Effects of low dose radiation on immune cells subsets and cytokines in mice

Abstract

Whole-body exposure to low-dose radiation due to diagnostic imaging procedures, occupational hazards and radiation accidents is a source of concern. In this study, we analyzed the effects of single and long-term low-dose irradiation on the immune system. Male Balb/c mice received a single whole-body dose of irradiation (0.01, 0.05, 0.2, 0.5 or 1 Gy). For long-term irradiation, mice were irradiated 10 times (total dose of 0.2, 0.5 or 1 Gy) over a period of 6 weeks. Two days after single or long-term irradiation, the numbers of splenic macrophages, natural killer cells and dendritic cells were reduced, and the spleen organ coefficient was decreased. At 2 Days after long-term low-dose irradiation, the number of white blood cells in the peripheral blood of the mice decreased. Between 7 and 14 Days after long-term low-dose irradiation, the number of immune cells in the thymus and spleen began to increase and then stabilized. Th1/Th2 cytokines and reactive oxygen species-related proteins first decreased and then increased to a plateau. Our results show a significant difference in the effects of single and long-term low-dose irradiation on the immune system.

Introduction

Broadly speaking, the concept of low-dose radiation (LDR) is not clearly defined, with different standards and interpretations for different application purposes and occasions. For investigation of the biological effects of radiation, LDR refers to the disturbance or slight damage caused by the absorption of radioactive energy by key parts of the cell, which can be corrected and repaired by cellular self-repair mechanisms. In the 2010 annual repific Committee on Atomic Radiation Effects (UNSCEAR), LDR were defined as doses <200 mGy and dose rates <0.1 mGy·min⁻¹ (average dose rates of <1 h or >1 h) and external γ-ray exposure dose. In the LDR risk assessments published in the USA and other countries in 2006, the equivalent dose is <100 mSv, and the dose rate <0.1 mSv·min⁻¹ is collectively referred to as LDR [1].

The immune system is one of the most important defense mechanisms against various environmental factors and is greatly affected by the ionizing radiation. A large number of experimental and epidemiological studies clearly demonstrate the deleterious and immunosuppressive effects of high-dose radiation [2–4]; however, the impact of LDR on the immune system remains unclear and controversial [5]. On the one hand, LDR has been shown to induce genetic and epigenetic changes and is associated with a range of physiological disorders, including changes in the immune system, abnormal brain development leading to cognitive impairment, cataract occurrence [6], abnormal embryogenesis, circulatory diseases, weight gain, premature menopause in females, tumorigenesis and shortened lifespan. On the other hand, studies have demonstrated the stimulatory effects of LDR including increased cell growth [7], prolonged survival [8], reduced tumor incidence [9] and enhanced immune system function [10, 11].

Therefore, the purpose of this study was to investigate and compare the effects of single and long-term LDR on several immune parameters in experimental animals. We investigated quantitative changes by determining time-dependent numerical changes in various lymphocyte subpopulations on Days 2, 7 and 14 after irradiation. The direct effects of radiation on body weight and organ coefficients in mice were also assessed. By measuring changes in cell number, we hoped to gain an insight into the regenerative kinetics of different cell types that affect humoral and cell-mediated immune function. The characteristics of cytokine secretion reflect the functional integrity of the immune system. Therefore, we studied the time-dependent changes in cytokine expression in animals after irradiation. Moreover, we also studied reactive oxygen species-related proteins, as the main mediators of radiation-induced cell damage.

Materials and Methods

Animals and irradiation

Male Balb/c mice (aged 6–8 weeks) used for whole-body LDR experiments were purchased from Beijing Weitong Lihua Laboratory Animal Center (China). For single LDR experiments, mice received a single dose (0.01, 0.05, 0.2 and 0.5 Gy) of ⁶⁰Co γ-radiation (dose rate: 3.93 cGy/min). For long-term irradiation, mice were irradiated 10 times (total dose of 0.2, 0.5 or 1 Gy) over a period of 6 weeks. (dose rate: 3.93 cGy/min). At the end of the experiments, animals were killed humanely by i.p. injection of a lethal dose of pentobarbital (200 mg/kg body weight).

All animal studies were conducted in accordance with the National Regulations of China and with the permission and guidance of the corresponding institutional and national ethical bodies.

