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”.

Despite FLASH-RT presents a promising technology that could improve the delivery of radiotherapy in the future, the limited availability of such devices cannot meet the fast-growing research demands of FLASH related biology studies. Furthermore, the underlying mechanism of "FLASH effect" was scarcely known. The expression of pro-in ammatory and anti-in ammatory cytokines regulated by FLASH and conventional irradiation in animal models was scarcely explored. The purpose of the present study was to determine whether FLASH and conventional irradiation would induce differential in ammatory cytokines expression.

Methods And Materials FLASH beam generation
Our modi ed linac setup was based on Lempart et al's. [18], with some changes to simplify its implementation. These changes reduced the equipment needs while ensuring a stable dose output. We used the analog signal detection port of the beam generation system reserved for engineers to identify and count the electron beam pulses. Unlike in Lempart's work [18], we, in principle, simpli ed the signal path of pulse identi cation to achieve a higher count accuracy by analyzing the picked signals. The test signal could be the magnetron current (MI) or the modulator's pulse forming network (PFN) with a sharp falling edge for each pulse, as seen in Fig. 1a. The test signal passed through a voltage comparator. At a preset number of pulses, the comparator's output was reversed and transmitted to the microcontroller to send a trigger to a high-speed relay, which was connected to the Function Keypad's (FKP) interrupt port. In doing this, we realized the beam termination at a single or any number of pulses as set. To set the delivered pulse number, an 8-bit timer was connected to a microcontroller unit (MCU, AT89C52, Atmel Corporation, San Jose, California, USA) (Fig. 1b), similar to the electrical control unit reported by Lempart et al. [18].
For mice irradiation, the "treatment head" of the linac, or the beam limiting device (BLD), was lifted and removed with the gantry set at 180 degrees. The sample was placed a short distance from the source to increase the dose output and the BLD safety interlocks bypassed. The lament current, magnetic eld, and charging current of the magnetron were adjusted to increase the radio frequency's (RF) power. The electron gun's output was ne-tuned while the RF's power was gradually increased so that the starting pulses were stable (Fig. 1a). The energy beam was slightly altered from the clinical 6 MeV beam.
To test if the dose output of each pulse was stable, a repeated single-pulse dose measurement was performed with a PTW Advanced Markus ion chamber (Type 34045, PTW-Freiburg) placed at a distance from the irradiation sample. In order to determine whether the output was stable, a single-pulse beam was repeated 25 times with 10 second intervals. Film measurement was compared with the chamber's measurement for consistency checks.
Till now, the optimum FLASH parameters are uncertain. Here, we tested two strategies to obtain an ultrahigh dose rate by either a manual repeated single pulse beam with a trigger interval of 20 seconds in between (Fig. 1c upper) or multiple pulses with a 10 millisecond (ms) interval triggered once. The pulse width was 3.3 microsecond (µs) and the PRF was 12.5 Hz for single pulse delivery and 100 Hz for multiple pulse delivery (Fig. 1c [19] demonstrated response differences to EBT3 lm between four electron beams with energies between 6 and 16 MeV was within 0.5%, so we decided to perform optical density (OD) calibration with a 10 MeV electron beam (Elekta Synergy linac) with a 40 mm x 40 mm eld size, 100 cm SSD, at the maximum dose depth. The dose points for calibration were from 1 to 15 Gy with 0.5 Gy increments.
Two calibration curves were generated, one for quick reading to ensure the correct dose was to be delivered to the samples, and one for reading the lm scanner (Epson Expression 11000XL, Seiko Epson Corporation, Nagano, Japan) 24-hours post-irradiation. Beam percentage depth dose curve (PDD) and absolute dose at the maximum dose depth of the FLASH beam were measured by two lms positioned perpendicularly in the mice applicator (depth 16 mm) (Fig. 2a).
Irradiation lms were read out with the lm scanner 24-hours post-irradiation and analyzed with MEPHYSTO mc² (Medical Physics Tool) (MEPHYSTO mcc 3.3, PTW-Beijing).

