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Abstract
International Journal of Molecular Sciences Article Interleukin-1 Receptor Antagonist Modulates Liver Inflammation and Fibrosis in Mice in a Model-Dependent Manner 1 , 2 , 1 1 1 Raphael P. H. Meier * , Jeremy Meyer , Elisa Montanari , Stephanie Lacotte , 1 2 3 3 , 4 Alexandre Balaphas , Yannick D. Muller , Sophie Clément , Francesco Negro , 1 1 1 Christian Toso , Philippe Morel and Leo H. Buhler Visceral and Transplant Surgery, Department of Surgery, Geneva University Hospitals and Medical School, 1211 Geneva, Switzerland; [email protected] (J.M.); [email protected] (E.M.); [email protected] (S.L.); [email protected] (A.B.); [email protected] (C.T.); [email protected] (P.M.); [email protected] (L.H.B.) Transplant Surgery, University of California San Francisco, San Francisco, CA 94143, USA; [email protected] Division of Clinical Pathology, Geneva University Hospitals and Medical School, 1211 Geneva, Switzerland; [email protected] (S.C.); [email protected] (F.N.) Division of Gastroenterology and Hepatology, Geneva University Hospitals and Medical School, 1211 Geneva, Switzerland * Correspondence: [email protected]; Tel.: +41-22-372-33-11; Fax: +41-22-379-51-86 Received: 22 January 2019; Accepted: 6 March 2019; Published: 14 March 2019 Abstract: Background: Interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra) have been proposed as important mediators during chronic liver diseases. We aimed to determine whether the modulation of IL-1 signaling with IL-1Ra impacts on liver fibrosis. Methods: We assessed the effects of IL-1 on human hepatic stellate cells (HSC) and in mouse models of liver fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride treatment (CCl-4). Results: Human HSCs treated with IL-1 had increased IL-1 , IL-1Ra, and MMP-9 expressions in vitro. HSCs treated with IL-1 had reduced -smooth muscle actin expression. These effects were all prevented by IL-1Ra treatment. In the BDL model, liver fibrosis and Kuppfer cell numbers were increased in IL-1Ra KO mice compared to wild type mice and wild type mice treated with IL-1Ra. In contrast, after CCl-4 treatment, fibrosis, HSC and Kupffer cell numbers were decreased in IL-1Ra KO mice compared to the other groups. IL-1Ra treatment provided a modest protective effect in the BDL model and was pro-fibrotic in the CCl-4 model. Conclusions: We demonstrated bivalent effects of IL-1Ra during liver fibrosis in mice. IL-1Ra was detrimental in the CCl-4 model, whereas it was protective in the BDL model. Altogether these data suggest that blocking IL-1-mediated inflammation may be beneficial only in selective liver fibrotic disease. Keywords: liver fibrosis; interleukin-1 receptor antagonist; interleukin-1; bile duct ligation; carbon tetracholoride; insulin 1. Introduction Interleukin-1 (IL-1) is a pro-inflammatory cytokine playing a key role in acute and chronic inflammation [1–4]. IL-1 refers to two similar cytokines which bind to the same receptor, one being mostly secreted (IL-1 ) and the other remaining intracellular (IL-1 ) [2]. The inflammasome promotes the maturation of IL-1 in response to infectious or non-infectious agents. IL-1 is produced by stimulated monocytes, macrophages and to a lesser extent by other cell types, such as neutrophils, Int. J. Mol. Sci. 2019, 20, 1295; doi:10.3390/ijms20061295 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 1295 2 of 18 epithelial and endothelial cells, smooth muscle cells and fibroblasts [5]. IL-1 activity is regulated by IL-1 receptor antagonist (IL-1Ra), a naturally anti-inflammatory cytokine that binds to type 1 IL-1 receptors and that is produced by activated myeloid cells, hepatocytes, fibroblasts [2] and or mesenchymal stem cells (MSCs) [6,7]. IL-1 has been proposed as an important