Abstract
Macrophages (Mϕ) with the M2b phenotype (Pheno2b-Mϕ) in bacterial translocation sites have been described as cells responsible for the increased susceptibility of mice with gastrointestinal acute radiation syndrome to sepsis caused by gut bacteria. In this study, we tried to reduce the mortality of mice exposed to 7–10 Gy of gamma rays by controlling Pheno2b-Mϕ polarization in bacterial translocation sites. MicroRNA-222 was induced in association with gamma irradiation. Pheno2b-Mϕ polarization was promoted and maintained in gamma-irradiated mice through the reduction of a long noncoding RNA growth arrest–specific transcript 5 (a CCL1 gene silencer) influenced by this microRNA. Therefore, the host resistance of 7–9-Gy gamma-irradiated mice to sepsis caused by bacterial translocation was improved after treatment with CCL1 antisense oligodeoxynucleotide. However, the mortality of 10-Gy gamma-irradiated mice was not alleviated by this treatment. The crypts and villi in the ileum of 10-Gy gamma-irradiated mice were severely damaged, but these were markedly improved after transplantation of intestinal lineage cells differentiated from murine embryonic stem cells. All 10-Gy gamma-irradiated mice given both of the oligodeoxynucleotide and intestinal lineage cells survived, whereas all of the same mice given either of them died. These results indicate that high mortality rates of mice irradiated with 7–10 Gy of gamma rays are reducible by depleting CCL1 in combination with the intestinal lineage cell transplantation. These findings support the novel therapeutic possibility of victims who have gastrointestinal acute radiation syndrome for the reduction of their high mortality rates.
Introduction
Whole-body gamma irradiation induces severe damages to the gastrointestinal (GI) tract (1–4). In our previous studies in a model of sepsis caused by bacterial translocation (5), mice exposed to a nonlethal dose (5 Gy) of whole-body gamma irradiation (5-Gy WBI-mice) were shown to be highly susceptible to sepsis stemming from bacterial translocation. Gut bacteria–associated sepsis, however, does not develop in healthy mice (5, 6) because pathogens invaded from intestinal tracts are rapidly eliminated by host defense effector cells at the translocation sites (mesenteric lymph nodes [MLNs] and lamina propria [LP]) (3). A major host antibacterial effector cell against bacterial translocation has been characterized as an IL-12+CD38+iNOS+ F4/80+ cell (Pheno1-Mϕ) (5, 6), and sepsis stemming from Enterococcus faecalis oral infection does not develop in healthy mice with interchangeable macrophages (Mϕ) in the bacterial translocation sites (5, 6). Interchangeable Mϕ easily switch to Pheno1-Mϕ under bacterial Ag stimulation (5–7). However, the pathogens that translocate from the intestinal tracts are not eliminated by the Mϕ distributed in the LP and MLNs of 5-Gy WBI-mice (5), although Mϕ are highly resistant against gamma irradiation (8). Mϕ isolated from the MLNs of 5-Gy WBI-mice are characterized as CCL1+IL-10+miR-27a+ F4/80+ cells (Mϕ with the M2b phenotype [Pheno2b-Mϕ]) that inhibit the Mϕ switching to Pheno1-Mϕ (5). The increased susceptibility of 5-Gy WBI-mice to E. faecalis oral infection has been completely improved after elimination of Pheno2b-Mϕ by CCL1 antisense oligodeoxynucleotide (ODN), an inhibitor of CCL1 (5), which is an essential chemokine on the prolongation of Pheno2b-Mϕ (9).
In the current study, we tried to reduce the mortality of mice with GI acute radiation syndrome (GIARS) (GIARS-mice; mice exposed to 7–10 Gy of whole-body gamma irradiation) through controlling Pheno2b-Mϕ polarization. As in 5-Gy WBI-mice, Pheno2b-Mϕ were isolated from the LP/MLNs of 7–10-Gy GIARS-mice. Unlike 5-Gy WBI-mice, 7-Gy GIARS-mice died within 3 wk of irradiation without any infections of external pathogens. These mice did not die after decontamination with an antibiotic mixture, but they were shown to be very susceptible to enterococcal oral infection, a model used for gut bacteria–associated sepsis. This indicates that the mortality of 7-Gy GIARS-mice, not exposed to external pathogens, is associated with bacterial translocation. Therefore, 7–9-Gy GIARS-mice survived for a month after gamma irradiation when Pheno2b-Mϕ were eliminated from these mice by treatment with CCL1 antisense ODN. However, the ODN treatments did not prevent the deaths of 10-Gy GIARS-mice. Severe GI damage (measured through the decreased crypt number and crypt regeneration in the ileum) was observed in 10-Gy GIARS-mice, and the damage was markedly improved after the transplantation of intestinal lineage cells differentiated from murine embryonic stem cells (ES-ICs). ES-ICs were a mixture of enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. The 10-Gy GIARS-mice subjected to ES-IC transplantation and the ODN treatment in combination did not die for a month or more. These results indicate that high mortality rates of 7–10-Gy GIARS-mice can be reducible by the CCL1 antisense ODN treatment, with higher Gy levels needing an ES-IC transplantation in addition.