Mice irradiation
This study included 69 3 to 5 week old female FvB mice (Jackson Laboratory, Sacramento, CA). This study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center with the approval number of L102042019080P.
The FVB mice were anesthetized by iso urane and immobilized in the prone position in a customdesigned applicator, which consisted of lead blocks to position the head, 15 mm of thick silica gel and a mouse holding plate (Supple. Fig. 1a). Films were placed between the mice and the silica gel to calculate the dose irradiated to the mouse's ventral skin.
For FLASH irradiation, the applicator was placed 2 cm above the frame of the ionization chamber (SSD 15 cm). For conventional irradiation, the SSD was 95 cm in order to uniformly irradiated two to three mice at a time. The beam rst traversed the plastic applicator's 1mm thick base and the silica gel build-up before reaching the mice, which ensured that the mice's ventral skin was positioned at the maximum of the depth dose curve (Supple. Fig. 1b).

Statistical analysis
Single dose per pulse, mean dose per pulse, mean dose rate, intra-pulse dose-rate and total delivery time were calculated as follows: Single dose per pulse = Mean dose per pulse = Mean dose rate = mean dose per pulse x pulse repetition frequency (mean dose rate for FLASH treatment was modulated by the set PRF) Intra-pulse dose-rate = Total delivery time = pulse width x pulse number + pulse interval time x (pulse number-1) Data were analyzed by using GraphPad Prism. Student's t-test or ANOVA were used to compare the differences between two groups or among more groups. The data were presented as mean ± standard error. Statistical signi cance was denoted by P values. Degrees of signi cance were *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.
Beam pro les and percentage depth dose curves Successive three pulse delivery parameters at a distance from the ion chamber of 0 cm, 1.5 cm, 3 cm and 5 cm were used. Total doses were read out from the lms, then the dose per pulse was calculated by the total dose divided by three (Fig. 2b). Successive three, four and ve pulse delivery at a distance from the ion chamber of 0 cm, 1.5 cm, 3 cm and 5 cm were used. Total doses were read out from the lms (Fig. 2c). For the irradiated area of the mice, the beam's range of atness was 97%-105% (Supple. Figure 2b. blue line), and the maximum dose was at 2 mm subcutaneous depth (Supple. Figure 3a. red line).

Dose measurement by lm
Forty-four mice were irradiated using three dose levels of 6, 9, 12 and 15 Gy with a 20 second pulse interval. The mean pulse number was 3.64 (range: 2-5 pulses). The mean dose per pulse was 3.08 Gy (range: 2.68-3.48 Gy/pulse), with each pulse delivered with a beam-on time of 3.3 us, resulting in a mean intra-pulse dose-rate of 9.3 x 10 5 Gy/s, while the mean dose rate was 38.5 Gy/s (range: 33.5 ~ 43.5 Gy/s) with a PRF of 12.5 Hz. The mean irradiation dose for the 41 lm measurements was 11.3 Gy (range: 5.9-15.4 Gy) (irradiation lms for three mice were accidentally forgotten). Deviations between the prescribed dose and the lm measurement were 2.47% on average (-11.93% ~ 13.77%), and 78.0% of the data (32/41) were within ± 5%.
Taken together, FLASH irradiation caused transient elevation of pro-in ammatory cytokines changes and reduction of anti-in ammatory cytokine but gradually recovered to the non-irradiated group level within one month, however, no sign of recovery was seen in the CONV group.
In ammatory cytokines response reversely in low dose and high dose CONV and FLASH irradiation When mice were divided into low dose group (13 Gy) and high dose group (17)(18)(19), different cytokine expression between groups were distinguished. Even low dose CONV irradiation could induce higher level of pro-in ammatory cytokines and lower level of anti-in ammatory cytokine than high dose FLASH irradiation. However, high dose FLASH irradiation induced lower level of anti-in ammatory cytokine compared with non-irradiation group and low dose FLASH irradiation (Fig. 4a-d).
FLASH radiotherapy caused less intense immune response in peripheral blood than conventional radiotherapy It has been reported that IFN-γ was released from CD8 + T cells [20]. Since IFN-γ was only signi cantly increased in CONV group, we performed ow cytometry to test the CD8+/CD45 + ratio in three groups. We found that the CD8+/CD45 + ratio in the blood were higher in the CONV-RT than in FLASH-RT (one week post irradiation) (Fig. 5), in accordant with the high level of serum INF-γ in CONV group, indicating that more toxicities induced by CONV-RT was as a result of over-activated immune response, which was absent in FLASH-RT.