Preparation of splenocytes and thymocytes

Animals were sacrificed at the indicated times after irradiation and the spleen and thymus were harvested. The tissues were mechanically disaggregated and the cells were resuspended in RPMI 1640 culture medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin B. Single cell suspensions of splenocyts and thymocytes were used for subsequent immunological measurements.

Phenotypic analysis of splenocytes by flow cytometry

Immunophenotyping of splenocytes was carried out using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). Isolated splenocytes in flow cytometric buffer were incubated with purified anti-mouse CD16/CD32 (mouse BD Fc Block™) at 4°C for 15 min to minimize non-specific binding of antibodies to Fc-receptors. Next, cells were stained with fluorescein isothiocyanate (FITC)-or allophycocyanin (APC)-or Phycoerythrin (PE)-conjugated monoclonal antibodies simultaneously for 1 h at 4°C. The cells were then incubated with the following antibodies: CD40-APC, CD80-FITC CD86-APC, CD25-FITC, CD69-FITC, CD68-PE, CD4-PE, CD28-PE CD8-PERCP-CYTM5.5, CD44-FITC, F4/80-PE, NK1.1-PE, CD11C-PERCP-CYTM5.5, CD3-FITC, CD19-Alexa Flour® 647. All antibodies were purchased from BD Biosciences. Results were analyzed using FlowJo software (v.10, FlowJo, LLC, Ashland, OR, USA).

Hematological analysis

Peripheral blood samples were collected by retroocular plexus bleeding with heparinized capillary tubes from individual mice at the indicated times irradiation radiation, and complete blood cell counts were obtained using a HEMAVET 950 hematology system (Drew Scientific, Oxford, CT, USA) as according to the manufacturer’s instructions.

Real-time quantitative reverse transcription PCR

Total RNA was extracted from spleen or thymus tissue using TRIzol Reagent (Sigma, USA), and cDNA was reverse transcribed using miRcute Plus miRNA First-Strand cDNA Synthesis Kit (TIANGEN BIOTECH, Beijing, China). The miRNA was subjected to quantitative PCR in a Bio-Rad Real-Time PCR System (California, USA) using the miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN BIOTECH, Beijing, China). The mRNA was reversed transcribed using PrimeScriptTM RT Master Mix (Takara, China), and mRNA was quantified by SYBR Green Taq Mix (Takara). Calculation of miRNA and mRNA was expressed using 2^−△△Ct method. The sequences of primers used in the real-time quantitative RT-PCR reactions are shown in Table 6.

Table 6.

Sequences of primers used for real-time RT-PCR

Name	Sequence
IL-4	forward:5′-GTTCTTCGTTGCTGTGAGGAC-3′
	reverse: 5′-TGTACCAGGAGCCATATCCAC -3′
IL-5	forward:5′-AATGAGACGATGAGGCTTCC-3′
	reverse: 5′-CCCACGGACAGTTTGATTCT-3′
INF-γ	forward:5′-TAACTCAAGTGGCATAGATGTGGAAG-3′
	reverse: 5′-GACGCTTATGTTGTTGCTGATGG-3′
IL-12	forward:5′-ACAGCACCAGCTTCTTCATCA-3′
	reverse: 5′-TCTTCAAAGGCTTCATCTGCA-3′
IL-10	forward:5′-AGAAGGACCAGCTGGACAACAT-3′
	reverse: 5′-CAAGTAACCCTTAAAGTCCTGCAGTA-3′
18 s	forward 5′-CGAAAGCATTTGCCAAGAAT-3′
	reverse 5′-AGTCGGCATCGTTTATGGTC-3′

Western blot analysis

Balb/c mice (male, 20 g) in an SPF-class animal feeding environment were taken, and three mice in each group were taken for thymus and spleen. Tissues were rinsed with saline. In total, 3 mL of lysis buffer was added per 0.3 g of tissue, and protease inhibitor cocktail (×100) was added at 100:1 (v/v). Tissues were homogenized on ice for ~30 min and the upper layer was discarded. The homogenate was centrifuged at 13 000 rpm for 20 min at 4°C. After discarding the precipitate, the protein concentration in the supernatant was measured by mixing 25 μl 5 × loading buffer (20 μl 6 × loading buffer) with 100 μl of protein solution. After and boiling for 5 min, separation of proteins, transfer tot PVDF (polyvinylidene fluoride) membrane, incubation with primary and secondary detection antibodies (specificity, dilution factor). Western blot analysis of ROS (reactive oxygen species)-related protein expression was performed and protein bands were visualized by electrochemiluminescence (ECL) development.

