Fractionated alpha and mixed beam radiation promote stronger pro-inflammatory effects compared to acute exposure and trigger phagocytosis

Frontiers in Cellular Neuroscience 01 frontiersin.org Fractionated alpha and mixed beam radiation promote stronger pro-inflammatory effects compared to acute exposure and trigger phagocytosis MostafaKarimi Roshan 1, SergeyBelikov 1, MelissaIx 2,3, NicolettaProtti 4,5, ClaudiaBalducci 6, RichardDodel 2,3, J.AlexanderRoss 2,3 and LovisaLundholm 1* 1 Department of Molecular Biosciences, Centre for Radiation Protection Research, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden, 2 Therapy Research in Neurogeriatrics, Chair of Geriatric Medicine, University Duisburg-Essen, Essen, Germany, 3 Department of Geriatric Medicine, Center for Translational and Behavioral Neuroscience, University Duisburg-Essen, Essen, Germany, 4 Department of Physics, University of Pavia, Pavia, Italy, 5 Pavia Unit, National Institute of Nuclear Physics INFN, Pavia, Italy, 6 Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy Introduction and methods: Aiming to evaluate safety aspects of a recently proposed approach to target Alzheimer’s disease, wemimicked a complex boron neutron capture therapy field using a mixed beam consisting of highand low-linear energy transfer (LET) radiation, 241Am alpha particles (α) and/or X-ray radiation respectively, in human microglial (HMC3) cells. Results: Acute exposure to 2 Gy X-rays induced the strongest response in the formation of γH2AX foci 30 min post irradiation, while αand mixed beam-induced damage (α:X-ray = 3:1) sustained longer. Fractionation of the same total dose (0.4 Gy daily) induced a similar number of γH2AX foci as after acute radiation, however, αor mixed irradiation caused a higher expression of DNA damage response genes CDKN1A and MDM2 24 h after the last fraction, as well as a stronger decrease in cell viability and clonogenic survival compared to acute exposure. Phosphorylation of STING, followed by phosphorylation of NF-κB subunit p65, was rapidly induced (1 or 3 h, respectively) after the last fraction by all radiation qualities. This led to IL-1β secretion into the medium, strongly elevated expression of pro-inflammatory cytokine genes and enhanced phagocytosis after fractionated exposure to αand mixed beam-irradiation compared to their acute counterparts 24 h post-irradiation. Nevertheless, all inflammatory changes were returning to basal levels or below 10–14 days post irradiation. Discussion: In conclusion, wedemonstrate strong transient pro-inflammatory induction by daily high-LET radiation in a microglia model, triggering phagocytosis which may aid in clearing amyloid beta, but importantly, from a safety perspective, without long-term alterations. KEYWORDS radiation, microglia, inflammation, DNA damage, cGAS-STING, phagocytosis 1 Introduction Radiation therapy (RT) is one of the most common tools for cancer treatment, used either alone or in combination with surgery, hormone therapy, chemotherapy, and immunotherapy (Pereira etal., 2014). Technological advances of the last decades enable the delivery of RT to tumors with great precision; however, the dose tolerance of the healthy tissue remains an obstacle OPEN ACCESS EDITED BY Miou Zhou, Western University of Health Sciences, UnitedStates REVIEWED BY Tarun N. Bhatia, Emory University, UnitedStates Shun-Ming Ting, University of Texas Health Science Center at Houston, UnitedStates *CORRESPONDENCE Lovisa Lundholm [email protected] RECEIVED 29 May 2024 ACCEPTED 07 November 2024 PUBLISHED 09 December 2024 CITATION Karimi Roshan M, Belikov S, Ix M, Protti N, Balducci C, Dodel R, Ross JA and Lundholm L (2024) Fractionated alpha and mixed beam radiation promote stronger pro-inflammatory effects compared to acute exposure and trigger phagocytosis. Front. Cell. Neurosci. 18:1440559. doi: 10.3389/fncel.2024.1440559 COPYRIGHT © 2024 Karimi Roshan, Belikov, Ix, Protti, Balducci, Dodel, Ross and Lundholm. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 09 December 2024 DOI 10.3389/fncel.2024.1440559 Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 02 frontiersin.org on the path to maximal eradication of tumor cells. An obvious approach is to use new modes of RT deposition as well as the employment of heavy particles. Low-LET radiation-induced double-strand breaks (DSBs) generally undergo rapid repair, primarily through the mechanism of non-homologous end joining (NHEJ), and are less toxic to cells (Lees-Miller and Meek, 2003). High-LET radiation focuses energy along particle trajectories, causing localized and clustered DNA damage and, thus, is more lethal than similar doses of low-LET radiation (Asaithamby and Chen, 2011). Phosphorylation of the histone variant H2AX at Ser-139, leading to the formation of γH2AX foci, represent an early cellular response to the induction of DSBs. DNA lesions also induce the activation of DNA damage response genes including CDKN1A (p21), MDM2, and FDXR (see Chen etal., 1994; O'Brien etal., 2018; Jebelli etal., 2022 for review). One type of particle therapies currently in use for head and neck cancers and glioblastoma multiforme is boron neutron capture therapy (BNCT). BNCT principles were proposed as early as 1936, only 4 years after the discovery of neutrons. BNCT relies on 10 B-containing compounds. It involves external irradiation with low energy (thermal) neutrons, resulting in the creation of a de novo complex beam containing high-and low-LET radiation (α-particles as well as lithium nuclei, and gamma rays, respectively) through reactions between neutrons and 10 B in the target (tumor) cells (Malouff etal., 2021). In theory, exclusive boron delivery to target cells enables precise radiotherapy (RT) due to the sharp Bragg peak of high-LET particles with a range of about 10 microns. However, 10 B uptake by normal tissues remains a challenge (Miyatake etal., 2016). In BNCT, the cumulative dose depends on the 10B concentration. To avoid adverse effects, the 10 B concentration in tumors must besignificantly higher than in healthy tissues (ratio ≥ 3). For the aims of the present study and for the experiments described later on, it is useful to note that 10 B concentration in normal brain tissue during glioblastoma treatment typically ranges from 3.6 to 16.8 ppm (Elowitz etal., 1998; Bergenheim etal., 2005; Shimosegawa etal., 2016). Brain tissue consists of various cells including neural cells, microglial cells, astrocytes, and oligodendrocytes, each with distinctive characteristics and functions. One and the same radiation quality is able to influence them differently and activate different mechanisms and pathways accordingly. In the adult brain, microglia, as innate immune cells in the central nervous system, contribute to the maintenance of homeostasis, immune surveillance, and regulation of neuroinflammation (Stupp etal., 2005). They play critical roles in brain health and disease, including cognitive processes. Microglia can undergo changes in response to various stimuli toward a range of phenotypes from anti-inflammatory to pro-inflammatory, which shape neuroinflammatory responses. Pro-inflammatory microglia promote neuroinflammation and neurotoxicity by releasing inflammatory cytokines and chemokines, whereas the antiinflammatory microglia stimulate anti-inflammatory cytokines, healing, and neuroprotection (Paolicelli etal., 2022). To overcome the problem of restricted availability of primary human microglia cells, Tardieu lab established the human microglial clone 3 cell line (HMC3) by employing SV40 immortalization of human embryonic microglia cells. Since then, HMC3 cells have been comprehensively characterized and validated to present a relevant and robust model system for in vitro studies of brain cells, as the only commercially available immortalized human microglia cell model (Dello Russo etal., 2018). Up to the present, BNCT has been almost exclusively used for the treatment of various forms of cancer. Recently, weproposed adapting NCT by 10 B and 157 Gd for Alzheimer’s disease (AD), the most common cause of dementia, within the EU-funded NEutron Capture-enhanced Treatment of neurotoxic Amyloid aggRegates (NECTAR) project. Existing AD treatment options are primarily symptomatic and provide only moderate benefits. Wepropose to employ the synergy between an external beam of low-energy neutrons with 10B and 157 Gd-bearing compounds as amyloid beta (Aβ)-targeting agents, which allow for a boost in the radiation dose and switch the main quality of irradiation to high LET, specifically in Aβ-sites. Weanticipate that this strategy GRAPHICAL ABSTRACT Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 03 frontiersin.org could provide a bimodal treatment of the disease. Firstly, by local depolymerization of Aβ aggregates by high-LET particles, secondly, through a long-distance stimulation of the brain immune cells (microglia cells) by penetrating photons resulting from neutrons (Kim etal., 2020a; Kim etal., 2020b) and/or radiation from the high-LET particles. Wereasoned that for a hypothetical treatment against AD, the 10 B concentration in the brain tissue adjacent to the amyloid