Tlrl 3pelps

LPS (InvivoGen; tlrl-3pelps), alum (InvivoGen; tlrl-alk), recombinant murine IL-5 (PEPROTECH; 210-13), recombinant murine IL-1β (R&D Systems; 401-ML), SecinH3 (Abcam; ab145048), Brefeldin A (Wako; 022-15991), Cytochalasin B (Wako; 14930-96-2), OVA (MilliporeSigma; A2512-1G), HDM (Dermatophagoides pteronyssinus; LSL; LG-5449), and aluminium hydroxide gel (Wako; 012-24241) were purchased. Cytokines were measured by ELISA with mouse IL-1β (R&D Systems; MLB00C), mouse IL-5 (R&D Systems; M5000), mouse IL-13 (R&D Systems; M1300CB), mouse OVA-specific IgG1 antibody (Cayman Chemical; 500830), and mouse OVA-specific IgE antibody (BioLegend; 439807) kits according to the manufacturer’s protocols. Mouse monoclonal antibodies against β-actin (MilliporeSigma; A5441), caspase-1 (R&D Systems; MAB6215), IL-1β (Cell Signaling Technology; 3A6), and a rabbit polyclonal antibody against ASC (AdipoGen; AG-25B-0006) were purchased. Primary airway macrophages were isolated from BALF and were grown in RPMI 1640 with 10% FBS, 65 μg/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml fungizone.

hiPSC cultures and microglial differentiation methods were as previously described (54 (link)). Fully differentiated microglial cells were placed into phenol-free Iscove’s modified Dulbecco’s medium (IMDM; Thermo Fisher Scientific, 21056023); 1 day later, cells were pretreated with 5 nM β-estradiol (Sigma-Aldrich, E8875) or control diluent for 4 hours and then exposed to 750 nM oligomerized Aβ (Anaspec, AS-20276), 150 nM oligomerized α-synuclein (Anaspec, AS-20276), and LPS (10 ng/ml; Invivogen, tlrl-3pelps) were incubated in the presence or absence of β-estradiol as previously described (55 (link), 92 (link), 93 (link)). For transient transfection of HEK293T cells (System Biosciences, LV900A-1) with plasmids encoding either WT C3 or mutant C3(C720A), Lipofectamine 2000 was used according to the manufacturer’s instructions (Thermo Fisher Scientific, 11668019).

PMφs, BMDMφs or RAW264.7 Mφs were cultured for 24 h with human medium oxidized low-density lipoprotein (100 μg/mL, Kalen Biomedical Cat#770202) or cholesterol (50 μg/mL, Sigma Cat#C3045), followed by ultrapure LPS stimulation (10 ng/mL, InvivoGen, Cat#tlrl-3pelps) for up to 8 h. Ethanol (0.5%) was used as a carrier control for cholesterol (-Chol). For inhibitor experiments, 2-DG (25 mM) (Sigma, D8375), Acriflavine (2.5 µM) (Sigma, Cat#01673) and TEPP-46 (100-900 µM) (Tocris, Cat#7809) were added 1 h prior to LPS stimulation.

BMDMφs and PMφs were cultured for 24 h with human medium oxLDL (100 μg/ml, Kalen Biomedical, no. 770202) or cholesterol (50 μg/ml, Sigma-Aldrich, no. C3045), followed by ultrapure LPS stimulation (10 ng/ml, InvivoGen, no. tlrl-3pelps) for up to 6 h, or CpG (300 nM) (InvivoGen, no. tlrl-1826), Pam₃CSK₄ (10 ng/ml) (Tocris Bioscience, no. 4633), or bpV (HOpic) (10 µM) (Sigma-Aldrich, no. SML0884) stimulation for up to 3 h. Ethanol (0.5%) was used as a carrier control for cholesterol. For inhibitor experiments, MK-2206 (3 µM) (Cayman Chemical, no. 11593), AKT2i (3 µM) (Sigma-Aldrich, no. 124029), Mito-TEMPO hydrate (25 µM) (Cayman Chemical, no. 16621), and bpV (HOpic) (10 µM) were added 1 h prior to LPS stimulation. For assessing the accumulation of oxLDL, a mixture of DiI-oxLDL and oxLDL (10 and 90 μg/ml, respectively) were added to PMφs for 24 h. In all experiments, Mφs were first cultured for 24 h with or without oxLDL or cholesterol. The groups without oxLDL or cholesterol served as controls. The control group for cholesterol loading was cultured with media containing the same concentration of ethanol as the cholesterol group. Cells were then stimulated with LPS from 0 up to 3 h. At each time point, the groups with versus without oxLDL or cholesterol were compared and changes over time were determined.