Materials and Methods
Mice and irradiation
BALB/c mice (9–12-wk-old pathogen-free male and female mice) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were exposed to 5 or 7–10 Gy of whole-body gamma irradiation with a [137Cs]-ray (0.662 MeV) irradiator (Mark I Model 30; J.L. Shepherd & Associates, San Fernando, CA) at a dose rate of 1.05 Gy/min, which was reduced from the 5.08-Gy/min via lead attenuators. Mice exposed to 5 Gy of gamma rays were referred to as 5-Gy WBI-mice, and mice exposed 7–10 Gy of gamma rays were referred to as GIARS-mice. Sepsis does not develop in 5-Gy WBI-mice without any infections of external pathogens, whereas it develops in GIARS-mice. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston (Institutional Animal Care and Use Committee approval no.: 0906044).
Reagents, cells, bacteria, and media
l-alanyl-2-phenylglycine-1,1-dimethylethyl ester, mitomycin C, and M15 feeder cells (a source of mesoderm-derived growth factors) were purchased from Sigma-Aldrich (St. Louis, MO). Murine embryonic stem cells (day 3.5 blastocysts from BALB/cJ mice) were obtained from Jackson Laboratory. E. faecalis (29212 strains), purchased from the American Type Culture Collection (Manassas, VA), was cultured in tryptic soy broth for the infection experiments (5, 6, 10). The cultures were centrifuged at 2000 × g for 15 min, and the bacterial pellet was suspended in PBS. The number of bacteria in the suspension was counted using a hemocytometer and adjusted to give the approximate desired inocula. The inocula were verified by serial 10-fold dilutions of the bacterial suspensions and plated on tryptic soy agar. Heat-killed E. faecalis was prepared by heating bacteria at 65°C for 30 min (5, 6, 10).
N-[(3,5-difluorophenyl) acetyl]-ES-ICs
The differentiation of murine embryonic stem cells to definitive endoderm (E-cadherin+Cxcr4+) was induced by activin A and FGF2. Briefly, murine embryonic stem cells were plated at 1 × 106 cells/ml in 100-mm cell culture dish precoated with an M15 cell feeder layer (3 d of cultivation at 7 × 104 cells/ml), which was previously treated with mitomycin C (10 μg/ml for 4 h) and washed with PBS 3 times. The cells were cultured in DMEM containing 20 ng/ml activin A, 50 ng/ml FGF2, 10% FBS, and 4500 mg/ml glucose for 5 d, as previously described (11). For differentiation to intestinal lineage cells, E-cadherin+Cxcr4+ cells sorted from these cells were further cultured on the same feeder cells in DMEM containing 5 μM bromoindirubin-3′-oxime (a Wnt/β-catenin activator), 10 μM 1-dimethylethyl ester (a Notch signaling pathway inhibitor), 10% knockout serum replacement, and 2000 mg/ml glucose for 10 d. Using flow cytometry, obtained cells were determined as ES-ICs consisting of enterocytes (Cdx2+), goblet cells (Muc2+), enteroendocrine cells (Chga+), and Paneth cells (Lyz1+) (11). Obtained ES-ICs were suspended in PBS at a concentration of 1 × 107 cells/ml, and then these cells (0.2 ml) were transplanted i.v. to 10-Gy GIARS-mice 2 and 4 d postirradiation.
Bacterial translocation and subsequent sepsis
The severity of infectious complications caused by spontaneous bacterial translocation in GIARS-mice was evaluated by 1) growth of bacteria in the blood, MLNs, liver, and kidneys and 2) the mortality rates of the test group mice in comparison with those of the control group mice. The quantity of bacteria in organ specimens was measured as previously described (5, 6). In some experiments, sepsis was induced by E. faecalis oral infection in GIARS-mice after antibiotic decontamination. Thus, GIARS-mice were decontaminated for 12 d (7 d before to 5 d after gamma irradiation) by an antibiotic mixture (4 mg/ml penicillin, streptomycin and bacitracin) in the drinking water (5, 6, 12, 13). When GIARS-mice were orally treated with the antibiotic mixture, the number of microbiota was dramatically reduced (102 or 4 × 104 CFU/g ileum of bacteria in mice 2 or 7 d after the end of the antibiotic treatment, Supplemental Fig. 1). In GIARS-mice without the antibiotic decontamination, 1010 CFU/g ileum or more of bacteria were detected 7 d postirradiation. For the stabilization of E. faecalis oral infection, decontaminated mice were injected orally with lansoprazole (a proton-pump inhibitor, 0.5 mg per mouse) on the day of the final antibiotic treatment (5, 6, 10). Two days after lansoprazole treatment, these mice were infected orally with 106 CFU per mouse of E. faecalis. Bacterial translocation and subsequent sepsis in decontaminated GIARS-mice (dGIARS-mice) were confirmed by using green fluorescent protein (GFP)-labeled E. faecalis. For the tracking of orally infected bacteria, pMV158–GFP plasmid construction was established in Escherichia coli STBL3 and then transferred to E. faecalis by electrotransformation, as previously described (14). E. faecalis expressing GFP was detected by electroporation with pMV158–GFP plasmid using a Bio-Rad Gene Pulser Electroporator (Bio-Rad Laboratories, Richmond, CA). The transformed E. faecalis was selected on tryptic soy agar supplemented with 4 μg/ml tetracycline for 16 h. The transformed cells from single colony were grown in tryptic soy broth for 5–10 h, and then GFP gene expression was induced by 2% maltose for 6 h (GFP-labeled E. faecalis). Two days postinfection with GFP-labeled E. faecalis or nonlabeled E. faecalis (1 × 106 CFU per mouse), the liver and kidneys were removed, and a half of each organ was treated with collagenase IV (1 mg/ml) for 20 min at 37°C. E. faecalis in the cell suspension was detected by flow cytometry. Also, the remaining half was embedded in optimal cutting temperature compound (Tissue-Tek; Sakura Finetek, Torrance, CA). Frozen tissue sections (5 μm) were prepared using Leica CM350 S cryostat (Leica Biosystems, Wetzlar, Germany) and analyzed by Olympus BX51 fluorescence microscope (Olympus, Melville, NY).