plaques should not exceed levels typical for normal brain tissue during glioblastoma treatment. Based on this and using a precautionary approach, a 10 B concentration equal to 14 ppm was chosen. At this concentration, the characteristics of the TRIGA reactor in Pavia, Italy (used for the NECTAR project), pre-determine the proportion of high-LET radiation of the BNCT beam as 75%, thus, motivating the use of the high/low LET in the ratio of 3:1 (Bortolussi etal., 2018). In this study, wereconstruct the complex BNCT beam to elucidate the effects of high and low-LET radiation alone or in combination, delivered via single or fractionated radiation treatment protocols to human microglial HMC3 cells in order to understand the distinctive responses to different radiation qualities and schemes in terms of toxicity and induction of inflammatory response. 2 Materials and methods 2.1 Cell culture The human microglial clone 3 (HMC3) cell line was purchased from ATCC (CRL-3304™), cultured in T75 flasks in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich, Germany) supplemented with 10% defined bovine serum (Sigma-Aldrich, Germany) and 1% penicillin–streptomycin (Sigma-Aldrich, Germany) and subcultured every 3 days. The cells were maintained at 37°C and 5% CO 2 and kept up to passage 20. Forty eight hours prior to irradiation cells were seeded on glass coverslips (ORSAtec, Germany) at the following densities (per well/coverslip): 2.0 × 10 5 cells for the γH2AX and phagocytosis assay, 2.5 × 10 5 cells for acute irradiation, and 1.0 × 105 for fractionated irradiation. To avoid cell overgrowth for fractionated irradiation, the cells were always subcultured 3 h after the third irradiation; and after the last fraction, the cells were replated in 6-well plates and 96-well plates for clonogenic and resazurin assay, respectively. A part of the cells was collected for gene expression, protein expression analysis and/or flow cytometry analysis 1, 3, 6, or 24 h after irradiation and kept at −80°C. The supernatant after cell pelleting was collected 24 h post-irradiation and kept at −80°C for ELISA assay. Part of the irradiated cells was maintained for further analysis at later time points (10 and 14 days), then harvested and kept at −80°C. 2.2 Irradiation protocols and sources Coverslips with growing cells were placed on a polyamide disk subsequently covered with a 2.5-um thick Mylar foil and placed in direct contact with the α-particle source using a motor device (see Staaf etal., 2012 for details). An Am-241 source was utilized for the α-particle irradiation, with a dose rate of 0.223 Gy/min and an average LET of 91 keV/μm. X-ray irradiation was performed using an X-ray tube which was operated at 190 kV, 4.0 mA without the inbuilt aluminum filter and with the dose rates of 0.068 Gy/min and 0.052 Gy/ min at the bottom-and top-shelf position, respectively. The mixed beam irradiation was performed by using both of the sources simultaneously. The X-ray tube was always switched on when the disk with coverslips was at the top position contacting the α-source (Staaf etal., 2012). 2.3 Cell viability and cell survival assay Three hours post-irradiation the cells were harvested from the coverslips by trypsinization and reseeded in triplicate at the concentrations of 600, 1,200, and 2,400 cells per well in a 96-well flat bottom plate and left in the incubator for 5 days. Resazurin reagent was added at a final concentration of 0.1 mg/mL and the plate was incubated for 4 h at 37°C in the dark. Subsequently, plates were analyzed using a microplate reader (BMG Labtech, Germany) to measure fluorescence, resazurin has ex/em of 530–560/590 nm. In parallel, HMC3 cells were replated in duplicate at a density of 200, 400, and 800 cells per well for non-irradiated and 800, 1,600, and 3,200 cells/well for irradiated cells using the fractionated scheme, and 200, 400, and 800 cells/well for singledose exposure scheme. Colonies were allowed to form for 10 days, then fixed with 1:3 acetic acid-methanol fixative and subsequently stained with 5% Giemsa in 25% methanol for 30 min. The colonies were counted using the ImageJ macro countPHICS (Brzozowska etal., 2019). 