Thymoquinone Rescues T Lymphocytes from Gamma Irradiation-Induced Apoptosis and Exhaustion by Modulating Pro-Inflammatory Cytokine Levels and PD-1, Bax, and Bcl-2 Signaling

Radiation is an important environmental factor with hazardous effects on health, including oxidative stress [1,2], hematopoietic system dysfunction [3], genetic mutations [4] and immune dysfunction [5]. Ionizing radiation (IR) administered during radiotherapy generates free radicals when it passes through living tissues. IR generates reactive oxygen species (ROS) as a result of water radiolysis. These ROS can induce oxidative damage to vital cellular molecules and structures, including DNA, lipids, proteins and membranes [6,7]. DNA damage can result in chromosomal abnormalities, gene mutations and cell death [8]. Products of lipid peroxidation such as malondialdehyde (MDA) can interact with and alter macromolecules, potentially causing disease [9,10]. Thus, the modification of lipids and proteins by ROS has been implicated in the etiology of radiation-induced physiological disorders and diseases.

The immune system is one of the most important defense mechanisms against environmental agents. IR induces impairment of the immune response as well as a persistent inflammatory status through the deregulation of cytokine production [11,12]. Furthermore, the dysregulation of immune homeostasis, particularly CD4⁺ T cell function, may influence health status and impede tissue repair after exposure to IR [13]. Dainiak et al. [14] proposed that IR induces a reduction in lymphocytes and halts the proliferation of hematopoietic progenitors, leading to the suppression of immune function and increasing the risk of infection. IR also induces pro-inflammatory processes in which tumor necrosis factor (TNF-α), interferon (IFN-γ) and interleukin levels are altered, eventually leading to inflammatory disorders [15].

Naturally occurring antioxidants may provide protection against irradiation and may have therapeutic potential when administered after irradiation. Several plant constituents possess considerable free radical-scavenging or antioxidant activity [16,17]. Thymoquinone (TQ) is an abundant Nigella sativa essential oil compound that is responsible for many of the seed's antioxidant and anti-inflammatory effects [18]. TQ also has promising antitumor effects [19,20]. Woo et al. [21] observed that TQ is a free radical and superoxide radical scavenger and preserves the activity of various antioxidant enzymes. The potential immunomodulatory effects of TQ include significantly enhanced splenocyte proliferation, macrophage function, and natural killer (NK) antitumor activity [22]. N. sativa and its component, TQ, are promising natural radioprotective agents against the immunosuppressive and oxidative effects of IR [23]. Oral administration of TQ significantly reduces levels of the pro-inflammatory mediators IL-1β, IL-6, TNF-α, and IFN-γ and increases IL-10 levels [24].

In this study, we aimed to investigate the radioprotective activity of TQ in rats irradiated by gamma -rays. We focused on determining the effects of TQ on radiation damage in the thymus, radiation-induced T cell imbalance, and on immune dysfunction induced by gamma-rays.

TQ was purchased from Sigma Chemical Company (St Louis, MO, USA). TQ was > 99% pure and was reconstituted in saline at a concentration of 5 mg/ml. This stock solution was stored at 4°C in 15-ml centrifuge tubes wrapped in aluminum foil to prevent dimer formation.

Whole-body gamma-irradiation was performed at the National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt, using a (¹³⁷cesium) Gamma Cell-40 biological irradiator. Animals were irradiated at a fractioned dose level of 4 Gy delivered at a dose rate of 0.012 Gy/s.

