Article Figures & Data
Figures
- FIGURE 1.
Enteroinvasive bacteria activates NF-κB in human colon epithelial cells. Human colon epithelial cell lines HT-29 (top), Caco-2 (middle), or T84 (bottom) were infected with the enteroinvasive bacterial strains S. dublin, Y. enterocolitica or E. coli O29:NM, the noninvasive bacterial strain E. coli DH5α, or stimulated with TNF-α as a control. NF-κB DNA binding activity was assessed by EMSA at the indicated times up to 60 min after infection. Background levels of NF-κB binding at time 0, immediately before infection, are shown immediately adjacent to the column for TNF-α stimulation. Immunoblots (IB) showing concurrent IκBα and IκBε levels under the same set of conditions are provided beneath each EMSA time point. The single major band for IκBα represents the nonphosphorylated molecule, whereas the second closely running band noted at 45 and 60 min after S. dublin infection of Caco-2 cells represents phosphorylated IκBα. IκBε exists as two closely related phosphoisoforms as most clearly shown for Caco-2 cells. Caco-2 cells show little, if any, response to TNF-α stimulation (44 ) and, consistent with this, TNF-α stimulation was a weak activator of IκBα degradation and NF-κB DNA binding in Caco-2 cells. The results are representative of three or more repeated experiments.
- FIGURE 2.
Enteroinvasive bacteria activate IKKα and IKKβ kinase activity. HT-29 (top), Caco-2 (middle), and T84 (bottom) cells were infected with the enteroinvasive bacterial strains S. dublin, Y. enterocolitica, or E. coli O29:NM, the noninvasive bacterial strain E. coli DH5α, or stimulated with TNF-α (20 ng/ml) as a control. Whole cell lysates were obtained before or up to 60 min after infection or TNF-α stimulation as indicated, and IKKα and IKKβ were isolated from cell lysates by immunoprecipitation as described in Materials and Methods. IκB kinase activity of each specific immunocomplex was determined using GST- IκBα (1-54) as a substrate. A GST-IκB-A32/36 fusion protein that contains alanine substitutions at residues 32 and 36 and is not phosphorylated by IKKα and IKKβ was used as a specificity control, and data using this substrate are shown in the right lane for lysates obtained 10 min after TNF-α stimulation (10AA). Consistent with the lack of response of Caco-2 cells to TNF-α stimulation, IKKα and IKKβ were not activated after TNF-α stimulation of those cells. WT, wild type.
- FIGURE 3.
An IκBα superrepressor inhibits the up-regulated expression of major components of the host’s endogenous epithelial cell proinflammatory gene program. HT-29 cells were infected with Ad5IκB-A32/36 which expresses an IκBα superrepressor or, as a control, Ad5LacZ which expresses β-galactosidase. Twenty-four hours after viral infection, cells were left untreated or were infected with S. dublin or stimulated with TNF-α as a positive control to up-regulate NF-κB target genes. Three hours later, the expression of genes known to be components of the proinflammatory program in bacteria-infected human intestinal epithelial cells, and control genes that are not components of that program, were assessed by RT-PCR. The IκBα superrepressor inhibited, partially to almost completely, the up-regulated expression of IL-8, GROα, MCP-1, ICAM-1, and COX-2 (Fig. 3) as well as NOS2 and TNF-α (not shown) which are components of the epithelial cell proinflammatory program. In contrast, the superrepressor did not inhibit expression of the control genes TGF-β1 or β-actin or TGF-α (not shown) in S. dublin-infected or TNF-α-stimulated cells. The results are representative of two or more repeated experiments.
- FIGURE 4.
