhibited B. vulgatus-induced NF-κB activity by blocking IκBα degradation. However, B. vulgatus-induced IRAK-1 degradation and RelA phosphorylation is not inhibited in IEC/lymphocyte co-cultures, whereas IκBα degradation is strongly suppressed. This suggests that the upstream signaling cascade leading to IκBα and RelA phosphorylation is not blocked by the presence of lymphocytes but rather points out an inhibition at the level of proteosome and/or ubiquitination. Interestingly, non-virulentSalmonella strains inhibit NF-κB activity by preventing IκB ubiquitination, possibly through inhibition of the IκB ubiquitin ligase (
      • Neish A.S.
      • Gewirtz A.T.
      • Zeng H.
      • Young A.N.
      • Hobert M.E.
      • Karmali V.
      • Rao A.S.
      • Madara J.L.
      ). Further studies would be required to dissect the precise mechanism of immune cell-mediated blockade of IκBα degradation. In addition, our recent studies
      D. Haller, L. Holt, R. B. Sartor, and C. Jobin, manuscript in preparation.
      show that the immunoregulatory cytokine TGF-β1 inhibits B. vulgatus-mediated NF-κB transcriptional activity in IEC through the induction of the Smad signaling pathway. This suggests that the responsiveness of IEC to luminal enteric bacteria in a complex environment such as the mucosal immune system may depend on a network of communication between immune and epithelial cells and their secreted mediators.
      Although various rodent models of inflammatory bowel diseases demonstrated the key role of enteric bacteria and bacterial products in initiating and perpetuating chronic immune-mediated intestinal inflammation in genetically susceptible hosts, the molecular mechanisms of bacterial signaling in the intestinal epithelium remain to be elucidated. Recent studies showed that NF-κB blockade in IEC by tissue-specific expression of the IκBαAA super-repressor exacerbates dextran sodium sulfate-induced experimental colitis (
      • Russo M.P.
      • Boubreau F.
      • Fengling L.
      • Panja A.
      • Traber P.G.
      • Sartor C.
      • Jobin C.
      ), suggesting that in contrast to previous observations of protective effects of NF-κB blockade in trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (
      • Neurath M.F.
      • Pettersson S.
      • Meyer Z.B.K.
      • Strober W.
      ), NF-κB-derived IEC gene expression may have a protective role during acute intestinal inflammation. This concept was further supported by the finding that p50 and p65 gene-deleted mice compared with wild type mice have more severe inflammation in a model of Helicobacter hepaticus-induced typhlocolitis (
      • Erdman S.E.
      • Fox J.G.
      • Dangler C.A.
      • Feldman D.
      • Horwitz B.H.
      ). In addition, blocking NF-κB activity with pharmacological inhibitors during the resolution phase of carrageenan-induced acute inflammation is deleterious to the host (
      • Lawrence T.
      • Gilroy D.W.
      • Colville-Nash P.R.
      • Willoughby D.A.
      ). This is in line with recent findings showing that activation of TLR9 signaling by unmethylated CpG bacterial DNA decreased dextran sodium sulfate- and hapten-induced colitis (
      • Rachmilewitz D.
      • Karmeli F.
      • Takabayashi K.
      • Hayashi T.
      • Leider-Trjo L.
      • Lee L.
      • Leoni L.M.
      • Raz E.
      ). Therefore, NF-κB activation may have a beneficial role in the early onset of inflammation, but at the same time may be detrimental if activation persists. We hypothesize that low levels of NF-κB activity induced by non-pathogenic commensal bacteria play a role in the maintenance of intestinal homeostasis in a normal host, whereas it may be deleterious in a genetically susceptible host. Alternatively, epithelial cell NF-κB activation may be protective, whereas NF-κB activation in lamina propria cells may induce activation. In summary, our data demonstrate that components of the Gram-negative non-pathogenicB. vulgatus have the potential to signal through the TLR4 system to activate NF-κB and gene expression in IEC, independent of viable bacterial adhesion and invasion. Therefore, a more complete understanding of bacterial signaling in IEC will provide additional insights into the mechanisms of mucosal homeostasis and injury and may yield new therapeutic targets for intestinal inflammation.