Characterization of MLN-Mϕ isolated from 7–10-Gy GIARS-mice
As an Mϕ population, F4/80+ cells were prepared from the MLNs of decontaminated normal mice (dNormal mice), decontaminated 5-Gy WBI-mice, and 7–10-Gy dGIARS-mice, as previously described (5, 6, 15). In the majority of experiments, MLN-Mϕ were isolated in 7-Gy dGIARS-mice 7 d postirradiation. As for Pheno2b-Mϕ, the numbers of CCL1+IL-10+ cells in the MLN-Mϕ population were determined by flow cytometry, as previously described (5, 6). Flow cytometry data were analyzed in FlowJo 10.2 software (Tree Star, Ashland, OR).
Gene expression analyses
To determine the role of GAS5 in Pheno2b-Mϕ polarization, MLN-Mϕ from 7-Gy dGIARS-mice 7 d postirradiation were transduced with GAS5 gene via a lentiviral vector, as previously described (16). Murine GAS5 cDNA was amplified from pCMV–Sport6–GAS5 plasmid and cloned into pLenti7.3/V5-TOPO vector (pLenti7.3-GAS5) and lentiviruses were prepared using HEK293FT cells, as described in the manufacturer’s protocol. Seventy-two hours after transfection, supernatants were filtered (0.45-μm filter), and the virus suspension was titrated onto HEK293FT cells by flow cytometry using GFP expression from pLenti7.3/V5-TOPO vector. Mock viruses were generated by the same procedure using otherwise identical vector lacking GAS5 cDNA and served as a control lentivirus. In experiments, MLN-Mϕ from 7-Gy dGIARS-mice were exposed to GAS5 lentivirus or control lentivirus and cultured for 2 d. Cells obtained were stimulated with 100 ng/ml LPS in culture plates that were previously coated with 100 μg/ml murine IgG (16). Five days after stimulation, cells were analyzed for Pheno2b-Mϕ (CCL1+IL-10+ cells) by flow cytometry.
Western blotting analysis
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Recover from GI damage
The cross-sections of ileum were obtained from 10-Gy GIARS-mice at 7 d postirradiation, paraffin-embedded, sectioned, and stained with H&E. Stained sections were analyzed under LEICA DMLB optical microscope (Leica, Wetzlar, Germany) at magnification 100× (for counting the number of crypts) and 200× (for measuring the length of the villi) in three different filed. The images were captured by a CoolSNAP-Pro digital camera (Media Cybernetics, MD). All measurements were performed with the program AnalySIS Docu (Soft Imaging System, Munchen, Germany). The number of crypts per circumference was counted in well-oriented transverse cross-sections of the ilea from three different mice. The length of the villi was measured from its basal region, which coincided with the top of the crypts. A line was drawn from point on the base toward the point at the apex of the villus. The length of the line provided by the image analyzer was taken as the length of the villus.
Statistical analysis
Data are presented as mean ± SE. Results were statistically analyzed by a Student t test. Kaplan-Meier curves were constructed, and a log-rank test was used to compare the survival of the groups. The results were considered to be significant if the p value was lower than 0.05.
Results
Sepsis and Pheno2b-Mϕ detected in 7-Gy GIARS-mice with (dGIARS-mice) or without (GIARS-mice) antibiotic decontamination
In our previous studies (5), mice exposed to 5 Gy of whole-body gamma irradiation (5-Gy WBI-mice) have been shown to be very susceptible to various infections. However, these mice did not die without any infections of external pathogens. In contrast, all 7-Gy GIARS-mice died without any infections of external pathogens. Therefore, a role of infectious complications on the mortality of 7-Gy GIARS-mice was examined using decontaminated 7-Gy GIARS-mice (dGIARS-mice). The decontamination of these mice was performed by the administration of an antibiotic mixture by drinking water, as previously described (5, 6, 10). In the results, all 7-Gy GIARS-mice died within 24 d of the gamma irradiation, whereas the same mice decontaminated with an antibiotic mixture survived (Fig. 1A). Progressive growth of bacteria was seen in the MLNs and liver of 7-Gy GIARS-mice 6–12 d after the irradiation, whereas the bacterial growth in organs was not seen in 7-Gy dGIARS-mice (Fig. 1B). After E. faecalis oral infection, however, all of these mice died within 12 d of the infection (Fig. 1C). E. faecalis grew progressively in the MLNs and liver of these mice (Fig. 1D). Bacterial translocation and subsequent sepsis were confirmed in 7-Gy dGIARS-mice after oral infection with GFP-labeled E. faecalis. The labeled pathogen was detected in the liver and kidneys of these mice flow cytometrically and fluorescence microscopically (Supplemental Fig. 2A, 2B). The results shown in Fig. 1A–D indicate that the mortality of 7-Gy GIARS-mice is associated with sepsis caused by bacterial translocation.
