tlr4 enhances tgf- signaling and hepatic fibrosis ... mice and fibrosis induction ccl4...
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TLR4 Enhances TGF-! Signaling and Hepatic Fibrosis
Ekihiro Seki1,2, Samuele De Minicis1,2, Christoph H. Österreicher1,2, Johannes Kluwe1,
Yosuke Osawa1, David A. Brenner2, and Robert F. Schwabe1
Mice and Fibrosis induction
CCl4 (Sigma-Aldrich, diluted 1:3 in corn oil) or vehicle (corn-oil) was administered by
gavage at a dose of 1 µl/g body weight every five days for a total of eight injections.
TAA-induced liver fibrosis was induced by administering TAA (Sigma-Aldrich) at a dose
of 300 mg/L in drinking water for 20 weeks. Some mice were infected with adenoviruses
expressing dnBAMBI or empty Adshuttle control virus (1x109 pfu/ml in PBS i.v./mouse)
48 hours before BDL. Some mice received liposomal clodronate (200 µl i.v.) one day
before BDL to deplete KCs. Liposomal clodronate was prepared as described 1.
Six hours after isolation, quiescent HSCs were treated with E.coli 055:B5 LPS (Sigma) at
a concentration of 100 ng/ml or vehicle (endotoxin-free H2O) for six hours (n=3 HSC
isolations). RNA was extracted by a combination of Trizol (Invitrogen) and RNeasy
columns (Qiagen). Microarray analysis was performed using mouse genome 430 2.0
array gene chips (Affymetrix) according to the manufacturer’s instructions. Briefly, first
strand and second strand synthesis were performed using the one-cycle cDNA synthesis
kit (Affymetrix) followed by cleanup of double-stranded cDNA using cDNA spin columns
(Affymetrix). Biotin-labeled cRNA was generated using one cycle IVT labeling kit
followed by cRNA cleanup using cRNA spin columns (both Affymetrix). cRNA
fragmentation which was confirmed by agarose gel electrophoresis. After hybridization to
gene chip 430, scanning with a genechip Scanner 3000 7G (Affymetrix) data analysis
were performed using Genespring GX 7.3 (Agilent). Analysis was performed as paired
analysis comparing untreated and LPS-treated HSCs from the same isolation. Genes
that displayed a significant (p
Reverse transcription PCR and real time quantitative PCR
RNA was isolated from mouse HSCs by the RNeasy kit, DNAse treated and was reverse
transcribed. PCR for BAMBI was performed using primers 5’-atggatcgccactccagctac-3’
and 5’-tcatatgaattccagctttccatgac-3’ at 95°C (45s), 60°C (45s) and 72°C (90s) for 33
cycles. Actin PCR was performed using 5’-GACGATATCGCTGCGCTG-3’ and 5’-
GTACGACCAGAGGCATACAGG-3’ for 27 cycles. Real time quantitative PCR was
performed for 40 cycles of 15 seconds at 95°C and 60 seconds at 60° C using an ABI
7000 sequence detection system (Applied Biosystems) and primer-probe sets from ABI.
Quantification was performed by comparing the Ct values of each sample to a standard
curve and normalization to 18s or !-actin. Values are expressed as fold induction in
comparison to untreated or sham controls.
Adenoviral construction and purification
Adenoviruses expressing I"Bsr, GFP, beta-galactosidase and NF-"B-driven luciferase
have been described previously and were amplified as previously described 2,3. Full-
length BAMBI (cloned from murine HSC cDNA library by RT-PCR), N-terminally deleted
dominant negative BAMBI 4 or BAMBI promoter-driven luciferase were constructed
using the AdEasy system (ATCC). Briefly, dominant negative BAMBI 4 was excised from
pcDNA 3.0 using KPNI and SmaI and ligated into CMV-Track and CMV-Shuttle using
KPNI and EcoRV. Full length BAMBI was ligated into CMV-Track and CMV shuttle using
HindIII and EcoRV. The human BAMBI promoter (-3382 to +82) 5,6 and luciferase were
excised from PGL3 basic by KPNI and NcoI, and NcoI and SalI, respectively, and ligated
into Shuttle plasmid using KPNI and SalI. Linearization, recombination and adenoviral
expansion were performed as previously described 3.
NF-"B, Smad and BAMBI reporter assays
For NF-"B reporter assays, HSCs (4x105 cells/well) were co-infected with AdNF"Bluc 3
and CMV-promoter driven Ad5LacZ 3 at at a multiplicity of infection (moi) of 100
particles/cell 12 hours after isolation for 12 hours followed by stimulation with various
concentrations of LPS for six hours. For TGF! reporter assays, HSCs were co-infected
with AdCAGA-Luc 7 at an moi of 100 and Ad5Shuttle,AdGFP, AddnBAMBI or AdBAMBI
12 hours after isolation using an moi of 300. 24 hours after isolation, cells were
pretreated with LPS (100 ng/ml) for 16 hours followed by treatment with recombinant
TGF!1 (R&D systems) at 300 pg/ml for six hours. For BAMBI reporter assays, HSCs
were co-infected with AdBAMBI-Luc and Ad5LacZ at an moi of 100 12 hours after
isolation. 18 hours after isolation, cells were treated with LPS (100 ng/ml) for 24 hours.
Luciferase activity was measured in a multiwell platereader (Fluostar Optima) and
normalized to !-galactosidase as described 3.
Measurement of hepatic collagen and hyaluronan content
Hydroxyproline content was measured as previously described 8. Hepatic collagen
content was analyzed by Sirius red staining of paraffin-embedded sections 8. The Sirius
red positive area was measured in at least six low power (40x) fields on each slide and
quantified using NIH imaging software. To determine the hepatic hyaluronan content,
liver was homogenized in 10mM Tris-Hcl (pH 7.4), 1mM EDTA, 150mM NaCl. Serum
and hepatic hyaluronan content were measured by ELISA (Echelon Biosciences Inc) as
suggested by the manufacturer.
Cell migration and cell adhesion assay
Cell migration assay was analyzed by the QCM Chemotaxis Cell Migration Assay (5 µm
pore size, Chemicon International). The lower chamber was filled with conditioned
medium from HSCs stimulated with LPS (100 ng/ml) or vehicle for 48 hours. KCs from
either TLR4-wild type or TLR4-mutant mice were placed into the upper chamber (105
cells/well) and incubated at 37°C for eight hours. Adhesion of KCs to HSCs was
determined cell adhesion assay as previously described 9. Briefly, HSCs were isolated
from TLR4 wild-type mice and incubated with LPS (100 ng/ml) or vehicle for 24 hours.
KCs were isolated from either TLR4-wild type or TLR4-mutant mice and labeled by
CellTracker Green CMFDA (Molecular Probes) and placed on HSCs for one hour. HSCs
were then gently washed and adherent KCs were counted in ten randomly chosen high-
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7. Dooley, S. et al. Smad7 prevents activation of hepatic stellate cells and liver
fibrosis in rats. Gastroenterology 125, 178-91 (2003).
8. Bataller, R. et al. NADPH oxidase signal transduces angiotensin II in hepatic
stellate cells and is critical in hepatic fibrosis. J Clin Invest 112, 1383-94 (2003).
9. Hellerbrand, Wang, S.C., Tsukamoto, H., Brenner, D.A. & Rippe, R.A.
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Hepatology 24, 670-6 (1996).
0 1 2 0 1 2
Vehicle TAA Vehicle
0 1 2 0 1 2
Oil CCl4 Oil
c d e f sham day5 day21
0 Tlr4+ Tlr4mut