Forty adult male rats (Rattus norvegicus) weighing 150 - 170 g were obtained from Theodor Bilharz Research Institute, Cairo, Egypt. All animal procedures were performed in accordance with the guidelines for the care and use of experimental animals established by the Committee for the Purpose of Control and Supervision of Experiments on Animals and the protocols of the National Institutes of Health. The Animal Ethics Committee of the Department of Radiation Biology, NCRRT, Atomic Energy Authority, Egypt, approved the protocol used in this study in accordance with the Declaration of Helsinki. Animals were allowed to acclimate for 2 weeks before the experiment and were housed in metal cages in a well-ventilated room. The animals were maintained under standard laboratory conditions (25°C, 60-70% relative humidity and a 12-hour light/dark cycle) and fed a standard commercial pellet diet and water. Animals were divided into three groups (10 animals per group). In the control group, rats were administered vehicle (saline) by gavage for 28 consecutive days. The irradiated group was exposed to γ-rays (4 Gy/14 days for 28 days). The TQ + irradiated group was exposed to gamma-rays (4 Gy/14 days for 28 days) and received TQ (5 mg/kg, gavage) 1 hour after irradiation for 28 consecutive days.

Lipid profiles were determined using BioMerieux kits and a standard assay method. Cholesterol levels were evaluated using the cholesterol esterase method. Triglycerides were measured using the lipase method. HDL, LDL, and chylomicrons were precipitated with phosphotungstic acid. The amount of cholesterol bound to HDL was determined using the cholesterol oxidase method and the phosphotungstate-magnesium salt method using a Cholesterol E-Test Kit (Wako, Osaka, Japan) according to the manufacturer's instructions.

Lipid peroxides are unstable indicators of oxidative stress in cells that decompose to form complex reactive compounds, such as MDA. Thiobarbituric acid-reactive substances (TBARS), the final products of lipid peroxidation, were assayed spectrophotometrically.

The levels of the plasma cytokines IL-6 and TNF-α were determined by ELISA using commercially available kits (R&D Systems, USA) according to the manufacturer's instructions. The cytokine concentrations were then calculated from a standard cytokine curve included on the same plate as the samples.

Cell surface antigen expression was determined by single-parameter fluorescence-activated cell sorter (FACS) analysis as previously described [25,26,27] using the following monoclonal antibodies (mAbs): FITC-conjugated anti-CD4, FITC-conjugated anti-CD8, and FITC-conjugated mouse isotype-matched control mAbs, all purchased from BD Biosciences. After incubation in culture for 4 hours, PBMCs were washed 3 times in PBS, washed 2 times with FACS buffer (1% BSA in PBS) and then double stained with FITC-conjugated anti-CD4 and FITC-conjugated anti-CD8 or -IgG2a isotype control at 10 µl of antibody/10⁶ cells at 4°C for 30 minutes. The cells were then washed with FACS buffer, resuspended and stored in 500 µl of 2% paraformaldehyde solution. A FACSCalibur flow cytometry instrument (Becton Dickinson) was used for data acquisition and analysis. After viable cell gating, 15000 events per sample were analyzed. For each marker, the threshold of positivity was defined beyond the nonspecific binding observed in the presence of a relevant isotype control mAb.

The percentage of lymphocytes undergoing apoptosis was determined by flow cytometry. Dead cells were identified using the Trypan blue exclusion test. To distinguish viable, early apoptotic, late apoptotic and necrotic cells, the cells were washed and incubated in PBS containing 30% human AB serum (4°C for 30 minutes) prior to staining with annexin V-FITC and propidium iodide (PI) (15 minutes at 25°C) using a commercial kit according to the manufacturer's instructions (Abcam, Canada). The cells were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson) within 1 hour of staining, and the percentage of cells undergoing apoptosis was determined. The expression of caspase-3, Bax and Bcl-2 was evaluated by staining with FITC-conjugated anti-caspase-3, PE-conjugated anti-Bax and PE-conjugated anti-Bcl-2 or -IgG2a isotype control at 10 µl of antibody/10⁶ cells.

PE-labeled anti-mouse PD-1 and isotype control IgG mAbs were purchased from BD Biosciences (San Jose, CA, USA). After washing with cold PBS, 1 × 10⁶ PBMCs were pelleted by centrifugation at 300 ×g for 10 minutes at room temperature. The cells were subsequently incubated with PE-labeled anti-mouse mAbs for 30 minutes at 4°C, followed by a final washing in cold 1X PBS. The cells were then washed twice and fixed in 1X PBS containing 2% paraformaldehyde. PD-1 expression on viable cells was quantified by flow cytometry analysis.

