Abstract
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.
Highlights
The most intensively studied radiobiological consequence of ionizing radiation was for long the induction of DNA damage and cell death as well as the various cellular pathways activated in response to DNA damage in the directly irradiated cells
The extracellular vesicles (EV) were further characterized by Western blot analysis following minimal required criteria suggested by Lötvall et al for EV identification [25]: a minimum of two EV-specific protein markers expected to be present in EV isolates and an endosomal protein not expected to be present in EVs were determined
Markers commonly used for exosome identification: the tetraspanin CD9, a protein highly enriched in EVs [26], and the TSG101, involved in multivesicular biogenesis [27], and were negative for calnexin, an endoplasmic reticulum marker (Figure 2E)
Summary
The most intensively studied radiobiological consequence of ionizing radiation was for long the induction of DNA damage and cell death as well as the various cellular pathways activated in response to DNA damage in the directly irradiated cells. Radiation-induced activation of pro- or anti-inflammatory pathways is a radiation response mechanism important at systemic level as DNA damage response at cellular level. Molecular pathways connecting radiation with inflammatory and immune responses are intensively studied. In a recent meta-analysis, several genes and pathways involved in immune response following ionizing radiation (IR) exposure were identified, such as transforming growth factor beta (TGFβ) signaling pathway, interleukin pathways, nuclear factor kappa B (NFκB) as the key transcription factor in the activation of immune system by IR, as well as regulation of DNA damage response by microRNAs (miRNA) [3]. The authors highlight the importance of toll-like receptors (TLRs) and the direct activation of inflammatory cytokine genes by NFκB and p53 [4]
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