Animals

C57BL/6J mice were obtained from Jackson Laboratories, housed and bred in well-ventilated cages under standard laboratory conditions on 12:12 h light–dark cycle with food and water ad libitum. Both male and female mice aged between 8 and 12 weeks were used unless otherwise mentioned. All animal experimental procedures were conducted in accordance with the animal care standards of the National Institute of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tennessee Health Science Center (UTHSC).

Lipopolysaccharide (LPS) treatment

Mice were randomized into experimental groups and received a single intraperitoneal injection of either endotoxin-free phosphate buffered saline (PBS) or LPS from E. coli O55:B5 (Sigma-Aldrich L2880) dissolved and diluted in endotoxin-free PBS (10 mg kg−1 body weight [23,24,25,26]). Mice were sacrificed at specific time points post injection.

Real time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using the Trizol method and cDNA synthesis was performed using the SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (Invitrogen, 11766050) according to the manufacturer’s protocols. All primer sequences were obtained from the primer bank [27,28,29]. RT-qPCR reactions were performed using 2× SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725271) on an Eppendorf Mastercycler Realplex2.

Exosome isolation, quantification

Isolation of exosomes was done using Exo-Quick serum exosome precipitation solution (EXOQ5A-1, Systems Biosciences, San Francisco, CA, USA) according to the manufacturer’s instructions. The pellets which were enriched in exosomes were dissolved in PBS. For quantifying the protein concentration of exosomes, either intact exosomes were used directly after resuspension into the PBS or were lysed in 2× radioimmunoprecipitation buffer and sonicated briefly. The lysates were then centrifuged at 12,000×g at 4 °C for 20 min. The supernatant was then quantified by using Pierce BCA protein assay kit (23225, Thermo Fisher Scientific, Waltham, MA, USA).

Transfusion of serum derived exosomes from donor to recipient mice

Whole blood (700–800 µl) from mice was collected by cardiac puncture. Blood was centrifuged at 2000×g for 10 min after sitting undisturbed at room temperature for 30 min to separate the serum. Purified exosomes from the sera were then resuspended in 200 μl of sterilized 1× PBS and passed through a 0.22 μm filter before intravenous (IV) tail vein injection to the recipient mice.

Cell culture

Immortalized microglial cells (BV-2) (Accegen, ABC-TC212S), astrocytes (C8-D1A) (ATCC, CRL-2541) and mouse endothelial cells (bEnd.3) (ATCC, CRL-2299), were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY, USA) with 10% fetal bovine serum (FBS; Gibco, NY, USA) in a 5% CO incubator at 37 °C. Cells were treated for 24 h with 0.1 mg/ml of exosomes derived from the serum of the donor mice injected with either LPS or PBS for 24 h.

Cytokine analysis

Exosome homogenates were prepared in RIPA buffer (Sigma, St. Louis, MO) by centrifuging (21,000×g, 30 min at 4 °C) and supernatants were removed for analysis. Cytokine levels in the supernatant were determined using the MesoScale Discovery (MSD, Rockville, MD) 96-well Mouse Pro-Inflammatory V-PLEX Assay (MSD, K15048D-1) according to the manufacturer’s instructions. Briefly, samples are incubated in wells containing an array of cytokine capture antibodies directed against Interleukins IL-6, KC/GRO, TNF-α, IL-12p70, IFN-γ, IL-1β, IL-2, IL-4, IL-5 and IL-10. Bound cytokines are detected immunochemically, and signal was read using an MSD Sector Imager 6000.

Statistics

All statistical analyses were conducted using GraphPad Prism v. 10 (GraphPad Software, San Diego, CA). Data are presented as mean ± standard error of the mean (SEM). The sample size for each experiment, and the statistical testing methods are reported in the figure legends.