Volume 122, Issue 4, Pages 1048-1057 (April 2002) Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation  Dan He, Stavros Sougioultzis, Susan Hagen, Jennifer Liu, Sarah Keates, Andrew C. Keates, Charalabos Pothoulakis, J.Thomas LaMont  Gastroenterology  Volume 122, Issue 4, Pages 1048-1057 (April 2002) DOI: 10.1053/gast.2002.32386 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of BHA on toxin A–induced IL-8 production and ROI generation from HT-29 cells. (A) Subconfluent HT-29 monolayers were incubated at 37°C with either medium alone or medium containing toxin A (10 nmol/L) for the indicated time points. In some experiments, cells were first exposed to the antioxidant BHA (200 μmol/L) for 30 minutes at 37°C before addition of toxin A. Culture supernatants were collected at different time points, and IL-8 protein levels were measured by enzyme-linked immunosorbent assay (see Materials and Methods). Toxin A stimulated a significant IL-8 release after 8 hours, and pretreatment with BHA inhibited this response. Values are the mean ± SEM of a representative experiment or 3 similar experiments, each with triplicate determinations. *P < 0.036, ** P < 0.001 vs. controls. †P < 0.05, ††P < 0.001 vs. toxin A. (B) HT-29 cells were cotransfected with an IL-8 reporter construct and the control vector pRL-TK. The IL-8 reporter gene contains 1521 base pairs of the IL-8 promoter linked to the PGL2-basic firefly luciferase expression vector. pRL-TK constitutively expresses renilla luciferase and served as a marker of transfection efficiency. Cotransfected cells were stimulated with toxin A (10 nmol/L) or first exposed to BHA and then stimulated with toxin A as in A, and luciferase activity was measured at different time points. The results are presented as mean ± SEM of the ratio between firefly and renilla luciferase activities from 4 experiments, each with duplicate determinations. *P < 0.05, **P < 0.001. (C) HT-29 cells were loaded with DHR 123, a fluorescent probe for ROIs, and then exposed to toxin A (10 nmol/L). Some cells were first exposed to BHA or BHT (200 μmol/L), an antioxidant, before addition of toxin A. At the indicated time points, cells were examined by cytofluorometry for estimation of ROIs. Toxin A stimulated increased ROI release, and this increase was inhibited by antioxidant pretreatment at 30 minutes. Gastroenterology 2002 122, 1048-1057DOI: (10.1053/gast.2002.32386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of BHA on toxin A–induced NF-κB activation. (A) HT-29 cells grown on cover slides were exposed to 10 nmol/L of toxin A. Cells were then fixed, permeabilized, and incubated with polyclonal antibodies directed against the p65 subunit of NF-κB. Cells were then incubated with FITC-labeled anti-rabbit IgG and examined with a confocal microscope. Nuclear translocation of NF-κB began 30 minutes after exposure to toxin A; by 3 hours, most of the nuclei were positively stained (arrows). Results shown in A and B are typical of 3 separate experiments. (B) Confluent HT-29 cell monolayers were either exposed to toxin A (10 nmol/L) or first exposed to BHA (200 μmol/L) for 30 minutes at 37°C before addition of toxin A. At the indicated time points, nuclear extracts were prepared and analyzed by electrophoretic mobility shift assay using a probe containing a consensus NF-κB binding element (left panel). Nuclear extracts from HT-29 cells exposed for 30 minutes to toxin A were incubated with either 100-fold excess of the specific unlabeled consensus NF-κB oligonucleotide (competitor) or antibodies against the NF-κB subunits p50, p52, and p65 (right panel). Toxin A increased NF-κB DNA binding after 30 minutes, and elevated binding was observed for up to 2 hours (Figure 2B). Supershift assays indicate that the toxin A–activated NF-κB complexes contain p50/p65 heterodimers and p50/p50 homodimers. Preincubation of HT-29 cells with the antioxidant BHA before exposure to toxin A resulted in diminished NF-κB DNA binding compared with toxin A alone. Gastroenterology 2002 122, 1048-1057DOI: (10.1053/gast.2002.32386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Toxin A causes degradation of IκB