Discussion
To our knowledge, this is the rst report to compare expression of circulating cytokines in terms of time variation and dose response in ultra-high dose rate electron FLASH-and conventional-irradiated mice.
Here, we demonstrated that pro-in ammatory cytokines of IL-6 and TNF-α were transiently increased and reduced to control level in FLASH, but kept augmenting in conventional group within one month. Another pro-in ammatory cytokine of IFN-γ was only signi cantly increased in CONV group, compared with nonirradiated group and FLASH group. Furthermore, anti-in ammatory cytokine of IL-10 was transitorily decreased in FLASH group while remained downregulated in CONV group. Last but not least, low dose conventional irradiation (13 Gy) could induce higher level of pro-in ammatory cytokines and lower level of anti-in ammatory cytokine than high dose FLASH irradiation (17)(18)(19).
Previous studies suggest that conventional irradiation could trigger a pro-in ammatory response mediated by uctuant expression of in ammation-regulating cytokines [13-16, 21, 22]. However, only one study reported the neuroprotective of hippocampus with lower pro-in ammatory cytokine levels induced by FLASH than in conventional irradiation 10 weeks post-irradiation [9], similar to the results in the present study. Although both the results point towards decreased in ammation response in FLASHirradiated mice compared to conventional-irradiated mice, the potential mechanism is still not completely clear.
To determine whether immune cells participated in cytokines regulation, we measured CD8+/CD45 + ratio in the blood, and found that CD8+/CD45 + ratio were higher in the CONV-RT than in FLASH-RT (one week post irradiation). It has been reported that IFN-γ was released from CD8 + T cells [20]. In the present study, IFN-γ was only signi cantly increased in conventional group, compared with non-irradiated group and FLASH group, indicating the ultra-high dose rate and conventional dose rate could possibly displayed different immunological responses.
In regard of the potential role cytokines play in the pathogenesis of radiation-induced normal tissue injury, the different cytokine response after FLASH and conventional irradiation would conjecturably lead to tissue dysfunction and failure, such as brosis, necrosis and vascular injury [22]. Radiation-induced early and late in ammatory responses have been observed in multiple organs and in vitro studies although the underlying regulating mechanisms are not fully understood [13][14][15][16][17][21][22][23][24][25]. Whether FLASH and conventional irradiation affect radiation-related toxicities differently regulating through in ammatory and immune factors remained to be systematically explored.
From a technical point of view, we successfully generated a FLASH electron beam delivery by increasing the number of pulses through bypassing the linac's ion chambers. Tuning of the linac's operation parameters achieved a dose per pulse (DPP) of 6 Gy at a certain height above the ion chamber to irradiate mice and cell line samples, which is equivalent to and in some cases higher than some previously reported results [7,10,11,18,26]. The intra-pulse dose-rate was around 1×10 6 Gy/s, and the mean dose rate was between 33.5-633 Gy/s using a PRF of 12.5 Hz or 100 Hz with a 3.3 µs pulse width.
Such high DPP allowed us to generate a higher dose rate in a shorter delivery time. At present, which parameters play a critical role in the "FLASH effect", including mean dose rate, intra-pulse dose rate, total dose, DPP, the interval between two pulses and so on are still under investigated. However, in our study, we modi ed the Elekta linac to generate similar parameters to most studies, thereby making it suitable for in vivo experiments.
To mimic skin irradiation, we designed a mouse applicator that provided a dose buildup for the targeted ventral skin and used a lead block to protect the head of the mice. We achieved a 97%-105% atness of the irradiation area (Supple. Figure 2b. blue line) and a maximum dose of 2 mm subcutaneous depth (Supple. Figure 3a. red line), which was su cient for mice ventral skin irradiation. During mice ventral skin irradiation, lms for verifying doses were placed under the mice (Supple. Figure 1), thus the lm's reading represented the dose to the mice's ventral skin. The average deviation from the prescribed dose determined by the lm measurement was 2.47% (-11.93% − 13.77%), and 78.0% (32/41), measurements were within ± 5% for the rst 44 mice using EBT3 lms. However, the deviation in the next 25 mice veri ed by EBT XD lms were less than 5%. This might mean that the effective range of reliable dose-response in the EBT XD lm is wider than in the EBT3 lm, thus more suitable for ultra-high dose veri cation. Buonanno et al. [27] and Petersson et al.
[28] tested different monitors and dosimeters (Gafchromic™ EBT3, TLD, Alanine pellets, Markus and a custom-made parallel plate ion chamber, and a methyl viologen dosimeter). They concluded that the dose-rate and a level of uncertainty was in the order of 5% for in-vivo dosimetry. We further compared the immediate dose reading and the reading after 24 hours, and observed that the EBT XD lms had more consistent results than the EBT3 lms (Fig. 2a).
The current work had certain limitations. Firstly, only four cytokines IL-6, IL-10, IFN-γ, and TNF-α was tested. This is partly because IL-6 and TNF-α were differentially regulated by FLASH and conventional irradiation in previous study [9] and partly because they are widely used as principal indicators of the in ammatory response, hence they were suitable to elucidate radiation-related in ammatory changes. Furthermore, the CD8+/CD45 + ratio in the blood cannot re ect all the distinct impact that the two radiation types have on expression of in ammation-regulating cytokines.
In conclusion, with novel setup and a rigorous laboratory procedure for murine FLASH RT, we veri ed a distinct circulating pro-in ammatory and anti-in ammatory cytokines were differentially regulated by electron ultra-high dose rate FLASH compared with conventional irradiation chronologically within one month after irradiation. The differential expression of in ammation-regulating cytokines associated with immune response may be the in uencing factors of reduced toxicities after FLASH irradiation. Further studies are necessary for elucidating how FLASH and conventional irradiation in uence these processes.

