Tlr ligands

BMDMs and thioglycolate-induced peritoneal macrophages were seeded in flat-bottom 24-well Corning Costar plates (Corning, NY, USA) at a density of 1 million cells/ml and incubated overnight to permit reattachment. Cells were then washed and given fresh medium containing ultra-purified LPS from Escherichia coli K12 (Ultrapure LPS-EK), IFNγ, Beta-1,3-glucan from Alcaligenes faecalis (Curdlan AL), or poly(deoxyadenylic-deoxythymidylic) acid sodium salt poly(dA:dT). TLR ligands were obtained from InvivoGen (San Diego, CA, USA). Cytokines and chemokines used in these studies were obtained from PeproTech. Experiments were performed with varying concentrations of ligand, but data is presented from the dose that gave the optimal response in WT controls.

All TLR ligands (InvivoGen, San Diego, CA) were resuspended in sterile, vaccination–grade physiological water. Twenty–four hours following TBI, mice were given an i.p. injection of physiological water (no treatment, NT), 2.5 mg/kg CpG–ODN 2395 (tlrl–2395), 2.0 mg/kg Flagellin FliC (vac–fla), 0.25 mg/kg MPLA (tlrl–mpla), 0.25 mg/kg FSL–1 (tlrl–fsl), 0.25 mg/kg Pam2CSK4 (tlrl–pm2s–1) or 0.25 mg/kg Pam3CSK4 (tlrl–pms). The total volume administered per mouse ranged from 50–70 μl based on the weight of the mouse and ligand concentration.

Dimethyl sulfoxide (DMSO) was purchased from Sigma. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (>99% purity) was originally obtained from Dow Chemical Co. (Midland, MI, United States). 6-Formylindolo[3,2-b]carbazole (FICZ), Indole-3-carbinol.
(I3C), 12-O-tetradecanoylphorbol-13-acetate (TPA) and other molecular biological reagents were purchased from Cayman Chemicals (Ann Arbor, MI, United States) and Applied Biosystems (Foster City, CA, United States). TLR ligands were purchased from InvivoGen (San Diego, CA, United States). RelB-specific polyclonal antibody from Active Motif (Carlsbad, CA, United States). AhR-specific polyclonal antibody from Enzo Life Sciences (Farmingdale, NY, United States). The traffic-related air pollution (TRAP)-related PM_2.5 was collected from an exposure facility immediately adjacent to a major freeway tunnel system in Northern California (Patten et al., 2020 (link)) via impaction-based filter sampling and extracted according to the protocols of Bein and Wexler (2014) (link) and Bein and Wexler (2015) (link). TRAP samples were collected before and during the Sonoma/Napa wildfire in 2017. The wildfire PM sample was collected during the Carr wildfire in Northern California in 2018. The dry PM extracts were resuspended in DMSO and sonicated immediately prior to treatment of the cells.

Plasmids encoding Myc-tagged IRF5 isoforms were a kind gift of Dr. Frank Neipel (Virologisches Institut - Klinische und Molekulare Virologie, Erlangen, Germany). Plasmids encoding Xpress-TRIM21 and GST-TRIM21 PRY/SPRY domain were a gift from Dr. David Rhodes (Cambridge Institute for Medical Research, Cambridge, UK). HA-ubiquitin wild type and mutants were a gift from Dr. James Burrows (Centre for Cancer Research and Cell Biology, Belfast, UK). Plasmids encoding FLAG-tagged IRF5 full length and deletion mutants were described previously [24] (link). Myc-MyD88 construct was a kind gift from Dr. Alberto Mantovani (Istituto Clinico Humanitas, Milan, Italy). pGL3-IFNA4 luciferase and pGL4-TK-Renilla were a kind gift from Dr. John Hiscott (Lady Davis Institute, Montreal, Canada) and Dr. Kate Fitzgerald (UMASS, Massachusetts, USA), respectively. Plasmids encoding shRNA targeting TRIM21 and scrambled negative control were described previously [16] (link). TLR ligands were purchased from InvivoGen (California, USA). Primary antibodies used were anti-FLAG (Sigma), anti-c-Myc and anti-β-Actin (Abcam), anti-GST (GE Healthcare), anti-Xpress (Invitrogen), anti-IRF5 (Cell Signaling) and anti-α-actinin, anti-HA and anti-TRIM21 (Santa Cruz).

Cells were incubated for 48 h in medium containing selected TLR ligands (all from InvivoGen, San Diego, CA), as follows: TLR3, Poly(inosinic acid):poly(cytidylic acid) (poly (I:C) (PIC)), a synthetic dsRNA (1 μg/ml); TLR4, ultrapure LPS (10 μg/ml); and TLR5, flagellin (1 μg/ml). After incubation, supernatants were collected and analyzed using a DIAplex Human Th1/Th2/Inflammation kit (Diaclone, Gen-Probe). DIAplex Human Th1/Th2/Inflammation is a multiplexed fluorescent bead-based immunoassay for the quantification of multiple human cytokines in serum and culture supernatants by flow cytometry: IFNγ, TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70 and IL-17A.

Rat and mouse HSCs were incubated with TLR ligands (InvivoGen) as detailed in figure legends. The ligands and their concentration in cell culture were (unless otherwise stated) - TLR2 (Lipoteichoic (LTA), 100 ng/ml), TLR3 (Poly (I∶C), 1 µg/ml), TLR4 (lipopolysaccharide (LPS), 100 ng/ml), TLR5 (flagellin, 1 µg/ml), TLR7/8 (Imiquimod, 1 µg/ml) and TLR9 (stimulatory CpG ODN, 10 µg/ml). IL-1α was used at a concentration of 2 ng/ml (Peptroech 211-11A), IFNγ at 100 ng/ml (Peprotech 315-05). Transcriptional inhibitors, actinomycin D and 5,6-Dichlorobenzimidazole Riboside (DRB) were purchased from Sigma Aldrich (A9415 and D1916 respectively). The C13-GT was used at a concentration of 20 µg/gram of body weight. Clodronate-liposomes were a kind gift from Professor Mirco Ponzoni and injected intraperitoneally at a concentration of 25 µg/gram of body weight.