The thymus tissue was isolated from control and treated animal groups and was transferred immediately to 10% formalin for 24 hr and dehydrated in ascending grades of ethanol (50-100%). Clearing was done in xylene and embedded in paraffin wax. Sections (4 μm thick) were prepared and then stained with hematoxylin and eosin (H & E). The preparations obtained were visualized using a light microscopy at a magnification of 100 X.

Results are expressed as the mean ± SEM. Variation between groups was measured by one way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Statistical significance was considered at P < 0.05.

Exposure to a high dose of gamma -irradiation induces dyslipidemia due to elevated serum triglyceride and cholesterol levels, increased levels of low-density lipoprotein (LDL) particles and decreased levels of high-density lipoprotein (HDL). This dyslipidemia induces immune cell exhaustion. We therefore monitored the lipid profiles of the 3 groups of animals. Serum levels of MDA, a marker of oxidative lipid damage and a major oxidative product of peroxidized polyunsaturated fatty acids, were significantly increased in irradiated rats compared with the control group (Fig. 1A). HDL-C levels were significantly lower in the irradiated group than the control group (Fig. 1B). By contrast, serum levels of LDL-C, triglycerides and cholesterol were significantly higher in irradiated rats than in the control group. Interestingly, a normal lipid profile was restored in irradiated rats following TQ treatment. We also monitored the plasma levels of various pro-inflammatory cytokines that can alter showed that immune cell function can be altered (Fig. 1C). The data acquired for eight individual rats per group are shown. In the irradiated group, we observed aberrant and significantly elevated levels of TNF-α and IL-6 compared with the control group, indicating prolonged pro-inflammatory conditions after exposure to gamma irradiation. By contrast, pro-inflammatory cytokine levels were significantly decreased in irradiated rats treated with TQ compared with untreated irradiated rats.

To further confirm that gamma-rays induce dyslipidemia and a sustained inflammatory condition, resulting in lymphocyte exhaustion, PD-1 expression was analyzed by flow cytometry (Fig. 2A). The results of one representative experiment out of eight revealed that, the percentage of PD-1 expression in stained PBMCs was low (14.54%) in cells isolated from control rats. By contrast, PBMCs isolated from the irradiated group were characterized by aberrant upregulation of PD-1 expression (41.89%). Interestingly, cells isolated from the irradiated group treated with TQ exhibited an obvious decrease in PD-1 expression (22.48%). The upregulation of PD-1 expression is an indicator of continuous activation and exhaustion. Data acquired from eight individual rats per group are presented as the mean MFI ± SEM for the expression of PD-1 (Fig. 2B) and revealed that PBMCs in the irradiated group were likely exhausted and that TQ treatment restored the functional state of the PBMCs.

To investigate the effects of TQ on irradiation-induced immune dysfunction, the numbers of CD4⁺ and CD8⁺ cells were measured using flow cytometry. The results of one representative experiment out of eight are shown in Fig. 3A. The mean percentage of CD4⁺ cells was significantly lower in the irradiated group (10.82%) than the control group (17.90%). By contrast, the irradiated group treated with TQ exhibited a significant increase in CD4⁺ cells (20%). Furthermore, as shown in Fig. 3B, the mean percentage of CD8⁺ cells was significantly lower in the irradiated group (6.04%) than the control group (14.60%). By contrast, the irradiated group treated with TQ exhibited a significant increase in CD8⁺ cells (16.50%). Data acquired from eight individual rats per group (Fig. 3C) revealed that the percentages of CD4⁺ and CD8⁺ cells were both significantly decreased in the irradiated group compared with the control group, indicating prolonged pro-inflammatory conditions after exposure to gamma irradiation. By contrast, TQ supplementation significantly increased the percentage of CD4⁺ and CD8⁺ cells compared with untreated irradiated rats, which indicates that TQ can ameliorate the radiation-induced decreases in T cell subsets and play important roles in immune system.

