Correction: Limited immune perturbations in mice exposed to sustained low-dose ionizing radiation

Ionizing radiation (IR) is widely used across various sectors; it powers vital medical diagnostics, fuels the engines of energy production, drives industrial tools and processes, and is wielded in weapons of mass destruction. As the utilization of this energy increases, the exposure of people, particularly among the growing occupationally exposed workforce, also increases. Therefore, understanding the interplay between ionizing radiation and cellular machinery is imperative. While the effects of high-dose ionizing radiation (HDIR) exposure are largely understood with scientific consensus, the biological effects associated with low-dose ionizing radiation (LDIR) exposure continue to be contested. LDIR is defined by the National Academies of Science as exposures resulting in absorbed doses below 0.1Gy (100 mGy) (1). Low-dose-rate radiation is typically defined as being under 0.5-0.6 mGy/hr (1,2). This level of exposure is significantly lower than exposures encountered in medical diagnostics or acute radiation syndrome (ARS) (3). Instead, it is more relevant to therapeutic procedures, natural background radiation, and occupational exposures, such as in the nuclear power industry or space travel (1). Despite efforts in both epidemiological and experimental studies, the field lacks a unified comprehension of the effects of LDIR on the immune system. HDIR is known to cause significant immune disruptions, including inflammation, hematopoietic syndrome, and lasting alterations in immune homeostasis (4)(5)(6)(7)(8)(9)(10)(11). However, it remains uncertain whether the effects of LDIR exhibit similar but more subtle changes or if they produce completely distinct outcomes. Without a thorough understanding of these effects, the potential role of the immune system in contributing to the reported positive excess relative risk ratios for cancer and other diseases following chronic occupational LDIR exposure remains uncertain (12)(13)(14)(15)(16)(17). In total body irradiation (TBI) studies using animals, exposure to low-doses of external X-ray or gamma radiation (<100 mGy) has been shown to cause modulations in immune cell populations. For instance, LDIR exposure has been associated with increased expression of Trp53 and Nrf2 (18)(19)(20). A single exposure to 100 mGy TBI can reduce circulating white blood cell counts and splenocytes in some models (19)(20)(21)(22). In other models, changes to cytokines, immunoglobulins, or even T cell receptor diversity have been explored as impacts of LDIR exposure, even in the absence of changes to immune cell numbers (20,21,(23)(24)(25). Other studies can be contrasting, for instance, LDIR in another model was shown to reduce apoptosis in the spleen (26,27). While the literature exhibits considerable variability, LDIR responses generally appear to be transient and of low magnitude, with their downstream implications for immune homeostasis remaining unclear. Furthermore, there is a growing appreciation for the cell-type specific responses to radiation exposure, but that has not been sufficiently explored in the low-dose range (6,21,(28)(29)(30). Studies suggest that LDIR can cause subtle and variable effects on the immune system. However, there remains little understanding of the molecular mechanisms underlying responses across diverse immune cell populations. Furthermore, most studies utilize high-dose rate single exposures; therefore, there exist minimal studies documenting responses to continuous LDIR exposure conditions. Due to subtle and heterogeneous effects, it has been recommended by the National Academy of Science charged with revitalizing low-dose radiation research, that future studies utilize high-resolution, high-throughput omic technologies and bioinformatic analysis to provide more detailed insights (1). The objective of this study was to characterize the effects of sustained LDIR on the murine immune system. Specifically, we sought to uncover and describe the mechanisms by which different immune cell populations respond to sustained whole-body radiation exposure. In collaboration with the Canadian Nuclear Laboratories (CNL), female mice were exposed to continuous low-dose-rate gamma rays for one week, reaching cumulative