Evaluation of the toxicity of iron-ion irradiation in murine bone marrow dendritic cells via increasing the expression of indoleamine 2,3-dioxygenase 1

Elevated JNK and IDO1 induced by Fe ion IR could result in dysfunction of BMDCs.

Abstract

High linear energy transfer radiation is known to deposit higher energy in tissues and cause greater toxicity compared to low-LET irradiation. Local immunosuppression is frequently observed after irradiation (IR). Dendritic cells (DCs) play important roles in the initiation and maintenance of the immune response. The dysfunction of DCs contributes to tumor evasion and growth. However, molecular mechanisms underlying the establishment of immune tolerance induced by heavy ion IR through this DC population are poorly understood. Therefore, here we report our findings on the dysfunction of bone marrow-derived dendritic cells (BMDCs) induced by 1 Gy iron ion radiation and promotions of expressions of JNK1/2/3, indoleamine 2,3-dioxygenase 1 (IDO1), p-ERK1/2 and p38/MAPK; and decrease of IDO2, MHC class II, CD40, CD80 expressions and IFN-γ and TNF-α secretion after total-body IR in mice. JNK⁺IDO1⁺ BMDCs showed up-expression of p-ERK1/2 and p-p38/MAPK, reduced expression of MHC class II and CD80, and were not able to effectively stimulate allogeneic spleen T cells. The inhibition of IDO1 expressions could partly restore the function of BMDCs. In all, our study shows that elevated JNK and IDO1 expression induced by Fe ion IR could result in dysfunction of BMDCs via p-p38/MAPK and p-ERK1/2 signal pathway, and it may represent a new mechanism in radiation-induced immune tolerance.

Introduction

Space travel beyond the low earth orbit exposes astronauts to radiation from solar particle events (SPE) and galactic cosmic radiation (GCR).1 While high-energy protons constitute a major part of sporadically occurring SPE, heavy ions are the major contributors to the dose equivalent in GCR, which is ubiquitous in space.2 Venturing beyond the Van Allen belt and into deep space, astronauts will encounter a significant amount of GCR which contains not only high-energy ¹H and alpha particles but also high-linear energy transfer (LET) radiation from high charge and high energy (HZE particles), such as ⁵⁶Fe.3 These high-LET HZE ions have a greater propensity for ionization and they deposit large amounts of energy along their tracks and thus have greater potential for causing damage to tissues. Importantly, unlike other radiation types in outer space, current shielding is unable to provide effective protection against energetic heavy ions raising further concerns about astronauts’ health during and after prolonged space travel.4 With increasing interest in space tourism, energetic heavy ion radiation exposure is of concern not only for astronauts but also for aspiring future space travelers at large.5 The current study is focused on the immunity system because cells that have a rapid turnover rate such as bone marrow and immune cells are particularly sensitive to the effects of radiation.6 Therefore, even a modest increase above the already high spontaneous incidence of impairment of the immune system after energetic heavy ion radiation exposure will have significant ramification not only for astronauts’ health risk estimates but also for future human deep-space exploration planning.

Epidemiological long-term studies could demonstrate that ionizing radiation may induce a dose-dependent impairment of the immune system as well as a persistent inflammatory status with deregulation of immunity cells. In addition, immunodeficiency not only contributes to the susceptibility to infections but also plays a crucial role in cancer pathogenesis and progression.7 Cancer cells evade immunosurveillance through several mechanisms including abnormalities in the number and function of macrophages, T cells, B cells, natural killer T cells and dendritic cells (DCs).7

