High-energy radiation reduces natural killer cells’ ability to clear aging intestinal cells in mice

We further examined NK cell retention at an early time point (5 days) and at 60 days after irradiation. The data show a significant reduction in LP-resident NK cells at both time points. Although NK cell numbers at 5 days were lower than at 60 days, the difference was not statistically significant (Fig. S2a, b). To assess a dose–response effect, we compared NK cell levels after exposure to 0.5 Gy or 4 Gy γ-rays and observed a significant reduction in NK cell retention at 4 Gy relative to 0.5 Gy and control groups (Fig. S2c, d). Consistently, similar reductions in LP-resident NK cells were seen when comparing 4 Gy γ-rays with 0.5 Gy ²⁸Si exposure (Fig. S2e). We further characterized maturation stages of NK cell subsets based on CD11b and CD27 expression (Fig. S1h, i). Marginal changes were observed after irradiation in CD11b^low/CD27^high NK cell subset (Fig. S3a), whereas a significant increase was observed in CD11b^high/CD27^low NK cell subset particularly after high-LET irradiation relative to control (Fig. S3b). In particular, the CD11b^high/CD27^high NK cell subset was significantly decreased in the high-LET group compared to the γ-irradiated and control groups (Fig. 1h). Taken together, high-LET irradiation selectively and markedly reduced LP-resident NK cells, while other myeloid populations remained largely unaffected. Immunostaining confirmed a radiation quality–dependent loss of NK cells in the LP, an effect that was tissue-specific and evident at both early and late time points. High-LET exposure also reshaped NK cell maturation dynamics, with reduced CD11b^high/CD27^high and increased CD11b^high/CD27^low subsets. These findings indicate that high-LET irradiation disrupts NK cell homeostasis within the intestinal microenvironment.

Radiation is known to activate p38 MAPK signaling as part of a stress-adaptive response. We previously reported the activation of p38 MAPK signaling in the mouse intestine following exposure to high-LET ²⁸Si irradiation [6]. To further investigate this, we performed immunoblot analysis of phosphorylated p38 and its downstream target, phosphorylated HSP27, 60 days after ²⁸Si exposure. The results confirmed sustained activation of the p38 MAPK pathway in the irradiated intestine (Fig. 5a, b). To validate these findings in situ, we conducted immunodetection of phosphorylated p38 and phosphorylated HSP27 in intestinal tissue sections. Both proteins showed significantly increased expression in ²⁸Si-irradiated tissues compared to controls, indicating persistent activation of the pathway (Fig. 5c, d, f, g). Additionally, immunoblot analysis revealed significant upregulation of Qa-1b in the intestines of high-LET irradiated mice (Fig. 5a, b). This increase was further confirmed by immunostaining, which showed enhanced Qa-1b expression specifically in IECs (Fig. 5e, h). Overall, these findings indicate that high-LET irradiation with ²⁸Si induces sustained activation of the p38 MAPK pathway and upregulates Qa-1b expression in the intestinal epithelium, potentially contributing to immune modulation within the irradiated tissue.

To determine whether suppression of Qa-1b via p38 inhibition enhances NK cell-mediated cytotoxicity, we repeated the co-culture experiments with senescent and non-senescent IECs pretreated with the p38 inhibitor. Caspase-3 activity was significantly increased in senescent IECs after p38 inhibition, suggesting enhanced NK cell killing, likely due to reduced Qa-1b expression (Fig. 7b). Further, we performed NK cell degranulation assays in the presence or absence of blocking antibodies. Pre-incubation of NK cells with an NKG2D-blocking antibody significantly reduced degranulation against senescent IECs compared to isotype control, confirming the importance of NKG2D-mediated activation. In contrast, blocking the inhibitory receptor NKG2A significantly enhanced NK cell degranulation in response to senescent IECs, suggesting that engagement of NKG2A by its ligand Qa-1b impairs NK cell function (Fig. 7c). Collectively, these findings demonstrate that IR-induced activation of p38 MAPK signaling promotes expression of Qa-1b in IECs, which suppresses NK cell cytotoxicity via NKG2A engagement. Inhibition of p38 signaling enhances NK cell-mediated clearance of senescent cells by downregulating Qa-1b, thereby restoring NKG2D-dependent immune surveillance (Fig. 7c).