Sepsis and Pheno2b-Mϕ demonstrated in 7-Gy GIARS-mice with or without antibiotic decontamination. (A) Mortality rates of 7-Gy GIARS-mice with (●, dGIARS-mice) or without (○, GIARS-mice) an antibiotic decontamination. **p < 0.001 (log-rank test). (B) Bacterial growth in the MLNs and liver of dGIARS-mice (●) and GIARS-mice (○) 1–12 d postirradiation. Data are displayed by the mean ± SE. *p < 0.01, **p < 0.001, Student t test. (C) Mortality rates of dGIARS-mice orally infected with (○) or without (●) E. faecalis. Seven days after gamma irradiation, dGIARS-mice were orally infected with 105 CFU per mouse of E. faecalis. **p < 0.001, log-rank test. (D) Bacterial growth in organs of dGIARS-mice infected with (○) or without (●) E. faecalis. Data are displayed by the mean ± SE. **p < 0.001, Student t test. (E) Appearance of Pheno2b-Mϕ in the MLNs of 5-Gy WBI-mice and 7–10-Gy GIARS-mice. MLN-Mϕ, isolated from various groups of mice 1–30 d postirradiation, were analyzed for CCL1+IL-10+ cells by flow cytometry. (F–H) MLN-Mϕ, isolated from dNormal mice and 7-Gy dGIARS-mice 7 d postirradiation were analyzed for the expression of typical Pheno2b-Mϕ biomarkers by real-time PCR; CCL1 mRNA (F), LIGHT mRNA (G), and miR-27a (H). Data are displayed as mean ± SE from three independent experiments. Data are representative of two (A–D) or three (E–H) independent experiments. Three (E–H), four to five (B and D), or ten (A and C) mice per group were used in each independent experiment. *p < 0.01, **p < 0.001 versus dNormal mouse MLN-Mϕ, Student t test.
In our previous studies (5, 6, 10), Pheno1-Mϕ have been characterized as host antibacterial effector cells against sepsis stemming from bacterial translocation, and Pheno2b-Mϕ are shown to be inhibitor cells on the Pheno1-Mϕ polarization (5, 17). Therefore, we tried to detect Pheno2b-Mϕ in the MLNs of 7–10-Gy GIARS-mice. All of these experiments were performed in mice decontaminated with an antibiotic mixture (dGIARS-mice). The time course of the appearance of CCL1+IL-10+ cells (Pheno2b-Mϕ) in the MLN-Mϕ of 5-Gy WBI-mice was shown as a control (5). As shown in Fig. 1E, Pheno2b-Mϕ were detected in the MLNs of 7–10-Gy dGIARS-mice 2–4 d postirradiation, when these Mϕ first appeared in the MLNs of 5-Gy WBI-mice 10 d postirradiation. MLN-Mϕ isolated from the MLNs of 7-Gy dGIARS-mice 7 d postirradiation were identified as Pheno2b-Mϕ by their biomarkers (Fig. 1F, CCL1 mRNA expression; 1G, LIGHT mRNA expression; 1H, miR-27a expression).
Pheno2b-Mϕ polarization in 7-Gy dGIARS-mice
We examined how normal Mϕ are switched to Pheno2b-Mϕ after the gamma irradiation. miR-222, a promoter of DNA damage repairing (18, 19), has been described as an inhibitor of GAS5 expression (20). GAS5 is known as a silencer of CCL1 (21), which is an essential chemokine for the Pheno2b-Mϕ polarization (9). A group of microRNAs drives the RNA degradation through the activation of nonsense-mediated RNA decay (NMD) pathway (the UPF1 phosphorylation) (22, 23). Therefore, the expression of miR-222 and GAS5 RNA in MLN-Mϕ from 7-Gy dGIARS-mice was investigated. In the results, the expression of miR-222 was shown to be minimal in the MLN-Mϕ of dNormal mice, whereas it was markedly increased (4-fold, Fig. 2A) in the MLN-Mϕ of 7-Gy dGIARS-mice. Similar increased expression of miR-222 was seen in MLN-Mϕ from 8–10-Gy dGIARS-mice (data not shown). As shown in Fig. 2B, the expression of GAS5 RNA was greatly reduced in the MLN-Mϕ of 7-Gy dGIARS-mice. GAS5 RNA expression is reduced through the UPF1 phosphorylation of the NMD pathway (16). Therefore, the phosphorylation of UPF1 in the total cellular proteins, derived from MLN-Mϕ from 7-Gy dGIARS-mice, were analyzed by Western blotting (Fig. 2C, left panel) and quantified by densitometric analysis (Fig. 2C, right panel). In the results, phosphorylated UPF1 increased (6.5-fold) in MLN-Mϕ from 7-Gy dGIARS-mice, as compared with those of dNormal mice. In the next experiments, a role of GAS5 on the prolongation of Pheno2b-Mϕ was investigated using Pheno2b-Mϕ from the MLNs of 7-Gy GIARS-mice after transduction with GAS5 gene. GAS5 gene transduction to Pheno2b-Mϕ was performed in cultures of Mϕ with GAS5 gene-encoded lentivirus for 2 d (16). In the results, Pheno2b-Mϕ switched to non–Pheno2b-Mϕ (IL-10−CCL1−Mϕ) after transduction with GAS5 RNA (Fig. 2D, left panel). Also, these Pheno2b-Mϕ transduced with GAS5 RNA did not switch back to Pheno2b-Mϕ, even when they were stimulated with a typical inducer of Pheno2b-Mϕ (Fig. 2D, right panel). These results indicate that the Pheno2b-Mϕ polarization is maintained through GAS5 RNA reduction influenced by miR-222, which is expressed in association with gamma irradiation.
