Volume 128, Issue 5, Pages 1258-1267 (May 2005) Interferon γ Induces Translocation of Commensal Escherichia coli Across Gut Epithelial Cells via a Lipid Raft--Mediated Process  Edwin Clark, Catherine Hoare, Jolanta Tanianis-Hughes, Gordon L. Carlson, Geoffrey Warhurst  Gastroenterology  Volume 128, Issue 5, Pages 1258-1267 (May 2005) DOI: 10.1053/j.gastro.2005.01.046 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Differential effects of IFN-γ on paracellular permeability and translocation of Escherichia coli C25 translocation in T84 monolayers. T84 cells were treated with the indicated concentrations of IFN-γ added basolaterally for 48 hours, followed by measurement of TEER (•), apical to basolateral permeability of LY (▵) (A), and apical to basolateral translocation of E coli C25 measured over a 4-hour period as described in Materials and Methods (B). Statistical analysis was performed by using Student t tests with significance indicated as follows: *P < .05; **P < .01. Data are the mean ± SD for 6 monolayers in each group. Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Dose dependence of IFN-γ effects on the tight junction proteins claudin 1 and occludin in T84 cells. Monolayers were exposed to 0, 1, or 100 IU/mL IFN-γ on the basolateral surface for 48 hours. At the end of this period, cells were harvested, and membranes were processed for immunoblotting as indicated in Materials and Methods. The figure shows the levels of the indicated protein in the detergent-soluble (S) and -insoluble (I) membrane fractions. Images are representative of those from 3 separate experiments. Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 IFN-γ stimulates C25 translocation in the absence of changes in paracellular permeability and in Caco-2 cells. Caco-2 monolayers were exposed to the indicated concentrations of IFN-γ on the basolateral surface for 48 hours, followed by measurement of TEER (•) and C25 translocation (■). Data show the mean ± SD for 6 monolayers in each group. *P < .05; **P < .01 Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Lack of effect of IFN-γ on tight junction proteins in Caco-2 cells. Monolayers were incubated for 48 hours in the absence or presence of 0–100 IU/mL IFN-γ on the basolateral surface. (A) Immunoblot for claudin 1 or occludin in detergent-soluble (S) and -insoluble (I) membrane fractions isolated from untreated or 100 IU/mL IFN-γ–treated cells is representative of 3 separate experiments. (B) Confocal analysis of untreated and IFN-γ–treated cells (1, 10, and 100 IU/mL) stained for occludin and processed for immunofluorescence as described in Materials and Methods. Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 IFN-γ increases the internalization of Escherichia coli C25 into Caco-2 cells. After treatment with 100 IU/mL basolateral IFN-γ, Caco-2 cell monolayers were incubated on the apical surface with 108 CFU/mL C25. After 4 hours, the numbers of bacteria associated with or internalized in the monolayers were assessed by using the gentamicin resistance assay described in Materials and Methods. Data are mean ± SD for 4 monolayers in each group. **P < .01. NS, not significant. Con, control. Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Inhibition of C25 translocation across Caco-2 monolayers by methyl-β-cyclodextrin (MCD) and filipin. After treatment for 48 hours with 100 IU/mL IFN-γ, monolayers were placed into HBSS and incubated on both surfaces with MCD (1–100 mg/mL; top) or filipin (0.1–10 μg/mL; bottom) for 15 minutes before the addition of C25 and measurement of translocation. The bars represent the level of C25 translocation in control monolayers and monolayers incubated with IFN-γ alone. Data are mean ± SD for 4 monolayers in each group. *P < .05; **P <.01. Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 IFN-γ treatment increases the levels of ganglioside GM1 in Caco-2 cells. The figure shows dot blot analysis of Caco-2 cell lysates (2 and 5 μg of protein) either from untreated cells or after exposure to IFN-γ (100 IU/mL; 48 hours). Blots were incubated with an HRP conjugate of the B subunit of cholera toxin to label GM1 as described in Materials and Methods. Lysates from 2 separate control and IFN-treated cultures are shown. Similar data were observed in a further 2 experiments. Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Filipin does not prevent apical to basolateral translocation of Shigella sonnei or its effects on barrier function in Caco-2 monolayers. Monolayers were exposed on the apical surface to HBSS alone or HBSS containing S sonnei (108 CFU/mL) for 4 hours, either with or without pretreatment for 15 minutes with 10 μg/mL filipin (Fil). After 4 hours, monolayer TEER and the number of S sonnei in the basolateral medium were measured. For comparison, data showing the effects of this concentration of filipin on barrier properties and C25 translocation in IFN-γ–treated monolayers (100 IU/mL) are also shown. Data are mean ± SD for 4 monolayers in each group. **P < .01. Gastroenterology 2005 128, 1258-1267DOI: (10.1053/j.gastro.2005.01.046) Copyright © 2005 American Gastroenterological Association Terms and Conditions