mediator of inflammation and tissue damage in chronic liver disease [8]. Indeed, elevated circulating levels of both IL-1 and IL-1Ra are detected in the serum of patients with chronic liver diseases, including alcoholic liver disease [9], chronic hepatitis B and C, and primary biliary cirrhosis [10–12]. More recently, IL-1 was shown to be implicated in the transformation of steatosis to steatohepatitis and liver fibrosis [13,14]. However, determining whether IL-1 is a contributor or a consequence to liver fibrosis pathogenesis demands further investigation. In this context, two functional polymorphisms in the IL-1 gene cluster were identified as related to development of acute-on-chronic liver failure [15]. Blocking IL-1 pathway during liver injury using IL-1Ra represents an interesting strategy to determine its role in liver diseases. We previously observed that IL-1Ra knockout (KO) mice had delayed liver regeneration after partial hepatectomy [16], which was consistent with observations from others [17,18]. More recently, we demonstrated that 23% to 36% of MSC subpopulations release IL-1Ra in vitro and in vivo, and that MSC-secreted cytokines show an anti-fibrotic effect in mice [19]. This MSC-IL-1Ra-mediated hepatoprotective effect was further confirmed by others in acute liver injury models [20,21]. The important role of IL-1 signaling in the progression from chronic liver injury to fibrosis was previously characterized in mice [8] and rats [22]. However, the mechanisms involving IL-1Ra and its potential protecting effect in hepatic fibrogenesis remain to be elucidated. In the present study, we aimed to investigate whether IL-1Ra is regulating, along with IL-1 , the progression of chronic liver injury to fibrosis. In vitro, we showed that IL-1 upregulated itself and IL-1Ra in human stellate cells (HSC); IL-1Ra prevented these effects. In vivo, we observed that IL-1 and IL-1Ra were strongly upregulated in mice liver after bile duct ligation (BDL) or carbon tetrachloride treatment (CCl-4) and further showed that IL-1Ra KO mice displayed increased fibrosis in the BDL model and reduced fibrosis in the CCl-4 model. We thus highlighted a dual effect of IL-1Ra that was dependent on the type of liver injury. 2. Results 2.1. Interleukin-1 Receptor Antagonist and Liver Fibrosis 2.1.1. IL-1 Reduce Stellate Cell Activation In Vitro -smooth muscle actin ( -SMA) expression by activated HSCs is a key feature of liver fibrosis [23]. Therefore, we investigated the effect of IL-1 on -SMA mRNA and protein expressions in human HSCs in vitro, using immunoblotting and RT-PCR. Treatment of HSCs with IL-1 modestly decreased -SMA protein expression levels in primary HSCs when compared to control (Figure 1A,B). This effect was prevented when IL-1Ra was added. Transforming growth factor- 1 (TGF- 1) (used as a positive control) induced increased -SMA mRNA expression under these conditions (Figure 1C). Of note, IL-1Ra was not able to reverse the effect of TGF- 1 (Supplementary Figure S1), suggesting that the IL-1Ra effect is specific to IL-1 induction. We then sought to analyze whether IL-1 and IL-1Ra are regulated in HSCs, the key cell in the transition from inflammation to fibrosis [24]. IL-1 and IL-1Ra mRNA expressions were significantly upregulated in HSCs after exogenous IL-1 treatment (by 3.2- and 1.4-fold, respectively) (Figure 1D,E). Treatment with IL-1Ra prevented IL-1 effects and further reduced IL-1 and IL-1Ra expressions below basal levels. No effect on IL-1 and IL-1Ra expression was observed following HSC treatment with TGF- (Figure 1D,E). Matrix metalloproteinases expression was previously demonstrated to be regulated by IL-1 in HSCs [25,26]. Therefore, we analyzed whether this regulatory effect persisted when using IL-1 . MMP-9 mRNA expression level was