The Radio-mitigative Effect of CpG-Oligodeoxynucleotides on Mice After Exposure to Carbon Ions Radiation.

Background: Heavy ion radiation constitutes a major health risk for astronaut in space ight, potential damage to healthy tissues surrounding the tumor target along its penetrating path should still be considered in hydrotherapy. Therefore, there is a demand for reliable countermeasure against heavy ions radiation. In this study, we will estimate the radiomitigative effect of CpG-ODN on immune tissues after carbon ions radiation (CIR). Methods: Firstly, the 30 days’ survival of mice was observed, peripheral blood cell was counted, the injury of three principal immune tissues (including bone marrow, thymus and spleen) was evaluated by histological examination, apoptosis and double strand breaks (DSB) were detected by TUNEL staining and γ-H 2 AX immunohistochemistry respectively, and cytokine (G-CSF, IL-6 and TNF-α) was measured by ELISA assay. Results: the 30 days’ survival improved, the injury of three principal immune tissues were obviously ameliorated, the number of γ-H 2 AX foci and TUNEL-positive nuclei decreased, and G-CSF, IL-6 and TNF-α expression increased by CpG-ODN treatment after CIR. Conclusion: CpG-ODN could enhanced mice survival, and ameliorate immune tissues injury, the mechanism may be that CpG-ODN induced cytokines production and inhibited the double strand breaks (DSB) and apoptosis in order to stimulate the generation and mobilization of the immune cells and reestablish immune system to combat bacterial infections.


Introduction
Heavy ions radiation is densely ionizing, which have a maximum dose deposition upon entry in a medium, the depth-dose distribution is characterized by a low dose plateau upon entrance and pronounced maximum, the radiant energy is not only deposited by the primary interaction but also by secondary electrons, which may travel considerable distances from the core. Therefore, it produces more irreparable DNA break and death to cells in comparison with X rays or γ rays [1][2][3][4]. In space ight, heavy ion radiation constitutes a major health risk for astronaut, because unlike other radiation types in outer space, current shielding is unable to provide effective protection again heavy ions radiation [5,6]. In hydrotherapy, although heavy ions radiation as an in innovative modality of high precision tool for cancer therapy, potential damage to healthy tissues surrounding the tumor target along its penetrating path should still be considered [1]. Given space radiation protection and cancer therapy, there is a demand for reliable countermeasure against heavy ions radiation.
Recently, more and more attention has been focused on Toll-like receptor (TLR) ligands as radiation countermeasure [7]. TLRs are the key sensor elements of innate immunity and are evolutionary conserved receptors,which recognize highly conserved structural motifs known as pathogen-associated molecular patterns (PAMPs). Stimulation of TLRs by PAMPs, initiates signaling cascades which lead to the activation of transcription factors, such as NF-κB. TLR signaling results in a variety of cellular response including the production pro-in ammatory cytokines and effector cytokines which direct the adaptive immune response [8]. TLR ligands are characteristic of large group of pathogens, and cannot be easily mutated, which mediated speci cally activation of TLRs. TLR ligands are appealing as potential radiation countermeasure since they have little effect besides activating TLRs [9].
Our previous research has shown that TLR9 ligand(CpG-oligodeoxynucleotides, CpG-ODN)could reduce bone marrow after γ-rays and protect RAW264.7 cell in vitro against heavy ions radiation [10]. However, the effect of CpG-ODN against injury induced by heavy ions radiation in vivo is still largely unknown.
Immune tissue injury is of great important parameter of radiation injury as they are potentially life threatening. Carbon ions radiation (CIR) is a speci c type of heavy ions radiation. In this regard based on ground experiments at accelerators, the present study has been undertaken to estimate the mitigative effect of CpG-ODN on immune tissue after exposure to CIR.

Mice
Male C57BL/6 (20~22 g), 10~12 weeks' old were purchased from SLAC laboratory animals Co. Ltd (SLAC, Shanghai, China), and were kept under standard laboratory conditions (a temperature of 23 ± 2 ℃ with 24 h cycles of fresh air and 12 h light/dark cycle). Food and water were sterilized by 60 Co γ rays and high pressure, respectively.

