Inhibition of Triggering Receptor Expressed on Myeloid Cells 1 Ameliorates Inflammation and Macrophage and Neutrophil Activation in Alcoholic Liver Disease in Mice

Tornai, David; Furi, Istvan; Shen, Zu T.; Sigalov, Alexander B.; Coban, Sahin; Szabo, Gyongyi

A quantitative in vitro macrophage assay of endocytosis of rho B-labeled HDL-mimicking lipopeptide complexes by J774 macrophage was performed as described. (18) (19) (20) Briefly, BALB/c murine macrophage J774A.1 cells (ATCC) were cultured at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (Cellgro Mediatech, Manassas, VA) with 2 mM glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (Cellgro Mediatech) and grown to approximately 90% confluency in 12-well tissue culture plates (Corning Costar, Corning, NY). After reaching target confluency, cells were incubated for 1 hour in medium with or without fucoidan (400 μg/mL), BLT-1 (10 μM), or cytochalasin D (40 μM). Cells were subsequently incubated for 4 hours and 22 hours at 37°C in medium containing 2 μM of rho B-labeled GF9-HDL or GA/E31-HDL (as calculated for rho B). Cells were washed twice using phosphate-buffered saline and lysed using Passive Lysis Buffer (Promega, Madison, WI). Rho B fluorescence was measured in the lysates with 544-nm excitation and 590-nm emission filters, using a Fluoroscan Ascent CF fluorescence microplate reader (Thermo Labsystems, Vantaa, Finland). Protein concentrations in the lysates were measured using Bradford reagent (Sigma-Aldrich) and an MRX microplate reader (Dynex Technologies, Chantilly, VA) according to the manufacturer's recommended protocol. animals C57BL/6 female mice (10-to 12-week-old) were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed at the University of Massachusetts Medical School (UMMS) animal facility. All animals received humane care in accordance with protocols approved by the UMMS Institutional Animal Use and Care Committee. Mice (n = 6-9/group) were acclimated to a Lieber-DeCarli liquid diet of 5% ethanol (EtOH) (volume [vol]/vol) over a period of 1 week, then maintained on the 5% diet for 4 weeks. Pair-fed (PF) control mice were fed a calorie-matched dextran-maltose diet. All animals had unrestricted access to water throughout the entire experimental period. In treated groups, mice were intraperitoneally treated 5 days/week with vehicle (empty HDL) or the TREM-1 inhibitory formulations GF9-HDL (2.5 mg of GF9/kg) or GA/E31-HDL (4 mg equivalent of GF9/kg) (SignaBlok, Shrewsbury, MA) from the first day on a 5% EtOH diet. At the end of all animal experiments, cheek blood samples were collected in serum collection tubes (BD Biosciences, San Jose, CA) and processed within an hour. After blood collections, mice were euthanized and liver samples were harvested and stored at −80°C until further analysis.