To further investigate the underlying mechanism, we performed functional causality experiments by silencing or overexpressing Qa-1b in IEC for NK cell cytotoxicity assay. Immunoblot analysis confirmed efficient Qa-1b knockdown using shRNA and robust overexpression following transfection with Qa-1b (Fig. 7d). To test whether Qa-1b modulation alters NK cell–mediated cytotoxicity, we conducted co-culture assays using senescent and non-senescent IECs in which Qa-1b was either overexpressed or silenced, with or without p38 inhibitor. Caspase-3 activity was significantly increased in senescent IECs following Qa-1b knockdown, indicating enhanced NK cell–mediated killing, likely due to reduced Qa-1b expression (Fig. 7e). In contrast, Qa-1b overexpression markedly suppressed caspase-3 activation. Notably, p38 inhibition did not significantly alter caspase-3 activity in senescent IECs regardless of Qa-1b overexpression or knockdown (Fig. 7e, f). Over all these results demonstrate that senescent IECs upregulate the inhibitory ligand Qa-1b through p38 MAPK activation, suppressing NK-cell cytotoxicity via NKG2A engagement. p38 inhibition reduces Qa-1b expression and restores NK-mediated killing. Qa-1b knockdown increases, whereas overexpression decreases NK-cell cytotoxicity, and p38 inhibition has no additional effect when Qa-1b levels are altered, indicating Qa-1b is the key downstream effector of p38. These findings show that p38-driven Qa-1b expression enables senescent IECs to evade NK-cell surveillance.

Male Lgr5-EGFP-IRES-CreERT2 mice (Stock No. 008875) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and housed at the Georgetown University (GU) Animal Care Facility as described earlier [6]. For this study, groups of Lgr5-EGFP-IRES-CreERT2 mice were subjected to whole-body irradiation with ²⁸Si ions (energy: 300 MeV/nucleon; LET: 69 keV/μm; dose: 0.50 Gy) at the NASA Space Radiation Laboratory (NSRL), Brookhaven National Laboratory (BNL). Precise dose delivery was achieved through beam calibration and dosimetry performed by NSRL physicists, with exposure variability kept within ± 2.5%. For additional information on the procedures for beam calibration and dosimetry, please refer to https://www.bnl.gov/nsrl/userguide/dosimetry-calibration.php↗ (accessed 15 August 2024). Additional groups received whole-body γ irradiation (0–4.0 Gy) using a ¹³⁷Cs source, while control mice were sham-irradiated. All mice were transported to the BNL Animal Facility one week prior to irradiation to allow for acclimatization and were returned to the GU Animal Facility the day following radiation exposure. Transportation was carried out in a temperature-controlled environment to maintain physiological stability and ensure animal welfare. At both facilities, animals were maintained under standardized conditions, including temperature-controlled rooms set at 22 °C, 50% humidity, and a 12-h light/dark cycle, with ad libitum access to irradiated food and filtered water. All animal procedures were approved by the Institutional Animal Care and Use Committees of BNL (Protocol #345, approved October 12, 2021) and GU (Protocol #2016–1129, approved August 1, 2021). All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, U.S. National Academy of Sciences).

Mice were euthanized and small intestines were incised and IECs were isolated as described earlier [6]. Briefly, tissues were incubated in 2 mM EDTA in PBS for 20 min, followed by two vigorous washes with PBS. The supernatant containing villus cells was discarded. Tissues were then treated with a dissociation solution consisting of 1 mg/mL STEMxyme® 2 Collagenase/Neutral Protease (Dispase) (Cat# LS004112↗, Worthington Biochemical Corporation, Lakewood, NJ) and 0.1 mg/mL DNase I (Cat# 89,836, Thermo Scientific, Waltham, MA) in HBSS (Cat# 14175103, Thermo Scientific), and incubated at 37 °C for 20 min. After removing the supernatant, cells were released by vigorous shaking in cold HBSS. The resulting cell suspension was filtered through a 70 μm strainer, centrifuged, and washed twice with cold HBSS before being resuspended in HBSS supplemented with 2% FBS for organoid culture. LP immune cells were isolated as described earlier [50]. The LP cells were then enriched with a percoll gradient. For myeloid cell staining, dead cells were excluded using SYTOX™ Blue (cat#S34857, Life Technologies) and non-specific binding was blocked with Fc block anti-mouse CD16/32 Antibody (Clone 93, BioLegend). Samples were then surface stained at 4 °C for 30 min with the antibodies. Surface markers CD45, CD11c, NKp46, CD27, CD11b, CD64, NKG2A, CD94, Ly49c, Cd226, CD16, NKG2D, FasL, TRAIL CD357, CD11b, CDH1, H60 and Rae1 were used for flowcytometry analysis. Antibody details are mentioned in Supplementary Table S1. Cells were then washed with ice-cold PBS (supplemented with 10% FCS and 2 mM EDTA) at 400 g for 10 min at 4 °C and acquired immediately on BD Fortessa flow cytometer running FACS-Diva software (BD Bioscience). Analysis was performed using FCS express software version 7 (De Novo Software, Pasadena, CA). Average percentage of specific immune phenotypes was calculated from at least 6 mice per group.

