H 151

Fibroblasts of a patient with DNase2 interferonopathy (D2I) and from healthy control subject were maintained at a density of about 2 × 10⁴ cell/cm² with complete medium (RPMI with 10% FBS, 100 U/mL Penicillin, 100 μg/mL Streptomycin, 2 mM L-Glutamine, all from EuroClone; Milan, Italy), supplemented with Normocin (100 μg/mL, InvivoGen, San Diego, CA, USA). For all experiments, fibroblasts were used at a density of 10⁴ cell/cm² with complete medium (with 10% low endotoxins FBS, Microtech, Naples, Italy).
Fibroblasts were treated for 1 h with scalar concentrations of LPS (0.5–1–5 μg/mL, E. coli—serotype 055:B5, Sigma-Aldrich, St. Louis, MO, USA).
For phospho-TBK1 evaluation after STING inhibition, fibroblasts were pre-incubated for 2 h with human STING inhibitor H-151 (2.5–5–10 μM, InvivoGen), and afterward treated for 1 h with LPS (0.5 μg/mL, Sigma-Aldrich).
For IFN signature assessment and PIK3AP1 expression (after 24, 48 and 72 h treatment with H-151 10 μM), fibroblasts were recovered for RNA extraction (High Pure miRNA Isolation Kit, Roche, Basel, Switzerland) and cDNA synthesis (SensiFAST™ cDNA Synthesis Kit, Bioline, London, UK), following the manufacturer’s instructions.

In brief, 5 ml ethanol was added slowly to 1 g of tamoxifen (Sigma-Aldrich) in a 50-ml Falcon tube. The tamoxifen and ethanol mix was sonicated at 40% amplitude in 20-s pulses until the tamoxifen was completely dissolved. Then, 50 ml of corn oil (Sigma-Aldrich) pre-heated at 50 °C was added immediately to the tamoxifen and ethanol mix to obtain a 20 mg ml⁻¹ tamoxifen stock solution. The tube was then vortexed and incubated for up to 12 h in an orbital shaker at 50 °C to ensure adequate solubilization. Aliquots of 5 ml were then stored indefinitely at −20 °C. H-151 (InvivoGen) was diluted in DMSO to obtain 20 mg ml⁻¹ or 4 mg ml⁻¹, and was further diluted in sterile phosphate-buffered saline (PBS) to obtain a 5% DMSO concentration before injection into mice. Aliquots of 2 ml were then stored at −20 °C. Before injection, the mixture was allowed to warm up to room temperature. Age-matched mice (between 10 and 12 weeks old) were used in all experiments. Each mouse received three doses of 2 mg tamoxifen by intraperitoneal injection. tamoxifen was administered every other day to allow mice to recover between doses. Mice received a daily dose of 7 mg per kg or 1.4 mg per kg H-151 by intraperitoneal injection.

ODN1826 (5′-tccatgacgttcctgacgtt-3′), fluorescein isothiocyanate (FITC)-labeled ODN1826, poly(I:C) HMW, FITC-labeled poly(I:C), ODN1585 (5′-ggggtcaacgttgagggggg-3′), c-di-GMP, and H-151 were purchased from InvivoGen (San Diego, CA, USA). CpG K3 (5′-atcgactctcgagcgttctc-3′), GpC K3 (5′-atgcactctgcaggcttctc-3′), modified CpG K3 with a phosphodiester backbone (CpG K3(O); 5′-atcgactctcgagcgttctc-3′), CpG D35 (5′-ggtgcatcgatgcagggggg-3′), and GpC D35 (5′-ggtgcatgcatgcagggggg-3′) were purchased from GeneDesign (Osaka, Japan). AS1411 (5′-tttggtggtggtggttgtggtggtggtgg-3′), FITC-labeled AS1411, CRO (5′-tttcctcctcctccttctcctcctcctcct-3′) and FITC-labeled CRO were synthesized at Hokkaido System Science (Hokkaido, Japan). Anti-nucleolin monoclonal antibody (clone: MS-3, catalog numbers: sc-8031) and phycoerythrin (PE)-labeled anti-nucleolin monoclonal antibody (clone: MS-3, catalog numbers: sc-8031 PE) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse IgG1 isotype control (clone: T8E5, catalog numbers: mabg1-ctrlm) was purchased from InvivoGen. PE-labeled mouse IgG1 isotype control antibody (clone: MOPC-21, catalog numbers: 400112) was purchased from BioLegend (San Diego, CA, USA).

STING was stimulated with 10 μg/mL cGAMP (InvivoGen, San Diego, CA, USA). Toll-like receptor 3 (TLR3) was stimulated with 10 ng/mL naked p(I:C) and Toll-like receptor 4 (TLR4) with 100 ng/mL LPS (Merck, Rahway, NJ, USA). As negative control, we used ssRNA (InvivoGen). STING was inhibited with the irreversible STING inhibitor H-151 (0.5 μM, InvivoGen) [27 (link)]. NFκB was inhibited with 5 μM BAY 11-7082 (Cayman Chemical Company, Ann Arbor, MI, USA). Irf3 expression was suppressed via siRNA silencing, using oligos from Thermo Fisher (Waltham, MA, USA) (Silencer™ Pre-Designed siRNA, Cat. No.: AM16708, siRNA ID: 184585). Autophagy was stimulated with serum deprivation. Autophagy was inhibited with 100 μM chloroquine treatment, as described in [28 (link)].

Alveolar macrophages (AM) and LEC were infected in vitro with B. abortus at multiplicities of infection (MOI) of 100 bacteria/cell (AM) or 200 bacteria/cell (LEC) as described previously (14 (link)). Bacteria were dispensed suspended in culture medium without antibiotics, followed by centrifugation of the plates and incubation for 2 hours (37°C, 5% CO₂ atmosphere). Bacteria not adhered or internalized into the cells were removed by three washes with sterile PBS (time 0 p.i.). To kill non-internalized bacteria the cells were incubated in culture medium containing 100 µg/ml of gentamicin (Sigma, USA) and 50 µg/ml of streptomycin (Sigma, USA). Culture supernatants were harvested for cytokine measurement 24 h after antibiotics addition. At the same time, cell lysates prepared using 0.2% Triton X100 were plated on TSA to enumerate CFU of intracellular bacteria. In some experiments, a STING inhibitor (H151, InvivoGen, 15 µM) or its vehicle (DMSO) were added to AM and LEC for 24 h before infection, and were maintained during the whole infection period.