DCs are powerful antigen presenting cells capable of inducing antitumor immune responses by linking the innate and adaptive immune systems.8 Effective antigen processing and presentation by DCs are essential for the induction of antitumor immunity. Immune-suppressive DCs also play a critical role in maintaining effector cell quiescence in the tumor microenvironment.9 The recently identified pathways to modulate T-cell response during allogeneic transplant rejection is through an immunosuppressive enzyme, indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan (Trp) into l-kynurenine (Kyn). IDO can be induced by interferon gamma (IFN-γ) in various cell types including DCs.10 IDO1-expressing DCs exert indeed broad and robust immunosuppressive effects such as (1) they directly suppress the proliferation and effector of functions of cytotoxic T lymphocytes, NK cells, and plasma cells11,12 and (2) they promote the conversion of naïve CD4⁺ T cells into CD4⁺CD25⁺FOXP3⁺ Tregs and activate them.11,12 Taken together, these observations reinforce the notion that IDO1 mediates robust immunosuppressive effects in both physiological and pathological scenarios. However, there were few pieces of evidence to show the exact mechanism of impairment of DCs induced by IR; IDO1, therefore, may be able to contribute to the dysfunction of DCs by iron ion IR. Here we demonstrated that iron ion IR could activate JNK/IDO1 in BMDCs; JNK⁺IDO1⁺ DCs showed up-regulation of p-ERK1/2 and p-p38/MAPK, low expression of MHC class II and CD80 and were not able to effectively stimulate T cell immune responses. It may represent a new mechanism in radiation-induced immune tolerance.

Experimental

Animals

All experimental animals were housed under specific pathogen-free conditions. C57BL/6J mice (10–12 weeks) which were half male and half female, weighing 20 ± 1 g, were provided by the Lanzhou University School of Medicine. They were randomly divided into 4 groups: control, IR, siRNA + IR, pcDNA + IR and inhibitor JNK + IR groups (20 in control, 40 in the IR group, 80 in siRNA (siRNA/con and siRNA/IDO1), 80 in pcDNA (pcDNA/con and pcDNA/IDO1) and 80 in the inhibitor group (DMSO and inhibitor)). The principles of laboratory animal care were followed and all procedures were conducted according to the guidelines established by the Institute of Modern Physics, and every effort was made to minimize suffering. This study was approved by the Animal Experiment Committee in the Institute of Modern Physics.

Experiment design and radiation exposure

The ⁵⁶Fe¹⁷⁺ ion beam was provided by Heavy Ion Research Facility in Lanzhou (HIRFL, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China). The animal was whole-body irradiated individually (energy of 160 MeV μm^–1, LET of 350.27 keV μm^–1 and a dose rate of 0.2–0.3 Gy min^–1), at doses of 0.5 or 1 Gy. This experiment was completed three times.

Generation of bone marrow dendritic cells (BMDCs)

24 hours after IR, mice were killed by cervical dislocation and bone marrow was isolated then. Bone marrow was flushed from the tibias and femurs; then using a 1-gauge needle cells were washed twice in RPMI 1640 medium supplemented with 10% v/v fetal bovine serum (HyClone), 100 U ml^–1 penicillin and 100 mg ml^–1 streptomycin (Invitrogen). Bone marrow cells (3 × 10⁶ cells, 5 ml) were cultured in T25 Nunc tissue culture flasks with recombinant mice granulocyte macrophage-colony stimulating factor (10 ng ml^–1, PeproTech EC), in combination with recombinant mice interleukin-4 (2 ng ml^–1, PeproTech EC). On days 3 and 5, half of the media was suctioned, followed by the addition of fresh media. On day 7, in order to obtain mature DCs cells were incubated for 24 h with 1 μg ml^–1 lipopolysaccharides (LPS) (Sigma-Aldrich) and we obtained about 1–3 × 10⁸ mature BMDCs per mouse at 90–95% purity using this method.13

Isolation and purification of spleen T cells

Single cell suspension of splenocytes from normal mice was prepared. Splenic T cells were purified by nonadherence and elution from nylon wool columns (Polysciences).

Small interfering RNA (siRNA) transfection and stable IDO-overexpressing transfectants of DC

Mature BMDCs were transfected with 250 nM control siRNA (siRNA/Con) and 250 nM IDO1-specific siRNA (siRNA/IDO1) using standard nucleofection.14 Briefly, 2 × 10⁶ DCs were transfected for each condition by using the U02 program. A mixture of four gene-specific siRNAs was used to silence IDO1 (Invitrogen). The target sequences were: (1) 5′-UCACCAAAUCCACGAUCAU-3′, (2) 5′-UUUCAGUGUUCUUCGCAUA-3′, (3) 5′-GUAUGAAGGGUUCUGGGAA-3′, and (4) 5′-GAACGGGACACUUUGCUAA-3′. Nontargeting pool siRNAs designed and tested for minimal targeting of mice genes were used as the negative control (by Invitrogen). The siRNA BMDCs were co-cultured with spleen T cells after 24 h transfection.