Carbon ions radiation CIR
Mice was placed inside a specially designed and well-ventilated acrylic container with dimensions of 8.0 cm ×3.5 cm ×3.5 cm. the acrylic container was placed into the beam path at the entrance plateau throughout the exposure. Whole-body radiation of mice was performed using CIR at initial energy of 250 MeV/u and the average LET of 31.3 keV/μm. Each mouse received 5 Gy of radiation at a dose rate of 0.8 Gy/min. CIR was supplied by Heavy Ion Research Facility at Institute of Modern physics, Chinese Academy of Sciences (HIRF, Lanzhou, China). The acquisition of data (preset beams converted by doses of radiation) was automatically calculated and controlled by a microcomputer.

Treatment and Ethics approval
Mice were treated with either 50 μg CpG-ODN (250 μg/ml) or 0.2 ml PBS via intraperitoneal (i.p) injection. CpG-ODN was given 30 min, 24 h and 48 h after CIR. The effective dosage and the time points of CpG-ODN treatment in this study was selected based on data from previous study [11] Mice were randomly divided into four groups as follows: a normal group (the non-irradiated mice with PBS treatment), a CpG-ODN group (the non-irradiated mice with CpG-ODN treatment), a CIR control group (the irradiated mice with PBS treatment) and a CIR plus CpG-ODN group (the irradiated mice with CpG-ODN treatment). All studies were performed under the guidelines and protocols of the Institutional Animals Care and Use Committee of the Lanzhou General Hospital of PLA (IACUC approval#15010936) according to the Guide for Care and Use of Laboratory Animals published by US NIH (publication No 96~101).

Survival
Irradiated mice were observed to monitor survival for 30 days after CIR. Moribund mice were euthanized.
On 31th day, surviving mice were euthanized by cervical dislocation. Data were expressed as percentage survival. 28 mice per groups were used for the experiments. The Mean Survival Time (MST) was calculated by Kaplan-Meier method.

Blood analysis
The peripheral blood was obtained from halothane-anesthetized mice and collected in tubes containing heparin, then mice were killed humanely by cervical dislocation. Whole blood samples were evaluated by automated hematology system (CA-700NVET, STAC Group, China). White blood cell (WBC), lymphocytes, granulocyte, and monocytes counts were determined. T cells and B cells were examined by immuno uorescence [23]. The speci c method is: 65 μl of 10 % formaldehyde was added to surplus 100 μl of blood and incubated for 10 min at room temperature, followed by the addition of 1ml Triton X-100 (diluted in PBS) to obtain 0.1 nal concentrations. After 30 min of incubation at room temperature, 1ml cold wash buffer was added. Samples were centrifuged, suspended in 1ml 50 % methanol diluted in PBS (stock stored at -20 °C), and incubated at 4 °C for a minimum of 10 min. All cells were stained blue by 4', 6-diamidino-2-phenylindole (DAPI, Sigma, USA). Rabbit polyclonal antibodies of CD3 (cat: 100203) and CD19 (cat: 115507) from Bio Legend were used to identify T cells and B cells respectively. These samples were examined and quanti ed under uorescent microscope (Nikon, Japan).
Bone marrow cell count and bone marrow histological examination two femurs were removed at the times of euthanasia. The bone marrow cells within one femur were ushed with PBS, and the number of live bone marrow cells were determined by a cell counter analyzer system (CASY, Innovates, Germany). The other femur was xed in 4 % paraformaldehyde for 24 h, treated with a formic acid sodium citrate decalci cation solution for 5 days, embedded in para n and stained with Hematoxylin and Eosin (H&E).

Organ index
Mice were weighted and then euthanized by cervical dislocation. The whole thymus and spleen were rapidly excised, their masses were measured. Relative organ index was calculated: relative organ index= organ mass (mg)/body mass (g).
TUNEL assay and γ-H 2 AX assay As description in M. Leonor Fernández-Murga's article [27] whole thymus and spleen were xed for 48 h in 10 % neutral buffered formalin, 5-μm section was prepared and stained with H&E. Apoptotic cells were detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining. 5-μm section was depara nized, permeabilized with proteinase K (20 μg/ml, Sigma) at 23 ℃ and rinsed 4 additional times with distilled water followed by incubation with a solution made up of TdT Unbound antibody was removed by several washing and goat anti-mouse antibody (1:100 Santa Cruz Santiago USA) was applied as a secondary labeled antibody for 30 min at room temperature. Slides were developed with DBA and stained with hematoxylin.

