Article Figures & Data
Figures
- Figure 1.
Improved survival of mice treated with total body irradiation (TBI) and Lactobacillus reuteri-IL-22 (n=10). Groups of 10 mice received 9.25 Gy TBI then 24 h later gavage of 100 μl of saline containing 109 Lactobacillus reuteri-interleukin (IL-22), or IL-22 protein delivered intraperitoneally at 20 mg/kg in 100 μl, or 100 μl cyclodextrin containing 20 mg/kg of the radiation mitigator JP4-039. Significant increase in survival was seen in irradiated mice treated with JP4-039 (p=0.0079), IL-22 protein (p=0.0428) or Lactobacillus-IL-22 (p=0.0014) but not control Lactobacillus (p=0.5021) compared to control irradiated mice.
- Figure 2.
Lactobacillus reuteri-interleukin (IL-22) gavage at 24 h after total body irradiation (TBI) rescues and preserves critical leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5+) cells in ileum of Lgr5+ green fluorescent protein (GFP)+ mice at day 7. A: Groups of 10 mice received 9.25 Gy TBI, then 24 h later gavage of 100 μl of saline containing 109 Lactobacillus reuteri-IL-22. At day 7, mice were sacrificed, ileum removed and fixed, as described in prior studies (5, 6), then 20 cross-sections of ileum were scored for number of Lgr5+ GFP+ intestinal stem cells. Results are the mean ± SEM. *Significantly different at p=0.0357. B: Photographs of Lgr5+ cells in ileum from control irradiated mice and irradiated mice treated with Lactobacillus-IL-22. Original magnification, ×1000.
- Figure 3.
Significant mitigation of 9.25 Gy total body irradiation (TBI) delivered to C57BL/6 female mice by gavage of Lactobacillus reuteri-interleukin-22-green fluorescent protein fusion protein (IL-22-GFP+) (109 bacteria in 100 μl saline) administered at 24, 48, or 72 h after TBI (n=12 animals per group). Compared to control irradiated mice, mice gavaged with Lactobacillus-IL-12-GFP at 24, 48 or 72 h after irradiation had increased survival (p=0.0001, 0.0047 and 0.0448, respectively.
- Figure 4.
pRSET-EmGFP plasmid map used to transform Escherichia coli. The mouse interleukin (IL-22) construct was inserted into the vector between the BamHI and NcoI restriction sites (blue arrow) forming pRSET-IL-22-EmGFP.
- Figure 5.
Gene sequence of interleukin-22-green fluorescent protein (IL-22–GFP) fusion protein inserted into pRSET plasmid. IL-22–GFP fusion protein. Blue: Cytokine sequence; green: GFP sequence.
- Figure 6.
Gene expression for interleukin-22 (IL-22) in clones of Escherichia coli-IL-22, as demonstrated by polymerase chain reaction. Five representative clones were positive for IL-22. N.C.: Negative control.
- Figure 7.
Gavage of Escherichia coli-interleukin-22-green fluorescent protein (IL-22-GFP+) increases survival of mice irradiated to 9.25 Gy. C57BL/6NTac mice were irradiated to 9.25 Gy total body irradiation (TBI). At 24 hours after irradiation, mice were gavaged with100 μl saline containing 109 Escherichia coli-interleukin IL-22-green fluorescent protein (IL-22-GFP+), or control Escherichia coli. Other mice were given a fecal microbiome transplant (FMT) of 109 bacteria from 30-day survivors of 9.25 Gy TBI, IL-22 protein dissolved in phosphate-buffered saline administered as an intraperitoneal injection or radiation mitigator JP4-039 dissolved in 30% (2-hydroxypropyl)-β-cyclodextrin and injected intramuscularly. Compared to control irradiated mice, administration of E.coli-IL-22 (p=0.0004), JP4-039 (p=0.0195), or IL-22 protein (p=0.0428) increased survival. FMT, and control E. coli did not increase survival (p=0.6876 and 0.8851, respectively).
- Figure 8.
Green fluorescent protein (GFP) expression in Escherichia coli-interleukin-22-green fluorescent protein (IL-22-GFP+) cells. A: Control Escherichia coli. B: IL-22-secreting E. coli clone. Original magnification, ×1000.
- Figure 9.
Histological localization of green fluorescent protein (GFP) to the ileum at 4 h after gavage of 109 Escherichia coli-interleukin-22-(IL-22)-GFP) cells at 24 h after total body irradiation (TBI) in C57BL/6J mice. A: Cross section of ileum. B: Inset from A showing localization of E. coli-IL-22-GFP+ fusion protein to the epithelial surface of the ileum. White arrow: Epithelial break (absence of red actin staining); green arrows: bacteria in villi near macrophages.
- Figure 10.
Survival of C57BL/6 mice gavaged at 24 h after 9.25 Gy total body irradiation (TBI) with different numbers of Lactobacillus reuteri-IL-22 (red), or Escherichia coli-IL-22 (green) (n=10 animals per group). Significant increase in survival compared with control irradiation only at *p=0.0139, and #p=0.303.
- Figure 11.
Secretion of interleukin-22 (IL-22) by Escherichia coli cells transformed with plasmid pRSET containing the IL-22-green fluorescent protein transgene (pRSET-IL-22-GFP) plasmid. A: Level of IL-22 detected in culture medium collected at 48 h. B: Level of IL-22 in cell lysates from E. coli-IL-22 and Lactobacillus reuteri-IL-22. C: Biological activity of IL-22 produced by Lactobacillus-IL-22 using the induction of IL-10 in Colo205 cells. Significantly different at **p<0.01 versus E. coli-GFP; #p<0.05 versus Colo205; and ^p<0.01 versus control Lactobacillus.