Statistical analysis

All results are expressed as the mean ± SEM. Mean values obtained from each group were compared by analysis of variance (ANOVA), followed by Fisher’ s least significant difference test. Non-parametric analyses using the Kruskal–Wallis and Mann–Whitney U tests were also used when appropriate; P < 0.05 was considered significant.

Results

Effects of single LDR on peripheral blood cell populations and mouse organs

To investigate the direct effect of single LDR in mice, we measured body weight before and after radiation exposure, and calculated the spleen and thymus organ coefficients. The peripheral blood of the mice was collected and analyzed.

There were no significant differences in the body weights of mice before and 24 h after irradiation with 0.01, 0.05, 0.2 or 0.5 Gy (Supplementary Fig. S1A). Furthermore, there were no significant changes in the body weight of mice before and 24 h after single LDR (Supplementary Fig. S1B). We then removed the mouse spleen and thymus and calculated the organ coefficient ratio (Table 1). The results showed that after a single dose of 0.5 Gy, the spleen organ coefficient decreased slightly, while the thymus organ coefficient did not change (Fig. 1A and B).

Table 1.

Organ coefficient of spleen and thymus after single low-dose irradiation

	0 Gy	0.01 Gy	0.05 Gy	0.2 Gy	0.5 Gy
Spleen organ coefficient	32.24 ± 3.54	31.90 ± 2.56	38.56 ± 23.29	33.56 ± 4.92	25.64 ± 4.12*
Thymus organ coefficient	18.77 ± 4.13	18.72 ± 2.88	17.31 ± 2.88	19.21 ± 4.70	15.95 ± 4.73

^*p < 0.05 vs. non-irradiated controls of each group.

Figure 1.

Analysis of organ coefficient of spleen cells and thymocytes after single low-dose irradiation. Mice received single low-dose irradiation and were sacrificed 24 h later. (A) Spleen, and (B) thymus tissues were removed and weighed. Data represent the mean ± SEM (n = 6 mice per group). ^*P < 0.05 vs. non-irradiated controls.

In addition, quantitative changes in the numbers of white blood cells, red blood cells, hemoglobin and platelets in the peripheral blood of mice were observed after irradiation, although these changes did not reach the level of statistical significance (Table 2). Because of the changes in the spleen organ coefficient observed after irradiation, we further investigated the effect of single LDR on the immune organs in mice.

Table 2.

Number of white blood cells in the peripheral blood of mice 24 h after single low-dose irradiation

	WBC (K/μl)	RBC (M/μl)	HGB (M/μl)	PLT (K/μl)
0 Gy	4.18 ± 1.18	9.25 ± 1.45	140.75 ± 24.83	515.92 ± 74.71
0.01 Gy	3.45 ± 1.48	9.51 ± 0.66	155.92 ± 11.17	578.58 ± 56.85
0.05 Gy	4.51 ± 1.05	10.05 ± 0.52	150.33 ± 10.25	568.25 ± 73.72
0.2 Gy	3.72 ± 2.94	9.98 ± 0.88	153.177 ± 14.96	527.5 ± 65.55
0.5 Gy	4.52 ± 0.90	10.09 ± 1.06	151.83 ± 18.39	542.25 ± 67.35

Effects of single LDR on immune organs in mice

The spleen is the largest peripheral immune organ and is also the main site at which lymphocytes respond to antigens. The thymus is a site of T cells differentiation, development and maturation, whereas B cells mature in the bone marrow.

The apoptotic rate of thymocytes did not change significantly with increasing dose at 24 h after single LDR (Supplementary Fig. S2C). In contrast, the rate of apoptosis in bone marrow cells increased after LDR at 0.2 and 0.5 Gy (Supplementary Fig. S2B). There were no significant changes in the total number of thymic T cells, mature T cells (CD4⁺ T cells/CD8⁺ T cells), and T lymphocyte activation markers after irradiation (Fig. 2C and D). There was also no change in the number of B cells in the bone marrow (Supplementary Fig. S2A). Interestingly, after irradiation at 0.01 and 0.5 Gy, the number of dendritic cells (DC) in the spleen decreased, and expression of the corresponding activation marker CD86 was also reduced. Similar changes were observed in the number of macrophages (MΦ) and CD68 expression in the spleen (Fig. 2A and B). The results of this series of experiments indicated that the immune system function of mice is inhibited by single LDR. We then analyzed the effects of long-term LDR at Days 2, 7 and 14 after the end of the radiation treatment for comparison with the effects of single LDR to evaluate the influence of differences in radiation patterns on regulation of the immune system.

