Cells and reagents
The human Mono Mac 6 cell line was obtained from DSMZ. Escherichia coli LPS 0111:B4 was purchased from List Biological Laboratories, and PMA from Sigma-Aldrich. pIFN-stimulated response element (pISRE)-Luc and pAP-1-Luc plasmids were from Clontech Laboratories. pTNF(-528)Luc, pTNF(-387)Luc, and pTNF(-120)Luc were generously provided by Dr. J. Economou (University of California-Los Angeles, Los Angeles, CA). Mouse and human TNF-α were measured by ELISA kits purchased from R&D Systems.
Mono Mac 6 cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 1× nonessential amino acid, and 1× penicillin-streptomycin (Invitrogen). The cells were incubated in 5% CO2
at 37°C and treated with 0, 50, or 100 mM EtOH for up to 6 days in a “master flask” in a chronic EtOH incubator system which has been previously described (12
). Cells were then removed and subcultured in a 24-well plate at a cell density of 1 × 106
/ml/well. For TNF-α
production from Mono Mac 6 cells, the cells were stimulated with 20 ng/ml LPS and 60 ng/ml PMA for various times and supernatants were collected for ELISA.
For primary mouse BM-derived macrophage culture, C57BL/6 and Trif−/− (Triflps2) male mice, 6-8 wk old, were purchased from The Jackson Laboratory and were kept under specific pathogen-free condition. The femurs of mice were removed and purified from the surrounding muscle tissue. The both ends of bones were cut with scissors and the marrow was flushed with the medium by using a 3-ml syringe with a 30G needle. After one wash, ~1 to 1.5 × 107 BM cells were obtained per femurs.
BM cells were grown in DMEM medium (Invitrogen) supplemented with 10% FBS, 1× nonessential amino acids (Invitrogen), 1× penicillin-streptomycin (Invitrogen), and murine GM-CSF (12 ng/ml; R&D Systems). Its density was adjusted to 4 × 106/4 ml/well in 6-well plates. On every 2 days, the culture media and floating cells were discarded; the fresh media with 12 ng/ml murine GM-CSF was added. On day 4, the BM cells were treated with 0 or 50 mM EtOH for another 5 days. On day 9, the cell density was regulated to 1 × 106/ml/well in 24-well plates. After culturing overnight, medium was changed and cells were incubated for additional 6 h with 0 or 100 ng/ml LPS (E. coli 0111:B4; List Biological Laboratories). The culture supernatants were collected and stored at −20°C for late analysis of TNF-α by ELISA. Macrophage preparations were also harvested and stained with 10 μg/ml anti-CD-11b (BD Biosciences). Cells were analyzed on a Becton Dickinson FACSAria flow cytometer. CD11b positivity in macrophage cultures was over 90%.
Real-time PCR analysis for TNF-α
Total RNA was isolated from Mono Mac 6 cells treated with and without EtOH by a single step method using TRIzol reagent as per manufacturer’s instructions. Thereafter, RNA was transcribed to cDNA and real-time PCR was performed according to the TaqMan Two-Step Real-Time PCR Master Mix Reagents Kit protocol supplied by the manufacturer (Applied Biosystems). Human TNF-α primers and probe were obtained from Maxim Biotech. This assay has a correlation coefficient of 0.98 over 6-logs of TNF-α RNA concentration.
TNF-α mRNA half-life assay
A total of 1 μg/ml actinomycin was added to the culture at 0, 30, and 60 min after stimulation with LPS/PMA. Mono Mac 6 cells were collected at each time point and TNF-α mRNA was measured by real-time PCR described previously.
Measurement of nuclear NF-κB p65, p50, FosB, and Egr-1
Mono Mac 6 cells culture in control or EtOH conditions were harvested 1 h after no stimulation or stimulation with LPS/PMA and nuclei were harvested using the TransFactor Extraction Kit (BD Biosciences). Total proteins was determined and equal amounts of nuclear protein were assayed for NF-κB p65, p50, FosB, and Egr-1 using specific Abs to these proteins in a sandwich ELISA (BD TransFactor assay).