Pheno2b-Mϕ polarization in 7-Gy dGIARS-mice. Mϕ from the MLNs of dNormal mice and 7-Gy dGIARS-mice 7 d postirradiation were tested for the expression of miR-222 [(A), real-time PCR], GAS5 RNA [(B), real-time PCR], and UPF1 phosphorylation [(C), Western blotting, left panel]. Phosphorylated UPF1 level was quantified by densitometric analysis [(C), right panel]. Data are displayed as the mean ± SE. *p < 0.01, **p < 0.001 versus dNormal mouse MLN-Mϕ, Student t test. (D) Pheno2b-Mϕ (MLN-Mϕ from 7-Gy dGIARS-mice) were exposed to GAS5 lentivirus or control lentivirus and cultured for 2 d (left panel). Cells obtained were stimulated by a mixture of LPS and immune complex (LPS/IC) for 5 d for the Pheno2b-Mϕ polarization (right panel). Cells obtained were analyzed for CCL1+IL-10+ cells by flow cytometry (left panel). Data are displayed as the mean ± SE. **p < 0.001 versus Pheno2b-Mϕ exposed to control virus, Student t test. (A–D) Data are representative of three independent experiments. Three mice per group were used in each independent experiment.
Survival rates and bacterial growth in the organs of 7–9-Gy GIARS-mice treated with CCL1 antisense ODN
In our previous studies (5), MLN-Mϕ from 5-Gy WBI-mice have been switched to non–Pheno2b-Mϕ after treatment with CCL1 antisense ODN, and the resistance of 5-Gy WBI-mice to E. faecalis oral infection improved markedly after treatment with the ODN. Therefore, we examined the effect of the ODN on the survival of 7–9-Gy GIARS-mice. Additionally, bacterial growth in the blood, MLNs, liver, and kidneys of the ODN-treated mice 10–12 d after the gamma irradiation was measured. In these experiments, various GIARS-mice were treated with CCL1 antisense ODN or scrambled ODN (control) twice daily until end of the experiments starting 3 d postirradiation, according to the results in the previous studies (5). Although all 7–9-Gy GIARS-mice treated with scrambled ODN died, all of the 7- and 8-Gy GIARS-mice and 70% of the 9-Gy GIARS-mice treated with CCL1 antisense ODN survived (Fig. 3, left panels). A large number of bacteria were detected in the blood and organs of 7–9-Gy GIARS-mice treated with scrambled ODN, whereas decreased bacterial growth in the blood and organs was seen in 7–9-Gy GIARS-mice treated with the ODN (Fig. 3, middle and right panels). These results indicate that the radiation-associated mortality of 7–9-Gy GIARS-mice are reduced after the Pheno2b-Mϕ elimination by CCL1 antisense ODN.
Survival rates and bacterial growth in the organs of 7–9-Gy GIARS-mice treated with CCL1 antisense ODN. GIARS-mice exposed to 7 Gy (A), 8 Gy (B), and 9 Gy (C) of gamma rays were treated s.c. with 25 μg per mouse of CCL1 antisense ODN (●) or scrambled ODN (○) twice daily until end of experiments starting 3 d postirradiation. The survival of these mice was monitored twice a day for 30 d [(A–C), left panels]. Data are representative of two independent experiments. Ten to fifteen mice per group were used in each independent experiment. Bacterial growth in the blood [(A–C), middle panels] and various organs [(A–C), right panels] of these mice were determined by the colony counting method. Data are representative of two independent experiments. Four to five mice per group were used in each independent experiment. Data are displayed by the mean ± SE. *p < 0.01, **p < 0.001, Student t test.
Recovery of GI damage and antibacterial resistance of 10-Gy GIARS-mice
In contrast, 10-Gy GIARS-mice died even when they were decontaminated with an antibiotic mixture (Fig. 4A) or treated with CCL1 antisense ODN (Fig. 4B). These results indicate that the mortality of 10-Gy GIARS-mice is not directly associated with gut bacteria–associated sepsis influenced by the Pheno2b-Mϕ polarization. As shown in Fig. 4D, severe damages to the crypt/villus units were demonstrated in the ileum of 10-Gy GIARS-mice. Therefore, we tried to heal the GI damage of 10-Gy GIARS-mice in the first step, and then the infection-associated mortality of these mice was reduced by the Pheno2b-Mϕ elimination using CCL1 antisense ODN. In the results, 7 d postirradiation, GI damage in the ileum of 10-Gy GIARS-mice was markedly improved after the transplantation of ES-ICs (2 × 106 cells per mouse, i.v., 2 and 4 d postirradiation) (Fig. 4D). ES-ICs were created from murine embryonic stem cells after the activation of Wnt/β-catenin and inhibition of Notch signaling pathways (Fig. 4C), as previously described (11). ES-ICs were shown to be a mixture of enterocytes (Cdx2+, 68.9%), goblet cells (Muc2+, 4.6%), enteroendocrine cells (Chga+, 16.7%), and Paneth cells (Lyz1+, 5.8%) (Supplemental Fig. 3). Also, the number of crypts and the length of villi were recovered in the ileum of 10-Gy GIARS-mice transplanted with ES-ICs (Fig. 4D, 4E). These results indicate that crypt/villus units are significantly renewed in 10-Gy GIARS-mice transplanted with ES-ICs. Therefore, in subsequent studies, the mortality rate of 10-Gy GIARS-mice was examined after the transplantation of ES-ICs and treatment with CCL1 antisense ODN in combination. As a result, all of the 10-Gy GIARS-mice given both CCL1 antisense ODN and ES-ICs survived, whereas mice given either of them died within 20 d of the gamma irradiation (Fig. 4F).