Figure 2.

Effect of single LDR on immune cell subsets in the spleen and thymus of mice. (A) Number of immune cell subsets in the spleen 24 h after irradiation. (B) Number of immune cell activation markers in the spleen 24 h after irradiation analyzed by flow cytometry. (C) Number of immune cell subsets in the thymus 24 h after irradiation. (D) Number of immune cell activation markers in the thymus 24 h after irradiation analyzed by flow cytometry. The subpopulations of cells in the irradiated group was compared to a subpopulation of cells in the unirradiated group (n = 6 mice per group). Data represent the mean ± SEM (n = 6 mice per group). ^*P < 0.05 vs. non-irradiated controls. DC, dendritic cells; NK cells, natural killer cells. MΦ, macrophages.

Effects of long-term LDR on peripheral blood cell populations

After a single LDR, there were no significant differences in the body weight of the mice in each irradiation group compared with those in the control group, although the spleen organ coefficients were slightly decreased. After long-term LDR, we also found that the mouse spleen organ coefficient showed a dose-dependent decrease on Day 2 (Fig. 3A). Next, we observed the changes in the peripheral blood of mice after long-term LDR. Changes in the number of peripheral blood cells directly reflect the changes in hematopoietic function of the bone marrow and spleen. Furthermore, hematopoietic organs are dysfunctional due to radiation damage, which can cause a reduction in the number of cells in peripheral whole blood that is closely related to the degree of damage. On Day 2 after long-term LDR, the number of white blood cells in the peripheral blood was significantly reduced (Table 3). On Days 7 and 14, there were no significant changes in the number of white blood cells in the irradiated group compared with those in the control group (Tables 4 and 5). It is worth mentioning that there were no significant changes in the numbers of red blood cells, hemoglobin and platelets on Days 2, 7 and 14 after irradiation.

Figure 3.

Analysis of organ coefficient of spleen cells and thymocytes after long-term LDR. The thymus and spleen of mice were sampled and weighed at 2 (A and D), 7 (B and E) and 14 Days (C and F) after long-term radiation (n = 12 mice per group). Data represent the mean ± SEM (n = 6 mice per group). ^*P < 0.05 vs. non-irradiated controls.

Table 3.

Number of white blood cells in the peripheral blood of mice on Day 2 after long-term low-dose irradiation

	WBC (K/μl)	RBC (M/μl)	HGB (M/μl)	PLT (K/μl)
0 Gy × 10	5.63 ± 2.19	12.98 ± 0.80	174.17 ± 27.63	528.27 ± 189.3
0.02 Gy × 10	4.52 ± 1.25	10.31 ± 1.70	183.42 ± 45.72	496.33 ± 200.26
0.05 Gy × 10	3.68 ± 0.34*	10.49 ± 1.20	177.25 ± 31.96	451.90 ± 168.39
0.1 Gy × 10	2.87 ± 0.69*	10.84 ± 1.88	184.25 ± 31.50	458.25 ± 185.55

^*p < 0.05 vs. non-irradiated controls of each group.

Table 4.

Number of white blood cells in the peripheral blood of mice on Day 7 after long-term low-dose irradiation

	WBC (K/μl)	RBC (M/μl)	HGB (M/μl)	PLT (K/μl)
0 Gy × 10	7.34 ± 2.93	8.74 ± 1.76	148.44 ± 31.01	838 ± 192.96
0.02 Gy × 10	6.88 ± 1.32*	9.05 ± 1.23	147.17 ± 21.07	661.33 ± 192.79
0.05 Gy × 10	6.06 ± 1.35	9.56 ± 0.95	155.75 ± 17.96	785.17 ± 137.23
0.1 Gy × 10	6.66 ± 1.95*	8.59 ± 2.15	147.08 ± 37.56	693.42 ± 237.18

^*p < 0.05 vs. non-irradiated controls of each group.