To investigate the protective effects of TQ on the survival of PBMCs after exposure to gamma irradiation, PBMCs isolated from control, irradiated, and irradiated + TQ-treated rats were stained with annexin V/PI and then analyzed by flow cytometry to determine the percentages of viable cells (lower left quadrant), early apoptotic cells (lower right quadrant), late apoptotic cells (upper right quadrant), and necrotic cells (upper left quadrant). The data of one representative experiment out of eight are shown in the dot plot (Fig. 4A), which reveals that 6.11% and 8.66% of cells underwent late apoptosis and early apoptosis, respectively, in the control group, compared with a significant increase 33.33% and 22.19%, respectively, in the irradiated group. By contrast, treatment with TQ significantly reduced the percentage of late apoptotic cells and early apoptotic cells to 21.27% and 16.46%, respectively, compared with the untreated irradiated group. Pooled data for eight individual rats in each group (Fig. 4B) indicated that treatment with TQ significantly (P ˂ 0.01) decreased apoptosis and necrosis in PBMCs induced by exposure to gamma irradiation.

To investigate the mechanism by which TQ attenuates radiation-induced apoptosis, Bcl-2, Bax and caspase-3 expression on PBMCs isolated from control, irradiated, and irradiated + TQ-treated rats were examined by flow cytometry. The results of one representative experiment out of eight are shown in Fig. 5A. These results demonstrated that the mean percentage of cells expressing Bcl-2 decreased significantly in the irradiated group (18.22%) compared with the control group (30.85%). Interestingly, the irradiated group treated with TQ exhibited a significant increase in Bcl-2 expression (33.88%). Moreover, as shown in Fig. 5B, the mean percentage of cells expressing Bax increased significantly in the irradiated group (22.76%) compared with the control group (10.62%). The irradiated group treated with TQ also exhibited a significant increase in Bax expression (13.48%). Furthermore, as shown in Fig. 5C, the mean percentage of cells expressing caspase-3 increased significantly in the irradiated group (20.60%) compared with the control group (9.97%). The irradiated group treated with TQ also exhibited a significant increase in caspase-3 expression (14.14%). The cumulative results for eight individual rats from each group (Fig. 5D) indicated that the percentages of cells expressing caspase-3 and Bax were markedly decreased in the TQ-treated irradiated group, whereas Bcl-2 expression was increased by TQ supplementation.

Because the thymus is the primary lymphoid organ in which T cells undergo differentiation and maturation, we next investigated whether TQ supplementation could reverse the histological changes in the thymus induced by exposure to gamma-rays. The normal thymus in the control group displayed incompletely separated lobules with a large, densely stained cortex and a narrow, well organized medulla (Fig. 6A). In the gamma ray-irradiated group, the thymus exhibited marked degenerative changes in the thickness of the cortex, with visible bodies of macrophages and medullary epithelial cells scattered throughout the cortex. The degenerated medulla displayed lymphocytic depletion, invading fibroblasts, and prominent hemorrhages scattered throughout the medulla (Fig. 6B). Treatment with TQ nearly restored the normal organization of the thymus in the irradiated group (Fig. 6C).

According to previous published works, it has been shown that radiation exposure is divided into low-dose (˂ 1Gy) and high-dose (> 1Gy) [28]. While, varying degrees of clinical toxicity from ionizing radiation may develop after a mild (1 - 2Gy), moderate (2 - 4Gy), severe (4 - 6Gy), very severe (6 - 8Gy), or lethal (>8Gy) exposure (International Atomic Energy Agency, 1998). In this context threshold dose of ionizing radiation exists for a biological effect remains debatable and classified the sub-lethal dose to be (2 - 4Gy) or (2 - 6Gy). It was provided a correlation between absolute lymphocyte count and ionizing radiation dose investigated that acute radiation syndrome on circulating lymphocytes varying from mild to lethal and (4Gy) is the critical dose to give a severe reduction in the lymphocyte [28]. Therefore, the researchers have focused to study the protective effects of natural antioxidants against (4Gy) gamma radiation-induced cellular damage to normal tissues [29,30].

