Animals
WT C57BL/6 mice were obtained from Experimental Animal Center of Zhejiang University (Hangzhou, China). Trem2-/- mice were kindly provided by Professor Macro Colonna from Washington University in St. Louis and bred as described [22]. For the study, 6- to 8-week old male mice (weighing 20 to 25 g) were used. Mice were housed using a 12-hour light-dark cycle (lights on at 7:00 AM) under constant temperature (21-23°C). All mice were maintained on a standard chow pellet diet and tap water ad libitum. All experiments using animals were performed under the approval of the Animal Care and Use Committee of Zhejiang University (Hangzhou, China).
LPS-induced Septic Shock Model
WT and Trem2-/- mice were intraperitoneally injected with LPS (Escherichia coli serotype O111:B4, 30 mg/kg; Sigma-Aldrich, USA) or an equal volume of the sterile saline vehicle to elicit septic shock as previously described [23]. Blood, hypothalamus and adrenal gland were collected postmortem 0, 3, 9 h later for analysis. The mice were randomly assigned to experimental conditions. All further experiments were blinded to murine genotypes and treatments.
Isolation of Bone Marrow-Derived Macrophages (BMDMs)
The murine BMDMs of WT and Trem2-/- mice were isolated from the femur and tibia in single cell suspension and seeded in 6-well plates (2.5 × 106 cells per well) with Dulbecco’s modified eagle medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, USA), 1% penicillin/streptomycin and 20 ng/ml mouse granulocyte macrophage-colony stimulating factor (PeproTech, USA). These cells were differentiated into macrophage for another 6~7 days with replacement of fresh growth media in every 3 days. Cells were maintained in a humidified incubator (Thermo Fisher Scientific, USA) with 5% CO2 at 37 °C. Then BMDMs were harvest or stimulated for further experiments.
Primary Adrenocortical Cell Culture
Primary mouse adrenocortical cells were isolated from WT mice as previously reported with some modifications [24, 25]. Briefly, adrenal glands of WT mice were excised and freed from perirenal fat. The adrenal was carefully halved and enucleated to eliminate medullas parts. Repeated digestions (thrice to quartic) were performed for 1 h in buffer containing 0.1% collagenase I (Gibco, USA), 1% hyaluronidase (Sigma-Aldrich, USA) and 0.1% bovine serum albumin (Sigma-Aldrich, USA) at 37 ºC on an orbital shaker. The adrenocortical suspension was pressed through a 70 μM cell strainer, centrifuged at 800 g for 5 min at 4 ºC and the cell pellet was resuspended in DMEM/nutrient mix F-12 (Gibco, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. Suspended cells were placed in culture dishes at 37 ºC for 1 h to enable selective attachment of fibroblasts. Cell viability (> 90%) was assessed by trypan blue exclusion test. Adrenocortical cells collected from supernatants were counted and seeded in a 24-well culture plate (Corning, USA) at a density of 1 × 105 cells/ml, the medium being changed every 48 h. Cells were then cultured in media for 5~6 days for the subsequent treatments.
Macrophage-Adrenocortical Cell Coculture
Differentiated BMDMs were co-cultured with matured adrenocortical cells using a trans-well plate (Corning, USA), with BMDMs cultured in the upper chamber (membrane pore = 0.4 μm) and primary adrenocortical cells in the lower chamber, allowing a contact-independent communication by secreted components. To evaluate the effect of WT and Trem2-/- BMDMs on adrenocortical cells in an inflammatory condition, the experimental system was stimulated with ACTH (100 nM; Bioss, China) and LPS (100 ng/ml) for 6 h. The culture medium was collected for corticosterone detection and adrenocortical cells were harvested for RNA extraction after washing with phosphate buffer saline (PBS) twice.
Purification of Macrophage-derived Exosomes
To harvest exosomes, WT and Trem2-/- BMDMs were changed for fresh media and stimulated with LPS (100 ng/ml) for 24 h. The cell culture supernatants were centrifuged sequentially at 1,500 g for 15 min and 16,500 g for 30 min to eliminate the cell debris and large vesicles. The pellet was washed once with PBS by ultracentrifugation for 2 h at 118,000 g at 4 °C in a swinging bucket rotor (Beckman Coulter, USA). The exosome-containing pellet was resuspended in 100~200 μl of PBS for further experiments.