The expression vector for mouse IDO was constructed by inserting full-size mouse IDO cDNA15 into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). Transfection into BMDCs was carried out as described previously.16 More than 20 stable clones were selected as IDO-overexpressing cells (pcDNA/IDO1). For a negative control, the control vector (pcDNA3.1) was transfected into BMDCs (pcDNA/Con). pcDNA/IDO1 cells and pcDNA/Con cells were cultured using G418 (Sigma-Aldrich) at 200 μg mL^–1 in MEM-alpha supplemented with 10% FCS.

Autologous mixed leukocyte reactions

The mature BMDCs (2 × 10⁵) were co-cultured with allogeneic spleen T cells at a ratio of 1 : 10. After 5 day co-culturing, T cell differentiation was measured by flow cytometry (FACSCalibur, Becton Dickinson). There were 3 parallel samples for each dose.17

Treatment with inhibitors

Mature BMDCs were treated with SP600125 (a selective inhibitor of JNK) for 24 h. SP600125 was also dissolved in DMSO (≥99.7%) and diluted (1 : 1000) in culture media to administer to treatment group cells. The same volume of DMSO was added to control group cells.

Protein expression

BMDCs were lysed and analyzed by western blot as previously described.18 Anti-β-actin, anti-IDO1, anti-IDO2, anti-c-Jun NH2-terminal kinases (JNK) 1/2/3, anti-ERK1/2 and anti-p38/MAPK antibodies were purchased from the Bioworld Technology Company. The anti-p-ERK1/2, p-cJUN and p-p38/MAPK antibodies were supplied by the Cell Signaling Technology Company. They were used according to the manufacturer's recommendations.

Enzyme activity of IDO1

l-Kynurenine levels were measured to determine IDO enzyme activity. Culture supernatants were harvested to quantify the concentration of l-kynurenine using spectrophotometric analysis as described previously, with minor modifications.19 Briefly, BMDCs were supplemented with 500 μM Trp (Sigma-Aldrich), and incubated. Supernatants were harvested after 4 h and mixed with 30% trichloroacetic acid (2 : 1), vortexed, and centrifuged at 8000g for 5 min. Subsequently, this solution was added to Ehrlich's reagent (1 : 1, Sigma-Aldrich) in a 96-well plate. Triplicate samples were run against a standard curve of defined Kyn concentrations (0–100 μM; Sigma-Aldrich). Optical density was measured at 490 nm, using a Multiskan EX microplate reader (Infinite M200, Tecan).

Enzyme-linked immunosorbent assay (ELISA)

IDO1 is induced by several pro-inflammatory cytokines, including interferon-γ (IFN-γ) and tumor necrosis factor (TNF-α);20,21 so supernatants from the BMDCs (1 × 10⁶ cells) were analyzed for their cytokine expression levels. The concentrations of IFN-γ and TNF-α were determined by ELISA (R&D). The user instructions were followed carefully. DCs without irradiation were used as the control.

RNA extraction and quantitative PCR

Expression of the cytokines IFN-γ and TNF-α in BMDCs was measured by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). cDNA was prepared using the ProtoScript Reverse Transcription Kit (New England Biolabs) according to the manufacturer's instructions. Five hundred ng of purified RNA was used for cDNA synthesis. Real-time quantitative RT-PCR was carried out using the Stratagene MX3005P cycler software (40 cycles) and SYBR RT-qPCR primers obtained from Biosciences (Qiagen, Valencia) according to the manufacturer's protocol. Mouse genomic cDNA was used as the positive control. β-Actin was used as the housekeeping gene. The following primer sets were used: β-actin (NM_007393) forward 5′-CGATGCCCTGAGGCTCTTT-3′, reverse 5′-TGGATGCCACAGGATTCCA-3′; TNF-α (NM_013693.2) forward 5′-GCC-ACCACGCTCTTCTGTCT-3′, reverse 5′-GGTCTGGGCCATAGAACTGATG-3′; and IFN-γ (NM_008337.3) forward 5′-GGTTGCTCCTCTTACCGTCTTT-3′, reverse 5′-CGTGGCACTTTTTACCACAGA-3′.