Accordingly, in the present study we choose (4Gy) as sub-lethal dose induced immune suppression and defect in T-cells. We also used fractionated dose of (4Gy) for several reasons: 1) to study the acute effect of sub-lethal gamma radiation dose induced immune imbalance which is in consistent with previous study showed that fractions of (4Gy) cause total peripheral T cell decline and immunosuppressive effect [31]; 2) according to Boston university committee about irradiation of rodents demonstrated that fractionation of irradiation dose (the total irradiation dose of lethal dose split into two equal parts of sub-lethal dose (4Gy) separated by 14 days time interval) is the optimum method in order to minimize morbidity and mortality of rats; 3) our preliminary results showed that gamma irradiated rats for 14 days is mainly the optimal period to induce hematopoietic syndrome by gamma radiation; and 4) we used fractionated sub-lethal dose as a simulative for what applied during radiotherapy where human patients take more fraction doses. Moreover, TQ was orally supplemented for 28 days according to previous studies revealed the immune-modulator effects of TQ for 28 consecutive days [32,33].

Exposure to IR results in DNA damage, which induces apoptosis in radiation-sensitive tissues, including lymphocytes [28]. Immune suppression contributes to alterations in peripheral lymphoid populations and the impairment of immune responses [34,35]. Natural antioxidants play central roles in enhancing the immune system through mechanisms dependent on oxidative stress, which seems to play significant roles in many human diseases. In this context, we previously demonstrated the beneficial effects of TQ in the induction of growth arrest of multiple myeloma cancer cells and its protective anti-diabetic effects [36,37,38,39]. Moreover, we provided clear evidence for the effects of other natural antioxidants (camel whey protein and bee propolis) as immune-modulators in accelerating the healing process of diabetic wounds in experimental animal models [40,41,42,43,44,45,46]. Most interesting, we demonstrated that natural antioxidants isolated from snake and ant venoms enhanced normal lymphocyte functions and exerted antitumor effects in different human and animal cancer cells [47,48,49,50,51,52,53].

The present results show that fractionated, sub-lethal doses of gamma-irradiation produce significant oxidative damage. Whole-body exposure of rats to high-energy radiation causes tissue damage in several organs, as assessed by increased lipid peroxidation [54,55]. Thus, the modification of lipids by ROS has been implicated in the etiology of radiation-induced physiological disorders and diseases [56]. In the present study, a significant increase in the levels of serum cholesterol, triglycerides, and LDL and diminished levels of serum HDL were observed post-irradiation, suggesting that IR-induced oxidative stress alters lipid metabolism and the levels of serum lipoproteins and supporting an association between IR-induced oxidative stress and elevated levels of lipid fractions [56]. Therefore, we suggest that oxidative stress might be an important contributor to altered lipid metabolism following radiation exposure. In the present study, administration of TQ effectively decreased MDA levels and attenuated the alterations in the lipid profiles induced by gamma-irradiation, clearly indicating that TQ effectively reduces IR-induced oxidative stress. TQ was previously suggested to preserve the activity of various antioxidant enzymes and act as a free radical scavenger [24].

In addition to increasing oxidative stress by increasing lipid peroxidation and altering lipid levels, exposure to gamma-irradiation also stimulates the production of pro-inflammatory cytokines (TNF-α and IL-6). Treatment with TQ reduced this inflammatory condition and decreased TNF-α and IL-6 levels. Radiation induces inflammatory responses [57,58]. The release of pro-inflammatory cytokines such as TNF-α and IL-6 aggravates the IR-induced inflammatory cascade and plays a role in the development of late radiation effects [59]. Serum TNF-α and inflammatory changes are markedly reduced in rats administered TQ [60]. Tekeoglu et al. reported that TQ has anti-inflammatory effects and decreases the levels of inflammatory cytokines in circulation [61]. Similarly, antioxidant and anti-inflammatory properties of TQ have been reported in several diseases [62]. The present study demonstrates that TQ treatment prevents inflammatory changes induced by gamma-irradiation in rats.