Paraffin-embedded intestinal tissue sections were first deparaffinized to remove embedding media, followed by rehydration through a graded series of ethanol solutions to restore tissue hydration. Antigen retrieval was then performed by heating the sections to boiling in citrate buffer (pH 6.0; Cat# 64,142–08, Electron Microscopy Sciences, Hatfield, PA, USA), a step that helps unmask epitopes and enhance antibody binding during subsequent staining procedures. Sections were then incubated overnight at 4 °C with primary antibodies specific for H60, phosphorylated p38 (p-p38), phosphorylated HSP27 (p-HSP27), and Qa-1b. Detection of antigen–antibody complexes was performed using the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit (Cat# AB236466↗, Abcam), following the manufacturer's instructions. Following staining, tissue sections were counterstained with hematoxylin to visualize nuclei, then sequentially dehydrated through graded alcohols and cleared prior to mounting. Coverslips were applied using Permount mounting medium (Cat# SP15-100, Fisher Chemical) to preserve the samples and enhance optical clarity. Brightfield images were subsequently acquired using an Olympus microscope under appropriate imaging settings. For immunofluorescence analysis, tissue sections were incubated overnight at 4 °C with the primary antibodies, followed by washing and incubation with species-specific secondary antibodies conjugated to Alexa Fluor dyes: 488 (green), 594 (red), or 647 (far-red) for 1 h at room temperature. Nuclei were counterstained with DAPI to enable fluorescent visualization of nuclear structures. Slides were then examined and imaged using an Olympus fluorescence microscope under appropriate excitation and emission settings.

Crypt cells were isolated and cultured in IntestiCult™ Organoid Media (Stem Cell Technologies, Catalog #06005) according to the manufacturer's instructions. Ten-day-old organoids were irradiated with 4.0 Gy γ rays, and cellular senescence was assessed at 2- and 5-days post-IR. For expression analysis and co-culture experiments, cells were treated with 0–20 µM SB 203580 (Thermo Fisher Scientific cat# 55–939-51MG) a selective p38 inhibitor. Non-irradiated cells were used as non-senescent controls, whereas senescent cells were collected 5 days after irradiation. Co-culture experiments were performed using Effector (NK cells)-to-Target (Senescent cells or non-senescent cells) at two ratios [(E:T); 10:1 and 20:1] as described earlier [48]. Cytotoxicity in target cells was assessed after 6 h of co-incubation by quantifying active caspase-3 activity using a spectrophotometer (EnzChek™ Caspase-3 Assay Kit; Cat# E13183↗, Thermo Fisher Scientific). Organoids incubated with medium only served as a negative control for baseline caspase-3 activity. Organoids treated with doxorubicin (1 µg/mL; Fisher Scientific) were used as positive controls. Alternatively, CD107a (lysosomal-associated membrane protein-1, LAMP-1) expression was used as a marker of NK cell degranulation, as described previously [48]. The degranulation data are presented as an index calculated as: (Sample Degranulation − Spontaneous Degranulation)/(Maximum Degranulation − Spontaneous Degranulation) × 100.

Coding sequence of mouse Qa-1b protein was synthesized and were inserted into mammalian expression plasmid (pcDNA3.1(+)-C-DYK) at HindIII/ApaI multiple cloning sites (Fig. S6) by GenScript, USA. The Qa-1 shRNA Plasmid (m) (sc-42923-SH) and Control shRNA Plasmid-A (sc-108060) procured from Santa Cruz Biotechnology. Mouse IECs were transfected with these constructs or control vectors (0.5 μg) using Effectene Transfection Reagent (Qiagen, 301,425) according to the manufacturer’s instructions. After 24 h, cultures were refreshed with new medium, and antibiotic selection was initiated after 48 h using puromycin (1.0 µg/mL; Santa Cruz, sc-108071) or neomycin (400 µg/mL; Santa Cruz, sc-255391). Following 5 days of selection, cells were returned to fresh growth medium, and Qa-1b overexpression or knockdown was confirmed by immunoblotting. IECs with Qa-1b knockdown or Qa-1b overexpression were further used for radiation exposure and cytotoxicity assay as described above.

Appropriate controls were included with all experimental samples to verify the specificity of immunostaining and to distinguish true signal from background staining. These controls included sections processed in parallel under identical conditions to ensure consistency and reliability of the staining procedure. For both immunohistochemistry and immunofluorescence, randomly selected regions of normal mucosa were imaged from each section using cellSens Entry v1.15 (Olympus Corp., Center Valley, PA, USA), as previously described [51, 52]. Multiple fields per section were captured to provide a representative assessment and minimize sampling bias. The number of technical and biological replicates is indicated in the figure legends. Statistical analyses were performed using Student’s t-test or one-way analysis of variance (ANOVA), or two-way ANOVA as appropriate, followed by Tukey’s post hoc test. All analyses were conducted using GraphPad Prism version 10.6.1 (GraphPad Software, Boston, MA, USA). All the experimental data are presented as mean ± standard error of the mean (SEM). A p-value of ≤ 0.05 used the determine the statistical significance between experimental groups.