Table 5.

Number of white blood cells in the peripheral blood of mice 24 h on Day 14 after long-term low-dose irradiation

	WBC (K/μl)	RBC (M/μl)	HGB (M/μl)	PLT (K/μl)
0 Gy × 10	6.44 ± 2.16	10.98 ± 1.22	170.58 ± 26.39	532.33 ± 114.57
0.02 Gy × 10	6.01 ± 1.91	10.53 ± 1.28	165.58 ± 31.11	508.67 ± 145.28
0.05 Gy × 10	6.06 ± 1.24	11.39 ± 1.47	169.33 ± 14.59	525.17 ± 125.13
0.1 Gy × 10	6.23 ± 1.53	11.97 ± 1.89	172.33 ± 13.70	511.75 ± 138.61

Effects of long-term LDR on immune cells in mice

We next investigated the effects of long-term LDR on DC, MΦ, natural killer (NK) cells and T immune cells. The number of DC, NK MΦ and T cells decreased within 2 Days after irradiation (Fig. 4A and G). Although the number of MΦ, NK cells and T cells no longer decreased after Day 7, the number of DCs increased (Fig. 4C and H). In addition, the proportion of MΦ increased until Day 14, while there were no differences in the other subpopulations at Day 14 (Fig. 4E and I). In accordance with previous results, analysis of the expression of activation markers such as CD25, CD28 (T cells), CD69 (NK cells), CD80/CD86 (DC) and CD68 (MΦ) showed that the ratio of CD86⁺ DC, CD68⁺ MΦ decreased on Day 2 after long-term LDR (Fig. 4B), increased on Days 7 and 14 (Fig. 4D and F). The ratio of CD69 cells to NK cells decreased on Day 2, although this effect was not statistically significant. The number of CD69 cells increased on Day 7 (Fig. 4D) and recovered on Day 14 (Fig. 4F). The number of CD28⁺ T cells was reduced on Day 2 (Fig. 4G), but plateaued between Days 7 and 14 (Fig. 4H and I). Taken together, these results indicated that the proportion of each immune cell type was reflected by the expression of the respective activation marker, and that spleen cells and thymocytes respond differently to LDR.

Figure 4.

Effects of long-term LDR on immune cell subsets in spleen and thymus of mice. The thymus and spleen of mice were harvested at 2 (A and B and G), 7 (C and D and H) and 14 Days (E and F and I) after long-term irradiation. The number of immune cell activation markers in the spleen and thymus 24 h after irradiation analyzed by flow cytometry. The subpopulations of cells in the irradiated group was compared to a subpopulation of cells in the unirradiated group (n = 6 mice per group). Data represent the mean ± SEM (n = 6 mice per group). *P < 0.05 vs. non-irradiated controls. DC, dendritic cells; NK cells, natural killer cells. MΦ, macrophages.

Effects of long-term LDR on ROS-related protein expression

The physiological effects of radiation are mainly due to the production of ROS [12]; therefore, we studied the expression of ROS-related proteins. NF-κB plays a key role in radiation-induced immune dysfunction by regulating various factors involved in DNA damage repair, cell death and cytokine production. iNOS is an important redox molecule in the immune system that produces nitric oxide free radicals for pathogen defense. Nuclear-factor-related factor 2 (Nrf2), which is a key transcription factor that regulates oxidative stress in cells, is regulated by KEAP1 and resists cytotoxic damage by interacting with the antioxidant response element, ARE. The strong adaptive response of HO-1 to various stimulating factors indicates its importance in preventing inflammatory processes and oxidative tissue damage.

On Day 2 after long-term irradiation, the expression of HO-1 and Nrf2 proteins in the spleen was downregulated (Fig. 5A). Only Nrf2 protein expression was downregulated in the thymus (Fig. 5D). On Day 7, HO-1 protein expression was slightly upregulated in the thymus and spleen (Fig. 5 B and E). On Day 14, there was a continued but slight increase in Nrf2 protein expression in the spleen (Fig. 5C), while upregulation of INOS and HO-1 proteins in the thymus was evident (Fig. 5F). The expression of these proteins after irradiation was also consistent with the changes in the number of immune cells, as described in section ‘Hematological analysis.’

Figure 5.