2.4 γH2AX assay Cells were seeded on square 22 mm glass coverslips 48 h prior to irradiation and fixed 10 min, 30 min, and/or 24 h after exposure in 70% EtOH. Treatment with 0.2% Triton X-100, 5 min was used for permeabilization of the cells on the coverslips. Subsequently, the cells were rinsed with PBS and stained with anti-phospho-Histone H2AX antibody Ser139 (16-202A, Sigma-Aldrich, Germany, 1:200) in 2% BSA in PBS at 37°C for 30 min followed by rinsing with PBS. Samples were incubated with secondary anti-mouse IgG fluorescein isothiocyanate (FITC) (Sigma-Aldrich, Germany, 1:800) in 2% BSA in PBS. Coverslips were counterstained with DAPI and mounted on an objective glass using VECTASHIELD® containing DAPI (Vector Laboratories, UnitedStates). Images were captured using a fluorescent microscope using a 100X objective (Nikon Eclipse E800; Nikon, Tokyo, Japan), and the total number of γH2AX foci per cell was scored using a macro for ImageJ version 1.43u, as previously described (Sollazzo etal., 2017). From each group, at least 50 cells were randomly selected for analysis, ensuring an equal number of cells per group. 2.5 Gene expression analysis RNA extraction was performed using the E.Z.N.A. Total RNA Kit I(Omega Bio-Tek, UnitedStates). cDNA was synthesized using a HighCapacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, UnitedStates). The reaction mix consisted of primers, cDNA, and 5x HOT FIREPol ® EvaGreen ® qPCR Supermix (Solis BioDyne, Estonia). Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 04 frontiersin.org Real-time PCR was carried out in 96-multiwell plates in duplicate using a LightCycler® 480, and the temperature protocol was starting at 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s. Primers were toward CDKN1A, MDM2, FDXR, IL-18, IL-12α, IL-10, IL-1β, CD163, and CD206 (see Sollazzo etal., 2017; Panda etal., 2021; López-Riego etal., 2023; Roshan etal., 2023 for primer sequences) (LGC Biosearch Technologies, Denmark). The data was normalized against 18S rRNA and GAPDH. The 2−∆∆Ct method was used to calculate the relative fold gene expression (Livak and Schmittgen, 2001). 2.6 ELISA assay Analysis of secretion of IL-1β protein in the media was carried out using a commercially available IL-1β ELISA kit (Sigma-Aldrich, Germany) based on an antibody sandwich method using microtiter plates, coated with the IL-1β cytokine. Supernatants from samples were concentrated using a vacuum centrifuge with the aim of concentrating IL-1β in samples, frozen samples (−80°C) were directly placed in the vacuum centrifuge and centrifuged for 3 h, in order to reduce the volume from 1,000 to 200 μL. Each sample was assayed in duplicate, according to the manufacturer’s instructions. Absorbance was measured by a microplate reader (BMG Labtech, Germany) at 450 nm. 2.7 Western blot Irradiated and non-irradiated cells were trypsinized, harvested, and lysed directly with loading buffer (10% SDS, 500 mM DTT, 50% glycerol, 500 mM Tris–HCL, and 0.5% bromophenol blue dye). Protein separation was achieved using 4–12% Bis-tris gradient gels in 1xMES running buffer (Invitrogen™, UnitedStates). The separated proteins were then transferred to a nitrocellulose membrane (Thermo Scientific, UnitedStates). Subsequently, the membrane was blocked using Odyssey® blocking buffer (Odyssey Blocking Buffer from LI-COR, UK) and Tris-buffered saline containing 0.05% Tween (TBST) in a 1:1 ratio at room temperature for 1 h. Probing was conducted using the following primary antibodies overnight at 4°C: Phospho-STING (Ser366, Thermo Fisher Scientific, 1:500), p65 (Sigma-Aldrich, 1:500) and P-p65 (Sigma-Aldrich, 1:500) as well as GAPDH (G8795, Sigma-Aldrich, 1:20,000). Subsequent to the primary antibody incubation, probing with secondary antibodies, infrared dye-conjugated goat anti-rabbit or donkey anti-mouse secondary antibodies (LI-COR, Cambridge, UK, 1:15,000) was carried out for 1 h at room temperature. The membranes were scanned, and the levels of proteins were analyzed using the Odyssey® S Infrared Imaging System (LI-COR) and quantified with Image Studio™ Lite version 5.2 (LI-COR, UK). 2.8 Phagocytosis assay Phagocytosis assay was conducted using a commercial phagocytosis assay kit (Sigma-Aldrich, Germany). Zymosan particles labeled with a red fluorophore, enabling detection and measurement through a fluorescent microscope, spectrophotometer, or flow cytometry, were used. Seventy twohours after the last fraction of irradiation, HMC3 cells were harvested and plated in a black 96-well plate with a clear bottom for spectrophotometry, and on square coverslips for fluorescent microscopy, suspended cells were removed after an hour of incubation. Subsequently, cells were treated with 5 μL of Zymosan and incubated overnight. The wells were rinsed with the provided phagocytosis buffer according to the manufacturer’s instruction. Quantification was performed in duplicate using a microplate reader (SpecraMax i3x, United States) at an ex/em wavelength of 540/570 from the bottom. In addition, the coverslips were immunostained with Anti-α-Tubulin−FITC (F2168, SigmaAldrich, Germany), counterstained with DAPI, and mounted on the objective glass slides for fluorescent microscopy. 