Hepatic stellate cells are responsive to stimulation by TLR ligands.

<p>(A) mRNA level of IL-6 was quantified by qRT-PCR in four separate preparations of activated rat HSCs (culture day 10) treated with TLR2 ligand (LTA, 100 ng/ml), TLR3 ligand (Poly(I∶C), 1 µg/ml), TLR4 ligand (LPS, 100 ng/ml) or TLR5 ligand (Flagellin, 1 µg/ml) for up to 24 hours (n = 4). Expression level was normalised to β actin. (B) Secreted IL-6 protein was measured by ELISA in conditioned media collected from activated HSCs treated with TLR ligands as in (A) following 2 h, 8 h or 24 h of stimulation (n = 4). (C) mRNA levels of IL-6, TIMP1, αSMA and collagen I were quantified by qRT-PCR in four separate preparations of activated rat HSCs (culture day 10) treated with TLR3 ligand (Poly(I∶C), 1 µg/ml) for up to 24 hours. Expression level was normalised to β actin. (#p<0.1, *p<0.05, **p<0.01***p<0.001).</p

Table of primers.

<p>Table of primers.</p

Interferon gamma and IL-6 expression are TLR3 mediated and loss of ability to induce IFNγ in aHSCs is associated with transcriptional repression by Polycomb complex.

<p>(A) WT and <i>tlr3</i>^−/− activated mHSCs were treated with Poly(I∶C) (1 µg/ml) for 8 and 24 hours; subsequently IFNγ mRNA expression was measured by qPCR. Data are normalised to GAPDH and expressed as fold induction relative to control. (B) IL-6 mRNA expression was measured in WT and <i>tlr3</i>^−/− activated mHSCs in response to increasing concentrations of Poly(I∶C). (C) Wild type qHSCs were treated with transcriptional inhibitors Act D (1 µM) and DRB (80 µM) for 24 hours and IL-6 mRNA expression analysed by qRT-PCR. Data are normalised to GAPDH and expressed as fold induction relative to untreated control. (D and E) One hundred micrograms of crosslinked chromatin from quiescent and activated rat HSC was incubated with 10 µg of anti-trimethyl and anti-dimethyl H3K27 antibody and ChIP assay performed. Data are expressed as fold enrichment relative to IgG control. (*p<0.05, **p<0.01, ***p<0.001).</p

Activation of HSC decreases TLR3 mediated interferon and cytokine response.

<p>(A,B,C) HSCs were isolated from control, acute CCl₄ treated rats (single injection), or chronic CCl₄ treated rats (4 weeks twice weekly injections); cells were seeded onto plates and treated with Poly(I∶C) (1 µg/ml) for up to 24 hours. Interferon α, β and γ were measured and normalised to β actin. Data are expressed as RLTD. (D) Interferon-inducible cytokines CXCL10, TNF-α, MxA, CXCL1/KC, IL-1β and indoleamine 2,3-dioxygenase were measured and normalised to β actin (n = 4). Data are presented as fold change, Poly(I∶C) stimulated to unstimulated. Secreted IFNγ was measured by ELISA in the media of cultured HSCs isolated from rats treated with either chlodronate-liposomes (E) or empty liposome control (F). (G) Expression of MHC class II on macrophages cultured in qHSC conditioned media. Flow cytometric analysis of control macrophages (untreated), or macrophages incubated with control media, untreated qHSC conditioned media, Poly(I∶C) treated qHSC conditioned media or 100 ng/ml recombinant IFNγ as positive control. (*p<0.05, **p<0.01, ***p<0.001).</p

Quiescent and activated HSCs express Toll like receptors.

<p>(A) mRNA levels of TLR1-13 were quantified by qRT-PCR in three separate preparations of primary rat qHSCs (day 0) and day 10 transdifferentiated myofibroblasts. Data are expressed as relative level of transcriptional difference (RLTD) to TLR1 mRNA expression (n = 3). (B) Thirty micrograms of whole cell protein extract from three separate preparations of quiescent rat HSCs (culture day 1) or activated myofibroblasts (culture day 10) were separated by SDS-PAGE and immunoblotted for TLR3 and GAPDH. (C) Toll-like receptor 3 was visualised in the cytoplasm of rat qHSCs (<i>ex vivo</i>) culture day 1 (bar represents 75 µm). (D) Quiescent rat HSCs were treated with Poly(I∶C) (1 µg/ml) or IL-1α (2 ng/ml) for up to 24 hours; IL-6 mRNA was measured and normalised to β actin (n = 3). (E) Thirty micrograms of whole cell protein extract from two separate preparations of quiescent HSCs or activated myofibroblasts (culture day 10) were separated by SDS-PAGE and immunoblotted for IRAK1, TRAF6 and GAPDH. (F) Activated rat myofibroblasts were treated with IL-1α (2 ng/ml) for up to 24 hours; IL-6 and TIMP1 mRNA were measured and normalised to β actin (n = 3). (*p<0.05, ***p<0.001).</p