      References

        • Sartor R.B.
        Am. J. Gastroenterol. 1997; 92: 5-11
        • Fiocchi C.
        Gastroenterology. 1998; 115: 182-205
        • Blumberg R.S.
        • Saubermann L.J.
        • Strober W.
        Curr. Opin. Immunol. 1999; 11: 648-656
        • Bhan A.K.
        • Mizoguchi E.
        • Smith R.N.
        • Mizoguchi A.
        Immunol. Rev. 1999; 169: 195-207
        • Elson C.O.
        • Sartor R.B.
        • Tennyson G.S.
        • Riddell R.H.
        Gastroenterology. 1995; 109: 1344-1367
        • Rath 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: 945-953
        • Taurog 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: 2359-2364
        • Rath H.C.
        • Wilson K.H.
        • Sartor R.B.
        Infect. Immun. 1999; 67: 2969-2974
        • Onderdonk A.B.
        • Franklin M.L.
        • Cisneros R.L.
        Infect. Immun. 1981; 32: 225-231
        • Kagnoff M.F.
        • Eckmann L.
        J. Clin. Invest. 1997; 100: 6-10
        • Blumberg 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: 2518-2524
        • Panja A.
        • Barone A.
        • Mayer L.
        J. Exp. Med. 1994; 179: 943-950
        • Hershberg 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: 204-215
        • McCormick B.A.
        • Colgan S.P.
        • Delp-Archer C.
        • Miller S.I.
        • Madara J.L.
        J. Cell Biol. 1993; 123: 895-907
        • Eckmann 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: 296-309
        • Huang G.T.
        • Eckmann L.
        • Savidge T.C.
        • Kagnoff M.F.
        J. Clin. Invest. 1996; 98: 572-583
        • Jung H.C.
        • Eckmann L.
        • Yang S.K.
        • Panja A.
        • Fierer J.
        • Morzycka- Wroblewska E.
        • Kagnoff M.F.
        J. Clin. Invest. 1995; 95: 55-65
        • Savkovic S.D.
        • Koutsouris A.
        • Hecht G.
        Am. J. Physiol. 1997; 273: C1160-C1167
        • Schulte R.
        • Wattiau P.
        • Hartland E.L.
        • Robins-Browne R.M.
        • Cornelis G.R.
        Infect. Immun. 1996; 64: 2106-2113
        • Tak P.P.
        • Firestein G.S.
        J. Clin. Invest. 2001; 107: 7-11
        • Jobin C.
        • Sartor R.B.
        Am. J. Physiol. 2000; 278: C451-C462
        • Zhang G.
        • Ghosh S.
        J. Clin. Invest. 2001; 107: 13-19
        • Tapping R.I.
        • Akashi S.
        • Miyake K.
        • Godowski P.J.
        • Tobias P.S.
        J. Immunol. 2000; 165: 5780-5787
        • Faure 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: 11058-11063
        • Beutler B.
        Curr. Opin. Immunol. 2000; 12: 20-26
        • Hatada E.N.
        • Krappmann D.
        • Scheidereit C.
        Curr. Opin. Immunol. 2000; 12: 52-58
        • Sizemore N.
        • Lerner N.
        • Dombrowski N.
        • Sakurai H.
        • Stark G.R.
        J. Biol. Chem. 2002; 277: 3863-3869
        • Sizemore N.
        • Leung S.
        • Stark G.R.
        Mol. Cell. Biol. 1999; 19: 4798-4805
        • Wang D.
        • Baldwin A.S.J.
        J. Biol. Chem. 1998; 273: 29411-29416
        • Bird T.A.
        • Schooley K.
        • Dower S.K.
        • Hagen H.
        • Virca G.D.
        J. Biol. Chem. 1997; 272: 32606-32612
        • Zhong H.
        • SuYang H.
        • Erdjument-Bromage H.