Nanoparticle Tracking Analysis
The size distribution of exosomes was measured by nanoparticle tracking analysis technology using ZetaView PMX 110 (Particle Metrix, Germany) and Software ZetaView 8.04.02 SP2. Samples were diluted in PBS to obtain an appropriated concentration and subsequently assessed according to the operating instructions.
Electron Microscopic Examination
Fresh exosomes samples were resuspended with 100 μl PBS and then placed on 200-mesh formvar-coated copper grids. Samples were stained with 2% aqueous uranyl acetate and air-dried. Transmission electron microscopy (Thermo Fisher Scientific, USA) was performed to view the physical characterization of exosomes.
Exosome Treatment ex vivo
For ex vivo treatment, WT or Trem2-/- BMDM-derived exosomes (20 μg/ml) were added to 1 × 105 cells/ml adrenocortical cells. After 6 h, adherent cells and culture medium were collected for corticosterone production and mRNA expression measurements, respectively.
Measurement of ACTH and Corticosterone Concentrations
The serum concentrations and cell culture supernatant levels of corticosterone (Cayman, USA) and ACTH (Mybiosource, USA) were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.
Analysis of Serum Cytokines
The serum cytokines tumor necrosis factor (TNF)-α, interleukin (IL) -6, IL-1β and IL-10 were measured by corresponding ELISA kits (all from eBioscience, USA) according to the manufacturer’s instructions.
Evaluation of Organ Functions
The serum concentrations of creatinine (CRE), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and plasma concentrations of lactic acid were determined using commercial assay kits (NanJing KeyGen Biotech, China).
Immunofluorescence Analysis
The adrenal gland was fixed in a 4% formaldehyde solution and embedded in OCT Tissue-Tek for 10-μm frozen sections. Frozen sections were mounted on slides, fixed and permeabilized in PBS-0.3% Triton X-100. Adrenal glands were incubated with primary antibodies for rat anti-mouse F4/80 (clone Cl:A3-1; 10 μg/ml; AbD Serotec, USA) and goat anti-mouse TREM2 antibody (10 μg/ml; Abcam, USA) overnight at 4°C, followed by incubation of Alexa Fluor 488 conjugated donkey-anti-rat IgG (4 μg/ml; Invitrogen, USA) for F4/80 and Alexa Fluor 594-conjugated donkey-anti-goat IgG (4 μg/ml; Invitrogen, USA) for TREM2 for one hour at room temperature. Coverslips were mounted with VECTASHIELD mounting medium with DAPI (Vector Labs, USA) to detect all cell nuclei. Images were evaluated using a Leica DM5000B microscope (Leica, Germany) or Olympus confocal laser scanning microscope (Olympus, Japan). At least 3 fields in a randomly selected section of each mouse were captured for analysis of F4/80+TREM2+ double-labeled cells in the adrenal gland.
Quantitative Real-time Polymerase Chain Reaction Analysis (qRT-PCR)
The mRNA was extracted from homogenates of the hypothalamus and adrenal cortex, and cultured cells using TRIzol reagent (Life Technologies, USA) as instructions described. qRT-PCR was applied to detect relative expression level of mRNA from tissue samples by using SYBR PCR Master Mix (TaKaRa, Japan) and standard oligonucleotides. β-actin was used as the endogenous control gene. Primers designed to quantify specific transcripts were listed in Table S1. Amplification data were analyzed using Roche 480 PCR System (Roche, USA). Data analysis was performed using the comparative threshold cycle method with arithmetic formulae (2-△△Ct), and the mRNA levels were expressed as a fold change compared to the control group.
Statistical Analysis
Statistical evaluations were employed by GraphPad Prism 6.00 (GraphPad Software Inc., USA). A two-tailed paired or unpaired Student’s t test was used to compare difference between two independent groups. The covariance efficiency factor was also assessed for each endogenous control (covariate) to ensure that there were no effects of treatment on the endogenous controls. Survival between two independent groups was analyzed using a log-rank (Mantel-Cox) test. All data shown in the figures and text are represented as mean ± standard error of the mean (SEM). Differences were accepted as statistically significant if a p value was less than 0.05.