2.9 Flow cytometry HMC3 cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Germany) using the CytExpert software. Cells in anti-inflammatory state were defined by CD206 (Alexa Fluor® 700 anti-human CD206 (MMR) Antibody, BioLegend, Koblenz) surface marker. Intracellular staining for TNF-α (APC anti-human TNF-α Antibody), IL-1β (FITC anti-human IL-1β Antibody) and IL-10 (PE/ Dazzle™ 594 anti-human IL-10 Antibody, all from BioLegend) was performed after treatment with Fixation/permeabilization Kit (BD Biosciences) according to the manufacturer’s manual. 2.10 Statistical analysis Data normality distributions were confirmed using the Shapiro– Wilk test. Therefore, statistical analysis was carried out using two-way ANOVA and one-way ANOVA considering the number of variables, and multiple comparisons were corrected by Bonferroni’s and Tukey’s tests, respectively (GraphPad prism ver. 10.1.0). p value < 0.05 was considered statistically significant when comparing α or mixed beams to X-rays (*), or these groups to themselves at the different time points. Comparisons to control ( # ) were performed as indicated in the figure legends. Cell viability data points for X-ray and α-particles and also the survival curve of X-ray irradiated cells were fitted to a linear quadratic equation ( ) 2 aD D Se β −+ = where D is the total radiation dose in Gy, α and β are fitting coefficients. The survival curve of α-irradiated cells and also residual γH2AX foci data points were fit to a linear equation ( ) Se D α − = , α is a fitting coefficient. 3 Results 3.1 Experimental design To mimic the effect of BNCT in healthy tissue we used a high-LET/low-LET, i.e., α/X-rays ratio equal to three based on the abovementioned consideration, and most of the experiments were conducted using this ratio. Two irradiation protocols were employed: acute (single dose) and fractionated as shown in Figure1A. Mixed beam irradiations were performed by simultaneous use of both high-and low-LET sources. Routinely, a 2 Gy dose was delivered to cells either in “one shot” (acute protocol) or in five consecutive daily fractions of 0.4 Gy (fractionated protocol). Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 05 frontiersin.org 3.2 DNA damage and γH2AX foci formation We first characterized the formation of double strand breaks (DSBs) in acutely irradiated HMC3 cells by analysis of γH2AX foci. Cells were irradiated with either α-particles or X-rays alone (0.5 to 2.0 Gy) or using a mixed beam with different proportions of low-and high-LET components, namely 25/50/75% high LET with a total dose of 2 Gy, as well as 50% of each with a total dose of 1 Gy. FIGURE1 (A) Schematic representation of the fractionated irradiation protocol. (B,C) Violin plots showing γH2AX foci numbers per cell after different irradiation modalities using all values from three experiments (50 cells per experiment) at 30 min (B) and 24 h (C) after acute irradiation of HMC3 cells. Dashed lines in violin plots represent median. (D,E) Plots illustrate the same data using mean ± SD for three independent biological experiments, after subtraction of control foci. Lines represent fitted lines for X-ray and alpha particle data points using linear regression. (F) Plot displays both dose–response curves of X-ray and alpha particles at different time points. (G) γH2AX foci formation at 24 h after the last fraction using a total dose of 2 Gy given by an acute or fractionated irradiation protocol displayed as a violin plot (as in B,C) or mean ± SD (as in D,E). (H) Representation of fluorescence microscopy of the nucleus of HMC3 after fractionated irradiation with different radiation qualities. Symbols are nudged for transparency. Asterisks represent significance at the levels of *** < 0.001 and **** < 0.0001. Statistical analysis was carried out using two-way ANOVA and multiple comparisons were corrected by Bonferroni’s test. Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 06 frontiersin.org Foci were quantified at 30 min and 24 h post irradiation. The results of γH2AX quantification are shown in Figures1B–F. At all radiation modalities and exposure levels weobserved a significant increase in the number of foci compared to non-irradiated samples. A clear tendency toward a higher number of foci was observed in cells 30 min after irradiation with X-rays compared to α-particles. The numbers of foci detected in cells irradiated with the mixed beam at different α:X-ray ratios were comparable but slightly higher than the numbers weobtained for samples that only received the dose of the X-ray component in a mixed beam. At 24 h after irradiation the number of foci for X-ray irradiated cells decreased, whereas the number of α-particle induced ones tended to increase as indicated in Figures1C,E,H. Weconcluded that the higher the proportion of α-particles in the mixed beam, the higher levels of γH2AX foci remained after 24 h. We also compared the DSB formation when using acute versus fractionated protocols. HMC3 cells received a total of 2 Gy of α-, X-ray, or mixed beam (α: X-ray = 3:1) radiation as a single dose or in five equal consecutive fractions. Mean foci numbers detected 24 h post irradiation by both protocols were very similar as shown in Figure1G. However, this mean for the mixed beam is slightly lower in the fractionated setup. Interestingly, analysis of the overall population using a violin plot reveals a statistically significant reduction when using fractionation for both X-ray and mixed beam. 3.3 Expression of DNA damage response genes The expression of three genes known to beamong the first responders to radiation-induced DNA damage was assessed. At 6 h post acute irradiation, weobserved a noticeable upregulation of all tested genes. The overall expression pattern indicated a trend toward elevated gene expression in samples that received doses with high proportions of α irradiation (1.5 α + 0.5 X-ray and 2.0 α) as shown in Figures2A,C,E. The tendency became even more pronounced 24 h post irradiation; the difference in RNA expression between samples irradiated with low-LET radiation (2.0 X-ray) and samples that received high proportions of α-radiation reached statistical significance for the CDKN1A and FDXR genes (Figures2A,E). Wealso assessed the mRNA levels of the selected DNA damage response genes in cells irradiated via the fractionation scheme 24 h and 10 days after the last irradiation. Similar to acute exposure, X-ray irradiated cells showed a moderate increase of mRNA levels (2–2.5-fold) 24 h post irradiation for all tested genes, while at 10 days post irradiation, expression levels returned to basal levels as indicated in Figures2B,D,F. In cells that received doses with high proportions of α-radiation (1.5 α + 0.5 X-ray and 2.0 α), gene expression was even more upregulated 24 h post irradiation and decreased to basal levels 10 days after the delivery of the last fraction. However, statistical difference between X-ray irradiated cells and those exposed to high proportions of α-irradiation was observed only for the MDM2 gene. Interestingly, the average fold changes in CDKN1A and MDM2 gene expression in response to mixed or α-irradiation approximately doubled at 24 h after fractionated versus acute exposure. 3.4 Radiation reduces cell viability and colony-forming ability of HMC3 cells and alters the cell and nuclear size Firstly, HMC3 cells were exposed to increasing single doses of αand X-ray radiation (acute protocol) to assess cell viability. Cell viability gradually decreased with the increase of X-ray radiation dose compared to the non-treated control cells. The decrease was somewhat more pronounced in cells exposed to α-particles. Clonogenic survival experiments showed the same tendency. As anticipated for high-LET irradiations, experimental data from α-irradiated samples could befitted with a linear model. However, as shown in Figure3A, the observed difference between two irradiation modalities was not as dramatic as could beexpected based on previous studies (Goodhead, 1999; Roobol etal., 2020). Interestingly, our data from HMC3 cells suggests a relative biological effectiveness (RBE) of α-radiation to X-rays for 20% survival being around one (see Figure 3C). This indicates that high-LET α-particle radiation is not more effective than low-LET X-rays in this experimental context, at least at the highest tested dose. Next, wecompared the effectiveness of the fractionated irradiation protocol against the acute one. Cells were exposed to 2 Gy of α-, X-ray, or mixed beam (total 2 Gy; α:X-ray = 3:1) radiation, and the results are presented in Figures3B,D. Both experimental approaches argue for minor differences in the cell viability and clonogenic survival following X-ray irradiation, only a tendency toward a sparing effect was evident from clonogenic survival. Contrary to this observation, fractionated irradiation with αand mixed beam radiation (75% of α-particles) resulted in a prominent decrease in both cell