GI damages and antibacterial resistance of 10-Gy GIARS-mice. The survival of 10-Gy GIARS-mice (○) and dGIARS-mice (●) was monitored twice a day for 30 d (A). Data are representative of two independent experiments. Ten mice per group were used in each independent experiment. GIARS-mice expose to 10 Gy of radiation were treated s.c. with 25 μg per mouse of CCL1 antisense ODN (●) or scrambled ODN (○) twice daily until end of experiments starting 3 d after the irradiation, and the survival of these mice was monitored twice daily for 30 d (B). Data are representative of two independent experiments. Fifteen mice per group were used in each independent experiment. ES-ICs (C), differentiated from murine embryonic stem cells, were transplanted (2 × 106 cells per mouse, i.v.) to 10-Gy GIARS-mice 2 and 4 d postirradiation. Light microscopic images (original magnification ×100) of the H&E ileum section were obtained from these mice 7 d postirradiation (D). Data shown in (C) and (D) are representative of three independent experiments. Three mice per group were used in each independent experiment. Light microscopic images obtained in (D) were digitized, and the number of crypts per circumstance and the length of villi were measured 7 d postirradiation by AnalySIS Docu software (E). Data are shown as mean ± SE. *p < 0.001 versus GIARS-mice treated with media, Student t test. (F) GIARS-mice exposed to 10 Gy of radiation were given ES-ICs alone (2 × 106 cells per mouse, i.v.) 2 and 4 d postirradiation (▲), 25 μg/mouse of CCL1 antisense ODN alone (●), or both (□). Untreated 10-Gy GIARS-mice served as a control (○). The survival of these mice was monitored twice a day for 30 d. Data are representative of two independent experiments. Ten mice per group were used in each independent experiment. *p < 0.001 versus control, log-rank test.
Discussion
Gut bacteria–associated sepsis is a serious concern in persons who suffer from GIARS caused by lethal doses (6–15 Gy) of gamma irradiation (1–4). In our previous studies, Pheno2b-Mϕ were predominantly isolated from the MLNs of mice exposed to a nonlethal dose (5 Gy) of gamma rays, and these mice were shown to be highly susceptible to E. faecalis oral infection, a model used for sepsis stemming from bacterial translocation (5). Pheno2b-Mϕ located in the bacterial translocation sites of mice exposed to 5 Gy of gamma rays were characterized as cells responsible for the increased susceptibility to E. faecalis translocation (5). However, these mice did not die without any infections with external pathogens (5). In contrast, without any infections with external pathogens, all GIARS-mice exposed to 7–10 Gy of gamma rays died within 2–3 wk of the gamma irradiation. Therefore, in this study, we tried to reduce the radiation-associated mortality of 7–10-Gy GIARS-mice. Infectious complications shown by bacterial growth in the blood and organs were demonstrated in 7–9-Gy GIARS-mice. After decontamination with an antibiotic mixture, however, these mice (dGIARS-mice) were shown to be resistant to radiation-associated mortalities. In our studies, bacterial load dramatically reduced to 102∼103 CFU/g ileum of normal mice 2 d after 7-d decontamination by an antibiotic mixture. Ten days after the end of decontamination, bacterial number in the intestine of these mice recovered up to 1010∼1011 CFU/g ileum (the same as compared with the levels shown before the antibiotic treatment). The composition of gut microbiota seemed to be altered by the antibiotic decontamination. In fact, the majority (70%) of bacteria in the ileum of mice just after the decontamination was shown to be Gram-negative, as compared with that of Gram-positive bacteria (65%) demonstrated in the ileum of normal mice. Gram-negative bacteria (68%) were counted as a majority of microbiota in the ileum of mice 10 d after the decontamination. Then, mice 10 d after the antibiotic decontamination were exposed to 7-Gy whole-body gamma irradiation. In the results, all of these mice did not die for a month, as compared with the same mice without decontamination, which died within 20 d of the gamma irradiation. These results suggest that the composition of gut microbiota influenced by an antibiotic mixture is not a very important factor in the decontamination-associated survival of 7-Gy GIARS-mice. Also, all of the 7-Gy dGIARS-mice died when they were subjected to a model of sepsis caused by bacterial translocation. These results indicate that the mortality of 7-Gy GIARS-mice is associated with bacterial translocation. In addition, we measured the endotoxin levels in the sera of normal mice and 7-Gy GIARS-mice using an LAL Chromogenic Endotoxin Quantitation Kit. The concentration of endotoxin in the sera of normal mice was 0.65–1.1 EU/ml, whereas 1.4, 5.0, or 12 EU/ml endotoxin was detected in the sera of 7-Gy GIARS-mice 4, 7, or 10 d postirradiation. These results indicate that some infections occur in mice irradiated with gamma rays. Intestinal permeability is well known to be increased in mice exposed to whole-body gamma irradiation. We have previously measured the intestinal permeability of GIARS-mice by the FITC–dextran method. It was increased five times in 10-Gy GIARS-mice 7 d postirradiation, as compared with that of normal mice, whereas a slight increase of intestinal permeability (1.1–1.4 times) was demonstrated in 7-Gy GIARS-mice 7 d postirradiation. From these, the increased intestinal permeability seems to be involved in the bacterial translocation and sepsis developed in mice exposed to higher doses of gamma rays. The Pheno2b-Mϕ polarization occurred in the MLNs of 7–10-Gy GIARS-mice markedly earlier than in the MLNs of 5-Gy WBI-mice, and it was controlled in these mice by the administration of CCL1 antisense ODN. Pheno2b-Mϕ require CCL1 for their prolongation (9), and the ODN suppresses the CCL1 gene expression in Pheno2b-Mϕ (5, 6, 9, 17). Therefore, the radiation-associated mortality of 7–9-Gy GIARS-mice was dramatically reduced after treatment with CCL1 antisense ODN.