low absorbed doses of 10 mGy or 100 mGy. The total body dose of 100 mGy was chosen because it represents the upper limit of "low dose" radiation as defined in the literature (1). By setting this dose as the highest level of exposure, we aimed to investigate potentially, the most intense response within the low-dose range. We hypothesized that sustained low-dose radiation exposure would induce subtle changes to immune system homeostasis, including both cell-type specific and dose-dependent reactions to LDIR.Eighteen-week-old female C57BL/6 mice (Jackson Laboratories) were housed and irradiated at the CNL (Chalk River, Ontario, Canada). The entire facility, including the irradiation hall and the conventional animal room where control mice were housed, is Specific Pathogen Free. The mice were fed PicoLab Rodent 20 5K75 chow and provided water in bottles. Housing conditions were maintained at a temperature of 20-26°C, and humidity of 20-80%, with a 12-hour light/dark cycle. Three mice per cage were housed in Animal Care Systems Optimice cages with BlockParty bedding, and during acclimatization, the cages were individually ventilated on an Optimice rack system. During irradiation in the Gamma Beam Facility, the cages were placed on wooden racks, in static caging, with Biofresh™ Performance Bedding. Enrichment materials included an enviropack, nestlet, wooden block, and hut, with food enrichment of Cheerios provided after ear punching and cage changes. Whole-body gamma irradiation was administered using a Nordion (Ottawa, Ontario, Canada) Gammabeam 150C irradiator equipped with a Cobalt-60 source. The irradiation was continuous for 7 days in the pathogen-free irradiation hall of the CNL (31). Treatment groups were positioned at varying distances from the irradiator to achieve different dose rate exposures. Mice receiving a cumulative dose of 10 mGy (n=6) received an average dose rate of 0.0614mGy/hr. Mice receiving a cumulative dose of 100 mGy (n=6) received an average dose rate of 0.625mGy/hr. The positioning ensured the beam targeted the mid-level of each cage, centered between the two cages. Dosimetry was performed using an Exradin A8 ion chamber and a Supermax electrometer. Control mice (n=6) were housed separately in a conventional animal room. Following irradiation, mice were euthanized via exsanguination under Isoflurane gas anesthesia, followed by cervical dislocation. Blood samples were collected via cardiac puncture. Mice were then dissected to collect spleen, bone marrow and thymus. Blood samples were immediately analyzed using the Zoetis Vetscan MS5 hematology analyzer. The remaining organs were transported on ice for around 2.5 hours to the University of Ottawa (Ottawa, Ontario, Canada) for further analysis. All animal procedures were approved by the CNL Animal Care Committee and adhered to Canadian Council on Animal Care standards.Spleens were mechanically disrupted by pressing through a 70µm cell strainer (Bio Basic Canada Inc.) using a syringe plunger. The disrupted tissue was suspended in RPMI medium supplemented with 2% fetal bovine serum (FBS) (Gibco™, Canada) and kept on ice. The suspension was centrifuged at 1200 rpm for 10 minutes at 4°C. The pellet was resuspended in 1 mL of red blood cell (RBC) lysis buffer (Roche) to lyse erythrocytes. Post-lysis, cells were washed with RPMI containing 2% FBS and filtered through a nylon mesh to remove debris. Thymus samples underwent the same mechanical disruption and subsequent processing steps as spleens, with the omission of red blood cell lysis. Bone marrow cells were obtained from excised femurs. The femurs were cleaned of excess tissue and placed in cold PBS. They were then dried with a Kimwipe to remove remaining connective tissue. Their ends were removed, and marrow was flushed out with a 27g needle and 1 mL of FBS into a microcentrifuge tube. The marrow was collected by repeated flushing and scraping with the needle to ensure complete extraction, and the femurs were discarded when they appeared pale/white. Bone marrow was kept on ice. Cells were then centrifuged at 1200 rpm for 10 minutes at 4°C, resuspended in RBC lysis buffer, washed with RPMI containing 2% FBS, and filtered through nylon mesh.For ex vivo NK cell intra-cellular IFN-□□ measurements, freshly derived splenic leukocytes were stimulated