Statistical analysis
For the survival data, the difference in 30 days' survival between was analyzed by Kaplan-Meier plots. For other data, the difference between CIR control group and CIR plus CpG-ODN group was analyzed by the two-sided, non-paired Student's t-test. Data are expressed as mean ± standard error mean (SEM). The software analysis program SPSS 13.0 (release 12.0K; SPSS Inc. Chicago, USA) was used. All differences were considered signi cant if p was less than 0.05.

Effect of CpG-ODN on survival after CIR
Death is a typical endpoint in the study of radiation injury, which described as the most attributable consequence to immune system failure. Mice were irradiated with 5 Gy of CIR, it is important to note that in vivo C57BL/6 mice experiments where typically doses no more than 5 Gy of CIR are required to determine the sub-lethal dose [12]. Irradiated mice were monitored for 30 days, the number of mortalities in each group recorded daily. Exposure to 5 Gy of CIR caused mortality between 4 d and 14 d, the 30 days' survival only was 10.7 %. On the contrary, the mortality of irradiated mice treated by CpG-ODN occurred between 8 d and 12 d, the 30 days' survival was 57.1 %. This difference of survival was statistically signi cant ( g.1). To determine the radio mitigative effect of CpG-ODN, MST of mice was calculated. Compared with irradiated mice without CpG-ODN treatment, MST in irradiated mice with CpG-ODN treatment increased 11.1 days. these indicated that CpG-ODN enhanced mice survival after CIR.
Effect of CpG-ODN on WBC and bone marrow after CIR Peripheral blood WBC and bone marrow were not only the blood system, but also were immune principal components. It was generally agreed that radiation death in the sub-lethal dose range was due to reduction of WBC and impairment of bone marrow, which ultimately predisposed to infection, sepsis or even death [13]. Mice were irradiated with 5 Gy of CIR, WBC was detected at 1 d and 3 d. CIR lead to a rapidly decrease in number of WBC (including lymphocyte granulocyte and monocyte). Compared with irradiated mice without CpG-ODN treatment, WBC, lymphocyte, granulocyte and monocyte in irradiated mice with CpG-ODN treatment increased 1.38, 1.41, 1.30 and 1.40-fold at 1 d, and increased 3.00, 2.13, 3.83 and 2.00-fold at 3 d respectively ( g.2A, g.2B, g.2C and g.2D). Bone marrow histological examination showed that there was a signi cant reduction in bone marrow cell accompanied by typical apoptotic changes such as karyopyknosis, meanwhile, there were larger empty space and extensive interstitial hemorrhage with sinusoidal lling in bone marrow cavity at 1 d after CIR. In comparison, pathological alterations of irradiated mice with CpG-ODN treatment were less severe as evidenced by less bone marrow cell reduction, higher hemorrhage, and decreased edema. At 3 d, pathological changes in bone marrow cavity appeared 'empty'. There was a marked reduction in cell number with collapsed sinusoidal structures that were almost entirely destroyed, and extensive hemorrhage within bone marrow cavity ( g.2E). a nearly 1.5-fold raised in the number of bone marrow cell for irradiated mice with CpG-ODN treatment compared with irradiated mice without CpG-ODN treatment ( g.2F). These indicated that CpG-ODN increased the number of peripheral blood WBC and ameliorated bone marrow injury after CIR.

