Effect of solar particle event radiation and hindlimb suspension on gastrointestinal tract bacterial translocation and immune activation

Abstract

The environmental conditions that could lead to an increased risk for the development of an infection during prolonged space flight include: microgravity, stress, radiation, disturbance of circadian rhythms, and altered nutritional intake. A large body of literature exists on the impairment of the immune system by space flight. With the advent of missions outside the Earth's magnetic field, the increased risk of adverse effects due to exposure to radiation from a solar particle event (SPE) needs to be considered. Using models of reduced gravity and SPE radiation, we identify that either 2 Gy of radiation or hindlimb suspension alone leads to activation of the innate immune system and the two together are synergistic. The mechanism for the transient systemic immune activation is a reduced ability of the GI tract to contain bacterial products. The identification of mechanisms responsible for immune dysfunction during extended space missions will allow the development of specific countermeasures.

Figure 1. SPE-like proton or reference gamma radiation and hindlimb suspension increase circulating LPS levels.

Hindlimb suspension and proton or gamma radiation cause a breakdown in containment of gram negative bacterial products as measured by circulating LPS. Mice were placed in hindlimb suspension or not and irradiated with 2 Gy of protons at 50 cGy/min (A) or 2 Gy of gamma radiation at 44 cGy/min (B) 2 days later or not. Six hours (A, B) after radiation and prior to and 1 and 4 days (B) post irradiation (2, 3, or 6 days post suspension), serum was analyzed for LPS content by Factor C assay. Data shown are the averages for five mice per group analyzed in duplicate; error bars are SEM. Statistically significant differences between groups is noted in the inset table (B is 4 days post) with C = control, R = radiation, S = suspension as calculated with ANOVA analyses with post-hoc group comparisons using Bonferroni correction. Data shown are from a single experiment and are representative of three experiments.

Figure 2. SPE-like radiation and hindlimb suspension increase the acute-phase reactant LPS binding protein.

Mice were hindlimb suspended and/or irradiated with 2 Gy protons at 50 cGy/min. One (A) and 4 (B) days later, serum was obtained and analyzed for LPS binding protein content by ELISA. Data shown are the averages for five mice per group run in duplicate; error bars are SEM. Statistically significant differences between groups is noted in the inset table with C = control, R = radiation, S = suspension as calculated with ANOVA analyses with post-hoc group comparisons using Bonferroni correction. Data shown are from a single experiment representative of three experiments.

Figure 3. SPE-like or reference gamma radiation and hindlimb suspension increase sCD14 levels.

Mice were hindlimb suspended and/or irradiated with 2 Gy of protons at 50 cGy/min (A) or gamma radiation at 44 cGy/min (B). One (A) and 4 (B) days later, serum was obtained and analyzed for sCD14 protein content by ELISA. Data shown are the averages for five mice per group run in duplicate; error bars are SEM. Statistically significant differences between groups is noted in the inset table with C = control, R = radiation, S = suspension as calculated with ANOVA analyses with post-hoc group comparisons using Bonferroni correction. Data shown are from a single experiment representative of three experiments.

Figure 4. Radiation and hindlimb suspension induce a transient increase in circulating proinflammatory cytokines.

Mice were irradiated with 2 Gy of gamma radiation at a dose rate of 44 cGy/min. Six hr later, serum was obtained and analyzed for IFN-α (A), IL-6 (B), and TNF-α (C) by ELISA. Data shown are the averages for 5 mice per group run in duplicate; error bars are SEM. Statistically significant differences between groups is noted in the inset table with C = control, R = radiation, S = suspension as calculated with ANOVA analyses with post-hoc group comparisons using Bonferroni correction. Data shown are from a single experiment representative of three experiments.

Figure 5. SPE-like radiation and hindlimb suspension induces breaks in the GI epithelial barrier.

Terminal ileum obtained 4 days post irradiation and/or 6 days post hindlimb suspension or from control animals was stained for Claudin-3. A) Ileum from a control treated mouse. B) Ileum from a mouse irradiated with 2 Gy of 70 MeV protons. Black arrow indicates a region of tight junction incongruity. C) Ileum from a mouse subjected to hindlimb suspension. D) Ileum from a mouse treated with hindlimb suspension and irradiated with 2 Gy of protons. Original magnifications 200×. E) Quantitation of Claudin-3 breaks in terminal ileum columnar epithelium. The number of breaks or interruptions in claudin-3 staining per 1,000 enterocytes for each animal per treatment were averaged. The number of goblet cells per 1,000 enterocytes is given as a control. (a) SD, standard deviation for breaks in claudin-3 staining, within animals in a treatment. (b) p-values for breaks in claudin-3 staining determined by ANOVA analyses with post-hoc group comparisons using Bonferroni correction, F = 4.57, p = 0.008. (c) ns, not significant. Tissue from 10 animals was analyzed for each condition.

Figure 6. SPE-like radiation and hindlimb suspension induces the accumulation of LPS in subepithelial regions of the ileum.

Terminal ileum obtained 4 days post irradiation and/or 6 days post hindlimb suspension or from control animals was stained for LPS using a mouse mAb specific for E. coli LPS. A) Ileum from a control treated mouse. B) Ileum from a mouse irradiated with 2 Gy of 70 MeV protons. C) Ileum from a mouse subjected to hindlimb suspension. D) Ileum from a mouse treated with hindlimb suspension and irradiated with 2 Gy of protons. Original magnifications 200×. Tissue from 10 animals was analyzed for each condition.