The FASEB Journal • Research Communication
Increased dietary iron and radiation in rats promote oxidative stress, induce localized and systemic immune system responses, and alter colon mucosal environment Jennifer L. L. Morgan,* Lauren E. Ritchie,§ Brian E. Crucian,† Corey Theriot,储 Honglu Wu,† Clarence Sams,‡ Scott M. Smith,† Nancy D. Turner,§ and Sara R. Zwart¶,1 *Oak Ridge Associated Universities/National Aeronautics and Space Administration (NASA) PostDoctoral Fellowship Program and †Biomedical Research and Environmental Sciences Division and ‡ Space and Clinical Operations Division, Human Health and Performance Directorate, NASA Lyndon B. Johnson Space Center, Houston, Texas, USA; §Department of Nutrition and Food Science, Texas A&M University, College Station, Texas, USA; 储Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas, USA; and ¶Division of Space Life Sciences, Universities Space Research Association, Houston, Texas, USA Astronauts are exposed to increased body iron stores and radiation, both of which can cause oxidative damage leading to negative health effects. The purpose of this study was to investigate combined effects of high dietary iron (650 mg/kg diet) and radiation exposure (0.375 Gy cesium-137 every other day for 16 d) on markers of oxidative stress, immune system function, and colon mucosal environment in male Sprague-Dawley rats (nⴝ8/group). Control rats consumed adequate iron (45 mg/kg diet) and were not irradiated. Combined treatments increased liver glutathione peroxidase, serum catalase, and colon myeloperoxidase while decreasing total fecal short-chain fatty acid concentrations. The high-iron diet alone increased leukocyte count. Radiation decreased the T-cell CD4: CD8 ratio. Plasma iron was negatively correlated with cytokine production in activated monocytes. Genes involved in colon microbial signaling, immune response, and injury repair were altered by radiation. Genes involved with injury repair and pathogen recognition changed with dietary iron. These data demonstrate that dietary iron and radiation, alone and combined, contribute to oxidative stress that is related to
ABSTRACT
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; Con, control (treatment group); CRP, C-reactive protein; FDR, false discovery rate; FITC, fluorescein isothiocyanate; FSC, forward scatter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPX, glutathione peroxidase; Iron, high iron/sham treatment (treatment group); Iron ⫹ Rad, high iron/radiation (treatment group); LPS, lipopolysaccharide; MPO, myeloperoxidase; PBS, phosphate-buffered saline; PC5, phycoerythrin cyanin 5; PCR, polymerase chain reaction; PE, phycoerythrin; PMA, phorbol 12-myristate 13acetate; Rad, control/radiation (treatment group); ROS, reactive oxygen species; SCFA, short-chain fatty acid; SOD, superoxide dismutase; SSC, side scatter; TAC, total antioxidant capacity; TCR, T-cell receptor; TFF3, Trefoil factor 3; TLR, Toll-like receptor 1486
immune system alterations in circulation and the colon. The model presented may help us better understand the changes to these systems that have been identified among astronauts.—Morgan, J. L. L., Ritchie, L. E., Crucian, B. E., Theriot, C., Wu, H., Sams, C., Smith, S. M., Turner, N. D., Zwart, S. R. Increased dietary iron and radiation in rats promote oxidative stress, induce localized and systemic immune system responses, and alter colon mucosal environment. FASEB J. 28, 1486 –1498 (2014). www.fasebj.org Key Words: body iron stores 䡠 short-chain fatty acids 䡠 cytokines 䡠 injury repair 䡠 pathogen recognition As with many nutrients, a sensitive balance exists between iron status and the potential health risks of either a deficiency or an excess (1). Physiological adaptations to microgravity and an iron-rich food supply contribute to altered iron homeostasis during spaceflight (2– 4), including increased amounts of iron storage in the body. Elevated iron stores have been associated with a number of diseases, including cardiovascular disease, decreased bone density, and cancer (4 –7). Furthermore, reducing high levels of iron stores (e.g., through blood donation) has been associated with lower disease risk (8, 9). Thus, maintenance of iron homeostasis within normal ranges is extremely important for human health in space, as well as on Earth. In addition to the physiological effects of microgravity on the human body, radiation exposure is a major concern for space travel. The immediate generation of free radicals, including reactive oxygen species (ROS), 1 Correspondence: Mail Code SK3, 2101 NASA Pkwy., Houston, TX 77058, USA. E-mail:
[email protected] doi: 10.1096/fj.13-239418 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.
0892-6638/14/0028-1486 © FASEB
that occurs in cells after radiation exposure is responsible for some of the deleterious effects of radiation on cell function and survival, but the complex late effects, including bystander effects and bursts of inflammation, can be just as damaging to cells (10, 11). In animal models, exposure to ionizing radiation (ranging from 0.1 to 8 Gy) markedly increases oxidative damage in many tissues (12–14). In addition, direct effects of radiation exposure to critical biomolecules, particularly DNA, can lead to irreparable molecular changes that cause genetic and somatic effects. The potential risks associated with radiation exposure include carcinogenesis, central nervous system effects, and other degenerative tissue defects such as cataracts, circulatory diseases, and digestive diseases (15–17). Elevated iron stores and radiation are expected to promote oxidative stress and damage, as both generate ROS (18, 19). Elevated oxidative stress associated with increased body iron stores may increase radiation sensitivity by decreasing cellular oxygen radical scavenging capabilities of enzymes like superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (20 –22). Evidence also exists that some types of radiation exposure and oxidative stress can release ferrous iron (Fe2⫹) from ferritin, which can then participate in the production of hydroxyl radicals through Fenton chemistry and promote cellular damage (23, 24). One system that can be affected by oxidative stress is the immune system. The local immune response in the gut is an important defense against pathogens. The intestinal immune system must recognize commensal bacteria, while preserving its capacity to respond to pathogenic species (25). The gut monitors bacterial populations by recognizing pathogen-associated molecular patterns (such as bacterial ligands, including lipopolysaccharide, ligand presenting assembly, and flagellin), thereby inducing innate and adaptive immune responses (26, 27) that are mediated through Toll-like receptors (TLRs; refs. 28, 29) and downstream activation of NF-B. Colonic dysbiosis has long-term implications because an increased inflammatory tone is thought to play a role in the etiology of gastrointestinal disorders, such as inflammatory bowel disease, which promotes colon carcinogenesis (30 –32). Changes in the microbiota and their functional state can be monitored by measuring their metabolites, such as short-chain fatty acids (SCFAs; ref. 33). In addition to indicating changes in the microbiota population, the concentrations of SCFAs, such as butyrate, influence colon health and the ability to maintain epithelial barrier functions, because butyrate is the preferred substrate for colon epithelial cell metabolism (34, 35). Persistent alterations of the immune system may also have adverse systemic effects on immune function. Reactive species are produced as a normal consequence of immune activation or inflammation. In the immune system, these reactive molecules are actually beneficial, supporting pathogen elimination through respiratory bursts that are balanced by antioxidant production by immunocytes. Chronic inflammation, however, may overwhelm antioxidant defenses and lead to cellular damage, as described above. Persistent exposure to space radiation may induce chronic low-level inflammaINCREASED DIETARY IRON AND RADIATION IN RATS
tion in astronauts. Plasma cytokine data during longduration spaceflight suggest this, as levels of IL-8, CXC5, and IL-1Ra are significantly elevated for the duration of a 6-mo orbital flight (unpublished results). Oxidative stress also negatively affects T-cell function (36), and diminished T-cell function was recently documented to occur during space flight (37). Investigators have also found reactivation of latent herpes viruses during short-duration flight (38, 39), and oxidative injury is a component of several neurotropic viral infections (40). Dysregulation of other hematologic and immunological variables has also been found to occur immediately after spaceflight (41, 42), and altered cell-mediated immunity has occurred during long-duration spaceflight (43). Any persistent immune dysregulation during exploration deep-space missions might result in specific clinical risks for crewmembers (44). In this study, we characterized the combined effects of radiation exposure and high dietary iron on multiple physiological systems. Specifically, we investigated the effects of these treatments on iron status and oxidative stress markers, systemic immune responses, production of colon bacterial metabolites, and colon mucosal gene expression and inflammatory markers.
