Materials and instruments. All of the reagents and materials, including [18F]F− and [18F]FDG, were obtained commercially. HPLC (Elysia-raytest GmbH) was equipped with an UltiMate 3000 pump (DIONEX) and a B-FC-1000 flow counter radioactivity detector (Bioscan). Hypersil GOLD-C18 reversed-phase HPLC column (250⊆4.6 mm, 5-µm particle size; Thermo Scientific) was used to analysis. PET imaging was performed with a nanoScan PET/CT preclinical scanner (Mediso). Radioactivity counts were measured with a CRC-55tR radioisotope dose calibrator (Hitachi-IGC-7, CAPINTEC. Inc) and a γ-counter (WIZARD 2470–8020, Perkin-Elmer). A Pannoramic 250 FlashⅢ digital scanner (3dhistech Kft, Budapest, Hungary) was used to obtain images of tissue sections. CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences, USA) was used in flow cytometric sorting.
Chemistry and radiochemistry. The labeling precursor (compound 5) was synthesized by a four-steps reaction using 2-fluoro-4-methoxybenzaldehyde as the starting material and the purity is ≥ 95%. Non-radioactive reference ([19F]FBTA) was synthesized by a two-steps one-pot method using compound 5 as the starting material and the purity is ≥ 95%. More details of the experimental materials and methods are shown in Supplementary information.
For labeling [18F]FBTA, fluoride-18 was trapped on an anion exchange resin (20 mg) and eluted with a mixture of potassium carbonate (3 mg in 0.1 mL of deionized water) and Kryptofix[2, 2, 2] (14 mg in 0.1 mL of acetonitrile). The eluted mixture was dried though azeotropic distillation with anhydrous acetonitrile (0.5 mL × 3) under a stream of nitrogen at 110 ℃. After cooling to room temperature, compound 5 dissolved in 0.2 mL of anhydrous acetonitrile was added to the reaction system. The mixture was heated to 90 ℃ and reacted for 20 min. After cooling the mixture to room temperature, deionized water (4 mL) was added, and the mixture was passed through a Sep-Pak Plus C18 cartridge, which was then washed with deionized water (10 mL), followed by the elution of the cartridge with 1 mL of dichloromethane (DCM). Then trifluoroacetic acid (TFA, 0.5 mL) was added to the eluent (0.5 mL), reacted for 5 min at room temperature, the solvent was removed under nitrogen atmosphere at room temperature, and HPLC was used for separation and purification. The HPLC mobile phase gradient of [18F]FBTA and [19F]FBTA was: solvent A: water; solvent B: acetonitrile. 0 to 30 min: 35% B. Flow rate was 1 mL/min.
Relationship between UV area and amount of [19F]FBTA loaded. [19F]FBTA was dilute to different concentrations (ranges from 0.01 to 3 mg/mL) and the UV absorption peak area of different concentrations of [19F]FBTA was calculated by HPLC. The relationship between the concentration of [19F]FBTA (x-axis) and the absorption peak area (y-axis) was obtained by linear analysis. According to Fig. S7, the linear equation of the HPLC UV absorption standard curve of [19F]FBTA is y = 1592.7x + 5.28, R2 = 0.9999.
The octanol-to-water partition coefficient (log P ). The logP affects the absorption, distribution, transport, and metabolism of radiotracers. The general experimental steps were as follows: firstly, a 0.025 mol/L PBS buffer solution with pH = 7.4 was prepared. An equal volume of 1-octanol solution was added to the PBS, vortexed thoroughly, and then left to stand for more than one day. 1 mL of 1-octanol and 1 mL of PBS were added to a centrifuge tube containing 370 kBq of dried [18F]FBTA. The radiotracer and the solvent were thoroughly mixed and vigorously vortexed for 5 min, followed by centrifuging at 5000 rpm for 5 min. Add 100 µL of the 1-octanol phase to a new centrifuge tube containing 0.9 mL of 1-octanol and 1 mL of PBS, and the above steps were repeated for three times. Finally, 100 µL of the organic layer and 100 µL of the aqueous layer solution (n = 3) were taken and measured by an automatic γ-counter to calculate log P.
Stability of [ 18 F]FBTA. Two systems, physiological saline and ethanol, were selected for the study of the in vitro stability of [18F]FBTA. To monitor the stability, a certain amount of [18F]FBTA must be dissolved in physiological saline or ethanol, and incubated for 4 h at room temperature. During the incubation, the samples were analyzed by HPLC and the radiochemical purity of the compound was used to determine whether it was stable.
STING-binding studies. The THP-1 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells were cultured in DMEM/high-glucose medium (Gibco, Carlsbad CA, USA) containing 10% of fetal bovine serum (Gibco), 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Beyotime, Shanghai, China). All cells were placed in a 37 °C incubator with a moist atmosphere of 5% of CO2 and 95% of air.