Availability of supporting data
None.

Competing interests
The authors declare that they have no competing interests.   Successive three pulse delivery at a distance from the ion chamber of 0 cm, 1.5 cm, 3 cm and 5 cm. Total doses were read out from the lms, then the dose per pulse calculated by the total dose divided by three.
(c) Successive three, four and ve pulse delivery with a distance from the ion chamber of 0 cm, 1.5 cm, 3 cm and 5 cm. Total doses were read out from the lms.

Figure 4
In ammatory cytokines response reversely in low dose and high dose CONV and FLASH irradiation (a-c) At D31, low dose CONV irradiation signi cantly induced higher level of pro-in ammatory cytokines (TNF-α and IL-6) and lower level of anti-in ammatory cytokine (IL-10) than high dose FLASH irradiation.
However, high dose FLASH irradiation induced lower level of IL-10 compared with non-irradiation group and low dose FLASH irradiation (d). Results are plotted as mean ± SEM. Statistical signi cance of differences between treatment groups was determined using a one-way ANOVA with Fisher's LSD posthoc test. *P < 0.05, **P < 0.01 and ***P < 0.001. Note: L, low dose (13 Gy); H, high dose (17-19 Gy); red stars, compared with control; bule stars, compared with FLASH-L; black stars, compared with FLASH-H. FLASH radiotherapy caused less intense immune response in peripheral blood than conventional radiotherapy (a-d) Flow cytometry analysis of CD45+ cells and CD8+ T cells isolated from peripheral blood. (e) one week post irradiation, CD8+/CD45+ ratio was higher in the CONV-RT than in FLASH-RT.

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