MATERIALS AND METHODS Animals This project was approved by the National Aeronautics and Space Administration (NASA) Johnson Space Center Institutional Animal Care and Use Committee. Four groups (n⫽8/ group) of 4-mo-old Sprague-Dawley male rats (Charles River Laboratories, Wilmington, MA, USA) were fed a control iron diet (45 mg iron/kg diet) and exposed to sham (0 Gy) radiation (control/sham, designated Con group), fed a highiron diet (650 mg iron/kg diet) and exposed to sham radiation (high iron/sham; designated Iron group), fed the control iron diet and exposed to fractionated (0.375 Gy of 137 Cs every other day for 16 d for a total of 3 Gy) radiation (control/radiation; designated Rad group), or fed a highiron diet and exposed to fractionated radiation (high iron/ radiation; designated Iron⫹Rad group). After arrival at the animal test facility at Johnson Space Center, rats were acclimated to the environment and control diet (AIN-93G; Research Diets, New Brunswick, NJ, USA) for 20 d (d ⫺20 to d 0; Fig. 1). They were housed individually with a 12-h light-dark cycle, in a temperature- and humiditycontrolled room (21⫾1°C). After 20 d (defined as study day 0), half of the animals were given the high-iron diet (AIN-93G with 650 mg iron/kg; Research Diets). Food intake was determined 3⫻/wk to ensure that food consumption and animal weights were similar between groups and that pairfeeding was not required. On study day 14, the first radiation dose was administered to the radiation groups. Each animal in the sham-treatment groups and radiation groups was placed in a restraint tube (Battelle, Geneva, Switzerland) for ⬃10 min/session to be either irradiated or sham treated. The last irradiation was performed on study day 28. Treatments The dietary iron content of 650 mg iron/kg has been used by others as a moderately high iron intake known to induce 1487
Figure 1. Timeline describing the experiment design. oxidative damage (45). The fractionated radiation exposure protocol consisted of 8 individual doses of 0.375 Gy of ␥ radiation over a 16-d period. This low, fractionated dose of ␥ radiation was intended to provide enough total dose (3 Gy) to induce an oxidative stress signal, and also to model the upper limit of the possible exposure scenario expected over a long-duration space mission to Mars (46). On the basis of recent radiation measurements taken during the Mars Science Laboratory’s trip to Mars, the spacecraft that took the rover Curiosity to the surface of Mars in 2012, the calculated radiation exposures of a 360-d round trip to Mars are estimated to be 0.66 ⫾ 0.12 Sv (or ⬃0.66 Gy of ␥ irradiation; ref. 47). The exposures during spaceflight can be greater if a large solar particle event occurs during travel to or from Mars. This value also does not include exposure accumulated while on the surface of Mars and is highly dependent on mission design (46).
digestion vials with 2 ml of ultrapure nitric acid (SigmaAldrich, St. Louis, MO, USA). Samples were digested using a CEM Mars XP-1500 Plus microwave digestion system (CEM Corp., Matthews, NC, USA) using the method designed for bovine liver (ramp to 200°C over 15 min and hold at 200°C for 15 min). After cooling, samples were clear with low surface tension. Samples were transferred to centrifuge tubes, using 3 ml of 18-M⍀ water to rinse the Teflon digestion vessels, and weighed. Samples were diluted 1:50 with 10% nitric acid and 10% ethanol for mineral analysis on an inductively coupled plasma mass spectrometer (Sciex Elan DRC II; Perkin Elmer, Waltham, MA, USA). Copper, iron, selenium, and zinc were measured using gallium as the internal standard. Process blanks revealed that no significant contamination of Cu, Fe, Se, or Zn occurred during processing. Oxidative stress and iron status analyses
Tissue collection
Biochemical analyses
Whole-blood SOD and catalase were measured colorimetrically using commercially available kits (Cayman Chemical Co., Scottsdale, AZ, USA). Whole-blood GPX was measured on an Ace Alera autoanalyzer (Alfa Wassermann Inc., West Caldwell, NJ, USA) using the Randox GPX method and reagents (Randox, Crumlin, UK). Serum ferritin was measured using a commercially available rat ferritin ELISA assay (Alpco Immunoassay, Salem, NH, USA). Plasma iron was measured using a Beckman Coulter AU analyzer (Beckman Coulter, Miami, FL, USA). Plasma heme was measured using the Quantichrom heme assay kit (BioAssay Systems, Hayward, CA, USA). Serum C-reactive protein was measured using a Rat C-Reactive Protein ELISA kit (EIAab, Wuhan, China). Serum n-telopeptide was measured using a Rat NTX ELISA kit (Cusabio, Wuhan, China). Oxidized low-density lipoprotein was measured using a Rat OxLDL ELISA kit (Cusabio). Serum total antioxidant capacity was measured using Randox reagents on a Beckman Coulter AU analyzer, with a blank measurement being taken of each sample before reagents were added. This allowed subtraction of any contribution from urate, ascorbate, or albumin. Liver total protein was measured using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Hematocrit was measured on a Coulter LH750 hematology analyzer (Coulter, Brea, CA, USA).
