Modulation of apoptosis against P . falciparum by low dose radiation in human PBMCs

Background: Implications of low dose radiation (LDR) have been well reported in cancer therapy but data is scanty on the therapeutic application of LDR in infectious diseases. Methodology: Human peripheral blood mononuclear cells (PBMCs) were cultured and exposed to 0.07 Gy. P. falciparum infected RBCs were mixed with the PBMCs after five hours of irradiation. Thereafter, PBMCs were monitored for micronuclei and apoptosis. Results: The low dose pre-irradiated PBMCs which were subsequently challenged with parasite, showed a reduction in micronuclei frequency and apoptosis as compared to controls. Conclusion: LDR inhibited apoptosis against P. falciparum in human PBMCs.


Introduction
Infection with malaria parasite occurs with a disturbance of immune homeostasis.It is associated with severe immune mediated pathology due to excessive inflammatory responses [1].Malaria specific antibodies mediate inhibition of parasite cytoadherence, inhibition of erythrocyte invasion, and antibody dependent cytotoxicity [2].Cellmediated immune effector mechanisms include macrophage activation by IFN-to kill parasitized erythrocytes and inhibit parasite growth [3].However, Malaria parasites have evolved to acquire diverse immune evasion mechanisms which are critical for the ability of the parasite to evade the host immune response.Depletion of lymphocyte population is one of the important factors of immunosuppression which may be due to apoptosis.Parasite exotoxins and soluble S-antigen are released when infected RBCs rupture.These toxins may be responsible for the reduction in the number of lymphocytes during the malaria infection.Excessive damage in the DNA results in the cell death by apoptosis.Micronucleus is the DNA segment which becomes separated from the genome and acts as a biomarker of DNA damage.A direct relationship has been found between the occurrence of micronuclei and apoptotic cells [4].
The most lethal form of malaria caused by Plasmodium falciparum poses a major problem in treatment because of increasing resistance to antimalarial drugs.Of various alternatives available for the control of parasitic/bacterial diseases, LDR has been found to be beneficial.Some studies on the therapeutic application of LDR in non cancerous diseases such as diabetes, arthritis and infectious diseases are also available [5][6][7].Furthermore, it is also known that LDR exposure reduces apoptosis in human PBMCs.Circulating PBMCs primarily include T cells and monocytes, which play defensive roles against plasmodium infection.Therefore, we planned to test the hypothesis whether LDR plays any role in the modulation of apoptosis in healthy human PBMCs against P. falciparum.

Materials and methods
Human PBMCs (1 × 10 6 cells/ml) from healthy volunteers (fellow scientific workers who consented to provide 5 ml peripheral blood, aged 20-30 yrs, non-smokers and no medical history within the last one month) were cultured in RPMI-human AB + heat inactivated serum, 1 mg/ml glucose, 40 g/ml of gentamycin sulfate and 2 mg/ml sodium bicarbonate.These cells were stimulated with 10 µg/ml phytohemagglutinin.Cells were irradiated with a low dose (0.07 Gy) of 60 Coγ-rays (45 rad/min) after 22 hours of culture initiation.After five hours of irradiation, routine culture of P. falciparum strain MRC2 (NIMR, India) containing trophoziotes and schizonts [1×10 6 parasite (iRBCs)/ml] was added to PBMCs culture and fresh human RBCs were also added accordingly [8].The cultures were maintained in flat-bottom 96well culture plates and incubated in 5% CO 2 at 37°C.Student's t-test was used for statistical analysis of the data.

Results
Cultured PBMCs were monitored for the micronuclei frequency and apoptosis.Micronuclei frequency was significantly (p < 0.05) enhanced in PBMCs challenged with P. falciparum without preirradiation with LDR in all donors (Table 1).On the contrary, the level of micronuclei frequency was reduced (17.5-37.5%) in the LDR pre-exposed PBMCs cultured with P. falciparum (except in donors 3 and 4) as compared to their controls (Table 1).In a similar fashion, low dose pre-irradiation drastically reduced (25.6-68.2%)apoptosis in PBMCs of eight donors subsequently challenged with P. falciparum as compared to non pre-irradiated PBMCs (Table 2).The parasite induced the apoptosis more than two-fold in PBMCs which were not preexposed with a low dose of radiation.No effect of LDR was observed in the PBMCs of donors 3 and 4.

