Toll-like receptor 7-mediated type I interferon signaling prevents cholestasis- and hepatotoxin-induced liver fibrosis

Abstract

Toll-like receptor 7 (TLR7) signaling predominantly regulates production of type I interferons (IFNs), which has been suggested in clinical studies to be antifibrotic. However, the mechanistic role of the TLR7-type I IFN axis in liver fibrosis has not been elucidated. In the present study, liver fibrosis was induced in wild-type (WT), TLR7-deficient, and IFN-α/β receptor-1 (IFNAR1)-deficient mice and TLR7-mediated signaling was assessed in liver cells isolated from these mice. TLR7-deficient and IFNAR1-deficient mice were more susceptible to liver fibrosis than WT mice, indicating that TLR7-type I IFN signaling exerts a protective effect against liver fibrosis. Notably, the hepatic expression of interleukin-1 receptor antagonist (IL-1ra) was suppressed in TLR7- or IFNAR1-deficient mice compared with respective WT mice, and treatment with recombinant IL-1ra reduced liver fibrosis. In vivo activation of TLR7 significantly increased IFNa4 and IL-1ra expression in the liver. Interestingly, each cytokine had a different cellular source, showing that dendritic cells (DCs) are the responsible cell type for production of type I IFN, while Kupffer cells (KCs) mainly produce IL-1ra in response to type I IFN. Furthermore, TLR7 activation by R848 injection suppressed liver fibrosis and production of proinflammatory cytokines, and these effects were dependent on type I IFN signaling. Consistent with in vivo data, IFN-α significantly induced IL-1ra production in primary KCs.

Conclusion: TLR7 signaling activates DCs to produce type I IFN, which in turn induces antifibrogenic IL-1ra production in KCs. Thus, manipulation of the TLR7-type I IFN-IL-1ra axis may be a new therapeutic strategy for the treatment of liver fibrosis.

Fig. 1. TLR7-deficient mice are prone to BDL-induced liver fibrosis and inflammation

(A–F) TLR7-deficient and respective WT (Balb/c) mice underwent sham operation (n = 3 per group) or BDL (n = 8 per group). After 21 days, mice were evaluated as follows: Fibrillar collagen deposition was determined by Sirius red staining (A) and quantification of the Sirius red–positive area (C). (B) The expression of α-SMA was determined by immunohistochemistry. (D) Hydroxyproline levels were determined. (E) Expression of pro-fibrogenic markers was determined by qRT-PCR and shown as fold change compared with WT sham-operated mice. (F) Hepatic inflammatory cytokines were quantified by ELISA. The results are expressed as ng of each cytokine per mg of liver protein. Data are presented as means ± SEM per group. Two-tailed Student’s t-test, *P < 0.05, **P < 0.01. Original magnification, ×100 (Sirius red), ×200 (α-SMA).

Fig. 2. TLR7-deficient mice are susceptible to CCl₄-induced chronic liver fibrosis and inflammation

(A–F) TLR7-deficient and respective WT (Balb/c) mice were treated with corn oil (n = 3 per group) or with 36 injections of CCl₄ (2 μl/g body weight, 10% of CCl₄, three times per week; n = 10 per group). Fibrillar collagen deposition was determined by Sirius red staining (A) and quantification of the Sirius red-positive area (C). (B) The expression of α-SMA was determined by immunohistochemistry. (D) Determination of hydroxyproline levels. (E) Expression of pro-fibrogenic markers determined by qRT-PCR shown as fold change compared with WT corn oil-treated mice. (F) Quantification of hepatic inflammatory cytokines by ELISA. The results are expressed as ng of each cytokine per mg of liver protein. Data are presented as means ± SEM per group. Two-tailed Student’s t-test, *P < 0.05, **P < 0.01. N.S., not significant. Original magnification, ×100 (Sirius red, BDL groups of α-SMA), ×200 (α-SMA).

Fig. 3. TLR7 deficiency decreases the induction of type I IFN signaling in DCs

(A) mRNA levels of TLR7 was determined by qRT-PCR in isolated liver cell fractions representing HPs, KCs, DCs, and HSCs in WT. (B) mRNA of IFNa4 was determined by qRT-PCR in the WT liver at 30 min after injection of TLR7 ligand (R848). Depletion of DCs was achieved by diphtheria toxin injections (4 ng/g of BW) to CD11c-DTR mice for two consecutive days, and depletion of KCs was done by clodronate liposome injections (200 μl/mouse) for two consecutive days. (C) mRNA levels of IFNa4 and Pin1 were determined by qRT-PCR in isolated DC fraction. The results are shown as fold change compared with normal cells from WT mice. Values presented are from three independent experiments and each sample was assayed in duplicate. (D) Hepatic IFN-α and Pin1 were quantified by Luminex and ELISA, respectively. The results were expressed as ng of IFN-α per mg of liver protein. Data are presented as means ± SEM per group. Different letters indicate significant differences (p < 0.01) according to ANOVA and DMRT (B). Two-tailed Student’s t-test, *P < 0.05, **P < 0.01 (C–D).