Liver protein and mineral analysis
Immune system assays
For protein assays, a ⬃0.2-g section of liver was homogenized in buffer [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylene glycol tetraacetic acid, 90 mM mannitol, and 70 mM sucrose]. Samples were centrifuged at 3500 rpm (1780 g) for 15 min, and the supernatant was aliquoted for analysis of SOD, catalase, GPX, and total protein. Samples were stored at ⫺80°C until assays were performed. For mineral analysis, a ⬃0.2-g section of liver was dried at 70°C for 24 h (Fisher Scientific Isotemp oven; Fisher Scientific, Pittsburgh, PA, USA), and the dry samples were weighed and then placed in acid-washed Teflon microwave
Leukocyte subset analysis
At the time of termination (24 h after the last radiation exposure), all animals had been denied access to food for 12 h and were anesthetized with isoflurane before blood was collected by cardiac puncture. Samples of liver, colon mucosa, and feces were collected. The liver was removed, weighed, rinsed with phosphate-buffered saline (PBS), cut into small pieces, placed in cryovials, and flash-frozen in liquid nitrogen. All tissues and samples were stored at ⫺80°C until further processing. Samples of the blood were placed in lithium-heparin, ethylenediaminetetraacetic acid (EDTA), or serum separator tubes. Whole blood (EDTA) was used for erythrocyte count and leukocyte analyses. Samples were centrifuged at 3500 rpm (1780 g) for 15 min, and then portioned into aliquots for individual tests. The colon was resected, feces were removed and snapfrozen at ⫺80°C, and the colon was washed twice in RNasefree PBS and scraped on a chilled RNase-free surface. Half of the scraped mucosa was homogenized by pipetting with 500 l of denaturation solution (Ambion, Austin, TX, USA) and transferred to a 2-ml Eppendorf tube for RNA isolation (48). The other half was snap-frozen in liquid nitrogen, and both samples were stored at ⫺80°C.
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Total leukocyte count was determined, after erythrocyte lysis, using a Hemocue leukocyte analyzer (Hemocue, Brea, CA, USA) with a DNA intercalating dye. The major leukocyte subsets (granulocytes, lymphocytes, monocytes) were determined by analysis on a Beckman Coulter LH750 hematology analyzer. The percentages of T cells and CD4⫹ and CD8⫹ T-cell subsets were determined by flow cytometry. The antibody matrix and fluorescent labels were as follows: CD8, fluorescein isothiocyanate (FITC), CD3, phycoerythrin (PE),
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MORGAN ET AL.
CD4, and phycoerythrin cyanin 5 (PC5) (Beckman Coulter). Antibody staining and erythrocyte lysis were performed as described previously (38), and analysis was performed on a Beckman Coulter EPICS XL flow cytometer. Lymphocytes were resolved and gated from granulocytes and monocytes by scatter properties [forward scatter (FSC)/side scatter (SSC)]. T cells (CD3⫹) were resolved and gated from unstained cell populations by plotting CD3 vs. SSC from the lymphocyte gate. CD4⫹ and CD8⫹ T cells were plotted and enumerated from the T-cell gate. T-cell function T cells were activated by culturing 100 l of heparinized blood in 1.0 ml RPMI medium containing 100 ng/ml each of Staphylococcus enterotoxin A and B (Sigma-Aldrich) for 24 h (49). Cultured whole blood was then stained as described above, using an anti-CD25-FITC, CD3-PE, and CD4-PC5 antibody matrix. Lymphocytes were resolved and gated from granulocytes and monocytes by scatter properties (FSC/SSC). T-cell subsets were resolved and gated from unstained cell populations by plotting CD3 vs. CD4 from the lymphocyte gate. Finally, activated T cells were identified and quantified by expression of the CD25 cell surface marker for both the CD4⫹ and CD8⫹ (defined as CD4⫺) T-cell subsets after being plotted from the appropriate gates. Cytokine production profiles Whole-blood cultures (heparinized blood) were set up as described above. Cell-specific mitogenic stimulation to enable cytokine production was generated by the addition of 0.5 g/ml anti-CD3 and 0.5 g/ml anti-CD28 antibodies (T cells), 10 ng/ml phorbol 12-myristate 13-acetate (PMA) ⫹ 2.0 g/ml ionomycin (a stronger stimulus affecting all cell populations), or 10 g/ml lipopolysaccharide (monocytes). Cultures were incubated for 48 h to allow for cytokine production and accumulation. After 48 h, supernatants were removed and frozen until they were analyzed.
ToTALLY RNA kit (Ambion) followed by DNase treatment (DNA-free kit; Ambion; ref. 48). RNA quality was assessed using an Agilent Bioanalyzer before the treated samples were stored at ⫺80°C. First-strand cDNA was synthesized using random hexamers, oligo dT primer (Promega, Madison, WI, USA), and Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Real-time PCR was performed on select genes [TOLLIP, Trefoil factor 3 (TFF3), Slc16a1, IL-6, IL-1, TNF-␣, TNF␣1a, TNF␣1b, RipK1, SLC5A8, TGF, TLR9, TLR5, TLR4, TLR2, MyD88, NF-B, and COX-2 (PTGS2)] using TaqMan Array Micro Fluidic Cards (Applied Biosystems, Foster City, CA, USA) and an ABI 7900 HT thermocycler (Applied Biosystems). Expression levels were normalized to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Myeloperoxidase (MPO) assay MPO concentration was measured in scraped mucosa according to the manufacturer’s instructions (Cell Technology, Mountain View, CA, USA). Mucosa samples were weighed and homogenized in 250 l of 1⫻ assay buffer containing 10 mM N-ethylmaleimide, then aspirated through a 23-gauge needle twice and centrifuged at 12,000 g at 4°C for 20 min. The resulting proteinaceous pellet was homogenized in 500 l of 1⫻ assay buffer containing 0.5% hexadecyltrimethylammonium bromide (HTA-Br; w/v), sonicated (Sonic Dismembrator; Fisher Scientific), and subjected to 2 cycles of freezing and thawing before being centrifuged at 8000 g at 4°C for 20 min. The supernatant was incubated with 20 mM 3-amino1,2,4-triazole (Sigma-Aldrich) for 60 min, and then incubated in the dark at room temperature with 50 l of reaction cocktail for 60 min. Fluorescence was measured at 530- to 570-nm excitation and 590- to 600-nm emission wavelengths in a fluorescent plate reader with the 1⫻ assay buffer serving as a negative control. Statistical analyses
Cytokine analysis Concentrations of soluble cytokines, from either plasma or mitogen-stimulated culture supernatants, were determined by using bead-array technology and a Luminex-100 analyzer (Luminex Corp., Austin, TX, USA). The analysis array was obtained from R&D Systems (Minneapolis, MN, USA) and consisted of separate bead-based cytokine analysis reagents for IL-1, IL-6, TNF-␣, IL-2, IFN␥, IL-4, and IL-10. Fecal and colon analyses Short-chain fatty acid analyses Frozen fecal samples were ground, mixed with an internal standard (2-ethylbutyric acid), and extracted in 70% ethanol. After centrifugation, the supernatant was removed and mixed with a second internal standard, heptanoic acid, before it was injected into an HP-FFAP column (Agilent Technologies, Santa Clara, CA, USA) in a Varian 3900 gas chromatograph (Varian, Walnut Creek, CA, USA). Concentrations of SCFAs were calculated on the basis of comparison of sample peak areas with those produced by a commercially available mix of standards (50). Gene expression using quantitative real-time polymerase chain reaction (PCR) Total RNA was isolated from mucosal samples using Phase Lock Gel tubes (5 Prime, Gaithersburg, MD, USA) and the INCREASED DIETARY IRON AND RADIATION IN RATS
Statistical analyses were performed using Stata IC 12.1 software (StataCorp, College Station, TX, USA) and setting 2-tailed ␣ to reject the null hypothesis at 0.05. Some dependent variables required log transformation to satisfy the required statistical assumptions. The variables requiring transformation are indicated in the tables. Overly influential observations (i.e., statistical outliers) were excluded from analysis if the standardized residual exceeded ⫾ 1.96 U away from the mean (also identified in the tables). Data were analyzed by 2-way ANOVA with diet and radiation as the main factors. Pairwise comparisons of all possible pairs were made for all analytes. Pearson correlations were performed on selected analytes. For real-time PCR, 2-way ANOVA was run on ⌬CT relative to the reference gene GAPDH as the dependent variable, but data are presented as change in expression [(2⫺⌬CT)⫻1000]. On completion of all analyses, the uncorrected P values were submitted to a single multiple-testing correction (810 hypothesis tests) using the Krieger false discovery rate (FDR) method (51). The FDR was set at 10%, thereby accepting that up to 10% of the rejected null hypotheses might be a result of type I error. As this was a small study examining a wide range of analytes, the use of a more conservative FDR value or method (Bonferroni) is not warranted. Unadjusted P values are presented in the tables and text with an asterisk indicating that the results remained significant after adjusting for multiple comparisons. The residuals from Anti-CD3-IFN␥, PMA:IL-1, and plasma IFN␥, IL-10, IL-1, and IL-4 could not be normalized and therefore 1489
statistical analyses were not performed on these assays. Nonetheless, the data are presented in Table 1. All data are presented as means ⫾ sd.