Flow cytometric analysis of ICAM-1 expression by HT-29 cells. HT-29 cells were infected with Ad5IκB-A32/36 (Ad5IκB) which expresses an IκBα superrepressor (middle column), with Ad5LacZ which expresses β-galactosidase (right column), or were not infected with adenovirus (control, left column). Twenty-four hours after viral infection, cells were infected with S. dublin (top row) or Y. enterocolitica (second row) or stimulated with TNF-α (20 ng/ml, third row) or IFN-γ (40 ng/ml, bottom row). After 9 h, cells were detached from the plates, stained, and analyzed by flow cytometry as outlined in Materials and Methods. Shaded area in each panel represents ICAM-1 staining of cells that were not bacteria infected or stimulated with cytokines. The unshaded area outlined by the darker line represents ICAM-1 staining in bacteria-infected or cytokine-stimulated cells. As shown, the IκB superrepressor inhibited increased ICAM-1 expression in bacteria-infected or TNF-α-stimulated cells, but not in IFN-γ-stimulated cells.
Tables
mRNA Species 5′-Primer 3′-Primer Size of PCR Product (bp) IL-8 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′ 289 GROα 5′ACTCAAGAATGGGCGGAAAG-3′ 5′-TGGCATGTTGCAGGCTCCT-3′ 468 MCP-1 5′-TCTGTGCCTGCTGCTCATAGC-3′ 5′-GGGTAGAACTGTGGTTCAAGAGG-3′ 510 COX-2 5′TTCAAATGAGATTGTGGGAAAATTGCT-3′ 5′-AGATCATCTCTGCCTGAGTATCTT-3′ 305 TGF-α 5′-ATGGTCCCCTCGGCTGGACAGCTCGCC-3′ 5′-GATGGCCTGCTTCTTCTGGCTGGCAGC-3′ 300 TGF-β1 5′-GCCCTGGACACCAACTATTGCT-3′ 5′-AGGCTCCAAATGTAGGGGCAGG-3′ 161 TNF-α 5′-CGGGACGTGGAGCTGGCCGAGGAG-3′ 5′-CACCAGCTGGTTATCTCTCAGCTC-3′ 355 iNOS 5′-CGGTGCTGTATTTCCTTACGAGGCGAAGAAGG-3′ 5′GGTGCTGCTTGTTAGGAGGTCAAGTAAAGGGC-3′ 259 ICAM-1 5′-GATGCTGACCCTGGAGAGCA-3′ 5′-AGCACTTGCGGTCCACGATG-3′ 409 β-Actin 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ 5′-CTAGAAGCATTGCGGTGGACGATGGAGGG-3′ 661 - Table II.
Activation of reporter genes by enteroinvasive bacteria is inhibited by IκBα and IKKβ superrepressorsa
Luciferase Reporter Construct Superrepressor Stimulus Added S. dublin Y. enterocolitica E. coli O29:NM E. coli DH5α TNF-α IL-8 None 8.3 ± 1.9b 9.7 ± 1.5 5.8 ± 1.2 2.2 ± 0.8 9.5 ± 1.4 IκBα-AA 1.7 ± 0.4 1.5 ± 0.3 2.3 ± 0.8 1.4 ± 0.7 0.9 ± 0.2 IKKβ-AA 1.2 ± 0.3 1.3 ± 0.9 1.3 ± 0.9 1.1 ± 0.8 0.9 ± 0.3 ICAM-1 None 1.8 ± 0.1 2.1 ± 0.2 ND ND 2.1 ± 0.3 IκBα-AA 0.8 ± 0.2 0.7 ± 0.1 ND ND 0.6 ± 0.1 IKKβ-AA 0.7 ± 0.1 0.7 ± 0.1 ND ND 0.5 ± 0.1 2XNF-κB None 2.2 ± 0.4 3.8 ± 0.8 2.2 ± 0.2 1.5 ± 0.3 2.6 ± 0.4 IκBα-AA 0.7 ± 0.2 0.5 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.3 ± 0.1 IKKβ-AA 0.7 ± 0.2 1.6 ± 0.3 0.9 ± 0.2 0.9 ± 0.2 1.0 ± 0.3 β-Actin None 0.9 ± 0.1 1.1 ± 0.2 1.1 ± 0.1 1.2 ± 0.1 1.0 ± 0.2 -
a HT-29 cells were transfected with IL-8, ICAM-1, or NF-κB-luciferase transcriptional reporters together with IκBα-AA or IKKβ-AA expression vectors or a vector control (none), as indicated. From 30 to 36 h later, cells were infected with the indicated bacteria or stimulated with TNF-α for 8 h.