THP-1 cells saturation binding assay
2×105 of THP-1 cells were added to each centrifuge tube (1.5 mL) in same volume and incubated overnight. Then the cells were centrifuged and the medium was aspirated. [18F]FBTA were diluted to different concentrations (0.07–148 nM) and 100 µL of diluted [18F]FBTA was added to the cells. Another 200 µL of DMEM/high-glucose medium was added and incubated at 37°C for 1 h. The medium was removed after centrifugation, the cells were washed twice with ice-cold PBS (1 mL, pH = 7.4) and centrifuged twice, respectively, to obtain medium-free cells. Radioactivity was measured using a γ-counter. Graph-Pad Prism 5 was used to calculate the Kd value of [18F]FBTA in THP-1 cells.
Cell-specific binding assay
First, 1×105 of THP-1 cells were added to each centrifuge tube (1.5 mL) in same volume and incubated overnight. Then the cells were centrifuged and the medium was aspirated. [18F]FBTA (0.185 MBq/0.2 mL per well) was added to the cells and the cells were incubated at 37°C for 5, 15, 30, 60, 90 and 120 min. After incubation, cells and medium were separated by centrifugation, then cells were washed with 1 mL of PBS and centrifuged, washing and centrifugation were repeated twice. The cells were collected and the radioactivity of these cells was counted using a γ-counter. In order to determine the specificity of [18F]FBTA, the STING inhibitor H-151 (10 µL per well, 1 mg/mL) was added to THP-1 cells for pre-treatment. Then the cells were incubated with [18F]FBTA (0.185 MBq/0.2 mL per well) at 37°C for 5, 15, 30, 60, 90, and 120 min, followed by repeating the PBS washing (1 mL) and centrifugation twice. These cells were finally collected and a γ-counter was used for analysis.
Animals. All the animals involved in the experiments were purchased from the Guangdong Medical Laboratory Animal Center, and all animal experiments strictly followed the ethics committee regulations of the Fifth Affiliated Hospital of Sun Yat-Sen University (NO.00137).
Mice with ALI. ALI was induced in male Balb/c mice aged 6–8 weeks by inhaling lipopolysaccharide (LPS, 1 mg/mL) through the trachea, and the same volume of saline was inhaled as a control. Mice induced by LPS for 2, 12 and 24 h were called as ALI-2 h group, ALI-12 h group and ALI-24 h group, respectively.
Aspirin treatment of ALI mice. Male Balb/c mice aged 6–8 weeks were randomly assigned to the saline-treated or aspirin-treated group. Mice in the aspirin-treated group were injected with aspirin (3.3 mg/mL, dissolved in saline) (Sigma-Aldrich) intraperitoneally in advance, with a dose of 0.1 mg/g each time, injected once a day for 4 days. In the saline-treated group, the same volume of saline was injected by the same way each time. Two hours after the last injection of aspirin or saline, each mouse was inhaled with 40 µL of LPS (1 mg/mL, dissolved in saline), and administrated for PET imaging at 24 h post-induction.
Human lung tissues. Paraffin-embedded human lung tissues were provided by the First Affiliated Hospital of Sun Yat-Sen University. The ethics committee of the Fifth Affiliated Hospital of Sun Yat-sen University approved these studies (K102-1). Normal lung tissues (n = 10) and series of lung inflammation tissues (n = 7) were examined. Among these inflammatory tissues, causative factors for ALI were infection (n = 1), pneumonia (n = 4), contusion (n = 1), and puncture (n = 1).
Small-animal PET/CT imaging. The saline group, ALI-2 h group, ALI-12 h group, ALI-24 h group and saline or aspirin treatment group (n = 3 per group) were administered with 11.1 ~ 14.8 MBq of [18F]FBTA by intravenous (iv) injection, respectively, and 1-h dynamic PET scan and 10-min CT scan were performed immediately after the injection. For comparison, another ALI-24 h group (n = 3 per group) was also administered with 14.8 MBq of [18F]FDG by iv injection and 1-h dynamic PET scan and 10-min CT scan were also performed immediately after the [18F]FDG injection. Before PET imaging, each mouse was weighed, anesthetized with isoflurane (1.5% isoflurane in 100% oxygen), and positioned into a PET/CT scanner bed. PET images were reconstructed by using a 3D ordered subset expectation maximization (3D-OSEM) with attenuation correction. CT images were used as a guide to draw region of interest (ROI) and three-dimensional ROI were drawn over the major organs (heart, liver, kidney), muscle and inflammatory lesions of lungs. Imaging data were acquired and analyzed with the Nucline NanoScan 3.0 software (Mediso Medical Imaging System), and expressed as percentage injected dose per gram (%ID/g).