RESULTS Neither high dietary iron nor the radiation exposure changed food intake or body mass gain (data not shown). There were no differences between groups in liver weights at the time of termination [11.07⫾1.35 g
(Con), 10.72⫾2.02 g (Iron), 11.69⫾1.33 g (Rad), and 12.50⫾2.51 g (Iron⫹Rad)]. Iron metabolism was significantly altered as a result of both high dietary iron and radiation, as evidenced by both blood (Fig. 2A) and liver (Fig. 3A) analyses. Plasma iron concentration increased by 37% in the Iron ⫹ Rad group relative to the Con group (P⫽0.007). Liver iron concentrations increased as a result of diet alone (P⫽0.004), with a 17% increase in the Iron group relative to Con and a 36% increase in the Iron ⫹ Rad group relative to Rad. Liver iron and plasma iron were
TABLE 1. Immune markers Effect Test
Leukocytes (count)§ Granulocytes (%) Lymphocytes (%) Monocytes (%) CD4⫹ T cells CD8⫹ T cells CD4⫹/CD8⫹, ratio Staphylococcus enterotoxin T-cell function CD4⫹ T-cell function CD8⫹ Plasma concentration (pg/ml) IFN␥ IL-10 IL-1 IL-2§ IL-4 IL-6§ TNF␣§ Anti-CD3/CD28 (pg/ml supernatant) IFN␥ IL-10§ IL-1§ IL-2§ IL-4§ IL-6§ TNF␣§ PMA/ionomycin (pg/ml supernatant) IFN␥ IL-10§ IL-1 IL-2 IL-4§ IL-6§ TNF␣§ LPS (pg/ml supernatant) IFN␥§ IL-10§ IL-1 IL-2§ IL-4§ IL-6 TNF␣
Iron ⫹ Rad
Outliers
Con
Iron
Rad
0 0 1 1 0 0 0
4 ⫾ 1a 14 ⫾ 12a 76 ⫾ 18a 0.2 ⫾ 0.1a 62 ⫾ 6a 35 ⫾ 8a 2 ⫾ 1a
7 ⫾ 3b* 13 ⫾ 16a 90 ⫾ 8b* 0.4 ⫾ 0.9a 60 ⫾ 4a 37 ⫾ 4a 2 ⫾ 0a,b
3 ⫾ 1a 30 ⫾ 5b* 68 ⫾ 6a 0.5 ⫾ 0.8a 53 ⫾ 4b* 45 ⫾ 4b* 1 ⫾ 0b*
3 ⫾ 1a 28 ⫾ 12b 67 ⫾ 11a 0.6 ⫾ 0.8a 51 ⫾ 5b* 47 ⫾ 6b* 1 ⫾ 0b*
0 0
7.0 ⫾ 1.6a 1.9 ⫾ 0.6a
7.6 ⫾ 2.5a* 1.7 ⫾ 0.5a
4.3 ⫾ 2.4b 1.2 ⫾ 0.9a
6.4 ⫾ 2.7a,b 1.8 ⫾ 1.3a
0 0 0 0 0 0 0
0⫾0 0.2 ⫾ 0.5 15 ⫾ 37 3 ⫾ 2a 0.2 ⫾ 0.4 8 ⫾ 13a 4 ⫾ 4a
0⫾0 0.3 ⫾ 0.7 0⫾0 8 ⫾ 11a 0.0 ⫾ 0.0 21 ⫾ 42a 10 ⫾ 11a
0⫾0 3.2 ⫾ 6.2 2⫾4 6 ⫾ 5a 0.0 ⫾ 0.0 22 ⫾ 27a 7 ⫾ 10a,b
0⫾0 0.1 ⫾ 0.3 7 ⫾ 15 9 ⫾ 10a 0.1 ⫾ 0.1 6 ⫾ 11a 1 ⫾ 1b
0 0 0 0 0 0 0
3.9 ⫾ 8.0 4 ⫾ 4a 2 ⫾ 4a 28 ⫾ 13a* 0.2 ⫾ 0.2a* 62 ⫾ 44a* 3.6 ⫾ 4.2a
1.3 ⫾ 3.7 3 ⫾ 1a 1 ⫾ 2a 16 ⫾ 5a 1.7 ⫾ 3.8a 45 ⫾ 36a* 2.4 ⫾ 1.2a
Diet
⬍0.01* ⬍0.001*
81 ⫾ 26a* 65 ⫾ 40a*,c a 27 ⫾ 39 * 10 ⫾ 26b 13 ⫾ 19 10 ⫾ 20 857 ⫾ 330a 471 ⫾ 229b* 3 ⫾ 3a 1 ⫾ 1a,b a 255 ⫾ 287 162 ⫾ 346a,b 37.0 ⫾ 54.4a 12.3 ⫾ 16.4a,b LPS
18 ⫾ 7b 5 ⫾ 4a,b 0⫾0 259 ⫾ 75b* 0 ⫾ 0b*,c 34 ⫾ 23b* 4.7 ⫾ 4.0b*
38 ⫾ 36b,c 12 ⫾ 16a 0⫾1 428 ⫾ 394b* 0 ⫾ 0c* 53 ⫾ 39b 5.2 ⫾ 4.2b*
0 0 0 0 0 0 0
12 ⫾ 21a 0 ⫾ 0c* 40 ⫾ 16a 23 ⫾ 6b 60 ⫾ 13a 56 ⫾ 10a 17.3 ⫾ 12.2a 10.6 ⫾ 6.3a 0.2 ⫾ 0.2a* 0.4 ⫾ 0.9a 408 ⫾ 187a 322 ⫾ 138a 52 ⫾ 15a 58 ⫾ 11a
10 ⫾ 13b 22 ⫾ 7b* 52 ⫾ 9a 17.9 ⫾ 11.8a 0.7 ⫾ 0.4a 270 ⫾ 98a 66 ⫾ 12a
3 ⫾ 3a,b 18 ⫾ 13b* 47 ⫾ 16a 9.4 ⫾ 6.5a 1.4 ⫾ 1.8b* 338 ⫾ 297a 57 ⫾ 22a
Inter
⬍0.05
⬍0.001* ⬍0.001* ⬍0.01* ⬍0.05
IS IS IS IS ⬍0.05
53.7 ⫾ 56.8 4.7 ⫾ 12.0 IS 88 ⫾ 95b* 3 ⫾ 4a 22 ⫾ 25a 14 ⫾ 25a 58 ⫾ 53a* ⬍0.001* 9 ⫾ 13b b 5.3 ⫾ 5.5 1.1 ⫾ 2.0a* 1525 ⫾ 1849b 927 ⫾ 1233a,b 45.0 ⫾ 43.8b* 7.3 ⫾ 8.8a
1 0 0 0 0 0 0
Rad
⬍0.001*
⬍0.01* ⬍0.01* ⬍0.001*
IS ⬍0.001* ⬍0.05* ⬍0.001* ⬍0.01* ⬍0.05*
⬍0.05
⬍0.01* ⬍0.05*
⬍0.001* ⬍0.01* ⬍0.001*
Values are expressed as means ⫾ sd. Inter, interaction; IS, insufficient signal to analyze using an ANOVA. Values in each row with different superscript letters differ significantly (P⬍0.05; unadjusted pairwise comparison). *Pairwise and main effects that remained significant after adjusting for multiple comparisons. §Log transformed to achieve homogeneity of variance.
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Control Diet
High Fe Diet
Figure 2. Results from assays performed on blood samples collected at termination. Plasma iron (A), plasma catalase (B), serum ferritin (C), serum alanine transaminase (ALT; D), red blood cell GPX (E), and hematocrit (F). All data are expressed as means ⫾ sd. Significant radiation effect, *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 for radiation effect; ●●P ⬍ 0.01 for diet effect.
