Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence

Abstract

Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.

Figure 1. Absence of cGAS attenuates the senescence response.

(a) Proliferation curve of primary wild-type (WT) and cGAS knockout (cGAS KO) mouse embryonic fibroblasts (MEFs) cultured under 20% O₂. (b) Percentages of BrdU positive WT or cGAS KO MEFs after 27 days in culture are shown. (c) Left: Images of SA-β-Gal stained WT or cGAS KO MEFs at 27 days in culture. Scale bar: 200 µm. Right: Mean and s.d. of the percentages of SA-β-Gal-positive cells are indicated. (d) Heat maps of RNA-seq analysis of WT MEFs and cGAS KO MEFs (For each genotype one sample per day; MEFs collected at Day 33, 36, 42). Genes in WT MEFs exhibiting statistically significant (n=3 biological replicates; student t-test; P < 0.05), twofold or greater increases relative to cGAS KO MEFs are shown. Senescence markers are highlighted in green. (e, f) WT MEFs or cGAS KO MEFs were harvested after 3 weeks of culture and expression of depicted genes was measured via RT-qPCR or protein expression was analysed by immunoblotting. One representative experiment out of three (a, c (left)) or mean and s.d. of n=3 independent experiments (b, c (right)) or n=3 independent biological replicates (e) are shown. P values were calculated by unpaired t-test (* P < 0.05, *** P < 0.001, **** P < 0.0001). Unprocessed original blots are shown in Supplementary Fig. 7. Source data are available in Supplementary Table 1.

Figure 2. cGAS facilitates oxidative stress-induced senescence.

(a, b, c, d) WT MEFs, cGAS KO MEFs or STING KO MEFs were cultured under hyperoxic conditions (40% O₂) for 7 days and subsequently stained for SA-β-Gal activity (a), examined for percentages of EdU⁺ cells by immunofluorescence (b) or assessed for expression of Cdkn2a by RT-qPCR (c) or p16^Ink4a by immunoblotting (d). (e) WI-38 cells were exposed to 40% O₂ treatment for 9 days. On day 7 cells were transfected with a non-targeting control siRNA (si Control) or with siRNAs against cGAS (si cGAS #1, si cGAS #2). Expression of cGAS was assessed by immunoblotting on day 9. (f, g, h) WI-38 cells were treated as in (e). On day 9 SA-β-Gal activity was assessed by FACS (f) and expression of CDKN2A was examined by RT-qPCR (g) or p16^Ink4a was assessed by immunoblotting (h). Numbers in (f) indicate percentages of SA-β-Gal positive cells. Mean and s.d. of n=5 independent experiments (a) or n=3 biological replicates (b, c, g) or one representative experiment out of 3 (f) or 2 (d, e, h) independent experiments are shown. P values were calculated by one-way (a) and two-way (b, c, g) ANOVA (* P < 0.05, ** P < 0.01, ns = not significant). Source data are available in Supplementary Table 1. Unprocessed original blots are shown in Supplementary Fig. 7.

Figure 3. cGAS regulates the senescence-associated secretory phenotype.

(a, b) Conditioned medium (CM) was collected from WT MEFs or cGAS KO MEFs exposed to 40% O₂ for 7 days. Percentages of proliferating cells (WT MEFs) were assessed by BrdU incorporation assay (a) or induction of SA-β-Gal activity was determined by microscopy (b). (c) Cytokine profile of the CM from WT MEFs of cGAS KO MEFs exposed to 40% O₂ for 7 days. Some SASP factors are highlighted in red. (d) WT MEFs, cGAS KO MEFs or STING KO MEFs were incubated in 40% O₂ for 7 days and IL-6 was quantified by ELISA. (e) WI-38 cells were exposed to 40% O₂ treatment for 9 days. On day 7 cells were transfected with a non-targeting control siRNA (si Control) or with siRNAs against cGAS (si cGAS #1, si cGAS #2). Expression of IL-6 was determined by RT-qPCR. (f, g) WT MEFs and cGAS KO MEFs were cultured in the presence of recombinant IFN-β as indicated for 14 days. Cells were stained for SA-β-Gal (f) or cell cycle activity was assessed by EdU incorporation (g). (h, i, j) WT MEFs of IFNAR KO MEFs were cultured under 40% O₂ for 7 days and stained for SA-β-Gal (h), proliferative activity (BrdU incorporation) was assessed by FACS at day 4 (i) or expression of p16^Ink4a was determined by immunoblotting after 7 days (j). Mean and SEM of n=3 (a) or mean and s.d. of n=4 independent experiments are shown (h) or one representative out of 2 independent experiments is shown (c, j). Data shown in (b, d, e, f, g, i) are from n=2 independent experiments with the column representing the mean. P value was calculated by unpaired t-test (h) (ns = not significant). Source data are available in Supplementary Table 1. Unprocessed original blots are shown in Supplementary Fig. 7.

Figure 4. cGAS interacts with chromatin fragments in senescent cells.