Biodistribution. After the last in vivo imaging scans, blood samples were obtained from the animals before sacrifice. Shortly thereafter, all animals were perfused through the left ventricle with 0.4% of PFA (Sigma-Aldrich, Darmstadt, Germany) in 20 mL of cold PBS. Afterwards, organs were harvested and radioactivity was quantified with a γ-counter and the results are expressed as %ID/g.
Histology. Mice were sacrificed, perfused with 0.4% of PFA in 20 mL of cold PBS and rinsed in PBS to remove blood on the surface of the tissue. The clean lungs were fixed with 4% paraformaldehyde and embedded in paraffin and sectioned to 4 µm slices. The mice and human lung tissue sections were stained by hemotoxylin and eosin (H&E) and STING antibody to confirm the degree of inflammation and the expression of STING. Specifically, tissue sections were blocked with 5% BSA for 1 h at room temperature, and then incubated with primary antibody overnight at 4°C. For H&E staining, they were stained with hematoxylin and eosin, respectively. For STING staining, STING (D2P2F) rabbit monoclonal antibody (#1346, Cell Signaling Technology) was used as the primary antibody. After immersion in PBST for 3 times, the cells were incubated with enzyme-labeled goat anti-rabbit IgG polymer for 1 h at room temperature. After immersion in PBST for 3 times, DAB chromogenic solution was added dropwise. Finally, the nuclei were counterstained in hematoxylin solution for 2 min. A Pannoramic 250 FlashⅢ digital scanner was used to obtain images of tissue sections.
Flow cytometry. After execution, the lungs were harvested from the mice and the main bronchus were dissected. After rinsing with saline, the tissues were cut into 5 mm pieces and digested with digestion medium containing collagenase I, collagenase IV, dispase II and DNase I on a shaking bed at 37°C for 30 mins. Tissue fragments were then dissociated using a GentleMACS dissociator (Miltenyi Biotec, Germany) and filtered with a 70-mesh cell sieve. Erythrocyte lysis was performed using BD Pharm Lyse (BD Biosciences, CA) and the remaining lymphocytes were centrifuged for subsequent immunostaining.
Cells were stained to assess viability with dye Aqua (Invitrogen), followed by incubation with FcBlock (BD Biosciences) to block Fc Receptor. Surface staining was performed with a comprehensive mixture of fluorochrome-conjugated surface markers includes: CD45 clone 30-F11 PE/Cyanine7 (Biolegend), CD11b clone M1/70 APC (Biolegend), CD11c clone N418 Pacific Blue (Biolegend), Ly-6C clone HK1.4 Brilliant Violet 650 (Biolegend), Ly-6G clone 1A8 APC/Fire 750 (Biolegend), F4/80 clone BM8 PE/Cyanine5 (Biolegend), CD3 clone 17A2 APC (Biolegend). After surface staining, cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X100. For intracellular STING staining, cells were incubated with rabbit anti-STING antibody (19851-1-AP, Proteintech) followed by incubation with FITC conjugated goat anti-rabbit secondary antibody (ab6717, Abcam). Cells were run on a CytoFLEX Flow Cytometer and data analyses were performed using CytExpert software (Beckman Coulter Life Sciences, USA). Cell populations were differentiated using sequential gating strategy.
Western blot. Lungs were isolated from the mice and lysed with RIPA lysate containing protease inhibitor (P0013C, Beyotime) on ice for 30 min. The lysate was then centrifuged at 12000 rpm for 5 min at 4°C, and the concentration of proteins was measured by the BCA protein assay kit (P0010, Beyotime). 50 µg of protein was mixed with loading buffer (LT101S, EpiZyme) and heated in a metal bath at 100 ℃ for 10 min. After cooling on ice, the protein sample was loaded onto a 1.0 mm, 10% PAGE gel and run at 80 V for 30 min, then run at 120 V for 60 min. After the electrophoresis, the protein on the PAGE gel was transferred to the PVDF membrane (66485, BioTrace). The PVDF membrane was incubated in 5% milk at room temperature for 1 h, and then washed with TBST buffer, followed by incubation with the primary antibody STING (D2P2F) Rabbit mAb (#1346, Cell Signaling Technology) and GAPDH (#2118L, Cell Signaling Technology) at 4°C overnight. The next day, the membrane was washed 3 times with TBST and incubated with peroxidase-conjugated Goat anti-Rabbit IgG (ZB-2301, ZSGB-BIO) for 2 h at room temperature. ECL detection reagent was added dropwise to the membrane, and the protein was finally detected with iBright Imaging Systems (Invitrogen).
Statistical analysis. Statistical analysis was performed by Student’s t-test or one-way ANOVA analysis and P < 0.05 is considered a significant statistical difference between groups (P > 0.05, *P < 0.05, **P < 0.001 and ***P < 0.0001).