positively correlated for all groups (r⫽0.36, P⫽0.044, data not shown). In addition to the changes in iron status, several markers of plasma and liver oxidative stress were affected by the dietary iron and radiation exposures. High dietary iron increased plasma catalase (P⫽0.0008; Fig. 2B) and liver GPX (P⫽0.005; Fig. 3B). Liver GPX was positively correlated with liver iron for all groups (r⫽0.47, P⫽0.006; Fig. 4). Radiation exposure increased serum ferritin (P⫽0.007; Fig. 2C) and decreased alanine transaminase (ALT; P⫽0.002; Fig. 2D), erythrocyte GPX (P⫽0.02; Fig. 2E), hematocrit (P⫽0.03, Fig. 2F), and plasma total antioxidant capacity (TAC; P⫽0.03; Supplemental Table S1). Both aspartate transaminase (AST) and hemoglobin tended to be lower in the radiation-treated groups, but these did not reach statistical significance. Dietary iron and radiation had an interactive effect on liver TAC (P⫽0.03; Fig. 3C), in that liver TAC increased in the Iron group relative to the Con group (P⫽0.03), but dietary iron did not further increase liver TAC in the Iron ⫹ Rad
group beyond that which occurred with radiation alone (Rad group). Both radiation exposure and a high-iron diet resulted in alterations to both cellular distribution and functional aspects of the immune system (Table 1). Diet and radiation had an interactive effect on leukocytes (P⫽0.02). The percentage of leukocytes increased in the Iron group relative to the Con group (P⫽0.003); however, diet had no significant effect in the radiation group (Iron⫹Rad relative to Rad). Radiation induced alterations to the immune cell subset percentages, including an increase in the granulocyte percentage (P⫽0.001) and a decrease in total lymphocyte percentage (P⫽0.0009). A reduction in percentage of CD4⫹ T cells (P⬍0.0001) and an increase in percentage of CD8⫹ T cells (P⬍0.0001) corresponded to an overall decrease in the CD4⫹:CD8⫹ T-cell ratio (P⬍0.0001). There was a significant correlation between serum ferritin and CD4⫹ T cells (r⫽⫺0.44, P⫽0.01; Fig. 5), CD8⫹ T cells (r ⫽ 0.45, P ⫽ 0.01), and the CD4⫹:CD8⫹ ratio (r ⫽ ⫺0.45, P ⫽ 0.01).
Figure 3. Results from assays performed on liver samples collected at termination. Liver iron (A), liver GPX normalized to total protein (B), and liver total antioxidant capacity (TAC) normalized to total protein (C). All data are expressed as means ⫾ sd. Parentheses around significance footnote symbols indicate interactive effects using the values from the post hoc analysis. *P ⬍ 0.05 for radiation effect; ●P ⬍ 0.05, ●●P ⬍ 0.01 for diet effect. INCREASED DIETARY IRON AND RADIATION IN RATS
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Figure 4. Correlation between liver iron and liver glutathione peroxidase.
Diet had no main effect on these T-cell subset percentages. Generally, the plasma concentrations of the inflammatory and adaptive immunity cytokines that were measured were not altered. However, radiation did decrease the plasma concentration of TNF-␣ (P⫽0.03), which was decreased in the Iron ⫹ Rad group relative to the Con and Iron groups. As a measure of functional capability, cytokine production was determined after stimulating T cells by triggering the T-cell receptor (TCR) and costimulation (anti-CD3/CD28). Radiation induced a significant stimulatory effect on T-cell cytokine production. Cytokines IL-6 and TNF-␣ both increased as a result of radiation exposure after costimulation with anti-CD3/CD28 (P⫽0.002 and P⫽0.009, respectively). Production of IL-10 and IL-4 after costimulation showed an interactive effect (P⬍0.0001 and P⫽0.02, respectively): a significant increase occurred in IL-10 and IL-4 production in the Rad group relative to the Con group (P⬍0.0001 and P⫽0.006, respectively), but production of these cytokines in the Iron ⫹ Rad group was not significantly different from production in the Con or Iron group. The high-iron diet seemed to ameliorate the effect of radiation. However, after anti-CD3/CD28 stimulation, T-cell production of IL-2 actually decreased as a result of diet (P⬍0.001) and was reduced to the greatest extent in the Iron ⫹ Rad group.