Effects of long-term LDR on ROS-related proteins in spleen and thymus of mice. Following low-dose irradiation, expression of immune-related factors in mouse spleen was analyzed by western blotting. ROS-related Nrf2, HO-1 and iNOS expression and NF-κB activity were analyzed at day 2 (A and D), 7 (B and E) and 14 Days (C and F) after long-term LDR. Data represent the mean ± SEM (n = 6 mice per group). *P < 0.05 vs. non-irradiated controls.

Alterations in the cytokine expression profile of irradiated lymphocytes

Mice received cumulative doses of irradiation at 0, 0.2, 0.5 and 1 Gy prior to isolation of whole-cell RNA from spleen cells and thymocytes. The relative expression of various cytokine transcripts, including Th1 (IFN-γ and IL-12), Th2 (IL-4 and IL-10), were determined by quantitative RT-PCR using the primers listed in Table 6.

On Day 2 after long-term LDR, the expression of IFN-γ and IL-12 in the spleen was decreased (Fig. 6A), and the expression of IFN-γ and IL-4 in the thymus was decreased (Fig. 6D). Increased expression of IFN-γ, IL-12 and IL-4 was observed in the spleen at Day 7 after irradiation (Fig. 6B). Expression of IL-12 and IL-10 was increased in the thymus at Day 7 after irradiation (Fig. 6E). There were no changes in the expression of cytokines in the spleen and thymus at Day 14 after irradiation (Fig. 6C and F).

Figure 6.

Effects of long-term LDR on cytokines in spleen and thymus of mice. Cytokine in mouse spleen and thymus was analyzed at Days2 (A and D), 7 (B and E) and s 14 (C and F) after long-term LDR by quantitative RT-PCR. Data represent the mean ± SEM (n = 6 mice per group). *P < 0.05 vs. non-irradiated controls

Discussion

Perhaps the most controversial area of radiation biology is the biological effects of LDR. Anderson [13] first reported that ionizing radiation can cause serious damage to the immune system and lymphocytes. Subsequent studies showed that even local exposure can impair immune function [14].

In this study, we showed that there were no significant differences in body weight in mice at 24 h after single LDR in the dose range of 0.01–0.5 Gy. Furthermore, there were no changes in the body weight of mice before and after irradiation. This study also showed that when the irradiation dose reached 0.5 Gy, the spleen organ coefficient ratio decreased significantly compared with that of the control group, while irradiation had no significant influence on the thymus organ coefficient ratio. This difference may be related to the increased sensitivity of spleen cells to radiation compared with thymocytes.

Among the peripheral blood cell subpopulations, our results indicate that DC and MΦ cells in the spleen are the most sensitive to radiation. In the 0.01–0.5 Gy dose range, the number of DC cells and MΦ cells were significantly reduced compared with those in the control group (P < 0.05) and this trend was reflected by changes in the expression of corresponding cell activation markers. After irradiation, the number of NK cells and corresponding activation markers (CD69) also decreased. In accordance with our data, it has previously been reported that the number of spleen cells was significantly reduced following LDR in the 0.01–2 Gy [15]. There was no significant difference in the number of T lymphocytes and their activation markers in the thymus after single LDR. This also indicates that thymocytes are more tolerant to gamma-irradiation than spleen cells. Finally, we also observed that the apoptosis rate of bone marrow cells increased after LDR at 0.2 Gy and 0.5 Gy. Thus, our results indicate that the immune system function of mice is inhibited after single LDR.

We also investigated the effects of long-term LDR for comparison with our observation of the effects of single LDR. After long-term LDR, we also found that the mouse spleen organ coefficient showed a dose-dependent decrease on Day 2. Changes in the number of peripheral blood cells after long-term LDR also directly reflected the changes in hematopoietic function of the bone marrow and spleen. Hematopoietic organs are dysfunctional due to radiation damage, which can cause a reduction in the number of cells in peripheral whole blood that is closely related to the degree of damage. On Day 2 after long-term (multiple exposure) LDR, the number of white blood cells in the peripheral blood was reduced (Table 3). However, on Days 7 and 14, there were no significant differences in the number of white blood cells in the irradiated group compared with those in the control group (Tables 4 and 5). Peripheral blood leukocytes are one of the most important indicators of the immune system. Furthermore, radiation damage of hematopoietic organs is first reflected in the number of peripheral blood leukocytes, which is therefore the most direct and classic indicator of radiation damage. It is worth mentioning that there were no significant changes in the number of red blood cells, hemoglobin and platelets on Days 2, 7 and 14 after irradiation.