The remarkable dynamic changes in inflammatory cytokine levels and increases in oxidative stress suggest that these changes may contribute to the impairment of immune responses. PD-1 is a member of the immunoglobulin superfamily, and its expression is inducible in T cells, B cells, and activated monocytes [63]. PD-1 signaling was recently identified as an important mechanism of antigen-specific T-cell dysfunction in chronic infections in humans [64,65]. In the present study, PBMCs isolated from irradiated rats were functionally exhausted and were characterized by high PD-1 expression. Local upregulation of the PD-L1/PD-1 axis following exposure to IR suppresses immune responses and limits the full expression of antitumor immunity [66]. Fascinatingly, TQ supplementation regulated PD-1 expression, suggesting that TQ may provide a protective effect against irradiation-induced immunosuppression.

We observed a significant decrease in CD4⁺ and CD8⁺ T cells that was positively correlated with lymphocyte exhaustion in irradiated rats. Significant decreases in naïve T cells have been observed in atomic bomb survivors [67]. The decreased proportion of CD4⁺ naïve T cells were attributed to reduced T cell responses to mitogens and proliferation [68]. By contrast, TQ supplementation ameliorated the decrease in the proportions of CD4⁺ and CD8⁺ T cells following exposure to gamma-irradiation. Consistent with these results, treatment with N. sativa oil induces an increase in the CD4⁺ to CD8⁺ T cell ratio and an increase in NK cell function [69]. N. sativa seeds and their component, TQ, have a stimulatory effect on the proliferation of T cells and responses to different mitogens [70].

In this context, to determine the mechanism by which IR induces apoptosis, an Annexin V/PI assay was performed to quantify the apoptotic cell populations. Significant increases in both the early and late stages of apoptosis and necrosis were observed. Moreover, treatment with TQ decreased apoptosis induced by gamma-irradiation in PBMCs. To further analyze the possible mechanism underlying IR-induced apoptosis, we examined the expression of Bcl-2, Bax and caspase-3 in PBMCs after exposure to gamma-irradiation. In the irradiated group, the expression of Bcl-2 and caspase-3 increased significantly, whereas Bax expression decreased markedly. Consistent with these findings, it has been reported that cell apoptosis induced by radiation is regulated by a complex balance between pro-apoptotic factors, including caspase-3, c-caspase-3 and Bax, and anti-apoptotic factors, including the Bcl-2 family [71,72]. Recently, it has been shown that induction of apoptosis in T cells was associated with imbalance activation of caspase and Bcl-2 [73,74,75]. In the present study, we observed that TQ reduced radiation-induced caspase-3 and Bax activation and enhanced Bcl-2 expression. Indeed, N. sativa and its component TQ enhance innate immunity functions and decrease apoptosis in lymphocytes in peripheral blood [76]. Our results indicate that TQ can prevent radiosensitive cells from entering apoptosis. The structure of the thymus was also examined in the present study. In the irradiated group the cortex and medulla were seriously damaged and lymphocytes were reduced. These tissue structures were effectively protected by TQ supplementation. Our results indicate that TQ rescues T cells from IR-induced apoptosis and impairment of T cell maturation within the thymus.

In summary, this study demonstrates that the radio-protective effect of TQ on immune system is attributable to its antioxidant and anti-inflammatory capacities. TQ relieved the damage to the immune system induced by IR via reducing immune dysfunction by reducing the apoptosis of T cells either directly or by attenuating the destruction of the thymus as the site of T cell maturation.

The authors are grateful to the Department of Radiation Biology, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt, for providing support.

The authors declare they have no competing interest, state that the manuscript has not been published or submitted elsewhere, state that the work complies with the Ethical Policies of the Journal and state that the work has been conducted under internationally accepted ethical standards after relevant ethical review.