The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury

Abstract

Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure.

Figure 1. AIM2 inflammasome deficiency protects mice from SBI induced small intestine syndrome

(A–D) WT mice were cohoused with Casp1(11)^−/−, Casp1^−/−, Asc^−/− or Aim2^−/− mice for two weeks and then exposed to 14.2 Gy of subtotal-body irradiation (SBI). Kaplan-Meier survival analysis of Casp1(11)^−/− mice (A), Casp1^−/− mice (B), Asc^−/− mice (C), Aim2^−/− (D) mice and their cohabitated WT mice was performed. Each figure represents the pooled data from two to three independent experiments. The total number of mice in each group and the p value by log-rank comparison are indicated on the plots. (E) Representative pictures of H&E staining of the jejunum from Aim2^−/− mice and their cohabitated WT mice at day 0 and day 3.5 after 14.2 Gy of SBI. Scale bar=100 μm.

Figure 2. AIM2 inflammasome regulates intestinal radio-sensitivity through caspase-1 mediated epithelial cell death

(A) WT mice were cohoused with Casp1^fl/fl;VillinCre+ mice for two weeks and then exposed to 14.2 Gy of SBI. Kaplan-Meier survival analysis of Casp1^fl/fl;VillinCre+ mice and their cohabitated WT mice was performed on pooled data from two independent experiments. By log-rank comparison, ****P<0.0001. (B, C) Small intestines were harvested from Casp1(11)^−/−, Aim2^−/− and their cohabitated WT mice 24 hours post 14.2 Gy SBI, and cell death was analyzed by TUNEL staining. Epithelial cells stained positively for TUNEL showed green fluorescence. Nuclei were stained with PI (red) or DAPI (blue). Scale bar=100 μm. Numbers of TUNEL-positive cells per crypts were quantified (n=3–5 mouse/group, at least 20 crypts of each mouse were counted) and representative pictures were shown. Results are expressed as mean ± SEM, *P<0.05 by Student’s t test.

Figure 3. AIM2 inflammasome mediates radio-sensitivity of hematopoietic cells in response to dsDNA damage

(A, B) Kaplan-Meier survival analysis was performed on Aim2^−/− mice (A), Casp1^−/− mice (B) and their WT controls exposed to 7 Gy of total-body irradiation (TBI). Each figure represents the pooled data from two independent experiments. N=10–20 mice/group. (C) Caspase-1 activation in WT and Casp1^−/− BMDMs 4 hours after exposure to indicated doses of radiation was assayed by immunoblotting of the cleaved form of caspase-1 (p10 subunit). (D) Supernatant was collected from un-irradiated or 80 Gy-irradiated LPS-primed WT and Aim2^−/− BMDMs, and IL-1β concentration was measured by ELISA. (E, F, I, J) WT, Casp1^−/− or Aim2^−/− BMDMs were treated with different doses of ionizing radiation or drugs inducing dsDNA breaks, and cell death was measured by the amount of lactate dehydrogenase (LDH) released into the supernatant. (G, H) WT and Aim2^−/− BMDMs were treated with different doses of ionizing radiation or UV radiation, and cell death was quantified by trypan blue staining. Determinations were performed in triplicate and expressed as the mean ± SEM. * P<0.05 by Student’s t test.

Figure 4. Ionizing radiation induces the formation AIM2 specks in the nucleus

Primary macrophages from AIM2-Flag mice (A, B, C, D) or AIM2-Flag/ASC-HA double knock-in mice (E) were left un-irradiated or exposed to 80 Gy ionizing radiation. For immunofluorescence microscopy, cells were fixed at 4 hours (A, B, C) or at indicated time points (E) after radiation. AIM2 was stained with anti-Flag antibody (red in A; green in B, C, E), and co-stained with nuclear envelope protein NUP98 (red, B) or gamma-H2AX (red, C) or ASC (using anti-HA antibody, red, E). Cell nuclei were visualized by DAPI (blue) in A and E. Co-localization of AIM2-Flag specks and gamma-H2AX foci was indicated by white arrowheads in C. Scale bar=5 μm. Figures represent results from three independent experiments and at least 100 cells were analyzed for each condition. (D) Co-immunoprecipitation (co-IP) of gamma-H2AX with AIM2-Flag in irradiated macrophages using anti-Flag M2 agarose beads. The immunoprecipitates (Flag IP) or the total lysates were analyzed by immunoblotting with antibodies against gamma-H2AX(Ser139) or the Flag tag. Samples from untagged WT mice were used as controls to determine the specificity of immunoblots. IB, immunoblotting. Non-specific band is indicated with an asterisk. Data represent two independent experiments.