Figure 5. Correlation between serum ferritin concentration and CD4⫹ T cells. 1492
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Figure 6. Correlation between plasma iron and IL-10 production after LPS stimulation.
Cytokine profiles were also determined after panleukocyte activation using a more powerful pharmacologic stimulus (PMA-I) consisting of PMA (a protein kinase C activator) and ionomycin (a calcium ionophore). A suppressive effect was observed after radiation. Radiation decreased IFN␥, IL-4, IL-6, and TNF-␣ (P⫽0.0005, P⫽0.0005, P⫽0.01, and P⫽0.001, respectively). There was an interactive effect of diet and radiation for IL-10 and IL-2 production (P⫽0.01 and P⫽0.01, respectively). IL-10 and IL-2 decreased significantly in the Iron group relative to the Con group (P⫽0.007 and P⫽0.01, respectively), but no decrease in the Iron ⫹ Rad group occurred relative to Rad. IL-2 decreased in both Rad and Iron ⫹ Rad relative to Con (P⬍0.0001 and P⫽0.007, respectively). After monocyte stimulation with bacterial endotoxin [lipopolysaccharide (LPS)], production of IFN␥ and IL10 was decreased with both high dietary iron and radiation exposure (P⫽0.0002, P⫽0.005; P⫽0.006, and P⫽ 0.02, respectively), whereas IL-4 production increased with radiation exposure (P⫽0.0006). An increase in plasma iron was correlated with a decrease in IL-10 production after LPS stimulation (r ⫽ ⫺0.54; P ⫽ 0.002, Fig. 6). The feces and colon mucosal analyses showed significant changes in SCFAs in response to dietary iron and radiation (Fig. 7A–C). Both diet and radiation had significant main effects on SCFAs; both treatments caused a decrease in total SCFAs (P⫽0.04), which was greatest in the Iron ⫹ Rad group relative to the Con group. In addition, dietary iron decreased fecal butyric acid (P⫽0.051), and dietary iron and radiation combined decreased isovaleric acid concentration (P⫽ 0.005). Dietary iron and radiation had significant effects on expression of selected gene targets in the scraped colon mucosa (Table 2). Reduction in expression of TLR9 (P⬍0.001), TNF-␣ (P⫽0.001), and IL-6 (P⬍0.001) occurred with radiation exposure. Dietary iron decreased expression of TLR4 (P⫽0.03) and TFF3 (P⫽0.04). Expression of TNF␣1b increased with dietary iron (P⫽0.01) but decreased with radiation exposure (P⬍0.001). Slc5a8 expression was affected by iron and radiation interactively (P⫽0.004). Expression of Slc5a8 decreased in the Iron group relative to the Con group, but no decrease occurred as a result of diet in the Iron ⫹
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Figure 7. Results from assays performed on fecal samples or intestinal mucosa (A–C) collected at termination (D). A) Total SCFAs. B) Butyric acid. C) Isovaleric acid. D) MPO. All data are expressed as means ⫾ sd. *P ⬍ 0.05, **P ⬍ 0.01 for radiation effect; ●P ⬍ 0.05 for diet effect.
Rad group relative to the Rad group. Activity of MPO, an inflammatory marker in the colon mucosa, increased with radiation exposure (P⫽0.04), being significantly higher in the Iron ⫹ Rad group than in the Con group (P⫽0.04; Fig. 7D).
mented a relationship between iron stores, oxidative damage, and bone health in astronauts (4), along with animal studies of radiation and bone health (52–54). We report here an animal model for the combined effects of iron and radiation, with implications for hepatic and immune system function, and gastrointestinal health.
DISCUSSION Iron status The combined effects of increased dietary iron and radiation have implications for astronauts in longduration spaceflight. Recent reports have docu-
The level of iron overload in the animals in this study was comparable to the level observed among crewmem-
TABLE 2. Change in gene expression in colon relative to GAPDH Effect Test
TLR2 TLR4 TLR5 TLR9 MyD88 IB NFB TNF␣ TNF␣1␣ TNF␣1 Tollip Ripk1 Tff3 TGF IL-1 IL-6 Ptgs2 (COX-2) Slc16a1 Slc5a8
Outliers
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
Con
Iron
Rad
Iron ⫹ Rad
1.39 ⫾ 0.68a 1.37 ⫾ 0.71a 1.27 ⫾ 0.41a 1.26 ⫾ 0.42a 10.93 ⫾ 2.69a* 11.18 ⫾ 3.45a* 6.98 ⫾ 1.14b 10.63 ⫾ 3.39a 4.23 ⫾ 1.75a 5.38 ⫾ 2.94a 4.70 ⫾ 1.55a 4.09 ⫾ 2.21a 0.44 ⫾ 0.25a 0.65 ⫾ 0.30a 0.13 ⫾ 0.06b* 0.18 ⫾ 0.15b* a a a 14.16 ⫾ 2.54 11.99 ⫾ 1.13 13.26 ⫾ 2.33 12.27 ⫾ 2.33a 17.89 ⫾ 4.10a 16.89 ⫾ 2.26a 17.10 ⫾ 4.01a 15.26 ⫾ 2.23a a b a,b 22.24 ⫾ 3.78 18.23 ⫾ 1.45 20.16 ⫾ 4.12 19.70 ⫾ 3.96a,b 0.42 ⫾ 0.41a,c 0.83 ⫾ 0.80a 0.10 ⫾ 0.15b* 0.12 ⫾ 0.10b,c a a a 20.15 ⫾ 7.06 18.06 ⫾ 4.01 17.78 ⫾ 4.68 17.83 ⫾ 2.55a 1.35 ⫾ 0.53a 1.85 ⫾ 0.50c* 1.17 ⫾ 0.18a,b 1.03 ⫾ 0.21b a a a 8.86 ⫾ 1.78 8.13 ⫾ 0.83 8.18 ⫾ 1.74 8.53 ⫾ 1.19a 8.46 ⫾ 1.45a 8.93 ⫾ 1.09a 8.86 ⫾ 1.42a 7.99 ⫾ 0.85a a a a 684.85 ⫾ 97.83 564.48 ⫾ 151.12 748.23 ⫾ 259.02 596.01 ⫾ 129.14a 2.36 ⫾ 0.92a 2.93 ⫾ 0.84a 2.73 ⫾ 0.83a 2.29 ⫾ 1.10a a a a 0.32 ⫾ 0.17 0.53 ⫾ 0.39 0.32 ⫾ 0.11 0.28 ⫾ 0.11a 0.10 ⫾ 0.04a,b 0.14 ⫾ 0.05a* 0.06 ⫾ 0.05c 0.06 ⫾ 0.02b,c a a a 1.62 ⫾ 0.53 1.39 ⫾ 0.39 1.41 ⫾ 0.41 1.17 ⫾ 0.35a 88.49 ⫾ 14.16a 85.91 ⫾ 10.50a 86.85 ⫾ 12.60a 81.77 ⫾ 6.62a 30.43 ⫾ 7.40a 17.09 ⫾ 3.30b* 27.84 ⫾ 7.35a 27.53 ⫾ 5.39a
Diet
Rad
Inter
⬍0.05 ⬍0.001*
⬍0.01* ⬍0.05* ⬍0.001* ⬍0.05 ⬍0.001* ⬍0.01*
Values are expressed as means ⫾ sd. Statistical analyses were performed on ⌬CT relative to GAPDH [(2⫺⌬CT)⫻1000]. Inter, interaction. Values in each row with different superscript letters differ significantly (P⬍0.05; unadjusted pairwise comparison). *Pairwise and main effects that remained significant after adjusting for multiple comparisons.
