C di gmp

Enzymatic in vitro assays were performed and analyzed as described previously [15 (link),30 (link)]. To test a DGC activity, 5 µM purified His6-CdgE_gh, His6-RmdA_gh^GGDEF, His6-RmdA_gh, and His6-CdgA_gh in cyclase buffer [50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM MgCl₂] were individually incubated with 200 µM GTP. To test a PDE activity, 5 µM purified His6-RmdA_gh, His6-CdgA_gh, and Trx-His6-CdgA_gh^AAL were individually added to a reaction mixture containing 200 µM c-di-GMP, 50 mM Tris-HCl [pH 8.0], 50 mM NaCl, and 10 mM MgCl₂. Following 2 h incubation at 37 °C, reactions were stopped first by adding 10 mM CaCl₂ and then by heating at 70 °C for 10 min. Reaction products were analyzed by LC–MS. Standards were prepared by using purchased c-di-GMP (InvivoGen, San Diego, California, USA) and GTP (Sigma-Aldrich, St. Louis, MI, USA).

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).

BMDCs were isolated from mouse bone marrow cells as previously described (31 (link)). Briefly, bone marrow cells were isolated and cultured in RPMI 1640 medium containing 20 ng/mL recombinant GM-CSF (Peprotech), 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS. Petri dishes containing 2 × 10⁶ cells in 10 mL were incubated at 37°C in 5% CO₂. At day 3 of incubation, an additional 10 mL of fresh complete medium containing GM-CSF was added, and 10 mL of medium was replaced with fresh medium supplemented with GM-CSF on day 6. Immature BMDCs were collected on day 7 for experiments. The BMDCs were treated with 2′,3′-cGAMP (5 μg/mL, In vivoGen, California, USA) or c-di-GMP (5 μg/mL, In vivoGen, California, USA) in vitro for 24 h. Cells were treated with lipopolysaccharide (LPS, 1 μg/mL, Sigma–Aldrich) as a positive control or sterile PBS as a negative control. Cytokine (IFN-β and TNF-α) levels in the supernatant were quantified by ELISA. The stimulated BMDCs were stained with the specific antibodies described below and analyzed by using FlowJo software.

Dendritic cells in lamina propria were freshly prepared and sorted by flow cytometry (CD11c⁺MHCII⁺ as gate for DCs). Murine STING agonists (c-di-GMP) (Invivogen, San Diego, CA, USA) in designated concentration were incubated with DCs (3.5 × 10⁴/well) in 96-well plates for 3 days. Lipofectamine 2000 (Life Technologies, Invitrogen, Carlsbad, CA, USA) was used for transfection of c-di-GMP. Same volumes of c-di-GMP and Lipofectamine 2000 (i.e., 1:1 volume ratio) were mixed and incubated in vitro for 5 min at room temperature. The complex was subsequently added into cell culture system for transfection according to the manufacturer’s instruction. The cell pellet was collected for gene expression measurement.
CD4⁺ T cells freshly prepared from SILP and sorted by FACSCalibur (BD Bioscience) (CD3⁺ as gate for T cells) were cultured (2.5 × 10⁵/well) and stimulated with plate-bound anti-CD3 antibody (10 μg/ml) and soluble anti-CD28 antibody (1 μg/ml) for 3 days. The supernatant and cell pellets were collected for protein and gene expression measurement, respectively.