Volume 126, Issue 5, Pages 1358-1373 (May 2004) DNA from probiotic bacteria modulates murine and human epithelial and immune function  Humberto Jijon, Jody Backer, Hugo Diaz, Helen Yeung, David Thiel, Conor McKaigney, Claudio De Simone, Karen Madsen  Gastroenterology  Volume 126, Issue 5, Pages 1358-1373 (May 2004) DOI: 10.1053/j.gastro.2004.02.003

Figure 1 Representative agarose gel of isolated DNA. DNA was isolated from bacteria as detailed in Materials and Methods. DNA was incubated with 5 μg/mL DNase I overnight at 37°C in the presence of 5 mmol/L MgCl2. The average band length ranged from 100 to 500 base pairs. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 2 Effect of bacterial and calf thymus DNA on IL-8 secretion from HT-29 monolayers. Bacterial DNA (50 μg/mL) was placed on the apical surface of HT-29 cells and IL-8 measured in the supernatant after 6 hours. HT-29 cells secreted IL-8 in response to (B) E. coli, (C) S. dublin, and (D) S. typhimurium. Monolayers did not respond to DNA from (A) E. faecalis, (E) VSL3, or (F) calf thymus DNA. Treatment with DNase abolished the response. ∗P < 0.05 compared with control. Experiments were performed twice with n = 3 at each concentration. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 3 Effect of DNA from individual bacterial strains found in VSL3 on IL-8 secretion from HT-29 monolayers. Bacterial DNA (50 μg/mL) was placed on the apical surface of HT-29 cells and IL-8 measured in the supernatant after 6 hours. L. delbrueckii subsp. bulgaricus MB 453 stimulated IL-8 secretion, whereas B. longum, B. infantis, and B. breve significantly inhibited basal IL-8 secretion. Treatment with DNase abolished these responses. Experiments were performed twice with n = 3 at each concentration. ∗P < 0.05 compared with control. La, L. acidophilus MB 443; LdB, L. delbrueckii subsp. bulgaricus MB 453; Lc, L. casei MB 451; Lp, L. plantarum MB 452; Bl, B. longum Y10; Bi, B. infantis Y1; Bb, B. breve Y8; St, S. salivarius subsp. thermophilus MB 455. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 4 Representative agarose gels of IL-8 mRNA. Salmonella DNA (sd) (50 μg/mL) and TNF-α (10 ng/mL) but not VSL3 DNA (50 μg/mL) increased levels of IL-8 mRNA in HT-29 cells. VSL3 DNA inhibited IL-8 mRNA production in response to Salmonella DNA but not TNF-α. (A) Agarose gel. (B) Quantification by comparing the IL-8 band with that of β-actin reverse-transcription polymerase chain reaction product from the same multiplex reaction. ∗P < 0.05 compared with control. Experiments were conducted twice with measurements performed in triplicate. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 5 HT-29 cells were either pretreated with VSL3 DNA at concentrations of 10, 25, or 50 μg/mL for 2 hours and then exposed to S. typhimurium DNA (50 μg/mL) for 2 hours or coincubated with VSL3 DNA (25 μg/mL) and S. typhimurium DNA (25 μg/mL) for 2 hours. VSL3 DNA inhibited an S. typhimurium–induced release of IL-8 in a dose-dependent fashion. ∗P < 0.05 compared with control; +P < 0.05 compared with S. typhimurium DNA alone. Experiments were performed twice with n = 3 at each concentration. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 6 HT-29 cells responded to live S. typhimurium (107 CFU/mL) and to TNF-α (10 ng/mL) with release of IL-8. Pretreatment of cells with VSL3 DNA (50 μg/mL) for 2 hours significantly inhibited both S. typhimurium– and TNF-α–induced release of IL-8. In contrast, pretreatment with S. typhimurium DNA before treatment with TNF-α resulted in an additive effect on IL-8 release. ∗P < 0.05 compared with control; #P < 0.05 compared with TNF-α alone; ∗∗P < 0.02 compared with TNF-α alone; +P < 0.02 compared with S. typhimurium alone. Experiments were performed twice with n = 3 for each condition. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 7 Effect of DNA from individual bacterial strains found in VSL3 on IL-8 secretion from HT-29 monolayers in the presence of TNF-α. HT-29 cells were treated with bacterial DNA (50 μg/mL) for 2 hours and then stimulated with TNF-α (10 mg/mL) for 30 minutes. IL-8 release into the supernatant was measured after 6 hours. VSL3 DNA, S. salivarius, B. infantis, B. breve, and B. longum strains significantly inhibited IL-8 secretion in response to TNF-α. L. delbrueckii subsp. bulgaricus had an additive effect with TNF-α. Treatment with DNase abolished these responses. ∗P < 0.05 compared with control. Experiments were performed twice with n = 3 at each concentration. La, L. acidophilus MB 443; LdB, L. delbrueckii subsp. bulgaricus MB 453; Lc, L. casei MB 451; Lp, L. plantarum MB 452; Bl, B. longum Y10; Bi, B. infantis Y1; Bb, B. breve Y8; St, S. salivarius subsp. thermophilus MB 455. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 8 HT-29 cells were treated with bacterial DNA (50 μg/mL) for 2 hours and then stimulated with TNF-α (10 mg/mL). (A) TNF-α induced a rapid activation of NF-κB as measured by electrophoretic mobility shift assay, (B) which correlated with the disappearance of IκB and the phosphorylation of p38. (B) Pretreatment with VSL3 DNA resulted in a delay in NF-κB activation in response to TNF-α (15-minute time points), increased levels of IκB protein at time 0 and reduced levels of degradation (30 and 45 minutes), and reduced the degree of phosphorylation of p38. (C) A dose-dependent increase in TNF-α–induced luciferase induction following 24-hour incubation. (D) VSL3 DNA alone had no effect on luciferase, whereas TNF-α induced a 6-fold increase. VSL3 DNA had no effect on luciferase induction in the presence of TNF-α. Experiments were repeated twice and even loading confirmed by protein assays and Ponceau S staining (Western blotting only). Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 9 VSL3 DNA inhibits proteasome activity. (A) HT-29 cells were treated with VSL or S. dublin DNA (50 μg/mL) for 2 hours and proteasome activity measured as detailed in Materials and Methods. VSL3 DNA, but not Salmonella DNA, inhibited proteasomal activity. (B) A dose-dependent inhibition by VSL3 DNA as assessed at 60 minutes. Experiments were repeated twice with triplicate measurements. ∗P < 0.05 compared with control. Values are means ± SEM. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 10 Effect of bacterial DNA on IFN-γ secretion in colonic tissue. Colons from wild-type (n = 4) and IL-10–deficient mice (n = 6) were incubated with VSL3 (50 μg/mL) or Salmonella DNA (50 μg/mL). Wild-type mice responded to DNA from S. typhimurium with a significant increase in IFN-γ secretion. The addition of VSL3 DNA inhibited this response. Colons from IL-10–deficient mice secreted significantly higher levels of IFN-γ compared with colons from wild-type mice (P < 0.05). In contrast to the response in wild-type mice, the treatment of colonic tissue from IL-10–deficient mice with VSL3 DNA (50 μg/mL) resulted in a significant inhibition of IFN-γ secretion both in the presence and absence of S. typhimurium DNA. S. typhimurium DNA alone had no significant effect on IFN-γ secretion in IL-10–deficient mice. Values are means ± SEM. ∗P < 0.05 compared with basal secretion; +P < 0.05 compared with S. typhimurium DNA. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)

Figure 11 IFN-γ release from spleen cells isolated from wild-type and IL-10–deficient mice. Spleen cells were isolated from adult mice and cultured for 48 hours in media alone or with DNA (50 μg/mL) from VSL3, E. coli, or calf thymus in the (A) absence or (B) presence of microbial sonicates derived from B. vulgatus. Spleen cells were stimulated with plate-bound anti-CD3 as a positive control. In the absence of bacterial sonicates, spleen cells from wild-type mice responded to E. coli DNA, but not to VSL or calf thymus DNA, while spleen cells from IL-10–deficient mice also responded to VSL3 DNA, albeit to a smaller degree than E. coli DNA. In the presence of B. vulgatus sonicates, the addition of E. coli DNA to both wild-type and IL-10 splenocytes resulted in enhanced IFN-γ secretion (B). In contrast, VSL3 DNA attenuated IFN-γ secretion in response to B. vulgatus sonicates. Values are means ± SEM from triplicate samples for animals (n = 6–8) in each group. ∗P < 0.05 compared with basal; +P < 0.05 compared with VSL3. aP < 0.05 compared with wild-type B. vulgatus alone; bP < 0.05 compared with wild-type and VSL3; cP < 0.05 compared with IL-10–deficient B. vulgatus alone; dP < 0.05 compared with IL-10 deficient and VSL3. Gastroenterology 2004 126, 1358-1373DOI: (10.1053/j.gastro.2004.02.003)