Effect of CpG-ODN on thymus after CIR
The thymus is also an immune principal component and most sensitive to radiation [14]. CIR caused thymus wastage. The thymus index decreased from 2.19 to 1.25 at 1 d and 0.66 at 3 d after CIR. Compared with irradiated mice without CpG-ODN treatment, the thymus index in irradiated mice with CpG-ODN treatment raised 8 % at 1 d and 26 % at 3 d. This difference was statistically signi cant at 3 d ( g.3A). Histological examination showed that the thymus cortices structure had almost disappeared, and medulla has severely atrophied, the thymocytes were decreased in number and scattered in the reticulum of medulla after carbon ions radiation. the medullar of irradiated mice with CpG-ODN treatment was thicker, which contained much more thymocytes that of irradiated mice without CpG-ODN treatment ( g.3B). Apoptosis and DSB of thymocytes were detected by TUNEL staining and γ-H2AX immunohistochemistry after CIR. As g.3C and gh.3D shown, compared with irradiated mice without CpG-ODN treatment, the number of γ-H2AX foci and TUNEL-positive nuclei were obviously decreases by CpG-ODN treatment. In addition, T cell in peripheral blood from thymus was detected by labeling CD3 ( g.3E). The number of T cell decreased rapidly and showed a reduction of 24.0 % and 79.0 % at 1 d and 3 d after CIR, CpG-ODN produced signi cant sparing effect on the irradiation-induced decrease, the number of T cell increased 1.21-fold at 1 d and 2.31-fold at 3 d after CIR ( g.3F). These indicated that CpG-ODN ameliorated thymus injury induced by CIR.
Effect of CpG-ODN on spleen after CIR The spleen is known to play an important role in the immune system, which is one of the major injury sites after radiation. CIR caused spleen signi cantly atrophy, the spleen index of irradiated mice without CpG-ODN treatment decreased from 3.73 to 2.31 at 1th and 1.73 at 3 d. Compared with irradiated mice without CpG-ODN treatment, the spleen index in irradiated mice with CpG-ODN treatment raised 35 % at 1 d and 28 % at 3 d after CIR. This difference was statistically signi cant at 1 d and 3 d ( g.4A). Histological examination showed that the area of white pulp was obviously smaller, germinal centers shrunk, and lymphatic sinus were expanded and congested after CIR. Although there was reduction of white pulp in irradiated mice with CpG-ODN treatment, the reduction was less severe than in irradiated mice without CpG-ODN treatment ( g.4B). The area of white pulp was increased 10 % at 1 d and 8 % at 3 d by CpG-ODN treatment ( g.4C). Apoptosis and DSB of spleen were detected by TUNEL staining and γ-H 2 AX immunohistochemistry. Compared with irradiated mice without CpG-ODN, the number of γ-H 2 AX foci and TUNEL-positive nuclei in white pulp of spleen was decreased obviously in irradiated mice with CpG-ODN treatment ( g.4D and g.4E). In addition, B cell in peripheral blood from spleen was detected by labeling CD19 ( g.4F). The number of B cell decreased also rapidly and showed a reduction of 24.0 % at 1 d and 79.0 % at 3 d after CIR. CpG-ODN boosted the number of B cells, the number of B cell increased 1.72-fold at 1 d and 9.65-fold at 3 d after CIR ( g.4G). These results suggested that CpG-ODN ameliorate spleen injury induced by CIR.

Effect of CpG-ODN on cytokines after CIR
Cytokine stimulated the generation and mobilization of the immune cells and reestablish immune system after radiation to combat bacterial infection. Some radiation countermeasures that prevent against radiation injury do so in large part by stimulating expression of cytokines [15] . CIR stimulated the transient elevation of G-CSF, IL-6 and TNF-α within 8 h that might be associated with stress reaction, and then these cytokines expression drastically decreased. Compared with irradiated mice without CpG-ODN treatment, the expression of G-CSF signi cantly increased in all time points, was doubled in maximal value, and maintained high level within 24 h in the irradiated mice with CpG-ODN treatment ( g.5A). As g.5B showed that the expression of IL-6 also signi cantly increased by CpG-ODN treatment, maximal value of IL-6 level in the irradiated mice with CpG-ODN treatment was more twice as high as the irradiated mice without CpG-ODN treatment. TNF-α have similar expression trend with IL-6. Although the expression of TNF-α started declining at 2 h after CIR, CpG-ODN treatment showed much stronger ability in increasing TNF-α expression at all time points ( g.5C). There was signi cant difference between two groups. These indicated that CpG-ODN induces G-CSF, IL-6 and TNF-α expression after CIR.