The mortality of 10-Gy GIARS-mice, however, was not effectively reduced by the ODN treatment. It has been shown that the lethal doses of gamma irradiation cause the depression of intestinal stem cells (24–26), which exist at the intestinal crypt base. The loss of self-renewing crypt/villus units causes the diminished length of villi in the intestine. As such, the number of crypts and the length of villi were severely reduced in the ileum of 10-Gy GIARS-mice 7 d postirradiation, as compared with those of the ileum in normal mice. Such GI damage in the ileum of 10-Gy GIARS-mice was not seen in mice exposed to a nonlethal dose (5 Gy) of gamma rays; thereby, gut bacteria–associated sepsis was not commonly developed in these mice (5). When 10-Gy GIARS-mice were transplanted with ES-ICs, both the number of crypts and the length of villi recovered. ES-ICs were a mixture of enterocytes, goblet cells, enteroendocrine cells, and Paneth cells, which were differentiated from murine embryonic stem cells (day 3.5 blastocysts from BALB/cJ mice) under the cultivation with a Wnt/β-catenin activator and a Notch signaling pathway inhibitor for 10 d, as reported previously (11). GIARS-mice exposed to 10 Gy of radiation did not survive even when they were transplanted with ES-ICs alone or treated with CCL1 antisense ODN alone; however, all of the 10-Gy GIARS-mice given both ES-IC transplantation and CCL1 antisense ODN treatment survived for a month postirradiation. These results indicate that the mortality rates of 7–10-Gy GIARS-mice are reduced by treatment with CCL1 antisense ODN for 7–9-Gy and in combination with ES-IC transplantation for 10 Gy. In the ES-IC preparation used in this study, Lgr5+ intestinal stem cells were not included. To determine the paracrine effects of the ES-IC preparation on the recovery of crypt numbers and the length of villi in the ileum of 10-Gy GIARS-mice, further studies will be needed by the tracking of each cell component in ES-ICs after transplantation.
In our previous studies (5, 6, 10), Pheno1-Mϕ distributed in the bacterial translocation sites have been characterized as a major host defense effector cell against infectious complications stemming from gut-associated bacteria. Mϕ have been described to be radioresistant (8) and highly plastic and to undergo reprogramming with the emergence of a spectrum of distinct functional phenotypes in response to diverse signals derived from pathogens and injured/infected tissues (27–31). In our current study, following the stimulation with a bacterial Ag, dNormal mouse Mϕ switched to Pheno1-Mϕ in cultures, whereas 7-Gy dGIARS-mouse MLN-Mϕ did not. Thus, Pheno2b-Mϕ possess very low plasticity. The properties of Pheno2b-Mϕ were maintained for 7 d or more in cultures not supplemented with any cytokines or growth factors (9). Pheno2b-Mϕ disappeared when CCL1 was depleted from their cultures (9). CCL1 is an essential chemokine for prolonging the life of Pheno2b-Mϕ (9). Because Pheno2b-Mϕ are CCL1 producer cells (32, 33), they do not change their properties as long as CCL1 is present.
