Electron Ultra-High Dose Rate FLASH And Conventional Irradiation Induce Distinct Regulations of Inflammatory Cytokines And CD8 T Lymphocytes Ratio In Mice

Purpose: Ultra-high dose rate FLASH irradiation has been shown to cause less normal tissue damage compared with conventional irradiation, also termed “FLASH effect”. However, the underlying mechanism was scarcely known. The purpose of the present study was to determine whether FLASH and conventional irradiation would induce differential inflammatory cytokines expression. Materials and methods: Female FvB mice were randomly assigned to three different groups: non-irradiated control, conventional (CONV) and FLASH groups. Mice were irradiated at 6 to 19 Gy of CONV (0.1 Gy/s) or FLASH (38.5-600 Gy/s) irradiation using an Elekta Synergy linac (6 MeV). Mice were immobilized in prone position in a custom-designed applicator with dosimetry films positioned under the body. Dose were verified by Gafchromic films. Enzyme linked immunosorbent assay (ELISA) were performed in serum samples of the mice at 6, 18 and 31 days after irradiation for four inflammatory cytokines: tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and IL-10. Flow cytometry using antibodies for CD3, CD8, CD4 and CD45 in blood were performed pre- and 1-week post irradiation. Results: At D6 (18-19 Gy), both IL-6 and TNF-α were elevated, and IL-10 was reduced in FLASH and CONV group, while IFN-γ was only significantly increased in conventional group, compared with control group. At D18 (10 Gy) and D31 (13-19 Gy), conventional RT significantly elevated levels of IL-6, IFN-γ and TNF-α and reduced IL-10 level compared with FLASH group and control group. Additionally, even low dose conventional irradiation (13 Gy) could induce higher level of pro-inflammatory cytokines and lower level of anti-inflammatory cytokine than high dose (17-19 Gy) FLASH irradiation at D31. Flow cytometry showed that the CD8+/CD45+ ratio in the blood were higher in the conventional than in FLASH. These data indicate that minor inflammatory cytokine levels of serum in FLASH could be result of the absent of immune overactivation induced by conventional irradiation. Conclusions: Ultra-high dose rate electron FLASH caused less inflammatory cytokine levels of serum which might be a result from less CD8+/CD45+ ratio in the blood. Thus, differential cytokines and CD8+ T cell expression between FLASH and conventional irradiation would be a potential mechanism for “FLASH effect”.