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bers during spaceflight, in that liver iron increased 17% in animals supplemented with iron; this is the same percentage increase that was observed for body iron (estimated from ferritin and transferrin receptors) in the first 2 wk of spaceflight in astronauts (4). Increased iron stores and liver iron content reflect an increase in total body iron, as has been established in humans (55). This relationship is important because some of the markers of oxidative stress observed in this study are not limited to effects on liver tissue alone. The increase in serum iron observed in the high-iron diet groups further indicates that these groups’ iron load increased. Serum iron was also increased in the Rad group, an effect that could be caused by inhibition of erythrocyte formation. It is known that hematopoietic stem cells are radiosensitive and are easily disrupted by radiation exposure (56, 57), and the lower hematocrit levels and decreased erythrocyte GPX in the radiation-exposed animals provided evidence for this. If radiation prevents new erythrocytes from forming, then the age of circulating erythrocytes will increase, and, as a result, the cellular GPX will decrease, which was observed in this study. Oxidative stress markers Increasing dietary iron increased iron status, and the increase was correlated with some markers of oxidative stress. Liver iron was positively correlated with serum catalase and with liver GPX (Fig. 4), indicating that a greater oxidative load on the liver was associated with increased iron stores. Because this study only focused on one time point after chronic radiation exposure, the responses only indicate a snapshot of a possible inflammatory process. Ferritin was elevated in response to radiation exposure, a result that may either indicate an acute-phase response or reflect altered hematopoietic stem cell survival and increased iron stores. The lack of changes in other acute-phase proteins and cytokines supports the latter explanation; however, the timing of any change in expression of other acute-phase proteins, such as C-reactive protein (CRP), may have been missed in this study with only one time point, or CRP may behave differently when the radiation dose is fractionated over 16 d (58 – 60). Systemic immune response The immunological monitoring for this study consisted of determining basic leukocyte distribution and plasma cytokine levels, to monitor both in vivo immune activation and various mitogen-stimulated cytokine production profiles and to detect dysregulation of cellular functional capacity. The data indicated that the Iron group alone had an elevated leukocyte count but a normal subset distribution relative to the Con group. Both radiation groups, independent of diet, had an increase in granulocyte percentage, which may mitigate radiation injury (61, 62). Both radiation groups had a decrease in the total percentage of lymphocytes and a decrease in the T-cell CD4:CD8 ratio. A ⬃10% decrease in CD4⫹ T cells corresponding to ⬃3%/Gy (63) was 1494
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expected because of the radiation dose given (3 Gy). The decrease in CD4⫹ T cells also correlated with an increase in serum ferritin concentrations (Fig. 5), indicating that iron stores may affect T-cell distribution. The absence of change in plasma cytokine expression suggests that constitutive immune activation did not change, which does not support an acute-phase response at the time point used for this study. The lack of a change in constitutive immune activity may have diminished cellular functional capacity, resulting in an increased health risk. Previous studies showed that radiation alone has a negative effect on early blastogenesis of T cells but a positive effect on T-cell function in a murine model (64, 65). For the present study, wholeblood cultures were stimulated with cell-specific mitogen to produce in vitro cellular activation, and supernatant cytokine levels were measured as an indicator of functional capacity. Whole-blood culture is perceived to more closely represent in vivo conditions, as all potential cell-cell interactions are preserved and plasma soluble factors are present. T cells were stimulated by triggering the TCR and costimulatory molecule with CD3/CD28, modeling in vivo activation by an antigen-presenting cell. Elevated dietary iron reduced T-cell IL-2 production, whereas radiation increased T-cell production of TNF-␣ and IL-6 after stimulation. The RAD group showed an increase in IL-4 and IL-10, but in the Iron ⫹ Rad group, this increase was negated. This diet effect was not observed in the sham group. Thus, a high-iron diet seems to inhibit both constitutive immune functional responses and the elevated functional responses observed after radiation treatment. Radiation exposure resulted in alterations in leukocyte cytokine production after mitogenic stimulation with PMA-I. This mitogen, consisting of a phorbol ester/protein kinase C activator and calcium ionophore, bypasses much of the signal transduction used by surface molecule mitogens, and it is more powerful than other stimulations. Radiation resulted in decreased production of TNF-␣, IL-6, IFN␥, and IL-4, a grouping that spans inflammatory, Th1, and Th2 cytokine categories. The decrease in TNF-␣ and IL-6 in these cells, again, parallels the observations from the colon, suggesting that radiation has systemic, as well as localized effects on immune function in this fractionated protocol. Production of some cytokines (IL-2 and IL-10) was also diminished in the Iron group compared to the Con group, although this diet effect was not observed when the Rad and Iron ⫹ Rad groups were compared. LPS generally serves as an activator of monocytes and B cells. After culture of whole blood in the presence of LPS, IL-10 and IFN␥ production decreased as a result of radiation exposure and high dietary iron. Production of IL-4 actually increased as a result of radiation exposure. Plasma iron concentration was negatively correlated with IL-10 after LPS stimulation. These data generally indicate that adaptive Th1 and Th2 responses (IFN␥ and IL-10, respectively) may be diminished after either radiation exposure or increased dietary iron. With respect to radiation exposure, however, the elevation in IL-4 production may be considered discordant with this conclusion. Dimin-
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ished cytokine production profiles are consistent with recent reports of spaceflight-associated immune dysregulation in astronauts (50). Colon responses The total concentration of all SCFAs analyzed (acetic, propionic, isobutyric, butyric, isovaleric, and valeric acid) decreased in all other subject groups relative to the Con group, with a maximum decrease occurring in the Iron ⫹ Rad group. Changes in the concentration of fecal SCFAs could be caused by alterations in the bacterial populations and/or changes in bacterial metabolism after an increase in dietary iron or exposure to radiation. To our knowledge, no studies have shown an effect of high dietary iron or radiation on intestinal bacterial metabolism, although it is known that butyrate-producing bacteria strongly depend on iron as a cofactor for butyrate production (66). Independent studies have reported that radiation therapy (4.3–5.4 Gy) and dietary iron depletion variably promote and inhibit the microbiota, including taxa that are known butyrate producers (67, 68). It has been reported that cecum SCFA concentrations and the proportion of butyrate-producing bacteria can be increased when iron-depleted animals are fed diets fortified with iron (68); however, no reports describe the effect of high dietary iron on the microbiota or bacterial metabolism. Another potential explanation for the observed reduction in fecal SCFA is an alteration in the absorption of SCFAs into colonocytes, which was not directly assessed in this experiment. However, we observed alterations in gene expression of SCFA transporters (described below), which may contribute to the observed fecal SCFA concentrations. Colonic