upregulated in IL-1 -treated human HSCs (by 2.1-fold) (Figure 1F). This effect was prevented when cells were treated with IL-1Ra. Again, TGF- had no effect. MMP-2 and collagen type I expression was not significantly modified following IL-1 and L-1Ra treatment (Figure 1G,H). Overall, these results Int. J. Mol. Sci. 2019, 20, 1295 3 of 18 indicated that IL-1 reduces stellate cell activation and enhances IL-1 , IL-1Ra and MMP-9 production Int. J. Mol. Sci. 2019, 20, x 3 of 18 in vitro. FIGURE 1 Untreated HSCs HSCs treated with IL-1β HSCs treated with IL-1β and IL-1Ra AB +IL-1β NS Control +IL-1β +IL-1Ra NS α-SMA (47 kDa) 1.2 GAPDH (37 kDa) 1.0 0.8 0.6 Untreated HSCs HSCs treated with IL-1β 0.4 HSCs treated with IL-1β and IL-1Ra HSCs treated with TGF-β 0.2 0.0 NS 2.0 NS NS 1.5 NS NS 1.0 0.5 0.0 FIGURE 1 (continued) ** NS ** NS NS NS DE 4 NS 2.0 NS * * * * 1.5 3 Untreated HSCs HSCs treated with IL-1β HSCs treated with IL-1β and IL-1Ra HSCs treated with TGF-β 2 1.0 1 0.5 0 0.0 NS NS NS ** NS NS NS 3.5 2.0 NS NS G H 2.5 NS NS ** NS NS 3.0 NS NS 2.0 NS NS 1.5 2.5 1.5 2.0 1.0 1.5 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0.0 Figure 1. IL-1 reduces human stellate cell activation and increases the expression of IL-1 , IL-1Ra and matrix metalloproteinase (MMP) 9 in vitro. (A) Total protein extracts from human untreated HSCs MMP-9 mRNA IL-1B mRNA (fold change) (fold change) aSMA mRNA (fold change) MMP-2 mRNA IL-1Ra mRNA (fold change) (fold change) aSMA/GAPDH (fold change) Coll1a mRNA (fold change) Int. J. Mol. Sci. 2019, 20, x 4 of 18 Int. J. Mol. Sci. 2019, 20, 1295 4 of 18 Figure 1. IL-1β reduces human stellate cell activation and increases the expression of IL-1β, IL-1Ra and matrix metalloproteinase (MMP) 9 in vitro. (A) Total protein extracts from human untreated HSCs (control), HSCs treated with IL-1β, and HSCs treated with IL-1β and IL-1Ra simultaneously were obtained. Protein extracts were subjected to SDS-PAGE, transferred to nitrocellulose and blotted (control), HSCs treated with IL-1 , and HSCs treated with IL-1 and IL-1Ra simultaneously were with anti-α-SMA and GAPDH. (B) α-SMA signals were quantified by densitometry and normalized obtained. Protein extracts were subjected to SDS-PAGE, transferred to nitrocellulose and blotted using GAPDH signals as a loading control. α-SMA (C), IL-1β (D), IL-1Ra (E), MMP-2 (F), MMP-9 (G), with anti- -SMA and GAPDH. (B) -SMA signals were quantified by densitometry and normalized and collagen type 1 (H) mRNA expression levels were measured by RT-PCR. Data are mean values using GAPDH signals as a loading control. -SMA (C), IL-1 (D), IL-1Ra (E), MMP-2 (F), MMP-9 (G), obtained from four independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, comparing two groups and collagen type 1 (H) mRNA expression levels were measured by RT-PCR. Data are mean values as indicated. obtained from four independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, comparing two groups as indicated. 2.1.2. IL-1β and IL-1Ra Expression Levels are Upregulated in Mice after BDL or CCl-4-Induced 2.1.2. IL-1 and IL-1Ra Expression Levels are Upregulated in Mice after BDL or CCl-4-Induced Liver Fibrosis Liver Fibrosis We analyzed liver IL-1β and IL-1Ra mRNA expression levels using RT-PCR, in both BDL- and We analyzed liver IL-1 and IL-1Ra mRNA expression levels using RT-PCR, in both BDL- and CCl-4-induced liver fibrosis models. In both models, liver IL-1β mRNA expression levels were CCl-4-induced liver fibrosis models. In both models, liver IL-1 mRNA expression levels were significantly upregulated in all mice groups following liver fibrosis induction (Figure 2A,B). IL-1β significantly upregulated in all mice groups following liver fibrosis induction (Figure 2A,B). IL-1 expression was the highest in either WT mice (BDL model) or in IL-1Ra