Discussion
Reduction in micronuclei frequency is the marker of enhanced DNA repair in the cells.In our earlier findings, induction in DNA repair was reported in 0.07 Gy exposed human PBMCs subsequently treated with 5.0 Gy of radiation [9].Here also, low dose preirradiation reduced micronuclei in PBMCs challenged with P. falciparum as compared to the controls.Therefore, low dose pre-irradiation might be responsible for enhanced DNA repair in the PBMCs cultured with a parasite.The correlation between micronuclei frequency and apoptosis has been reported as directly proportional; i.e., under conditions of reduced apoptosis, micronucleus formation was also reduced [4].Induction of apoptosis has been observed during P. falciparum infection in human lymphocytes, endothelial cells, hepatocytes and neurons [10][11][12].It may also be the cause of lymphopaenia which occurs in the course of malaria because acute malaria infection causes a depletion of lymphocyte populations in the peripheral blood [13,4].However, Riccio et al. suggested that there is no correlation of apoptosis with lymphopaenia in the progression of malaria [14].Interestingly, low dose pre-irradiation reduced apoptosis in the PBMCs challenged with parasite as compared to non-irradiated PBMCs as shown in Table 2.This reduction in apoptosis by LDR against parasite suggests a positive effect of LDR in the therapy of malaria.In the absence of LDR, induction in micronuclei and apoptosis appeared in PBMCs cultured with parasite (Tables 1 and 2).Two donors (3 and 4) showed no effect of LDR on micronuclei frequency and apoptotic cells in the presence of P. falciparum.This variation among the donors may be due to the variability of LDR response in the different individuals because it is a genetically governed phenomenon [15].
LDR activates T cells, NK cells, and B cells, resulting in activation of the immune system and reduction in apoptotic cells induced by high dose radiation in healthy individuals [16][17][18].At the molecular level, expression of key proteins involved in the process of apoptosis with LDR exposed human PBMCs is also being studied in our laboratory.Reduced expression of pro-apoptotic proteins and induction of pro-survival proteins was observed in low dose radio-adapted human PBMCs (data not shown).In another study, low dose UVA-induced suppression of cutaneous lesions has been reported in mice infected with Leishmania [19,7].These studies also reported that suppression of cutaneous development was due to the systemic and upregulation of IFN and TNFα.Thus, the present study provides a clue for the therapeutic efficiency of LDR against malaria infection.

Table 1 .
Micronuclei frequency in human PBMCs challenged with P. falciparum after low dose 60 Co--irradiation % decrease in micronuclei frequency in PBMCs challenged with P. falciparum (iRBCs) = (MN 0Gy -MN 0.07Gy ) × 100/MN 0Gy .PBMCs were stained with Hoechst 33342 and total 1,000 cells were scored per sample at 400X magnification under fluorescent microscope.* indicates significant (p 0.05) decrease in the number of micronuclei per 1,000 cells in low-dose irradiated cells with respect to their respective controls.(Gy-Gray; uRBC-uninfected red blood cell; iRBCinfected red blood cell)

Table 2 .
LDR-induced suppression of apoptosis in humanPBMCs cultured with P. falciparum has relevance in the restoration of immune function in malaria patients.Further experimental studies are needed on various model systems infected with parasites to ascertain the usefulness of LDR.Modulation of apoptosis in LDR pre-exposed human PBMCs P. falciparum % decrease in apoptosis in PBMCs challenged with P. falciparum (iRBCs) = (apoptosis 0Gyapoptosis 0.07Gy ) × 100/apoptosis 0Gy .PBMCs were stained with Hoechst 33342 and total 1000 cells were scored per sample at 400X magnification under fluorescent microscope.*indicates significant (p 0.05) decrease in the number of micronuclei per 1000 cells in low dose irradiated cells with respect to their respective controls.