Ethics Statement.
All the mice used in the experiments were 9–10 weeks old and on a C57BL/6 background. All experimental manipulations were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. CD4 T cells from healthy human peripheral blood was purchased from Bioscience. Lonza.com (Bend, OR, USA).
Animal CLP model.
All animals were housed in separate cages in a temperature-controlled room with 12-h light and 12-h darkness, experimental sepsis was induced by the CLP procedure. Some mice were intravenously injected with 15 mg/kg PARP-1 inhibitor (olaparib)(Selleck Chemical LLC, USA) for 4-h before CLP model. After anesthesia, the mice were fixed on the operating table, and the abdominal skin was disinfected with iodine and alcohol. A 1.5 cm incision was made along the midline of the abdomen, and the peritoneum was opened to find the free root of the cecum. The cecum was ligated at the middle and punctured twice with a 21-gauge (0.723 mm) needle to induce a moderate severity of sepsis. Sham-operated mice underwent the same surgical procedure except the ligation and perforation step.
Generation of STING-deficient mice.
Age and sex matched littermates were used as controls. Mice were housed in BioMedical Research Institute of Nanjing University, Nanjing, China. All procedures used were pre-approved by the Institutional Animal Care and Use Committee, Nanjing University, Nanjing, China. Two sgRNAs-targeting the exon2 of STING gene were constructed and transcribed in vitro. Cas9 mRNA and sgRNAs were coinjected into 0.5-day-old C57BL/6J mouse zygotes.
Isolation of splenic CD4 T cells.
All procedures used were pre-approved by the Institutional Animal Care and Use Committee, Nanjing University, Nanjing, China. Spleens were obtained from WT C57BL/6 mice, STING−/− mice and septic mice, and they were teased in 5 ml RPMI 1640. Mononuclear cells were isolated and then CD4 T cells were isolated from them. A MiniMACS™ Separator with a negative selection LS column by following the manufacturer’s instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The pellets of selected CD4 T cells were obtained by centrifugation (1500 rpm, 10 min), then the supernatant was discarded, and the cells were cultured in medium RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum (FCS).
PARP-1 depletion.
A total of 1×106 CD4 T cells were planted in twelve-well plates and cultured overnight. Predesigned siRNAs against mouse PARP-1was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA), and control scrambled siRNA was purchased from Abcam (Abcam, Cambridge, MA, USA). The transfection procedure followed the protocol of Santa Cruz Biotechnology, USA. 48 h after transfection, the depletion of PARP-1 was confirmed by Western blotting, and cells were used in subsequent experiments.
Flow cytometric analysis for cell death.
CD4 T cells were treated with the indicated concentrations of LPS (1µg/ml) for the indicated time points. CD4 T cells were harvested and washed using Annexin V buffer provided by the supplier (BD Biosciences, San Jose, CA, USA) and then stained with Annexin V. Next, Bv-421-CD4 antibody (CD4 T cell maker) was added and stained for 1 h. Cell death was also measured, 7-AAD was added at a final concentration of 5 µg/ml. The cells were then analyzed by flow cytometry. Acquisition was performed on 10000 events using a FACScalibur cytometer (BD Biosciences, San Jose, CA, USA) or BD LSR II (BD Biosciences, San Jose, CA, USA) with CellQuestPro (BD Biosciences, San Jose, CA, USA) and FlowJo-V10 software (Tree Star, Ashland, OR, USA).
Immunofluorescence confocal microscopy.
After treatment with LPS for 24 h, CD4 T cells were washed with PBS for three times, and fixed with 4% paraformaldehyde in PBS for 20 min, then permeabilized with 0.02% Triton X-100 for 20 min at room temperature. Sections were preblocked with 1% bovine serum albumin in PBS for 30 min and stained with anti- PARP-1 and PAR antibody (Abcam, Cambridge, MA, USA) (1:200) overnight at 4°C. Anti-AIF antibody (Invitrogen, USA) were stained overnight at 4°C. After being washed in PBS for three times, CD4 T cells were stained with goat anti-mouse IgG H&L (Alexa Fluor® 647) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, Cambridge, MA, USA) as the second antibody for 1 h at room temperature followed by 3×PBS washes. After being washed, the nuclei were stained with Heochst (Sigma-Aldrich, USA) for 5 min. The cells were observed with a laser scanning confocal microscope.
Western blotting.
Western blot was performed to determine expressions of PARP-1, PAR polymer, COXIV, STING in CD4 T cells. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Grand Island, NY, USA). Proteins of CD4 T cells were separated by SDS PAGE and then transferred electrophoretically onto polyvinylidenedifluoride (PVDF) membranes. The membranes were probed with anti-PARP-1 antibody, anti-PAR polymer antibody (Abcam, Cambridge, MA, USA), anti-COXIV antibody (Invitrogen, USA), anti-STING antibody (Abcam, Cambridge, MA, USA) and polyclonal anti-actin antibodies (Santa Cruz Biotechnology, USA). Protein bands were detected using Odyssey System from LI-COR Biosciences, USA.
Measurement of NAD + and ATP levels.
CD4 T cells were seeded into 96-well plates (1×104 cells/well) with or without transfecting siRNA-PARP-1 for 48 h. The cells then were treated with LPS (1µg/ml) for 24h. The cellular NAD+ levels were measured using the Fluoro NAD/NADH detection kit (Cell Technology, USA) according to the manufacturer’s instruction. Additionally, CD4 T cells in 96-well plates were treated with LPS (1µg/ml) for the indicated intervals. Cellular ATP levels were measured using the Enzylight ATP assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions. The ATP assay kit provided a rapid method to measure intracellular ATP. Luminescence was measured on BioTek Synergy Mx luminometer (BioTek Instruments, Winooski, VT, USA) and quantitated to ATP standards.
Enzyme-linked immunosorbent assay (ELISA).
IL-6 level in culture supernatants was determined with ELISA kits (R&D System Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.
Statistical analysis.
Data were represented as mean ± standard deviation (SD). Data sets were examined by one-way ANOVA, and individual group means were then compared with Student paired t test. All statistical tests were two sides and a P value of 0.05 or less was considered to indicate statistical significance.