KO mice (CCl-4 model). IL- expression was the highest in either WT mice (BDL model) or in IL-1Ra KO mice (CCl-4 model). IL-1 1β expression was the lowest in IL-1Ra treated mice in both models. As expected, IL-1Ra expression expression was the lowest in IL-1Ra treated mice in both models. As expected, IL-1Ra expression was was absent in IL-1Ra KO mice in both fibrosis models (Figure 2C,D). In line with gene expression absent in IL-1Ra KO mice in both fibrosis models (Figure 2C,D). In line with gene expression findings, findings, corresponding elevations of serum IL-1β and IL-1Ra were found in the BDL model (Figure corresponding elevations of serum IL-1 and IL-1Ra were found in the BDL model (Figure 2E,F) (not 2E,F) (not done in the CCl-4 model). Comparison between WT and KO baseline IL-1β and IL-1Ra done in the CCl-4 model). Comparison between WT and KO baseline IL-1 and IL-1Ra mRNA levels mRNA levels in sham animals without fibrosis induction is provided in Supplementary Figure S3. in sham animals without fibrosis induction is provided in Supplementary Figure S3. Overall, these Overall, these results showed that IL-1β and IL-1Ra are upregulated during BDL-or CCl-4-induced results showed that IL-1 and IL-1Ra are upregulated during BDL-or CCl-4-induced liver fibrosis. liver fibrosis. A C ** *** ** BDL BDL ** *** ** 0 0 CCl CCl 4 4 Sham WT IL-1Ra KO WT, IL-1Ra treated Figure 2. Cont. IL-1B mRNA IL-1B mRNA (Fold change) (Fold change) IL-1Ra mRNA IL-1Ra mRNA (Fold change) (Fold change) Int. J. Mol. Sci. 2019, 20, x 5 of 18 Int. J. Mol. Sci. 2019, 20, 1295 5 of 18 E F *** *** BDL BDL Figure 2. IL-1 and IL-1Ra expression levels are upregulated in mice after bile duct ligation- or carbon tetrachloride-induced liver fibrosis. IL-1 (A,B) and IL-1Ra (C,D) liver mRNA levels were measured Figure 2. IL-1β and IL-1Ra expression levels are upregulated in mice after bile duct ligation- or carbon by RT-PCR in control mice and mice following 2–4 weeks BDL or 6 weeks CCl-4 treatment. BDL and tetrachloride-induced liver fibrosis. IL-1β (A,B) and IL-1Ra (C,D) liver mRNA levels were measured CCl-4 groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 and n = 4) and by RT-PCR in control mice and mice following 2–4 weeks BDL or 6 weeks CCl-4 treatment. BDL and IL-1Ra treated mice (50 mg/kg/day) (n = 10 and n = 13). Sham treated mice were used as control for CCl-4 groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 and n = 4) and IL- the BDL and CCl-4 groups (n = 8 and n = 4). Serum levels of IL-1 (E) and IL-1Ra (F) were measured 1Ra treated mice (50 mg/kg/day) (n = 10 and n = 13). Sham treated mice were used as control for the by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, comparing two groups as indicated. BDL and CCl-4 groups (n = 8 and n = 4). Serum levels of IL-1β (E) and IL-1Ra (F) were measured by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, comparing two groups as indicated. 2.1.3. IL-1Ra Has Bivalent Effects on Liver Fibrosis in BDL and CCl-4-Induced Liver Fibrosis To investigate the contributing role of IL-1Ra in hepatic fibrogenesis, we quantified liver fibrosis 2.1.3. IL-1Ra has Bivalent Effects on Liver Fibrosis in BDL and CCl-4-Induced Liver Fibrosis in WT, IL-1Ra KO and IL-1Ra treated mice over time after BDL or CCl-4 injection. Quantification of To investigate the contributing role of IL-1Ra in hepatic fibrogenesis, we quantified liver fibrosis collagen on sirius red stained sections showed that hepatic fibrosis was significantly increased in IL-1Ra in WT, IL-1Ra KO and IL-1Ra treated mice over time after BDL or CCl-4 injection. Quantification of KO mice following BDL and decreased in IL-1Ra KO mice following CCl-4 injections (Figure 3A–D). collagen on sirius red stained sections showed that hepatic fibrosis was significantly increased