264 - The Role of Macrophage Inflammatory Signaling in Skin Responses to Fractionated X Ray FLASH Radiotherapy

Purpose/Objective(s): The skin-sparing effect of FLASH radiotherapy is well validated in electron beams and has also been observed with X-rays. Although the NLRP3 inflammasome is known to play a critical role in radiation-induced tissue damage, its involvement in the protective effect of X-ray FLASH remains unclear. Furthermore, whether fractionated delivery modulates this pathway, remain unknown.

Materials/Methods: Eight-week-old male Balb/c mice were subjected to X-ray FLASH irradiation, delivered in either a single dose or fractionated regimens. Conventional irradiation served as the control. Gross and pathological skin responses were continuously monitored within 3 months to evaluate the acute and chronic skin toxicity. Subsequently, to investigate the radiation effects, bone marrow-derived macrophages(BMDMs) of mice received the same irradiation regimen. We assessed alterations in cell viability, polarization status, and key signaling molecules of the lysosomal membrane permeabilization and NLRP3 inflammasome activation pathways. Pharmacological interventions targeting intracellular ROS, NLRP3 activation, and lysosomal membrane stability were performed respectively to evaluate their impact on the X-ray FLASH effect in macrophages.

Results: In X-ray FLASH radiotherapy, 2- and 3-fraction regimens significantly reduced early and late skin damage in mice compared to conventional irradiation, with effects comparable to those of single-dose FLASH at equivalent biologically effective doses. Cellular studies identified macrophages as key mediators of this protective effect. Mechanistically, conventional irradiation triggered a damaging cascade involving ROS burst, lysosomal membrane permeabilization, and NLRP3 inflammasome activation, leading to pro-inflammatory macrophage polarization and cell death. Conversely, X-ray FLASH irradiation attenuated initial ROS production, thereby limiting lysosomal membrane permeabilization and subsequent NLRP3 inflammasome activation, ultimately driving macrophages toward a protective anti-inflammatory phenotype. Notably, this suppressed inflammatory cascade was consistently observed in both single and fractionated X-ray FLASH regimens. Interventional experiments abolished the X-ray FLASH protective effect, further confirming causality.

Conclusion: Fractionated irradiation does not abrogate the skin protective effect of X-ray FLASH radiotherapy. Furthermore, we identify the lysosomal membrane permeabilization–NLRP3 inflammasome activation axis in macrophages as a critical mechanism underlying this protective effect. The elucidation of these key physical and biological determinants provides a foundational rationale for accelerating the clinical translation of FLASH radiotherapy.