Fig. 4. BDL-induced fibrosis is exacerbated in IFNAR1-deficient mice compared with WT mice

(A–F) IFNAR1-deficient and respective WT (C57BL/6) mice underwent a sham operation (n = 3 per group) or BDL (n = 6 per group). After 21 days, mice were evaluated as follows: (A) Fibrillar collagen deposition was determined by Sirius red staining and (C) quantification of the Sirius red–positive area. (B) The expression of α-SMA was determined by immunohistochemistry. (D) Hydroxyproline levels were determined. (E) Expression of pro-fibrogenic markers was determined by qRT-PCR and shown as fold change compared with WT sham-operated mice. (F) Hepatic inflammatory cytokines were quantified by ELISA. The results are expressed as ng of each cytokine per mg of liver protein. Data are presented as means ± SEM per group. Two-tailed Student’s t-test, *P < 0.05, **P < 0.01. Original magnification, ×100 (Sirius red), ×200 (α-SMA).

Fig. 5. TLR7- and IFNAR1-deficiency reduce IL-1ra production in KCs

(A) Hepatic IL-1ra in BDL and CCl₄ murine models were quantified by ELISA. The results are expressed as ng of cytokine per mg of liver protein. (B) mRNA of IL-1ra was determined by qRT-PCR in the WT liver at 2 h after injection of TLR7 ligand (R848). Depletion of DCs was achieved by diphtheria toxin injection (4 ng/g) to CD11c-DTR mice for two consecutive days, and depletion of KCs was done by clodronate liposome injection (200μl/mouse) for two consecutive days. (C) mRNA levels of IL-1ra were determined by qRT-PCR in normal KCs, and in vivo activated (21 days post-BDL) KCs from WT and TLR7-deficient mice. (D) mRNA levels of IL-1ra were determined by qRT-PCR in normal KCs, and in vivo activated (21 days post-BDL) KCs from WT and IFNAR1-deficient mice. Values presented are from three independent experiments and each sample was assayed in duplicate. The results are shown as fold change compared with normal cells from WT mice. (E) KCs isolated from WT mice were stimulated with recombinant murine IFNa (500IU/ml), and mRNA and protein levels of IL-1ra were measured with qRT-PCR and ELISA, respectively. Data are presented as means ± SEM per group. Two-tailed Student’s t-test, *P < 0.05, **P < 0.01 (A, C, D). Different letters indicate significant differences (B, p < 0.01; E, p < 0.05) according to ANOVA and DMRT.

Fig. 6. Administration of recombinant IL-1ra ameliorates the liver fibrosis induced by TLR7 or IFNAR1 deficiency

(A–F) Recombinant IL-1ra (Anakinra®) or saline-injected TLR7 and IFNAR1-deficient mice versus respective WT mice, underwent BDL (n = 8 per group). After 21 days, mice were evaluated as follows: (A and D) Fibrillar collagen deposition was determined by quantification of the Sirius red-positive area. (B and E) Hydroxyproline levels were determined. (C and F) Expression of pro-fibrogenic markers was determined by qRT-PCR and shown as fold change compared with WT sham-operated mice. Data are presented as means ± SEM per group. Two-tailed Student’s t-test, *P < 0.05, **P < 0.01. N.S., not significant.

Fig. 7. R848-induced protection of liver fibrosis is dependent on type I IFN signaling

(A–E) R848 or Saline-injected WT and IFNAR1-deficient mice underwent BDL (n = 12–14 per group). After 21 days, mice were evaluated as follows: (A) Fibrillar collagen deposition was determined by Sirius red staining and (C) quantification of the Sirius red–positive area. (B) The expression of α-SMA was determined by immunohistochemistry. (C) Hydroxyproline levels were determined. (D) Expression of pro-fibrogenic markers was determined by qRT-PCR and shown as fold change compared with saline-treated mice. (E) Hepatic IL-1ra was quantified by ELISA and expressed as ng of IL-1ra protein per mg of liver protein. (F) Gene expression was determined by qRT-PCR analysis of KCs isolated from WT or IFNAR1 KO mice and stimulated for 6 h by conditioned media from vehicle- or R848-treated WT NPCs. Values are presented from three independent experiments and each sample was assayed in duplicate. Data are presented as means ± SEM per group. Two-tailed Student’s t-test, *P < 0.05, **P < 0.01. Original magnification, ×100 (Sirius red), ×200 (α-SMA).