Chromatin IP (ChIP) assay for human IRF-3
Mono Mac 6 cells culture in control or EtOH conditions were plated to 24-well plate at a cell density of 2 × 106 of cells/ml/well, and stimulated with LPS and PMA for 1 h as stated above. Then the cells were crosslinked with 1% (v/v) formaldehyde at 37°C for 20 min. Following crosslinking, the cells were resuspended in SDS lysis buffer with protease inhibitors (Upstate Cell Signaling Solutions). Total cell lysate was isolated, and the genomic DNA was sheared to sizes between 200 bp and 1000 bp by sonication on ice. The sample was precleared with Salmon Sperm DNA/protein A-agarose beads for 30 min at 4°C to reduce nonspecific background. ChIP assay was performed with a rabbit polyclonal Ab to human IRF-3 (Santa Cruz Biotechnology) or with normal rabbit IgG (Santa Cruz Biotechnology) as a negative control according to the protocol of Upstate Cell Signaling Solutions. The immunocomplex was heated at 65°C for 4 h to reverse the crosslinking between DNA and proteins. DNA was purified by repeated phenol/chloroform extraction and EtOH precipitation. The purified DNA (designated as bound) was dissolved in 20 μl of TE buffer. The DNA isolated using the same procedure with omission of the IP step was designated as the input DNA. Both bound and the input DNA were analyzed by PCR (35 cycles) with primers that amplify a 310-bp fragment of the human TNF promoter region, circumventing the IRF sequence. The PCR condition was 95°C for 5 min, 95°C for 45 s, 58°C for 45 s, 72°C for 45 s, and 72°C for 10 min. The resulting product of 310 bp in length was separated by 1.6% agarose gel electrophoresis. The primer pairs used for human TNF promoter were: forward 5′-ACTACCGCTTCCTCCAGAT GAG-3′; reverse 5′-TCATGGTGTCCTTTCCAGGG-3′. ChIP assays were also validated by real-time PCR using the same primes and SYBR green and expressed as ΔΔCt in relation to a no template control.
Generation of lentiviral reporter vectors
To generate lentiviral reporters, the firefly luciferase gene was amplified by PCR from pISRE-Luc (BD Clontech) and cloned into pENTR/D-TOPO (Invitrogen). ISRE, AP-1, NF-κ
B, TNF-528, -387, and -120 were then each cloned into pENTR 5′-TOPO. To construct the lentivirus destination vector with luciferase and the appropriate reporter element, a LR recombination reaction was performed between pENTR/D-TOPO luciferase vector, pENTR 5′TOPO reporter vector, and pLenti6/R4R2/V5-DEST (Invitrogen). The resulting plasmids were then cotransfected using CaPO4
with ViraPower Packaging Mix (Invitrogen) in 293T cells. The virus was purified 60-65 h after transfection, concentrated, and quantified by real-time PCR for luciferase. To investigate the putative IRF-3 site within the TNF promoter, nucleotides AAGAAACCGAGACAGAAGG were deleted from the TNF-528 promoter using PCR as described by Higuchi (16
Cell transduction with lentivirus and luciferase assay
Mono Mac 6 cells were transduced by lentivirus with ISRE, AP-1, and TNF-α promoters driving luciferase as a reporter. Cells (5 × 105/ml) mixed with viral supernatants in the presence of 8 μg/ml polybrene in a 5-ml tube were centrifuged at 1800 g for 3 h at 32°C. After overnight culture with viral supernatants, cells were washed and transferred to a 24-well plate and incubated in fresh culture medium. To generate a stable cell line, 3 μg/ml blasticidin (Invitrogen) was added to the cells, and medium was changed every 3 days with blasticidin until drug-resistant clones appeared. BM cells were transduced overnight by lentivirus with ISRE, AP-1 driving luciferase as a reporter on day 3 before treating EtOH, and followed with EtOH and LPS as described above. After stimulation by LPS/PMA (for Mono Mac 6 cells) or LPS (for BM cells) for 6 h, the cells were harvested and ISRE, AP-1, or TNF-α promoters activities were analysis using the Luciferase Reporter Assay System (Promega). The luciferase activities were normalized by total cellular proteins.
BM-derived macrophages profile was determined by flow cytometry. After blocking with 1% BSA in PBS, the macrophages were labeled with a rat anti-mouse CD11b Ab (10 μg/ml) and then fixed with 1% formaldehyde. The CD11b-positive cells in BM-derived macrophages were over 90%.
Experimental model of alcoholic hepatitis
Female 6- to 8-wk-old WT or Trif−/− mice (The Jackson Laboratory) were acclimated to isocaloric to EtOH containing Lieber-DeCarli diet (Bio-Serv) for 2 wk followed by 4 wk of 36% EtOH-derived calories in the EtOH group. After 4 wk, to induce hepatitis, mice were administered 1.0 μg/g weight E. Coli LPS IP. Mice were euthanized at 2 h for serum TNF and liver histology or at 6 h for serum alanine aminotransferase (ALT) levels. All studies were reviewed and approved by the Children’s Hospital of Pittsburgh Animal Research Review Committee.
Measurement of mouse serum TNF-α and ALT levels
TNF-α and ALT level from mouse serum were determined by ELISA (R&D Systems) and ALT kit (Biotron Diagnostics), respectively, according to the manufacturer’s instructions.
Formalin-fixed tissues were paraffin embedded, sectioned, and stained with H&E at Histo-Scientific Research Laboratories. Steatosis was scored on 10 random low powered fields using a scoring system of 0-3 as described by Brunt and colleagues (17
All data are presented as mean ± SEM. Significance was estimated using ANOVA followed by Tukey’s Multiple Comparison Procedure with p < 0.05 being considered significant.