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b Data are the mean fold induction in luciferase activity relative to uninfected or unstimulated controls. Values are the mean ± SEM of three or more experiments.
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- Table III.
Activity of IL-8 transcriptional reporter deletion mutants in HT-29 cells infected with enteroinvasive bacteria or stimulated with TNF-αa
IL-8-CAT Construct (bp) S. dublin Infected Y. enterocolitica Infected TNF-α Stimulated −1481 4.9 ± 1.8b 7.1 ± 2.1 9.0 ± 3.0 −546 3.4 ± 1.0 4.9 ± 1.3 5.5 ± 1.4 −272 3.4 ± 0.7 3.4 ± 0.8 4.8 ± 1.8 −98 8.6 ± 1.5 10.7 ± 1.9 14.3 ± 5.5 −8 0.6 ± 0.1 0.9 ± 0.2 0.6 ± 0.1 -
a HT-29 cells were transiently transfected with the various 5′-deletion mutant IL-8-CAT transcriptional reporters, as indicated, and infected with S. dublin or Y. enterocolitica or stimulated with TNF-α (20 ng/ml) as described in Materials and Methods.
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b CAT activity is given as fold increase over unstimulated cells. Data are means ± SEM of three or more experiments.
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- Table IV.
Ad5IκB-AA superrepressor inhibits IL-8 and GROα secretion by S. dublin-infected or TNF-α-stimulated HT-29 cellsa
Additions to Culture IL-8 (ng/ml) GROα (ng/ml) Ad5IκB-AA Ad5LacZ Ad5IκB-AA Ad12LacZ None 1.2 ± 0.7 1.2 ± 0.7 0.7 ± 0.1 1.1 ± 0.1 S. dublin 8.6 ± 2.5 89.6 ± 1.6 1.3 ± 0.1 11.6 ± 0.5 TNF-α 3.0 ± 1.8 143.0 ± 20.3 1.0 ± 0.1 36.6 ± 0.3 -
a Monolayers of HT-29 cells were infected with Ad5IκB-AA or Ad5LacZ as indicated. Twenty-four hours later, cells were infected with S. dublin (5 × 108 bacteria/well) or stimulated with TNF-α (20 ng/ml) as described in Materials and Methods. Culture supernatants were collected 10 h later, and cytokine concentrations were determined by ELISA. Results are the mean ± SEM of triplicate cultures from a single experiment. Similar results were obtained in two additional experiments.
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- Table V.
Activation of IL-8 luciferase reporter gene by enteroinvasive bacteria is inhibited by NIK, TRAF2, and TRAF5 superrepressorsa
Superrepressorsc Stimulus Added S. dublin Y. enterocolitica E. coli O29:NM TNF-α None 7.44 ± 2.32b 9.23 ± 1.1 6.35 ± 1.07 6.69 ± 0.64 NIK 2.85 ± 0.02 5.99 ± 0.30 2.49 ± 0.93 4.59 ± 0.28 TRAF2 2.46 ± 0.29 2.87 ± 0.47 1.63 ± 0.59 1.72 ± 0.27 TRAF5 2.69 ± 0.28 4.39 ± 0.25 1.19 ± 0.03 3.69 ± 0.65 -
a HT-29 cells were transfected with an IL-8-luciferase transcriptional reporter together with an expression vector for a catalytic mutant of NIK, or expression vectors for superrepressors of TRAF2 or TRAF5 or a vector control (none), as indicated. From 30 to 36 h later, cells were infected with the indicated bacteria or stimulated with TNF-α for 8 h.
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b Data are the mean fold induction in luciferase activity relative to uninfected or unstimulated cells. Values are the mean ± SEM of data from three experiments.
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c In a separate experiment, expression of A20 was shown to inhibit both bacteria and TNF-α-induced activation of the IL-8 reporter. Mean fold induction in luciferase activity for S. dublin, Y. enterocolitica, E. coli O29:NM, and TNF-α-stimulated cells was 1.51 ± 0.08, 1.97 ± 0.12, 1.42 ± 0.01, and 1.08 ± 0.01, respectively.
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