BMDC surface marker analysis

BMDCs were stained with either fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mAbs (Biolegend) against mouse MHC class II (FITC) and co-stimulatory molecules (CD40-PE, CD80-FITC, and CD86-PE) or the appropriate isotype controls according to the manufacturer's instructions. After incubation in the dark for 30 minutes, the percentage of BMDCs stained positive for MHC class II, CD40, CD80, CD86 or an appropriate isotype control was determined by using an FACSCalibur system (LSRFortessa, BD Biosciences).

Analysis of T cell subpopulations

Purified T cells and BMDCs were prepared as described previously. After being co-cultured for 5 days, floating T cells were collected and analyzed by FACS. T cell suspensions were stained for cell surface expression of CD3-allophycocyanin (APC), CD4-PE and CD8-FITC (Biolegend) to characterize individual T-cell subsets. The cell surface expression of CD3 was used as a marker of T-cells.22 CD4 identifies T-helper cells while CD8 identifies cytotoxic T cells. A two-parameter analysis was performed to determine the percentages of CD4 positive T cells (CD4⁺) and CD8 positive T cells (CD8⁺). Briefly, T cells were pre-incubated with antibodies for 30 min at room temperature. Then, 2 ml lysing solution was added and maintained for 15 min at room temperature. After centrifugation at 1500 rpm for 5 min, the cells were washed with PBS and analyzed using FACS.

Regulatory T cell (Tregs) experiments

For the identification of Treg cells in spleen T cells, 1 × 10⁶ cells were incubated with anti-CD4-PE, CD25-FITC and FOXP3-APC mouse antibodies (Biolegend), according to the manufacturer's protocols. Appropriate isotype controls were included. T cells were collected and centrifuged at 1500 rpm for 10 min. The cells (10⁶ cells per tube) were stained in duplicate with PE-anti-CD4 and FITC-anti-CD25 at room temperature for 30 min, fixed with 1× FoxP3 Fix buffer (10 min/RT), and permeabilized with 1× working solution of FoxP3 buffer for 30 min at room temperature. After being washed with PBS, the cells were stained with APC-anti-FoxP3 for 30 min and characterized by flow cytometry analysis using FACS and FlowJo software (v5.7.2) (TreeStar).

Statistical analysis

Each value was expressed as the mean ± SD. An ANOVA analysis of variance was used to determine the level of any statistically significant differences among control, IR, and siRNA or inhibitor groups. The post-hoc test was performed after ANOVA. A p-level of 0.05 or less was selected as a criterion for a statistically significant difference.

Results

BMDC dysfunction induced by iron particle IR

We first investigated dysfunction of BMDCs, and whether Fe ion IR affected IDO1 expression. BMDCs without IR were used as control samples. IFN-γ, TNF-α secretion and expression of MHC class II, CD40, CD80 in BMDCs decreased with dose as shown in Fig. 1A and B in the IR group; no significant down-regulation of CD86 was observed (p > 0.05). These irradiated BMDCs could decrease CD4⁺ and CD8⁺ T cell percentage as shown in Fig. 1C, elevate CD4⁺CD25⁺Foxp3⁺ Tregs number and reduce CD4⁺CD25⁺ T cell number as shown in Fig. 1D. Moreover, IDO1, JNK1/2/3, p-ERK1/2 and p38/MAPK expressions were strongly up-regulated by iron ion IR as shown in Fig. 2A. The expression of IDO2 decreased with dose. Conversely, IR had no effect on total ERK1/2 expression. We concluded that iron ion IR could lead to BMDC dysfunction and IDO1 protein might play very important role in this deregulation.