On Day 2 after long-term LDR, the changes of DCs, NK cells and MΦ cells in the spleen of mice were similarly to those observed after single LDR. The number of DCs, NK cells and MΦ cells were reduced, with corresponding changes in the expression of the relevant cell activation markers CD86, CD69 and CD68, respectively. In the thymus, the total number of T cells was reduced. Specifically, on Day 2 after irradiation, the number of immune cells in the spleen and thymus was reduced.

Under normal physiological conditions, HO-1 expression and activity are low, but increase under stress conditions, such as heme, hydrogen peroxide (H₂O₂), endotoxin, heavy metals, ultraviolet radiation and high oxygen/hypoxia. Both HO-1 expression and Nrf2 can show significant antioxidant effects [16–18]. Li et al. [19] found that anthocyanin upregulated HO-1 expression via the by serine/threonine protein kinase (Akt) and extracellular regulatory protein kinase (ERK1/2)/Nrf2 signaling pathways to clear ROS, thereby playing a protective role in hepatotoxicity. Thus, Nrf2 expression reflects the amount of ROS production and the magnitude of the radiation effect. On Day 7 after irradiation, HO-1 expression was increased in the spleen and thymus. Several studies have demonstrated that LDI-induced upregulation of endogenous antioxidants, including Nrf2, superoxide dismutase, glutathione reductase, glutathione, catalase and thioredoxin, in animal tissues, which contribute to the suppressive effects of LDI on oxidative tissue injury [12, 20–22]. For instance, LDI-induced Nrf2 and HO-1 expression may assist in preventing diabetic renal damage and dysfunction [12, 23]. On Day 14 after irradiation, in addition to the observed increase in HO-1 and Nrf2 levels, there was also an increase in the expression of iNOS. NF-κB also plays a crucial role in radiation-induced immune regulation by modulating multiple effector genes involved in DNA damage repair, cell death and cytokine production [24].

Since cytokines are the most important molecules in the immune system and their production is a common feature of all lymphocyte subpopulations, we investigated the effect of long-term LDR on the expression of several cytokines. We isolated whole-cell RNA from spleen cells and thymocytes and the relative amounts of cytokine transcripts were determined by quantitative RT-PCR. Th1 cells secrete mainly IL-2 and IFN-γ, enhance the toxic effects of killer T cells, promote macrophage activation and participate in the regulation of cellular immunity, which facilitates the differentiation of cytotoxic T cells and mediates the cellular immune hypersensitive response. Th2 cells secrete IL-4, IL-6 and IL-10, which mediate humoral immunity and promote antibody production [25, 26]. On Day 2 after long-term LDR, the expression of cytokines and proteins in spleen thymus was consistent, and the expression of Th1 and Th2 was decreased (the expression of IFN-γ, IL-12 and IL-4 was significantly decreased). Bogdandi et al. [27] also reported reduced Th1 and Th2 cytokine expression profiles at an early time point (72 h) after LDR at 0.01–0.1 Gy, and the immune response was milder than that of the 2 Gy high-dose radiation-induced immune response. Th1/Th2 imbalance is also a cause of radiation-induced immunosuppression [28, 29]. On Days 7 and 14 after LDR, the levels of cytokines in the thymus and spleen began to increase and gradually stabilized. The changes in cytokine expression after long-term irradiation also indicated that the immune system function of the mouse was inhibited during the period after the irradiation.

Conclusion

To the best of our knowledge, we are the first to compare the biological effects of single and long-term LDR. Systemic single LDR and long-term LDR have substantial effects on the quantitative and functional immune parameters of mouse spleen cells and thymocytes. In the short-term, LDR impairs immune function, but does not cause such damage in the long-term.

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (Grant No. 81530085, 81773359, 31470827).

Author Contributions

Y.Q. and P.-K.Z. designed the experiments; X.L. and Z.L. performed the experiments and analyzed data; X.L. interpreted the results and prepared figures; X.L., D.W., Z.L.; Y.H., S.Hu., Y.X., Y.L. participated in the discussion of the research progress, assisted with the experiments. Y.G. and P.-K. Z. supervised the research and reviewed the manuscript.