Pheno2b-Mϕ were originally demonstrated under costimulation of quiescent Mϕ via TLR4 and Fc receptors together (34). Subsequently, other stimuli including apoptotic neutrophils and PGE2 have been described as stimulators for the Pheno2b-Mϕ polarization (35). In the case of fungal infections, several components of the fungal cell wall have been shown as stimulators for the Pheno2b-Mϕ polarization via dectin-1 activation (36). Pheno2b-Mϕ inhibit the switching of Mϕ to Pheno1-Mϕ by suppressing the expression of KLF6, a critical molecular switch regulating the Mϕ polarization (37). With these stimulations, Mϕ influenced by the irradiation were shown to be switched to Pheno2b-Mϕ. Mϕ influenced by the irradiation expressed miR-222 excessively, and the Pheno2b-Mϕ polarization was promoted under the lesser expression of GAS5 RNA. miR-222 suppressed the GAS5 expression through the activation of the NMD pathway. Thus, the reduced expression of GAS5 RNA was shown to be responsible for the predominance of Pneno2b-Mϕ in GIARS-mice. The reduction of GAS5 RNA expression is maintained in Mϕ throughout the lifetime (∼3 wk), and mice with Pheno2b-Mϕ are susceptible to sepsis caused by gut-associated bacterial translocation. Therefore, we think that the increased susceptibility of the gamma-irradiated mice is maintained until the appearance of new Mϕ with GAS5 RNA. We have recently demonstrated that the level of HMGB1 increases (5- to 8-fold) in the sera of mice exposed to 7–9-Gy of gamma rays. This protein has been shown to induce miR-222 expression (38), which can bind directly to GAS5 and reduce its RNA level (20). miR-222 expression is minimal in normal mouse Mϕ, whereas the expression of miR-222 increases (4- to 7-fold) in the same Mϕ cultured with 100 ng/ml HMGB1 for 24 h. miR-222 expression was greatly inhibited by glycyrrhizin, an HMGB1 antagonist, in Mϕ cultures supplemented with HMGB1. Glycyrrhizin has been proved to bind directly to HMBG1 (Kd ∼150 μM, as shown by nuclear magnetic resonance and fluorescence studies) and interfere with the binding of HMGB1 to DNA in living cells (39, 40). Also, the interaction of HMGB1 with its receptors (RAGE and R ) is shown to be blocked by glycyrrhizin (41). Therefore, we will hypothesize that HMGB1 plays a role on the induction of miR-222 expression in GIARS-mice. This hypothesis will be examined in the following studies. In our studies, BALB/c mice are more susceptible to radiation-associated injury, as compared with C57BL/6 mice. BALB/c mice exposed to 9 Gy of gamma rays died within 2 wk of the irradiation. However, 20% of C57BL/6 mice did not die after the same gamma irradiation. The BALB/c strain of mice has a double-strand DNA relative repair defect (polymorphism in DNA-dependent protein kinase catalytic subunit) (42), and this defect seems to be involved in the enhanced radiation susceptibility. In this study, miR-222 gene expression in Mϕ was stimulated after exposure to a lethal dose of gamma rays, and GAS5 NMD was activated by miR-222. This resulted in the reduced GAS5 RNA in Mϕ. Because the reduction of GAS5 RNA causes the Pheno2b-Mϕ polarization influenced by LPS and immune complex in combination (16), the degree of radiation-induced miR-222 expression may differ between BALB/c mice and C57BL/6 mice. In our future studies, these questions will be examined.
Mϕ isolated from a bacterial translocation site organ (MLNs) were used in this study. As shown in Supplemental Fig. 4, peritoneal Mϕ from 7-Gy GIARS-mice also produced enough CCL1 and IL-10 (biomarkers of Pheno2b-Mϕ) in their cultures. However, Mϕ from the spleen of these mice produced a minimum number of these biomarkers. This indicates that Mϕ in the spleen of 7-Gy GIARS-mice are not completely polarized to the M2b phenotype. Mϕ in the bacterial translocation site organs seem to be the first host defense effector cells against invaded pathogens. Splenic Mϕ may play a role as host defense effector cells after spreading the invaded bacteria throughout the whole body.
Radiation-associated mast cell hyperplasia and activation have been demonstrated in the rectal tissues of patients treated with radiotherapy (43) and mice exposed 27 Gy of gamma rays on the colorectal region (43). Histamine released from activated mast cells increases vascular permeability. Mast cells and vasoactive intestinal peptide (a peptide hormone) have been described to be involved in the increased translocation of commensal and pathogenic live bacteria in colonic epithelial tissues from patients with irritable bowel syndrome (44). Therefore, we think that mast cells and some gut hormones play a role on the increased bacterial translocation in irradiated victims, in addition to Pheno2b-Mϕ. To clarify this, further studies will be required.
We have demonstrated that the mortality of 7–9-Gy GIARS-mice was associated with infectious complications stemming from gut microbiota. Pheno2b-Mϕ were predominantly isolated from the bacterial translocation sites (MLNs) of these mice. The mortality rates associated with bacterial translocation was markedly reduced in these mice after the elimination of Pheno2b-Mϕ. Although 10-Gy GIARS-mice died even after decontamination by the antibiotic mixture or CCL1 antisense ODN treatment, severe GI damage was considered to play a role on their mortalities. GI damage was markedly improved after transplantation with ES-ICs, and all of the 10-Gy GIARS-mice given both CCL1 antisense ODN and ES-ICs in combination survived for a month after the irradiation. Infectious complications were not severely developed in 10-Gy GIARS-mice subjected to both ODN treatment and ES-IC transplantation. In conclusion, the mortality rates of 7–10-Gy GIARS-mice are reducible by the CCL1 antisense ODN treatment, with higher Gy levels needing an ES-IC transplantation in addition.
Disclosures
The authors have no financial conflicts of interest.
Footnotes
This work was supported by National Institutes of Health Grant U01 AI107355 (to F.S.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- dGIARS-mice
- decontaminated GIARS-mice
- dNormal mice
- decontaminated normal mice
- ES-IC
- intestinal lineage cell differentiated from murine embryonic stem cell
- GAS5
- growth arrest–specific transcript 5
- GFP
- green fluorescent protein
- GI
- gastrointestinal
- GIARS
- GI acute radiation syndrome
- GIARS-mice
- mice with GIARS
- 5-Gy WBI-mice
- mice exposed to a nonlethal dose (5 Gy) of whole-body gamma irradiation
- LP
- lamina propria
- Mϕ
- macrophage
- miR
- microRNA
- MLN
- mesenteric lymph node
- NMD
- nonsense-mediated RNA decay
- ODN
- oligodeoxynucleotide
- Pheno2b-Mϕ
- Mϕ with the M2b phenotype.
- Received November 1, 2017.
- Accepted April 20, 2018.
- Copyright © 2018 by The American Association of Immunologists, Inc.