in IL- IL-1Ra treatment in WT mice caused decreased fibrosis in the BDL model and increased fibrosis in the 1Ra KO mice following BDL and decreased in IL-1Ra KO mice following CCl-4 injections (Figure 3A– CCl-4 model. Of note, IL-1Ra KO mice were rescued following IL-1Ra treatment (Supplementary Figure D). IL-1Ra treatment in WT mice caused decreased fibrosis in the BDL model and increased fibrosis S2). Quantification of liver mRNA expression of Collagen type 1 (Coll1 ) confirmed these results in the CCl-4 model. Of note, IL-1Ra KO mice were rescued following IL-1Ra treatment (Figure 3E,F). Comparison between WT and KO baseline Coll1 mRNA levels in sham animals without (Supplementary Figure S2). Quantification of liver mRNA expression of Collagen type 1α (Coll1α) fibrosis induction is provided in Supplementary Figure S3. Altogether, these results demonstrated that confirmed these results (Figure 3E,F). Comparison between WT and KO baseline Coll1α mRNA the effects of IL-1Ra knockout and treatment during liver fibrosis are model specific. While IL-1Ra levels in sham animals without fibrosis induction is provided in Supplementary Figure S3. deficiency increases liver fibrosis in the BDL model, it decreases liver fibrosis in the CCl-4 model. Altogether, these results demonstrated that the effects of IL-1Ra knockout and treatment during liver 2.1.4. Hepatic Stellate Cell Activation fibrosis are model specific. While IL-1Ra deficiency increases liver fibrosis in the BDL model, it decreases liver fibrosis in the CCl-4 model. To investigate whether the increased liver fibrosis induced by BDL or CCl-4 was paralleled by HSC activation, we determined -SMA expression using immunofluorescence and RT-PCR on livers from the three groups (Figure 4). Quantification of -SMA by immunofluorescence on liver sections and RT-PCR in liver showed that -SMA expression remained globally unchanged in the BDL group (Figure 4A–C). In the CCl-4 model, -SMA varied along with fibrosis intensity. WT mice and WT mice treated with IL-1Ra had significantly more -SMA positive cells and liver -SMA mRNA expression compared to IL-1Ra KO mice (Figure 4D–F). Comparison between WT and KO baseline -SMA mRNA levels in sham animals without fibrosis induction is provided in Supplementary Figure S3. These results confirm the model-specific variations in liver fibrosis intensity following IL-1Ra Knockout or treatment. IL-1Ra (pg/ml) Int. J. Mol. Sci. 2019, 20, 1295 6 of 18 Int. J. Mol. Sci. 2019, 20, x 6 of 18 BDL BDL in IL-1Ra KO BDL +IL-1Ra A 14 ** *** BDL D *** *** CCl4 CCl4in IL-1Ra KO CCl4+IL-1Ra 12 ** CCl Sham WT IL-1Ra KO WT, IL-1Ra treated * * BDL CCl Figure 3. Differential and model-dependent variation of liver fibrosis following IL-1 signaling modulation Figure 3. with Differential and IL-1Ra knockout model-depend versus treatment. ent variation Mice livers of liver f wereibrosi fixedsin foll formalin owing IL and -1embedded β signaling in paraffin and collected for mRNA extraction and RT-PCR following 2–4 weeks BDL or 6 weeks CCl-4 modulation with IL-1Ra knockout versus treatment. Mice livers were fixed in formalin and embedded treatment. BDL and CCl-4 groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 in paraffin and collected for mRNA extraction and RT-PCR following 2–4 weeks BDL or 6 weeks CCl- and n = 4) and IL-1Ra treated mice (50 mg/kg/day) (n = 10 and n = 13). Sham treated mice were used 4 treatment. BDL and CCl-4 groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n as= 7 and control n for = 4) theand IL-1Ra BDL and CCl-4 treated mice groups ( (50 n = 8 mg/kg/day) ( and n = 4, histology n = 10 and not nshown). = 13). Sham treated mice were Liver sections stained byusirius sed as red control (A). for the BDL a On sirius staining nd CCl-4 g liver par roups ( enchyma n = 8 