Radiotherapy-Induced Changes in the Systemic Immune and Inflammation Parameters of Head and Neck Cancer Patients.

Though radiotherapy is a local therapy, it has systemic effects mainly influencing immune and inflammation processes. This has important consequences in the long-term prognosis and therapy individualization. Our objective was to investigate immune and inflammation-related changes in the peripheral blood of head and neck cancer patients treated with radiotherapy. Peripheral blood cells, plasma and blood cell-derived RNA were isolated from 23 patients before and at two time points after radiotherapy and cellular immune parameters, plasma protein changes and gene expression alterations were studied. Increased regulatory T cells and increased CTLA4 and PD-1 expression on CD4 cells indicated an immune suppression induced by the malignant condition, which was accentuated by radiotherapy. Circulating dendritic cells were strongly elevated before treatment and were not affected by radiotherapy. Decreased endoglin levels in the plasma of patients before treatment were further decreased by radiotherapy. Expression of the FXDR, SESN1, GADD45, DDB2 and MDM2 radiation-response genes were altered in the peripheral blood cells of patients after radiotherapy. All changes were long-lasting, detectable one month after radiotherapy. In conclusion we demonstrated radiotherapy-induced changes in systemic immune parameters of head and neck cancer patients and proposed markers suitable for patient stratification worth investigating in larger patient cohorts.

Extracellular Vesicles Mediate Radiation-Induced Systemic Bystander Signals in the Bone Marrow and Spleen.

Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects, and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms.

The effect of ionizing radiation on the homeostasis and functional integrity of murine splenic regulatory T cells.

OBJECTIVE: Radiotherapy affects antitumor immune responses; therefore, it is important to study radiation effects on various compartments of the immune system. Here we report radiation effects on the homeostasis and function of regulatory T (Treg) cells, which are important in down-regulating antitumor immune responses. METHODS: C57Bl/6 mice were irradiated with 2 Gy and alterations in splenic lymphocyte fractions analyzed at different intervals. RESULTS: Total CD4+ numbers showed stronger decrease after irradiation than CD4+Foxp3+ Tregs. Tregs were less prone to radiation-induced apoptosis than CD4+Foxp3- T cells. The ratio of CD4+Foxp3- and CD4+Foxp3+ fractions within the proliferating CD4+ pool progressively changed from 74:26 in control animals to 59:41 eleven days after irradiation, demonstrating a more dynamic increase in the proliferation and regeneration of the Treg pool. The CD4+Foxp3+ fraction expressing cell-surface CTLA4, an antigen associated with Treg cell activation increased from 5.3 % in unirradiated mice to 10.5 % three days after irradiation. The expression of IL-10 mRNA was moderately upregulated, while TGF-ß expression was not affected. On the other hand, irradiation reduced Treg capacity to suppress effector T cell proliferation by 2.5-fold. CONCLUSION: Tregs are more radioresistant, less prone to radiation-induced apoptosis, and have faster repopulation kinetics than CD4+Foxp3- cells, but irradiated Tregs are functionally compromised, having a reduced suppressive capacity.

Effects of low-dose radiation on the immune system of mice after total-body irradiation.

The effects of acute exposure to low- and high-dose radiation on the quantitative and functional parameters of the immune system were analyzed. C57BL/6 mice were irradiated with different doses of γ radiation (0.01, 0.05, 0.1, 0.5 and 2 Gy) and splenocytes were isolated at various times. Alterations in the distribution and surviving fraction of splenocyte subsets such as CD4(+) and CD8(+) T lymphocytes, regulatory T cells (Treg), natural killer (NK) cells, dendritic cells (DCs) and B lymphocytes were analyzed by flow cytometry. Apoptosis frequency was quantified by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method 4 h after irradiation. Cytokine expression was investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). Low doses decreased apoptosis in the splenocyte subpopulations studied most prominently in NK cells and DCs. Exposure to 2 Gy increased apoptosis in all splenocyte subpopulations; B cells were the most sensitive and NK cells and DCs the least sensitive. The lowest cell numbers were measured 3 days after irradiation, with minor changes by day 7. CD8(+) and B cells were rather resistant to low doses but were very sensitive to 2 Gy, while NK cells, DCs and Treg cells were much more resistant to high doses. Expression of the T-helper 1 (Th1)- and helper 2 (Th2)-type cytokines decreased after low doses and increased after high doses. Interleukin 6 (IL-6) reacted at early times and IL-10 at later times. IL-5 levels were consistently elevated. These data highlight the differences in the responses of different splenocyte subpopulations to low- and high-dose radiation.