(a) Lysates from WT MEFs and senescent MEFs were probed for the expression of lamin B1 by immunoblot. (b) MEFs (left panel) or WI-38 cells (right panel) were incubated at 40% O₂ as indicated and expression of lamin B1 in individual cells was determined by confocal fluorescent microscopy (CTCF = Corrected Total Cell Fluorescence). (c) Confocal fluorescent microscopy images of WT MEFs cultured for 7 days under 40% O₂ stained for lamin B1 (red) and DAPI (grey). (d) cGAS KO MEFs carrying a Dox-inducible vector expressing FLAG-tagged cGAS were cultured in the presence of Dox overnight. Cells were stained with DAPI (grey) or anti-FLAG antibody (green) and imaged by confocal microscopy. (e, f) MEFs were transfected with a non-targeting siRNA (si Control) or Lmnb1-targeting siRNAs (si Lmnb1 #1, si Lmnb1 #2). After 72 h mRNA levels of indicated genes were analysed by RT-qPCR (e) or protein expression was determined by immunoblotting after 48 h. (g) MEFs carrying a Dox-inducible vector expressing FLAG-tagged cGAS were transfected with siRNAs as indicated. 24 h later cells were treated with Dox overnight and stained for lamin B1 (red), cGAS-FLAG (green) and DAPI (grey). One representative experiment out of 3 (f, g) or 2 (a, b, c, d) is shown. Mean and s.d. of n=20 individual MEFs at Day 0; n=20 individual MEFs at Day 7; n=42 individual WI-38 cells at Day 0; n=33 individual WI-38 cells at Day 7 are shown in (b). Mean and s.d. of n=3 independent experiments are shown in (e). P values were calculated by unpaired t-test (b) or one-way ANOVA (e) (* P < 0.05, **** P < 0.0001, ns = not significant). Scale bar: 20 µm. Source data are available in Supplementary Table 1. Unprocessed original blots are shown in Supplementary Fig. 7.

Figure 5. Engagement of cGAS is a common feature of multiple senescence triggers.

(a) MEFs with the indicated genotypes were exposed to 12 Gy ionizing irradiation or stimulated with Palbociclib. Expression levels of Cxcl10 or protein levels of IL-6 were quantified by RT-qPCR or ELISA, respectively. (b) WI-38 cells (left panel) were exposed to 12 Gy ionizing irradiation or WI-38 ER:RAS cells (right panel) were treated with 4-OHT (500 nM) for 7 days. At day 7 (irradiated cells) or at day 3 (WI-38 ER:RAS cells) were transfected with a non-targeting control siRNA (si Control) or with siRNAs against cGAS (si cGAS #1, si cGAS #2). Expression of depicted genes was determined by RT-qPCR. (c) Expression of lamin B1 in individual WT MEFs exposed to irradiation (left panel) or treated with Palbociclib (right panel) for 7 days was determined by confocal fluorescence microscopy (CTCF = Corrected Total Cell Fluorescence). (d) Expression of lamin B1 in individual WI-38 cells exposed to irradiation (left panel) or WI-38 ER:RAS cells treated with 4-OHT was assessed by confocal fluorescence microscopy (CTCF = Corrected Total Cell Fluorescence). (e) cGAS KO MEFs carrying a Dox-inducible lentiviral vector containing a FLAG-tagged murine cGAS construct were irradiated (12 Gy) or treated with Palbociclib. 2 days post treatment cells were cultured in the presence of Dox overnight and then stained for FLAG (green) and nuclei (DAPI, grey). One representative experiment out of 2 experiments is shown (e) or mean and s.d. of n=51 individual control MEFs compared with n=48 individual irradiated MEFs (c, left); n=50 individual control MEFs compared with n=58 individual Palbociclib treated MEFs (c, right); n=177 individual control WI-38 cells compared with n=158 individual irradiated WI-38 cells (d, left); n=151 individual control WI-38 cells compared with n=87 individual 4-OHT treated WI-38 cells (d, right) are shown. Data shown in (a) and (b) are from n=2 independent experiments with the column representing the mean. P values were calculated by unpaired t-test (c, d) (**** P < 0.0001, ns = not significant). Scale bar: 20 µm. Source data are available in Supplementary Table 1.

Figure 6. cGAS contributes to cellular senescence in vivo.

(a) Depicted mRNA levels of lungs from WT, cGAS KO and STING KO mice 16 weeks after irradiation are shown. (b) Representative liver stainings of Nras-, p21-, and SA-β-Gal 6 days after intrahepatic delivery of Nras^G12V or Nras^G12V/D38A into WT or cGAS KO mice are shown. Arrows indicate positive staining. (c, d) Quantification of Nras- or p21-positive cells (c) or expression of IL-6 protein levels or Cxcl10 mRNA levels (d) from (b) are shown. Mean of n=2 (WT mice untreated, cGAS KO mice) or mean and s.d of n=3 (WT mice irradiated, STING KO mice) (a), mean and s.d. of or n=4 (cGAS KO mice) or n=5 (WT mice) (c, d) or representative images (b) are shown. P values were calculated by two-way ANOVA (comparing genotypes or treatments) (* P < 0.05, ** P < 0.01, ns = not significant). Scale bar: 100 μm. Source data are available in Supplementary Table 1.