viability and clonogenic survival compared to acute exposure. These findings appeared somewhat contradictory to γH2AX foci results (see Figure1G) which demonstrated that mean foci numbers, i. e. the levels of DNA damage detected 24 h after irradiation via either of the protocols were relatively similar, yet the long-term effects at the gene expression and cell viability/survival level clearly differed. Interestingly, wenoticed a change in the nuclear and cell size of HMC3 cells after fractionated radiation exposure, particularly more pronounced after high-LET compared to low-LET radiation, as depicted in Figures3E,F. Additionally, both cell and nuclear sizes exhibited a similar trend, with the most notable increase observed after mixed beam radiation. An increase in cell size has previously been reported as a feature of activated microglia (Davis etal., 2017). Furthermore, our microscopic observations showed an increased number of microglial cells with amoeboid morphology in the alpha and mixed beam irradiated groups (data not shown), which aligns with elevated levels of pro-inflammatory cytokines and enhanced phagocytosis. However, confirming microglial activation requires further investigation using specific markers. Activation of cGAS-STING and NF-κB pathways in irradiated HMC3 cells. To investigate the cyclic GMP-AMP synthase (cGAS)—stimulator of interferon genes (STING) and NF-κB pathway activation in HMC3 cells after exposure to fractionated irradiation and the kinetics of the activation, phosphorylation of STING, as well as phosphorylation and protein expression of the p65 subunit of NF-κB were analyzed at 1, 3 and 6 h after the last fraction of irradiation (Figure4A). Our results regarding phosphorylation of STING indicated that the activation of STING occurred earlier and also at a higher magnitude compared to the NF-κB pathway (Figure4B). Furthermore, the phosphorylation of Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 07 frontiersin.org FIGURE2 mRNA expression of DNA damage/repair genes. Relative levels of CDKN1A, MDM2, and FDXR mRNA in HMC3 cells were assayed 6 and 24 h after acute exposure to radiation beams comprising different proportions of α-particles and X-rays as indicated (A,C,E), or 24 h and 10 days after fractionated irradiation (B,D,F). Bars represent mean results and symbols represent individual values from independent experiments (n = 3–4). Error bars signify SD. Asterisks represent significance at the levels of * < 0.05 and ** < 0.01. Statistical analysis was carried out using two-way ANOVA and multiple comparisons were corrected by Bonferroni’s test. Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 08 frontiersin.org FIGURE3 Radiation reduces cell viability and clonogenic survival and enlarges the nuclear and cell size of HMC3 cells. Cell viability plots are depicted after acute (A) or acute versus fractionated (B) exposures, and clonogenic survival after acute (C) or acute versus fractionated (D) exposures. (E) Nuclear size box (Continued) Karimi Roshan et al. 10.3389/fncel.2024.1440559 Frontiers in Cellular Neuroscience 09 frontiersin.org p65 showed a slight increase time independently compared to non-irradiated cells (Figures4C,E). Only minor changes were seen in the total protein expression of p65 (Figure4D). Moreover, the ratio of P-p65/p65 after exposure to mixed beam and α-irradiation appear to peak later in comparison with low-LET, analogous to the delayed DNA damage response. Full blots are shown in Supplementary Figure S1. It is important to note that, although trends and minor alterations in the protein expression and activation of NF-κB and STING were observed, these changes did not reach statistical significance. The semi-quantitative nature of the Western blot method, as well as the variability in how cells are hit by alpha radiation, are likely contributors to the difficulties in reaching significance for these relatively low-magnitude changes. 