Fig. 1. Dysfunction of BMDCs induced by iron ion IR. IFN-γ and TNF-α (A) mRNA expression and concentration in the BMDCs exposed to iron ion IR. A typical dot-plot profile of FSC vs. SSC and a histogram of surface molecule expression of BMDCs (B). Median of MHC class II, CD40, CD80 and CD86 expressions in the IR group (B). FACS analysis of CD3, CD4 and CD8 expression on spleen T cell numbers indicates the percentage of T cells that fall within the indicated quadrants (C). Surface marker expressions of CD4 and CD8 on CD3⁺ spleen T cells co-cultured with BMDCs in the IR group (C). Population of CD4⁺CD25⁺ T cells, CD4⁺FOXP3⁺ T cells and CD4⁺CD25⁺FOXP3⁺ Tregs distribution analysis in the IR group (D). ^aaap < 0.001, ^aap < 0.01 and ^ap < 0.05 compared to the control.

Fig. 2. The relative optical density of protein expression of IDO1, IDO2, JNK1/2/3, p-cJUN, p38/MAPK, p-p38/MAPK, ERK1/2 and p-ERK1/2 in mice BMDCs. (A) IDO1, IDO2, JNK1/2/3, p38/MAPK, ERK1/2, and p-ERK1/2 expression analysis in BMDCs. (B) Protein expression analysis of IDO1, IDO2, JNK1/2/3, p-cJUN, p38/MAPK, p-p38/MAPK and p-ERK1/2 in the absence and overexpression (C) of IDO1. IDO1 expression analysis in the absence of JNK1/2/3 (D). ^aaaP < 0.001, ^aaP < 0.01 and ^aP < 0.05 compared to control, ^bbbP < 0.001, ^bbP < 0.01 and ^bP < 0.05 compared to siRNA/Con group, ^cccP < 0.001 and ^cP < 0.05 compared to control group.

BMDC dysfunction via the IDO1 signal pathway

To further elucidate how IDO1 is regulated in BMDCs, we used siRNA and plasmid to inhibit or increase IDO1 expression. We measured IDO2, JNK1/2/3, p-cJUN, p38/MAPK, p-p38/MAPK and p-ERK1/2 protein levels in BMDCs in the absence or overexpression of IDO1. Moreover, the protein expression between the IR group and siRNA/Con or pcDNA/Con group did not differ significantly (p > 0.05). From Fig. 2B, one can observe that there were no significant changes of IDO2, JNK1/2/3, p-cJUN and p38/MAPK expressions in the siRNA/IDO1 group compared to siRNA/Con. However, p-ERK1/2 and p-p38/MAPK expressions decreased or increased because of deletion or over-expression of IDO1 as shown in Fig. 2B and C. Otherwise, the absence of JNK1/2/3 could inhibit the high-level expression of IDO1 as shown in Fig. 2D. As shown in Fig. 3, Kyn concentration increased after IR (p < 0.001). We found that siRNA/Con and DMSO did not change the Kyn level when compared with the IR group (p > 0.05), and the level of Kyn decreased with the absence of IDO1 or JNK1/2/3. Moreover, this study revealed that the IFN-γ and TNF-α secretion of BMDCs have no significant differences between IR and siRNA/Con, or siRNA/Con and siRNA/IDO1 groups as shown in Fig. 4 (p > 0.05). Taken together, IDO1 expression and function are strongly modulated by heavy ion IR via expression of JNK1/2/3 and might have no relation to IFN-γ and TNF-α secretion, and up-regulated IDO1 expression and function could lead to high level of p-ERK1/2 and p-p38/MAPK expressions.

Fig. 3. l-Kynurenine release of BMDCs in the IR, siRNA + IR and inhibitor JNK + IR groups. ^aaap < 0.001 compared to the control, ^bbbp < 0.001 compared to the siRNA/Con group, ^cccp < 0.001 compared to the DMSO group.

Fig. 4. IFN-γ (A) and TNF-α (B) secretion levels in BMDCs in the siRNA + IR group.