in HT-29 cells and human colonic mucosa. (A) HT-29 cells were treated with 10 nmol/L toxin A, and whole-cell extracts were prepared at the indicated times. Proteins were separated in 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred to membranes, and blotted with an antibody directed against IκB. IκB was markedly reduced 30 minutes after exposure to toxin A and absent after 60 and 120 minutes. Data shown in A and B are representative of 3 independent experiments. (B) Normal human colonic mucosal sheets were exposed in Ussing chambers to 10 nmol/L toxin A for 15 and 30 minutes. Tissues were then fixed, stained with anti-IκBα antibody, and examined under a fluorescence microscope (see Materials and Methods). Intense cytoplasmic staining is evident in untreated colonic mucosa (0). Staining is diminished after 15 minutes and nearly absent after 30 minutes of exposure to toxin A. Gastroenterology 2002 122, 1048-1057DOI: (10.1053/gast.2002.32386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Electron microscopy of (A) control and (B) EB-treated HT-29 cells. HT-29 cells were incubated in buffer or in 10 μg/mL EB for 28 days and then examined by electron microscopy. (A) In control cells, the cytoplasm of HT-29 cells contained typical mitochondria (M) ranging from small to large that were electron dense with many cristae. There were opaque areas in the center of some (normal) mitochondria with no matrix or cristae. (B) Most cells treated with EB had no structures that were identifiable as mitochondria. In a small number of cells, there were few structures that had discernable cristae and were identifiable as mitochondria (M). The cytoplasm of HT-29 cells treated with EB also had numerous lysosomes (L). (Original magnification 11,183×; bar = 2 μm.) Gastroenterology 2002 122, 1048-1057DOI: (10.1053/gast.2002.32386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 ATP, ROIs, and IL-8 production is decreased in mtDNA-deficient HT-29 cells treated with toxin A. (A) Wild-type and mtDNA-deficient HT-29 cells (generated by treatment with EB as described in Materials and Methods) were challenged with 10 nmol/L toxin A for 30 minutes, and ATP was measured by the bioluminescence technique (see ATP Assay section). Production of ATP by EB-treated cells is markedly diminished. *P < 0.04. (B) Wild-type and EB-treated HT-29 cells were exposed to 10 nmol/L toxin A or IL-1β for 24 hours. IL-8 was measured in the supernatants by enzyme-linked immunosorbent assay as described in Materials and Methods. The values are the mean ± SEM of a representative experiment, each with triplicate determinations. Toxin A–induced IL-8 production by EB-treated HT-29 cells is decreased compared with wild-type HT-29 cells. In contrast, no difference in IL-8 release by wild-type or EB-treated HT-29 cells was observed when IL-1β was used as a stimulus. Gastroenterology 2002 122, 1048-1057DOI: (10.1053/gast.2002.32386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Diminished toxin A–induced NF-κB nuclear translocation in mtDNA-deficient HT-29 cells. EB-treated HT-29 cells grown on cover slides were exposed to 10 nmol/L toxin A for 30 minutes and 3 hours. Cells were then fixed, permeabilized, and incubated with a polyclonal antibody directed against the p65 subunit of NF-κB. Cells were then incubated with FITC-labeled anti-rabbit IgG and examined with a confocal microscope. Nuclear staining for NF-κB is diminished at both time points compared with wild-type HT-29 cells (Figure 2A). Results shown are representative of 3 experiments. Gastroenterology 2002 122, 1048-1057DOI: (10.1053/gast.2002.32386) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Rho glucosylation by toxin A. There was no change in Rho glucosylation by toxin A (10 nmol/L) from 0 to 120 minutes. Rho glucosylation by toxin A HT-29 cells was measured indirectly by inhibition of C. botulinum C3 exoenzyme adenosine diphosphate ribosylation as described in Materials and Methods. No change was observed over 2 hours of observation. Gastroenterology 2002 122, 1048-1057DOI: (10.1053/gast.2002.32386) Copyright © 2002 American Gastroenterological Association Terms and Conditions