Fig. 7

Extracellular self-dsDNA is key to silica-induced inflammatory response in macrophages, not in DCs. a–c Extracellular DNase I treatment (1 µg/mL) was applied 3 h prior and 1 h after silica exposure (250 µg/mL) or transfection with c-di-AMP (6 µg/mL; cDN) for 18 h in bone marrow-derived macrophages (b) and dentritic cells (c). b Macrophage concentration of extracellular dsDNA and CXCL10 in culture supernatant. Ifnα and Ifnβ transcripts measured by real-time PCR on cell fractions and IFN-α and IFN-β protein concentrations determined in culture supernatants by multiplex immunoassay. c Dendritic cell IFN-α and IFN-β protein concentrations determined in culture supernatants by multiplex immunoassay. d–i Mitochondrial DNA replication was inhibited using a low concentration of EtdBr (150 ng/mL) on day 7 of bone marrow cell culture. On day 11, bone marrow-derived DCs (e–h) and macrophages (i) were unstimulated, stimulated with silica (250 µg/mL), or transfected with c-di-AMP (6 µg/mL; cDN) for 18 h. Fold change of (e) mitochondrial DNA (mMitoF1 and mMitoR1) and (f) nuclear DNA (mB2MF1 and mB2MR1) in untreated versus EtdBr-treated DC exposed to silica, as compared to untreated unstimulated cells. g Immunoblot showing cGAS, phospho-STING, STING protein expression, and dimerization in DCs after EtdBr treatment, with β-actin as a reference. h CXCL10 level in DC supernatant quantified by ELISA and IFN-α and IFN-β concentrations determined by multiplex immunoassay. Tmem173 transcripts measured by real-time PCR. i CXCL10 level in macrophage supernatant quantified by ELISA and IFN-α and IFN-β concentrations determined by multiplex immunoassay. Tmem173 transcripts measured by real-time PCR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Mann–Whitney U analysis). Data are presented as mean ± SEM and are representative of two independent experiments with n = 4 independent cultures. Immunoblots are representative of n = 2 samples per condition (g). Source data are provided as a Source Data file