        • Tempst P.
        • Ghosh S.
        Cell. 1997; 89: 413-424
        • Wang D.
        • Westerheide S.D.
        • Hanson J.L.
        • Baldwin A.S.J.
        J. Biol. Chem. 2000; 275: 32592-32597
        • Madrid L.V.
        • Mayo M.W.
        • Reuther J.Y.
        • Baldwin A.S.J.
        J. Biol. Chem. 2001; 276: 18934-18940
        • Madrid L.V.
        • Wang C.Y.
        • Guttridge D.C.
        • Schottelius A.J.
        • Baldwin A.S.J.
        • Mayo M.W.
        Mol. Cell. Biol. 2000; 20: 1626-1638
        • Schmitz M.L.
        • Bacher S.
        • Kracht M.
        Trends Biochem. Sci. 2001; 26: 186-190
        • Cario E.
        • Podolsky D.K.
        Infect. Immun. 2000; 68: 7010-7017
        • Cario E.
        • Rosenberg I.M.
        • Brandwein S.L.
        • Beck P.L.
        • Reinecker H.C.
        • Podolsky D.K.
        J. Immunol. 2000; 164: 966-972
        • Cario E.
        • Brown D.
        • McKee M.
        • Lynch-Devaney K.
        • Gerken G.
        • Podolsky D.K.
        Am. J. Pathol. 2002; 160: 165-173
        • Kim S.C.
        • Tonkonogy S.L.
        • Balish E.
        • Warner T.
        • Sartor R.B.
        Gastroenterology. 2001; 20: A82
        • Cong Y.
        • Brandwein S.L.
        • McCabe R.P.
        • Lazenby A.
        • Birkenmeier E.H.
        • Sundberg J.P.
        • Elson C.O.
        J. Exp. Med. 1998; 187: 855-864
        • Jobin C.
        • Haskill S.
        • Mayer L.
        • Panja A.
        • Sartor R.B.
        J. Immunol. 1997; 158: 226-234
        • Haller D.
        • Bode C.
        • Hammes W.P.
        • Pfeifer A.M.A.
        • Schiffrin E.J.
        • Blum S.
        Gut. 2000; 47: 79-87
        • Haller D.
        • Serrant P.
        • Peruisseau G.
        • Hammes W.P.
        • Bode C.
        • Schiffrin E.J.
        • Blum S.
        Microbiol. Immunol. 2002; 46: 195-205
        • Kotani K.
        • Ogawa W.
        • Hino Y.
        • Kitamura T.
        • Ueno H.
        • Sano W.
        • Sutherland C.
        • Granner D.K.
        • Kasuga M.
        J. Biol. Chem. 1999; 274: 21305-21312
        • Jobin C.
        • Panja A.
        • Hellerbrand C.
        • Iimuro Y.
        • Didonato J.
        • Brenner D.A.
        • Sartor R.B.
        J. Immunol. 1998; 160: 410-418
        • Jobin C.
        • Bradham C.A.
        • Russo M.P.
        • Juma B.
        • Narula A.S.
        • Brenner D.A.
        • Sartor R.B.
        J. Immunol. 1999; 163: 3474-3483
        • Jobin C.
        • Holt L.
        • Bradham C.A.
        • Streetz K.
        • Brenner D.A.
        • Sartor R.B.
        J. Immunol. 1999; 162: 4447-4454
        • Bradham C.A.
        • Hatano E.
        • Brenner D.A.
        Am. J. Physiol. 2001; 281: G1279-G1289
        • Galang C.K.
        • Der C.J.
        • Hauser C.A.
        Oncogene. 1994; 9: 2913-2921
        • Jobin C.
        • Hellerbrand C.
        • Licato L.L.
        • Brenner D.A.
        • Sartor R.B.
        Gut. 1998; 42: 779-787
        • Schwabe R.F.
        • Brenner D.A.
        Am. J. Physiol. 2002; 283: G204-G211
        • Bocker U.
        • Schottelius A.
        • Watson J.M.
        • Holt L.
