(B) Exosomes containing 20 g of proteins were used to detect exosome-specific proteins (CD9, CD63, and CD81) by Western blot
(B) Exosomes containing 20 g of proteins were used to detect exosome-specific proteins (CD9, CD63, and CD81) by Western blot. of the cytokines/chemokines in these two forms displayed different dynamics in the blood of mice challenged with LPS. Exosomes from septic mice induced the differentiation of Th1/Th2 cells, which was clogged by specific antibodies focusing on IL-12 and IL-4. In addition, these exosomes significantly augmented the proliferation and migration of T lymphocytes. Furthermore, preadministration of exosomes by intravenous injection restrained the inflammatory response, attenuated lung and liver tissue damage, and long term the survival of cecal ligation and puncture (CLP) mice. Our results indicate that exosomes enriched with cytokines/chemokines play crucial functions in T cell differentiation, proliferation and chemotaxis during the sepsis process and have a protecting effect on cecal ligation and puncture (CLP) mice. Therefore, these findings not only strengthen our understanding of the part of sepsis via exosomes but also provide potential focuses on for restorative applications. O111:B4 (Cat.L2630) were from Sigma-Aldrich (St. Louis, MO) and CellTrace? CFSE Cell Proliferation Kit (Cat.”type”:”entrez-nucleotide”,”attrs”:”text”:”C34570″,”term_id”:”2370711″C34570) was from Invitrogen (OR, USA). Transwell? polycarbonate membrane cell tradition inserts (Cat. CLS3422) was provided by Corning (Corning, NY, USA). Fetal bovine serum (FBS) (Cat.10099141) and Dulbecco’s Modified Eagle Medium (DMEM) (Cat.C11995500BT) were from Thermo Fisher (Cambridge, MA, USA). Recombinant Murine Exodus-2 (CCL21) (Cat. 250-13) was from PeproTech (Rocky Hill, NJ, USA). The MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Cat. MCYTOMAG-70K) was from Millipore (Burlington, MA, USA). Polymyxin B (PMB) (Cat.1405-20-5) was purchased from Calbiochem (San Diego, CA, USA). Mice and Sepsis Modeling C57BL/6 mice were purchased from your experimental animal center of Southern Medical University or college (Guangzhou, China), and TLR4 knockout C57BL/6 mice were kindly provided by Dr. T.R. Billiar (Division of Surgery, University or college of Pittsburgh, USA). Male mice were housed in a specific pathogen-free facility. Male C57BL/6 WS3 mice aged 8C9 weeks (weighing 21.6 0.8 g) were intraperitoneally injected with LPS (10 mg/kg) in sterile phosphate-buffered saline (PBS) to reproduce the sepsis magic size. All animal experiments were authorized by the Animal Welfare and Ethics Committee of Southern Medical University or college, Guangzhou, China. Isolation of Exosomes From Septic Mouse Serum Relating to a previously Rabbit Polyclonal to DIL-2 explained method (22), exosomes were isolated using WS3 a altered differential ultracentrifugation protocol (Beckman Coulter). The first step, centrifugation at 2,000 g for 20 min at 4C, was designed to get rid of large cell fragments or debris. The supernatant was collected and centrifuged at 12,000 g at 4C for 45 min to remove small cellular debris. Then, the supernatant was transferred to a new tube and ultracentrifuged at 120,000 g for 120 min at 4C to pellet the small vesicles. The supernatant was discarded, and the pellet was resuspended in a large volume of PBS and then filtered having a 0.22 m filter to remove potential pollutants. After ultracentrifugation at 120,000 g for 120 min at 4C, the pellets were resuspended in PBS, and protein quantitation was performed using a bicinchoninic acid (BCA) assay kit (Pierce, USA). Exosomes extracted from your blood of mice challenged with LPS for different amounts of time (0, 2, 12, 24, and 48 h) were defined as Exo-0, Exo-2, Exo-12, Exo-24, and Exo-48, respectively. Preparation of Exosome-Free Medium Exosomes were depleted from FBS-containing WS3 medium according to the protocol explained by Thery C (23). In brief, 10 ml of total DMEM supplemented with 20% FBS was prepared by centrifugation at 100,000 g immediately at 4C. The supernatant was sterilized having a 0.22 m filter unit driven by a vacuum system. Exosome Characterization Exosomes were purified from your sera of septic mice, and transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to confirm the quality of the exosome preparations. Western blot analyses were performed with specific antibodies (Abs) against exosome biomarker proteins, e.g., CD9, CD63, and CD81. Serum samples from mice challenged with LPS for different amounts of time and exosomes derived from the sera were analyzed with flexible multi-analyte profiling (xMAP) technology using a Luminex-200 system for cytokine quantitation (1, 24, 25). Isolation of Splenic Lymphocytes and Purification of T Cells The spleens from C57BL/6 mice were floor on 200-mesh sieves. The suspensions were centrifuged at 300 g for 8 min, and the pellets were mixed with 10 lysis buffer and incubated at 37C for 5 min to remove the red blood cells. The resuspended combination was centrifuged at 800 g for WS3 30 min in lymphocyte isolating answer, and the lymphocyte coating was collected. T cells were purified using the Pan.