mucosa gene expression and inflammatory markers Exposure to genotoxic stress, including ionizing radiation and oxidative species, can initiate physiological responses in the intestine, such as cell cycle arrest, diminished epithelial integrity, inflammatory infiltration, altered gene transcription, and a perturbed luminal environment by variably suppressing or supporting the growth of certain bacterial populations (67, 69 –78). The mucosal surface of the small and large intestine has shown increased propensity for radiation-induced mucosal injury because of its high proliferative capacity (69). Thus, we were interested in understanding the effect of high dietary iron and ␥ radiation exposure on gene expression in colonic mucosa for pathways associated with microbial signaling, and SCFA transport and repair. SCFAs are absorbed from the lumen passively through ionic diffusion, or through transporters such as Slc16a1 and Slc5a8 (79, 80). We observed a significant decrease in expression of Slc5a8 in the Iron group relative to the other 3 groups, whereas Slc16a1 showed minimal changes with either treatment. The highest Slc5a8 and Slc16a1 expression and SCFA concentration observed in the Con group may have resulted from the same factors that caused the reported promotion of INCREASED DIETARY IRON AND RADIATION IN RATS
Slc16a1 expression in a colonic epithelial cell line, where increased butyrate concentrations up-regulated Slc16a1 mRNA and protein levels (79). A decrease in SCFA production and absorption could be detrimental to astronauts’ gastrointestinal health, as these metabolites have been reported to have pleiotropic effects on colonocytes, including anti-inflammatory activity, the induction of gene expression, cellular differentiation, apoptosis, cell-cycle arrest, and interference with transcription factors critical for proinflammatory cytokine production (81– 86). Expression of 4 gene targets involved in bacterial recognition and inflammatory responses was reduced by radiation exposure, consistent with previous studies (87, 88). These targets were TLR9 (a bacterial recognition receptor); TNF-␣ (a cytokine involved in systemic inflammation) and its receptor, TNF␣1b; and IL-6 (an interleukin with both proinflammatory and anti-inflammatory effects). In addition, TLR4 and TFF3 expression decreased when rats were exposed to high dietary iron. The gene expression patterns indicate that changes occurred in the bacterial recognition signaling pathway (TLR) as a result of dietary iron and radiation, although the treatments had differential effects on mediators of this pathway. Alterations in TLR expression could also be attributed to changes in the composition of the intestinal microbiota, compromised barrier integrity, or a chronic inflammatory state, all of which have been documented to alter expression of these receptors (89 –91). The suppressed luminal SCFA concentrations, described above, may indicate that the microbiota was altered by radiation exposure and high dietary iron, which could have contributed to the differential effects on TLR4 and TLR9 expression with diet and radiation, respectively. Microbially derived signaling through pattern-recognition receptors affects gene expression, transepithelial electrical resistance, and colonocyte proliferation in normal tissues (92), and it plays a pivotal role in maintaining homeostasis between commensal microbiota and the host immune system (93). Alterations in the TLR signaling pathway also directly affect the immune response, specifically through downstream activation of the transcription factor NF-B (94). NF-B activation alters expression of genes for localized cytokines (IL-1, IL-10, IL-6, TNF-␣, IFN␥, and TGF-) and proinflammatory molecules (COX-2) during an inflammatory response (27). Although we did not observe a significant diet or radiation effect on expression of genes for NF-B or other proteins mediating the TLR signaling cascade, we did observe a significant reduction of TNF-␣ and IL-6 expression with radiation. The expression of TNF␣1b, IL-6, IL-1, and TNF-␣ was up-regulated in IRON rats. TNF␣1b signaling is known to activate the transcription of proinflammatory cytokines through NF-B (95), which could be one explanation for the up-regulation of IL-6 and the tendency for an up-regulation of IL-1, IL-6, and TNF-␣ expression in Iron animals relative to other groups. MPO, a biomarker of inflammation, is an enzyme found in neutrophils that conjugates bactericidal compounds in the phagosome (96). Increased infiltration of neutrophils into the epithelium occurs in response 1495
to an immune challenge, which could explain the observed increase in mucosal MPO concentration in Iron, Rad, and Iron ⫹ Rad animals in this study. The inflammation may result from the increased presence of ROS in the lumen and in the epithelial cells after radiation or from high exposure to oxidants. In addition, if the epithelial barrier is compromised by these environmental stresses, bacteria and antigens could translocate through the epithelial membrane and be responsible for an increased infiltration of neutrophils and activation of inflammatory mechanisms (74, 77). The decrease, with a high-iron diet, in expression of TFF3, which codes for a protein expressed on the apical side of the intestinal epithelial barrier and reported to be involved in epithelial repair and cellular migration (97), could suggest that the luminal environment resulting from a high-iron diet may have a detrimental effect on epithelial repair or barrier restitution after injury. Also, as mentioned previously, chronic inflammation and dysbiosis of the intestinal microbiota are known to persist in intestinal diseases, such as inflammatory bowel disease, and could increase an astronaut’s risk of getting colorectal cancers (30 –32). Moreover, the local colonic inflammatory responses to diet and radiation may further stimulate the systemic alterations in immune function and cytokines discussed earlier.
Krauhs for editorial assistance. This study was funded by the NASA Human Research Program’s Human Health Countermeasures Element.
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CONCLUSIONS
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Increased iron status and chronic, low-dose radiation exposure led to oxidative stress, immune system dysregulation, alterations of colon microbial metabolism, and changes in gene expression in the intestinal mucosa. Radiation exposure consistently induced dysregulation of both leukocyte distribution and functional capacity of the immune system. To a lesser degree, the elevated-iron diet also negatively influenced immunocyte functional responses, at times in an interactive fashion with radiation exposure. Significant radiation effects on localized mucosal expression of genes for TNF-␣ and IL-6 and reduced levels of the same cytokines in circulation could indicate that the intestinal tract, which harbors the largest and most complex immune cell population in the body, could have an effect on systemic acute or chronic inflammatory responses after radiation exposure. Astronauts are exposed to both increased iron stores and radiation during spaceflight, and this exposure raises concerns about crew health. These concerns will be greater on exploration missions beyond low Earth orbit, with longer durations and greater exposure to radiation. Studies such as these will be critical in understanding integrated physiological responses to spaceflight and ultimately in developing countermeasures to any pathological adaptations, particularly in light of the emerging field of osteoimmunology, which links perturbations in immune health with bone loss (98). The authors thank the staff of the NASA Johnson Space Center Nutritional Biochemistry Laboratory for their assistance in processing and analyzing the samples, and in all aspects of carrying out this project. The authors thank Jane 1496
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Received for publication September 9, 2013. Accepted for publication November 26, 2013.
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