Then, we analyzed the levels of surface molecule expression and capacity of antigen-presenting of BMDCs in the IR, siRNA/Con, and siRNA/IDO1 groups by means of FACS. In Fig. 5, one can observe that there were no significant changes between the siRNA/Con and IR groups (p > 0.05). There were significant increases of MHC class II and CD80 expressions in BMDCs, the numbers of CD4⁺ (p < 0.05), CD8⁺ (p < 0.01) and CD4⁺CD25⁺ T cells at a dose of 1 Gy in the siRNA/IDO1 group compared to the siRNA/Con group as shown in Fig. 6A–C. Furthermore, the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in the siRNA/IDO1 group was lower (p < 0.05) than the siRNA/Con group as shown in Fig. 6C. Overall, results indicated that IDO expression induced by iron ion IR could relate to the decline of the levels of surface molecule expression and capacity of antigen-presenting of BMDCs.

Fig. 5. The FACS results in the siRNA/Con + IR group. The expressions of cell surface molecules on BMDCs are shown in (A), the percentage of spleen CD4⁺ and CD8⁺ T cell in (B), and the status of CD4⁺CD25⁺FOXP3⁺ Tregs in spleen T cells co-cultured with BMDCs in (C).

Fig. 6. The FACS results in the siRNA/IDO1 + IR group. Median of MHC class II, CD40, CD80 and CD86 expressions are shown in (A), surface marker expressions of CD4 and CD8 on CD3⁺ spleen T cells co-cultured with BMDCs in (B), and the population of CD4⁺CD25⁺ T cells, CD4⁺FOXP3⁺ T cells and CD4⁺CD25⁺FOXP3⁺ Tregs distribution analysis in (C). ^bbp < 0.01 and ^bp < 0.05 compared to the siRNA/Con group.

Discussion

This study provides the first direct evidence that the JNK/IDO1 pathway functions in immune-suppressive BMDCs induced by iron ion IR. In this view, we have shown that iron ion IR could lead to BMDC immunosuppression; this suppression is related to IDO1 expression. Up-regulation of IDO1 being due to the signals of JNK1/2/3 could induce high expressions of p-ERK1/2 and p-p38/MAPK, then regulate the surface marker, and might influence BMDCs to drive T helper 1 type immune responses that have the potential to mediate tumor immune responses through multiple effectors, such as CD8⁺ cytotoxic T lymphocytes and CD4⁺ T helper cells.23,24

A large body of evidence shows that IDO1-expressing DCs could mediate immune tolerance.19,25 However, IDO1 expression is not a constitutive feature of DCs under homeostatic immunologic conditions, but it requires induction. In our experiments, we first observed promotion of IDO1 expression and enzymatic activity induced by heavy ion IR in BMDCs. IDO1 is an immunoregulatory enzyme that catalyzes the first and rate-limiting step of tryptophan metabolism along with l-kynurenine.26 Elevated IDO1 has indeed far-reaching consequences: if the essential amino acid tryptophan, necessary to synthesize proteins and antigen, is not present or present in a lower amount, T cell differentiation is inhibited.27 Regulation of IDO1 expression and enzymatic activity can be mediated by several cytokines and immunomodulating agents including TNF-α, IFN-γ, and LPS.19,28,29 Our results indicated that overexpression of IDO1 might not relate to IFN-γ and TNF-α secretion. Moreover, the down-regulation levels of IFN-γ and TNF-α were induced by Fe ion IR. It is reported that IR could induce suppression of immunity by the low level of cytokines in BMDCs.30 Otherwise, the results demonstrate that JNK1/2/3 could modulate the action mode of IDO1. The findings from Jung et al.31 indicated that LPS-induced IDO expression is mediated by JNK whereas IFN-γ-induced IDO expression is regulated by JAK. Our findings indicated that IR might induce LPS/JNK signaling pathway responses of IDO induction. Then, we evaluated the signaling pathways involved in the elevated IDO1 expression responses in BMDCs. We intended to demonstrate the possible basis of IR-induced IDO1 gene expression through the alteration of the cellular expression levels of the MAPK family. The mammalian MAPKs consist of ERK, p38, and JNK.32 Previous studies have demonstrated that MAPKs are involved in the regulation of some proteins including IDO1.33–35 The results indicate that p-ERK1/2 and p-p38/MAPK proteins could involve in IDO1 immunosuppressive effects, and up-regulation of IDO through regulating p-ERK1/2 and p-p38/MAPK expression might affect surface makers in BMDCs.35 Finally, our data support the concept that IDO2 is different from IDO1, and the results were suggesting that the IDO2 level might not relate to IDO1 expression.