3.5 Expression of pro-and anti-inflammatory markers in irradiated HMC3 cells To evaluate the effect of radiation on downstream cytokine release in HMC3 cells, mRNA expression of pro-and anti-inflammatory cytokine genes was examined. An increased expression of IL-1β, a key pro-inflammatory cytokine, was observed 6 h post-irradiation, across all radiation types. There was a clear trend of elevated IL-1β expression with a greater proportion of high-LET radiation, as depicted in Figure5A. After 2 Gy irradiation with mixed beam and α-particles, IL-1β expression was significantly higher than after exposure to X-rays alone. This trend continued 24 h later; however, statistical significance in IL-1β expression for α-particles alone was not reached (Figure5A). The fractionated irradiation scheme, illustrated in Figure 5B, revealed a strong induction of IL-1β expression across all radiation modalities 24 h post-irradiation. However, there was a clear trend of IL-1β expression returning to baseline levels 10 days later, and completely reverting to baseline within 2 weeks after the last irradiation. ELISA results showed an increased IL-1β secretion, particularly with high-LET radiation (Figure 5C). Notably, cells exposed to 2.0 Gy of α-particles exhibited significantly higher secretion compared to X-ray irradiated cells, supporting the correlation between radiation type and IL-1β secretion. The intracellular protein levels of IL-1β assayed with flow cytometry were also consistent with our mRNA and ELISA findings and showed an increase at 6 h, which returned to the basal level at 10 days (Figure5D). TNF-α, another well-known pro-inflammatory cytokine, showed the same pattern with a slightly higher magnitude compared to the IL-1β (Figure5E). We also analyzed the mRNA expression of IL-12α and IL-18 genes post-irradiation. At 6 h after acute exposure, only minor changes were observed (Figure5F), so the 24-h analysis was omitted. With the fractionated protocol (Figures 5G,H), a moderate increase in transcription of both cytokine genes was observed 24 h after the last fraction across all modalities. However, 10 days post-irradiation, transcription significantly decreased, exhibiting a V-shaped pattern, and reaching basal levels within 2 weeks. Subsequently, weshowed that phagocytic capacity of HMC3 cells following fractionated treatment displayed a statistically significant increase after αand mixed beam compared to X-ray irradiation (Figure5I). Next, we examined the expression of IL-10, a key antiinflammatory cytokine (Figure 6A). Minimal differences were observed in IL-10 mRNA levels at both six-and 24-h post-irradiation across all modalities, except for cells treated with 2.0 Gy of α-radiation, which showed a two-fold reduction in IL-10 expression at 6 h. With the fractionated protocol, a slight decrease in IL-10 expression was observed 24 h after the last fraction, but RNA levels returned to baseline 10 days post-irradiation (Figure6B). The intracellular level of IL-10 protein showed an increase compared to the unirradiated group at an early time point, i.e., 6 h, and returned to basal level 10 days after the last irradiation (Figure6C). Furthermore, gene expression analysis of CD163 and CD206 as anti-inflammatory markers at 10 days and 2 weeks post-fractionation showed no significant shift toward anti-inflammatory phenotype among the groups (Figure6D). Consistent with this result presence of surface marker CD206 and cell size 10 days post fractionated irradiation showed a similar level among all the groups, as depicted in Figures 6E,F. Figure 6G is summarizing the DNA damage and inflammatory response up to two weeks after fractionated high-LET radiation exposure. 4 Discussion Both low-and high-dose radiation therapy has been used to address conditions such as amyloidosis and more recently AD (Hall etal., 2022; Jebelli etal., 2022; Kaul etal., 2023). The use of NCT to target AD has been suggested as a potential treatment approach that allows utilization of both high-and low-LET radiation to eliminate amyloid plaques. However, in the case of AD where the targets of irradiation are extracellular molecules, aggregated amyloid peptide, the proposed procedure may affect the surrounding brain cells including neural and glial cells (Fukuda, 2021; Jin etal., 2022). In the current study, weinvestigated the inflammatory and toxicity responses of microglial cells to high-and low-LET radiation, as two components of the NECTAR-investigated alternative treatment for AD, alone and in combination, in the context of single-and multi-fraction irradiation schemes. γH2AX foci formation assay showed that X-rays and mixed beam (75% X-ray) delivered via acute scheme induced more foci and, thus, more DSBs 30 min after treatment, than α-irradiation. However, the decrease in number of γH2AX foci from 30 min to 24 h was much plot and the representative fluorescent microscopy of DAPI stained nucleus of HMC3 cells (100X objective). (F) Cell size (FSC-A; forward scatter area) and cellular granularity/complexity (SSC-A; side scatter area) of α, X-ray, and mixed beam-irradiated HMC3 cells 6 h after the fractionated scheme (n = 3). Error bars signify SD. 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