The GS-nitroxide JP4-039 improves intestinal barrier and stem cell recovery in irradiated mice

Total body irradiation (TBI) leads to dose- and tissue-specific lethality. In the current study, we demonstrate that a mitochondrion-targeted nitroxide JP4-039 given once 24 hours after 9–10 Gy TBI significantly improves mouse survival, and the recovery of intestinal barrier, differentiation and stem cell functions. The GI-protective effects are associated with rapid and selective induction of tight junction proteins and cytokines including TGF-β, IL-10, IL-17a, IL-22 and Notch signaling long before bone marrow depletion. However, no change was observed in crypt death or the expression of prototypic pro-inflammatory cytokines such as TNF-α, IL-6 or IL-1β. Surprisingly, bone marrow transplantation (BMT) performed 24 hours after TBI improves intestinal barrier and stem cell recovery with induction of IL-10, IL-17a, IL-22, and Notch signaling. Further, BMT-rescued TBI survivors display increased intestinal permeability, impaired ISC function and proliferation, but not obvious intestinal inflammation or increased epithelial death. These findings identify intestinal epithelium as a novel target of radiation mitigation, and potential strategies to enhance ISC recovery and regeneration after accidental or medical exposures.


Mice and Irradiation
JP4-039 was synthesized and formulated in F14 emulsion as described 1, 2 , and administered I.V.
to deliver a dose of 20 mg/kg (approximately 400 µg/mouse) 24 h after TBI. In brief, JP4-039 (4 mg/ml) + 10% Sesame Oil + 5% Soy Phosphatidyl Choline + 85% Dulbecco's Phosphate-Buffered Saline was placed in ice water with a stream of nitrogen blowing over top. The preparation was sonicated for 1-2 h, and then injected into mice intravenously, at 100 μl containing 400 μg drug or no drug (control) per mouse.
Bone marrow transplant was performed as described 3 . In brief, 7-9 week-old female C57BL/6 mice (The Jackson Laboratory) received 10 Gy total body irradiation the day before receiving transplantation of 1 X 10 6 whole marrow cells from 7-9 week-old male C57BL/6 donor mice.
Following transplantation, mice were allowed 8 weeks for recovery and engraftment of transplanted bone marrow. Sex-and age-matched mice without radiation and BMT were used as control for tissue analysis. C57BL/6NTac mice received 9.25 Gy TBI before BMT similarly to match JP4-039 studies.

TUNEL and BrdU staining
TUNEL (Terminal deoxynucleotidyl transferase dUTP nick-end labelling) staining was performed with the ApopTag In Situ Apoptosis Detection Kit (Millipore, Billerica, MA) according to the manufacturer's instructions.

Immunohistochemistry (IHC) and immunofluorescence (IF)
Mucin2 IF. 20% goat serum was used as blocking solution at room temperature for 1 hour.
ZO-1 IF. 20% goat serum was used as blocking solution at room temperature for 1 hour.
CD3 IHC. 20% goat serum was used as blocking solution at room temperature for 1 hour.

Gene
Primer Sequence Function        Quantification of crypt numbers, crypt depth, and villus length. Values are Mean ± SEM; n = 3 mice in each group. ++ P < 0.01, 1-way ANOVA followed by Tukey's multiple comparisons test.

Supplemental Figure legends
(C) Mucosal mRNA expression of indicated markers. cDNA was synthesized from RNA pooled from 3 mice/group. Expression was normalized to that on Day 0, prior to TBI. *P < 0.05, **P < 0.001, R vs. IR/BMT, unpaired 2-tailed Student's t test.