Discussion
Exposure to heavy ions radiation, immune organs atrophy and immune cells fall within a few days, which was likely to lead to considerable more serious immunological problems, with additional exposure to a bacterial challenge, leads to a very high level of morbidity which is equal to mortality. The bacterial challenge utilized bacterial known to be associated with infection, which are part of the normal bacterial ora of the month, skin and intestines [16]. In the mouse studies, these bacterial challenges are non-toxic to normal mice. But under the conditions of heavy ions radiation, mice were morbidity or mortality [17][18][19]. Therefore, morbidity or mortality could be prevented by relieving I immune tissues injury. Our previously study have documented that CpG-ODN relieved RAW264.7 macrophage cell damage induced by CIR. the present study was to estimate the mitigative effect of CpG-ODN on immune tissues after CIR by using C57BL/6 mice model. Mice were exposed to 5 Gy of CIR, it is important to note that in vivo C57BL/6 mice experiments where typically doses no more than 5 Gy of CIR are required to determine the sub-lethal dose. Radiation death in the sub-lethal dose range was due to immune tissues destruction, which ultimately predisposed to infection and sepsis [12]. In this study, the radiomitigative effect of CpG-ODN on immune tissue was manifested indirectly by signi cantly enhancing mice survival. Bone marrow, thymus and spleen are the immune principal components and the major sites of radiation injury. Compared with irradiated mice without CpG-ODN treatment, the numbers of WBC (including lymphocyte granulocyte and monocyte) in irradiated mice with CpG-ODN treatment signi cantly increased. the direct evidence from histological examination showed that the injuries of bone marrow, thymus and spleen were ameliorated by CpG-ODN treatment after CIR. DSB have a close correlation to DNA injury induced by heavy ions radiation, which are thought to be more di cult to repair and is probably the most lethal attack, with as little as one unrepaired DSB being capable of triggering apoptosis. Thus, DSB have been considered as highly signi cant biological endpoint [21,22]. The phosphorylation of the histone protein H 2 AX (γ-H 2 AX) has been used as a beacon of DSB and TUNEL staining was used to detect cell apoptosis. Many γ-H 2 AX foci and TUNEL-positive nuclei were observed in thymus and spleen of irradiated mice, but CpG-ODN provided a signi cant reduction in the elevation of γ-H 2 AX foci and TUNEL-positive nuclei. These results indicated that CpG-ODN ameliorated bone marrow, thymus and spleen injuries after CIR by inhibiting cell DSB and apoptosis, which were basically in agreement with our previous vitro experiment (Data not shown).
Although the effect of CpG-ODN against immune tissues injury induced CIR is not fully understood, the primary mechanism may be that CpG-ODN stimulates immune cell to secrete a group of cytokines. Published data suggested that TLR5 agonist ( agellin) could trigger immune tissues possibly via proin ammation cytokines, including IL-1, IL-6, IL-11, IL-12, G-CSF, IFN-α and TNF-α, which stimulated the generation and mobilization of the immune cells and reestablish immune system after radiation to combat bacterial infections [22]. The biological responses trigger immune cells by CpG-ODN display remarkable parallels to agellin.The result showed that CpG-ODN induced G-CSF, IL-6 and TNF-α production after CIR. We anticipate that CpG-ODN and agellin share operationally similar characteristics. Other possible mechanism of action is that NF-κB activation plays a pivotal role. The important radiomitigative strategy is to activate the NF-κB pathway TLRs are the key sensor elements of innate immunity, which are important for the defense against microbial infection. Stimulation of TLRs by special agonist initiates signaling cascades, and lead to the activation of NF-κB. The role of NF-κB regulates antiapoptotic genes coding for proteins especially the TNF receptor-associated factor (TRAF), checks the activates of the caspase enzyme family, inhibit DNA damage, and block major apoptotic pathways. The idea of using NF-κB pathway function to reduce radiation injury was initially tested using agellin, which activates NF-κB though TLR5. TLR9 is one of the members of TLR family, and expressed in peripheral blood leukocyte, thymus, spleen, and other lymphoid organs. Our previous study has demonstrated CpG-ODN enhance cell survival after radiation by activation NF-κB and inhibited Caspase pathway.
Overall, the present study demonstrated that the radiomitigative effect of CpG-ODN on immune tissues after CIR. The results showed that CpG-ODN could enhanced mice survival, and ameliorate bone marrow, thymus and spleen injuries by inhibiting DSB and apoptosis. The mechanism may be that CpG-ODN stimulates a group of cytokines production and NF-κB activation. A better understanding of mechanism will require further studies.

Conclusion
CpG-ODN could enhanced mice survival, and ameliorate immune tissues injury, the mechanism may be that CpG-ODN induced cytokines production and inhibited the double strand breaks (DSB) and apoptosis in order to stimulate the generation and mobilization of the immune cells and reestablish immune system All authors reached an agreement to publish the study in this journal.

Availability of data and material
All data generated or analyzed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no con ict of interest.