The FACS results indicate that the expression of IDO1 could result in the down-regulation of MHC class II and CD80 expressions. The antigen presenting function of DCs is dependent on their activation, which is characterized by the expression of MHC class II molecules and co-stimulatory molecules such as CD80. It is reported that the low level of MHC class II could reduce DC production of inflammatory cytokines in response to Toll-like receptor ligands. It also weakened DC ability to interfere with antigen-specific CD4⁺ T cell activity in regulatory T-cell development.36 Reduced CD80 expression has also been correlated with reduced IFN-γ levels by increasing the Th2 cytokine levels in a number of studies,31,37 and the suppression of the Th1 response38 in reflecting the negative influence of antigens on DCs. Here, we found that the lower level expression of MHC II and CD80 resulted in poor CD4⁺ and CD8⁺ T-cell stimulatory capacity and increased the proportion of CD4⁺CD25⁺Foxp3⁺ Tregs in CD4⁺CD25⁺ T cells.39 A decreased number of CD4⁺ and CD8⁺ T cells might be implicated in autoimmune and inflammatory disorders; many chronic diseases including cancer are linked to inflammation disorder.40 The balance between the different immunological cells is responsible for a normal immunological function to maintain immune homeostasis. In the present study, reduced populations of CD4⁺ CD25⁺ cells showed enhanced FoxP3 positivity. It is reported that fewer CD4⁺CD25⁺ cells interacted with higher number of active Tregs, and the resulting suppressive activity of Tregs might be higher.41 The others had reported that a higher number of Tregs could play a greater role in suppressing the activity of antigen-specific CD8⁺ T cells.42 Furthermore, enhancement of only this phenotype of immune-inhibitory cells (CD4⁺CD25⁺Foxp3⁺ Tregs) may not be the sole factor acting on immunosuppression. Suppression of different immune-activator cells (CD8⁺, CD4⁺, and CD4⁺CD25⁺ T cells) may also contribute to the overall immunosuppressive condition in vivo. These cumulative effects contribute extensively to systemic immunosuppression in many types of malignancies.42

Conclusion

In summary, our current results lead us to believe that exposures to iron ion radiation are potential risk factors for immunological disturbances and suppression including dysfunction of BMDCs and T cell differentiation. Knowledge of molecular events occurring after exposure to high-LET HZE radiation is essential for devising strategies to minimize human health consequences associated with space travel. In this study, results demonstrated for the first time that there was toxicity of Fe ion IR events on dendritic cells, and in co-culture experiments of DCs with T-cells, we could verify a significant effect of JNK⁺IDO1⁺ BMDCs on the T cell differentiation indicating that also exposure to Fe ion IR might cause suppression in part of the immune system.

Funding

This work was supported by grants from the Key Program of National Natural Science Foundation of China (U1432248), the Ministry of Science and Technology National Key R & D project (2016YFC0904602), the National Natural Science Foundation of China (11605260), and the Western Talent Program of Chinese Academy of Sciences.

Conflicts of interest

The authors declare that research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

DCs

Dendritic cells

BMDCs

Bone marrow-derived dendritic cells

IDO1

Indoleamine 2,3-dioxygenase 1

SPE

Solar particle events

GCR

Galactic cosmic radiation

HZE

High Z and energy

IR

Irradiation

LET

Linear energy transfer

Trp

Tryptophan

Kyn

l-Kynurenine

LET

Linear energy transfer

FACS

Flow cytometry

FITC

Fluorescein isothiocyanate

PE

Phycoerythrin

APC

Allophycocyanin

IFN-γ

Interferon-γ

TNF-α

Tumor necrosis factor

siRNA

Small interfering RNA

Tregs

Regulatory T cells

JNK1/2/3

c-Jun NH₂-terminal kinases