Fig 2: Cell death induction in monocytes by STING ligands. Purified monocytes were cultured in medium or were stimulated for 4 and 7 or 8 h with 100 ng/mL LPS or the indicated concentrations of STING ligands. Cell death was analyzed by flow cytometry following PI/annexin V staining and by Western blot analysis. (a) Contour plot of a representative experiment, left panel: 4 h, right panel: 8 h. (b) Summary of four experiments, displayed as proportion of viable (PI−/annexin V−) cells. Statistical significance was calculated by paired Student’s test. Red asterisks indicate the level of significance between stimulated and unstimulated (medium) samples. *p < 0.05, **p < 0.01. (c) Western blot analysis of death-associated proteins in monocytes after stimulation for 7 h with the indicated concentrations of STING ligands.

Fig 3: Responsiveness of monocytes to STING ligands. Purified monocytes were stimulated with 100 ng/mL LPS, 0.5 µg/mL Motolimod,100 nM diABZI, 25 µM MSA-2, or 15 µM cGAMP/cGAMP control as indicated. (a) STING expression in monocytes was analyzed by flow cytometry. Blue: FMO control, red: anti-STING-AF488. One representative out of five experiments. (b) Western blot analysis of monocyte lysates after 4 h stimulation. (c–h) Cytokines were quantified by ELISA in supernatants after 24 h. Each donor is identified by identical symbol and grey tone. (c) IFN-β (n = 5–9). (d) IP-10 (n = 3–5). (e) IL-1β (n = 7–18). (f) TNF-α (n = 10–22). (g) IL-10 (n = 8–20). (h) IL-19 (n = 5–12). Statistical significance was calculated for each group by comparing stimulatory conditions with medium by paired Student’s t-test or Wilcoxon signed rank test as appropriate. Red asterisks indicate the level of significance between stimulated and unstimulated (medium) samples. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Characterization of IL-10 producing monocytes. Purified monocytes were stimulated with low (1 nM diABZI, 2.5 µM MSA-2) or regular (100 nM diABZI, 25 µM MSA-2) concentrations of STING ligands in the presence or absence of 1 µM TBK1 inhibitor BX795. (a) TBK1 phosphorylation in response to 1 or 100 nM diABZI was determined by flow cytometry after 60 min. One representative out of three experiments is shown. (b) TBK1 phosphorylation in response to 2.5 or 25 µM MSA-2 was revealed by Western blot after 4 h. (c) IFN-β in supernatants collected after 4 and 24 h was quantified by ELISA (n = 5). (d) qRT-PCR analysis of IL10 and PRDM1 (Blimp-1) in monocytes cultured for 8 h in medium or with 100 ng/mL LPS, 1 nM diABZI, or 2.5 µM MSA-2 (n = 4). ΔΔCt values refer to the change versus freshly isolated monocytes. (e,f) Detection of intracellular IL-10 by flow cytometry. Purified monocytes were stimulated with 1 nM diABZI or 2.5 µM MSA-2. Intracellular expression of IL-10 was determined after 24 h. (e) One representative is shown. A gate on IL-10 expressing cells was set in relation to the FMO control. (f) Summary of three experiments. (g) Monocytes were stimulated with 1 nM diABZI or 2.5 µM MSA-2 in the absence or presence of 1 µM BX795. IL-10 was quantified in supernatants collected after 24 h (n = 4–5, or 2 [medium with BX795]). Statistical significance was calculated by paired Student’s test. In (c,d,f), significance was calculated between stimulated and unstimulated (medium) groups. In (g), significance was calculated between diABZI/MSA-2 and medium as well as between diABZI in presence versus absence of BXT795 as indicated by horizontal bars. Red asterisks indicate the level of significance between stimulated and unstimulated (medium) samples. *p < 0.05, **p < 0.01.

Fig 5: Responsiveness of M-CSF differentiated macrophages to STING ligands. Purified monocytes were cultured for 6 days in the presence of 50 ng/mL M-CSF. (a) M-CSF macrophages expressed STING as revealed by flow cytometry. Blue: FMO control; red: anti-STING-AF488. (b) M-CSF macrophages were stimulated with 100 nM diABZI or 25 µM MSA-2. After 60 min, phosphorylation of TBK1 was analyzed by flow cytometry using anti-pTBK1-PE antibody. Red: medium control; blue: lower dose of diABZI/MSA-2; beige: higher dose of diABZI/MSA-2. (c) Surface marker expression on paired samples of freshly isolated monocytes (left) and M-CSF macrophages (right). Blue: isotype controls; red: specific antibody. (a–c) Representative out of three experiments are shown. (d–f) M-CSF macrophages were cultured in medium only or were stimulated with 100 ng/mL LPS, 100 nM diABZI, 25 µM MSA-2 (left part), or the combination of LPS with STING ligands as indicated (right part). Supernatants were collected after 24 h, and cytokines were quantified by ELISA. (d) IL-10 (n = 5–8). (e) IL-1β (n = 6–8). (f) TNF-α (n = 3–6). Statistical significance between groups as indicated by the horizontal bars was calculated by paired Student’s test. Red asterisks indicate the level of significance between stimulated and unstimulated (medium) samples (left part) or between LPS plus STING ligands versus LPS only (right part). *p < 0.05, **p < 0.01.