        • Licato L.L.
        • Brenner D.A.
        • Sartor R.B.
        • Jobin C.
        J. Biol. Chem. 2000; 275: 12207-12213
        • Russo M.P.
        • Bennet B.L.
        • Manning A.M.
        • Brenner D.A.
        • Jobin C.
        Am. J. Physiol. 2002; 283: C347-C357
        • Lee K.Y.
        • Chang W.
        • Qiu D.
        • Kao P.N.
        • Rosen G.D.
        J. Biol. Chem. 1999; 274: 13451-13455
        • Elewaut D.
        • DiDonato J.A.
        • Kim J.M.
        • Truong F.
        • Eckmann L.
        • Kagnoff M.F.
        J. Immunol. 1999; 163: 1457-1466
        • Chen X.M.
        • Levine S.A.
        • Splinter P.L.
        • Tietz P.S.
        • Ganong A.L.
        • Jobin C.
        • Gores G.J.
        • Paya C.V.
        • LaRusso N.F.
        Gastroenterology. 2001; 120: 1774-1783
        • Wright S.D.
        J. Exp. Med. 1999; 189: 605-609
        • Abreu M.T.
        • Vora P.
        • Faure E.
        • Thomas L.S.
        • Arnold E.T.
        • Arditi M.
        J. Immunol. 2001; 167: 1609-1616
        • Swantek J.L.
        • Tsen M.F.
        • Cobb M.H.
        • Thomas J.A.
        J. Immunol. 2000; 164: 4301-4306
        • Suzuki N.
        • Suzuki S.
        • Duncan G.S.
        • Millar D.G.
        • Wada T.
        • Mirtsos C.
        • Takada H.
        • Wakeham A.
        • Itie A., Li, S.
        • Penninger J.M.
        • Wesche H.
        • Ohashi P.S.
        • Mak T.W.
        • Yeh W.C.
        Nature. 2002; 416: 750-756
        • Irie T.
        • Muta T.
        • Takeshige K.
        FEBS Lett. 2000; 461: 60-64
        • Mayo M.W.
        • Madrid L.V.
        • Westerheide S.D.
        • Jones D.R.
        • Yuan X.J.
        • Baldwin A.S.J.
        • Whang Y.E.
        J. Biol. Chem. 2002; 277: 11116-11125
        • Ghosh S.
        • Karin M.
        Cell. 2002; 109: 581-596
        • Saccani S.
        • Pantano S.
        • Natoli G.
        Nat. Immunol. 2002; 3: 69-75
        • Zhong H.
        • May M.J.
        • Jimi E.
        • Gosh S.
        Mol. Cell. 2002; 9: 625-636
        • Johnstone R.W.
        Nat. Rev. Drug Discov. 2002; 1: 287-299
        • Neish A.S.
        • Gewirtz A.T.
        • Zeng H.
        • Young A.N.
        • Hobert M.E.
        • Karmali V.
        • Rao A.S.
        • Madara J.L.
        Science. 2000; 289: 1483-1484
        • Russo M.P.
        • Boubreau F.
        • Fengling L.
        • Panja A.
        • Traber P.G.
        • Sartor C.
        • Jobin C.
        Gastroenterology. 2001; 20: 470
        • Neurath M.F.
        • Pettersson S.
        • Meyer Z.B.K.
        • Strober W.
        Nat. Med. 1996; 2: 998-1004
        • Erdman S.E.
        • Fox J.G.
        • Dangler C.A.
        • Feldman D.
        • Horwitz B.H.
        J. Immunol. 2001; 166: 1443-1447
        • Lawrence T.
        • Gilroy D.W.
        • Colville-Nash P.R.
        • Willoughby D.A.
        Nat. Med. 2001; 7: 1291-1297
        • Rachmilewitz D.
        • Karmeli F.
        • Takabayashi K.
        • Hayashi T.
        • Leider-Trjo L.
        • Lee L.
        • Leoni L.M.
        • Raz E.
        Gastroenterology. 2002; 122: 1428-1441