Figures & data
Figure 1. C. innocuum induces nucleotide-binding oligomerization domain containing 2 (NOD2) and receptor-interacting protein 2 (RIP2) expression. HT-29 cells were infected with C. innocuum at (A) multiplicity of infection (MOI) of 100 for 0–4 h, and (B) MOI of 0–100 for 4 h. Expression levels of NOD2 and RIP2 were determined using western blotting. The protein expression levels of NOD2 and RIP2 were quantified by densitometric analysis and normalized to β-actin level, respectively, and indicated at the bottom of each lane.
![Figure 1. C. innocuum induces nucleotide-binding oligomerization domain containing 2 (NOD2) and receptor-interacting protein 2 (RIP2) expression. HT-29 cells were infected with C. innocuum at (A) multiplicity of infection (MOI) of 100 for 0–4 h, and (B) MOI of 0–100 for 4 h. Expression levels of NOD2 and RIP2 were determined using western blotting. The protein expression levels of NOD2 and RIP2 were quantified by densitometric analysis and normalized to β-actin level, respectively, and indicated at the bottom of each lane.](/cms/asset/8e49cecd-9cd7-47ee-99ed-760c0f450380/kvir_a_2265048_f0001_b.gif)
Figure 2. C. innocuum recruits NOD2 to membrane rafts. HT-29 cells were infected with C. innocuum at MOI of 100 for 4 h. Cells were probed with (A and E) Hoechst 33,342 (blue), (B and F) anti-NOD2 (green), and (C and G) CTx-B (red), followed by performing confocal microscopy analysis. (E) arrowheads indicate the attached bacteria on the cell membrane. (D and H) yellow arrows indicate the area of confocal z-section analysis. Bars, 5 μm. White lines across the membrane shown in z-section indicate the distribution of fluorescence signals for C. innocuum (blue line), NOD2 (green line), and CTx-B (red line), which present as line intensity histograms in the right panels.
![Figure 2. C. innocuum recruits NOD2 to membrane rafts. HT-29 cells were infected with C. innocuum at MOI of 100 for 4 h. Cells were probed with (A and E) Hoechst 33,342 (blue), (B and F) anti-NOD2 (green), and (C and G) CTx-B (red), followed by performing confocal microscopy analysis. (E) arrowheads indicate the attached bacteria on the cell membrane. (D and H) yellow arrows indicate the area of confocal z-section analysis. Bars, 5 μm. White lines across the membrane shown in z-section indicate the distribution of fluorescence signals for C. innocuum (blue line), NOD2 (green line), and CTx-B (red line), which present as line intensity histograms in the right panels.](/cms/asset/aa828f29-87f0-465d-bc01-477e75bcc8ef/kvir_a_2265048_f0002_oc.jpg)
Figure 3. Live C. innocuum is essential for inducing cell death. HT-29 cells were treated with live C. innocuum, bacterial culture supernatant, heat-killed bacteria, and bacterial lysate for 24 h, followed by the (A) analysis of cell survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, (B) stained with annexin V/propidium iodide, and analyzed using flow cytometry. (C) percentage of apoptotic cells was quantitated. *, P < 0.05.
![Figure 3. Live C. innocuum is essential for inducing cell death. HT-29 cells were treated with live C. innocuum, bacterial culture supernatant, heat-killed bacteria, and bacterial lysate for 24 h, followed by the (A) analysis of cell survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, (B) stained with annexin V/propidium iodide, and analyzed using flow cytometry. (C) percentage of apoptotic cells was quantitated. *, P < 0.05.](/cms/asset/73b833ae-51a3-41a6-8283-939a98cb7c8e/kvir_a_2265048_f0003_oc.jpg)
Figure 4. Inhibition of cholesterol synthesis reduces C. innocuum-induced NOD2 expression in lipid rafts. HT-29 cells were pretreated with simvastatin (100 μM) for 1 h prior to C. innocuum infection. Cells were probed with Hoechst 33,342 (blue), anti-NOD2 (green), and CTx-B (red), followed by confocal microscopy analysis. Bars, 5 μm.
![Figure 4. Inhibition of cholesterol synthesis reduces C. innocuum-induced NOD2 expression in lipid rafts. HT-29 cells were pretreated with simvastatin (100 μM) for 1 h prior to C. innocuum infection. Cells were probed with Hoechst 33,342 (blue), anti-NOD2 (green), and CTx-B (red), followed by confocal microscopy analysis. Bars, 5 μm.](/cms/asset/9d822b09-f0bc-44e3-9fba-0b191ed8b831/kvir_a_2265048_f0004_oc.jpg)
Figure 5. Simvastatin attenuates C. innocuum-induced apoptosis. (A) HT-29 cells were pretreated with simvastatin or pretreated with simvastatin then replenished with cholesterol (400 μg/mL). Cells were infected with C. innocuum for 24 h and stained with annexin V/propidium iodide, followed by flow cytometry analysis. (B) percentage of apoptotic cells was quantitated. (C) HT-29 cells were co-transfected with the nuclear factor (NF-κB) and pGL3 luciferase reporters and treated with simvastatin (100 μM) for 1 h, followed by C. innocuum infection for 24 h. NF-κB promoter activity was determined and normalized to pGL3 luciferase activity. (D) IL-8 production was analyzed by using ELISA. *, P < 0.05.
![Figure 5. Simvastatin attenuates C. innocuum-induced apoptosis. (A) HT-29 cells were pretreated with simvastatin or pretreated with simvastatin then replenished with cholesterol (400 μg/mL). Cells were infected with C. innocuum for 24 h and stained with annexin V/propidium iodide, followed by flow cytometry analysis. (B) percentage of apoptotic cells was quantitated. (C) HT-29 cells were co-transfected with the nuclear factor (NF-κB) and pGL3 luciferase reporters and treated with simvastatin (100 μM) for 1 h, followed by C. innocuum infection for 24 h. NF-κB promoter activity was determined and normalized to pGL3 luciferase activity. (D) IL-8 production was analyzed by using ELISA. *, P < 0.05.](/cms/asset/5e3eb8f5-f434-4b49-8dde-f7f0b9086f6c/kvir_a_2265048_f0005_oc.jpg)
Figure 6. Hypothesized model illustrates C. innocuum-induced pathogenicity of intestinal epithelial cells. C. innocuum infection coalesces lipid rafts on the cell membrane, which then activates NOD2 pathway to promote NF-κB translocation in the nucleus, resulting in exacerbates cytotoxicity and inflammation of intestinal epithelial cells.
![Figure 6. Hypothesized model illustrates C. innocuum-induced pathogenicity of intestinal epithelial cells. C. innocuum infection coalesces lipid rafts on the cell membrane, which then activates NOD2 pathway to promote NF-κB translocation in the nucleus, resulting in exacerbates cytotoxicity and inflammation of intestinal epithelial cells.](/cms/asset/1249b8e8-1d10-417f-9b39-239cf971106b/kvir_a_2265048_f0006_oc.jpg)
Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article.