Age-Associated Activation of the cGAS-STING Pathway and Impairment of DNA Damage Repair in Human Primary Alveolar Type II Cells

Abstract

Homeostatic imbalance and lung function decline are central physiological characteristics of aging and susceptibility to respiratory diseases. Senescence contributes to tissue damage and alveolar epithelial cell injury and decreases reparative capacity. Alveolar type II (ATII) cells have stem cell potential and self-renew to regenerate the alveoli after damage. They were isolated from younger and older non-smoker and smoker organ donors to define their function in the lung. Smoking and older age increased ATII cell senescence as detected by high β-galactosidase activity and P21 levels by Western blotting and RT-PCR. Also, the number of ATII cells was the lowest in lung tissue in older smokers. This was associated with increased stress signaling, as shown by elevated 4-HNE and G3BP1 expression in ATII cells, and inflammation indicated by high IL-8 levels in BAL fluid. In addition, DNA damage and decreased repair were observed using the comet assay, especially in ATII cells isolated from older smokers. This was accompanied by the highest levels of cytosolic double-strand DNA in this group and correlated with the activated cGAS-STING pathway and increased IRF3 expression. Moreover, telomere shortening, accumulation of TERRA molecules, and increased ZBP1 protein expression in ATII cells were associated with smoking and older age. Reduced NRF2 and DJ-1 expression in ATII cells was detected by Western blotting, especially in older smokers, which suggests an antioxidant defense system dysfunction. Our study provides insights into the impaired interconnected signaling network, which can contribute to ATII cell senescence.

Figure 1.

ATII cell senescence, high oxidative stress, and pro-inflammatory response in older smokers. ATII cells and BALF were obtained from younger and older non-smokers (NS) and smokers (SM). (A) The representative purity of isolated ATII cells using SP-C antibody by immunofluorescence (scale bar - 10 µm). (B) β-galactosidase assay using ATII cells (scale bar - 10 µm). (C) P21 expression in ATII cells by Western blotting. (D) P21 mRNA levels were determined by RT-PCR. (E) Oxidative stress was analyzed using the 4-HNE antibody in ATII cells identified by SP-C staining in lung tissue sections (immuno-fluorescence, scale bar - 5 µm). (F) The percentage of ATII cells using SP-C antibody in lung tissue sections (immunofluorescence, scale bar - 50 µm). (G) IL-8 levels were analyzed in BALF by ELISA. Data are shown as means ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (Kruskal-Wallis test in B, C, E, F, G, H and a one-way ANOVA in D).

Figure 2.

Increased DNA damage, TREX1, and 8-OHdG levels in ATII cells in older smokers. BALF, ATII cells, and lung tissue were obtained from younger and older non-smokers (NS) and smokers (SM). ssDNA (A) and dsDNA (B) levels in BALF. (C) DNA damage was analyzed in ATII cells by comet assay (scale bar - 5 µm). Quantification of the Olive tail moment is shown. (D) ATII cells were treated with etoposide for 24h to induce DNA damage. (E) Recovery was determined using drug-free media for 24h. (F) dsDNA cytosolic levels were analyzed in ATII cells identified using SP-C antibody (scale bar - 5 µm). Quantification of fluorescence intensity is also shown. (G) TREX1 expression in ATII cells was detected using a proSP-C antibody (scale bar - 5 µm). (H) 8-OHdG levels were analyzed in ATII cells identified using a proSP-C antibody (scale bar - 5 µm). Data are expressed as means±SEM.*p<0.05; **p<0.01 (Kruskal-Wallis test).

Figure 3.

Protein and gene expressions in ATII cells. ATII cells were isolated from younger and older non-smokers (NS) and smokers (SM). (A) γH2AX levels and 53BP1 expression were analyzed in ATII cells by Western blotting. (B) 53BP1 levels were determined in ATII cells by RT-PCR. (C) SIRT1, LC3B-II, and multiubiquitin expression were analyzed in ATII cells by Western blotting. (D) SIRT1 mRNA and LC3B mRNA levels were defined by RT-PCR. (E) G3BP1 expression was determined in ATII cells by Western blotting. (F) NRF2 and DJ-1 protein expression were analyzed in ATII cells by Western blotting. (G) NRF2 mRNA and DJ-1 mRNA levels were determined by RT-PCR. Data are expressed as means±SEM. *p<0.05; **p<0.01; ***p<0.001 (Kruskal-Wallis test).

Figure 4.

Activation of the cGAS-STING pathway in ATII cells in older smokers. Lungs and ATII cells were obtained from younger and older non-smokers (NS) and smokers (SM). (A) cGAS and STING expression were analyzed by immunofluorescence in ATII cells in lung tissue sections (scale bar - 10 µm). Quantification of fluorescence intensity is also shown. (B) cGAS and STING mRNA levels were determined by RT-PCR. (C) p-ATM expression was analyzed in ATII cells (immunofluorescence, scale bar - 10 µm). (D) p-IRF3 and IRF3 levels were analyzed in ATII cells by Western blotting. (E) IRF3 expression was determined in ATII cells by RT-PCR. Data are expressed as means±SEM. *p<0.05; **p<0.01 (Kruskal-Wallis test in A, C, D, E, and one way ANOVA in B)

Figure 5.

Telomere shortening and increased TERRA levels in ATII cells in older smokers. ATII cells were isolated from younger and older non-smokers (NS) and smokers (SM). (A) Telomere length measurement by qPCR. (B) TERRA transcript levels from chromosomes 10q, 15q, and Xq/Yq were determined in ATII cells by RT-PCR. (C) ZBP1 protein expression was analyzed in ATII cells by Western blotting. Densitometric quantification is also shown. (D) ZBP1 mRNA levels were analyzed by RT-PCR. Data are expressed as means±SEM. *p<0.05; **p<0.01; ***p<0.001 (Kruskal-Wallis test).

Figure 6.

The model of dysregulated pathways in ATII cells associated with aging and smoking. ATII cells in older smokers have increased oxidative stress, impaired antioxidant defense system, telomere dysfunction, and dysregulated DNA damage response. This leads to decreased ATII cell repair capacity and exhaustion.