with either a combination of IL-2 (100U/ml, obtained from NCI Preclinical Repository, USA) and IL-12 (10 ng/ml) (eBioscience™), or with plate coated anti-NKp46 (BioLegend™) for 1 hr and then incubated in RP-10 medium containing 5 μg/ml brefeldin A (Invitrogen™) for 4 hrs, followed by intracellular staining. For ex vivo T cell intracellular IFN-□□ measurements, freshly derived spleen leukocytes were stimulated with anti-CD3/28 for 16 hrs and then incubated in RP-10 medium containing 5 μg/ml brefeldin A (Invitrogen™) for a further 4 hrs, followed by intracellular staining.Cell staining was conducted under cold conditions and protected from light to preserve cell integrity and prevent fluorophore photobleaching. Single-cell suspensions (1x10 6 cells) were aliquoted into a 96-well V-bottom plate and washed in staining buffer (PBS supplemented with 2% HI-FBS). Cells were incubated at 4°C for 10 minutes in α-CD16/32 (clone 2.4G2 from Bioexpress) to block non-specific binding. Cells were then stained with a cocktail of fluorophore-conjugated monoclonal antibodies in staining buffer and incubated at 4°C for 25 minutes. Post-staining, cells were washed with staining buffer and fixed with 2% paraformaldehyde (PFA) for 10 minutes at 4°C. Cells were then washed, centrifuged and resuspended in the staining buffer for acquisition. For intracellular proteins (Gzmb and CD107a), the Cytofix/Cytoperm kit was employed following surface staining. Briefly, cells were washed and centrifuged, then resuspended in BD Cytofix/Cytoperm buffer and incubated for 10-20 minutes at 4°C. Cells were washed twice with BD Cytoperm Wash buffer and centrifuged. The cells were then incubated with an intracellular antibody cocktail in BD Cytoperm Wash buffer for 25 minutes at 4°C. Following incubation, cells were again washed with BD Cytoperm Wash buffer and staining buffer, centrifuged, and resuspended in cold staining buffer for analysis. Flow cytometry was performed using an Attune NxT flow cytometer (Attune, ThermoFisher) and analyzed with Kaluza (Beckman Coulter). Antibodies used included: anti-TCRβ (H57-597), anti-CD8 (53-6.7), anti-CD49b (DX5), anti-CD11b (M1/70), anti-NKG2D (CX5) from eBioscience™; anti-CD19 (1D3), anti-CD4 (RM4-5), anti-F4/80 (T45-2342), anti-NK1.1 (PK136), anti-Ki-67 (B56), anti-CD69 (H1-2F3), anti-Ly6C (AL-21), anti-Gr1 from BD Biosciences™; anti-CD43 (1B11) from BioLegend™; and Live/Dead Fixable Yellow Dead Cell Stain from Invitrogen™. Bulk RNA-Sequencing and Bioinformatics RNA was extracted from isolated splenocytes using Trizol (Thermofisher). RNA integrity was ensured with a minimum RIN cut-off of 6.5. Poly-A enriched stranded cDNA libraries were generated and subjected to 100 bp paired-end sequencing on a NovaSeq6000 at Genome Quebec (Montreal, Canada). Samples were sequenced to a minimum depth of 23E6 reads. FASTQ files were pseudo-aligned to the mouse transcriptome assembly (mm10) using Kallisto (v.0.46.1). Gene-level quantification was performed with Tximport (v1.32.0). Normalization, differential expression, and principal component analysis were conducted using DESeq2 (v1.44.0). Differentially expressed genes were identified using an adjusted p-value threshold of <0.05. Gene set enrichment analysis (GSEA) was performed using fgsea (v1.30). Heatmaps were generated from variance-stabilized data. Lists of radiation-responsive genes were sourced from the Gene Ontology database, specifically from GO:0009314, GO:0071478, and GO:0010212.Plots were generated using Kaluza (Beckman Coulter), ggplot2 (v3.5.1), ComplexHeatMap (v2.20.0), Seurat (v5.1.0), cowplot (v1.1.3), and ggVenDiagram (v1.5.2). Hematology and flow cytometry statistical analyses were conducted with the Rstatix (v0.7.2) package. Bulk RNA-sequencing analysis was performed using DESeq2 (v1.44.0).In this study, 18-week-old female C57BL/6 mice were housed for 7 days in front of a 60 Co gamma beam source until cumulative whole-body absorbed doses of 10 mGy and 100 mGy were reached. An untreated (UT) control was housed separately without exposure to radiation. Immediately following the radiation exposure period, the mice were euthanized, blood samples were acquired, and immune cells were isolated from the spleen, thymus, and bone marrow for further study. 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