and appears n = 4, histo in light yellow logy not shown). Li and fibrotic arver sect eas appear ions in red. Morphometric quantification of fibrosis was performed on multiple liver sections and expressed stained by sirius red (A). On sirius staining liver parenchyma appears in light yellow and fibrotic as percentage of liver surface area for the BDL (B) and CCl-4 groups (D). Liver collagen type I mRNA areas appear in red. Morphometric quantification of fibrosis was performed on multiple liver sections levels were measured by RT-PCR in mice with BDL (E) or CCl-4 treatment (F). Scale bars, 400 m. and expressed as percentage of liver surface area for the BDL (B) and CCl-4 groups (D). Liver collagen * p < 0.05, ** p < 0.01, *** p < 0.001, comparing two groups as indicated. type I mRNA levels were measured by RT-PCR in mice with BDL (E) or CCl-4 treatment (F). Scale bars, 400 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, comparing two groups as indicated. 2.1.5. Kupffer Cell Activation 2.1.4. Hepatic Stellate Cell Activation We then investigated the activation of Kupffer cells during liver fibrosis in the two models by quantifying the ionized calcium binding adaptor molecule 1 (IBA-1) [27,28] on liver sections (Figure 5). To investigate whether the increased liver fibrosis induced by BDL or CCl-4 was paralleled by The results varied along with fibrosis intensity in each model, respectively. In the BDL model, the HSC activation, we determined α-SMA expression using immunofluorescence and RT-PCR on livers numbers of liver IBA-1 expressing cells were significantly increased in IL-1Ra KO mice compared to from the three groups (Figure 4). Quantification of α-SMA by immunofluorescence on liver sections WT mice or IL-1Ra treated mice (Figure 5A,B). The opposite situation was observed in the CCl-4 model; and RT-PCR in liver showed that α-SMA expression remained globally unchanged in the BDL group a lower level of activation was observed in the IL-1Ra KO group compared to the two other groups (Figure 4A–C). In the CCl-4 model, α-SMA varied along with fibrosis intensity. WT mice and WT (Figure 5C,D). Overall, these results are consistent with those observed in fibrosis level variations and mice treated with IL-1Ra had significantly more α-SMA positive cells and liver α-SMA mRNA further highlight model specific effects during IL-1Ra modulation. expression compared to IL-1Ra KO mice (Figure 4D–F). Comparison between WT and KO baseline α-SMA mRNA levels in sham animals without fibrosis induction is provided in Supplementary Figure S3. These results confirm the model-specific variations in liver fibrosis intensity following IL- 1Ra Knockout or treatment. Coll1a mRNA (fold change) Coll1a mRNA (fold change) Fibrotic surface area (%) Fibrotic surface area (%) Int. J. Mol. Sci. 2019, 20, x 7 of 18 Int. J. Mol. Sci. 2019, 20, 1295 7 of 18 FIGURE 4 BDL α-SMA+ BDL in IL-1Ra KO BDL +IL-1Ra 7 BDL ** CCl4 CCl4in IL-1Ra KO CCl4+IL-1Ra ** CCl Sham WT IL-1Ra KO WT, IL-1Ra treated *** C * 0 0 BDL CCl Figure 4. Differential and model-dependent variation in periportal hepatic stellate cell number and alpha-smooth muscle actin expression in fibrotic mice following IL-1Ra knockout or treatment. (A-F) Figure 4. Differential and model-dependent variation in periportal hepatic stellate cell number and Mice livers were fixed in formalin and embedded in paraffin and collected for mRNA extraction and alpha-smooth muscle actin expression in fibrotic mice following IL-1Ra knockout or treatment. (A-F) RT-PCR following 2–4 weeks BDL or 6 weeks CCl-4 treatment. BDL and CCl-4 groups were respectively Mice livers were fixed in formalin and embedded in paraffin and collected for mRNA extraction and as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 and n = 4) and IL-1Ra treated mice (50 mg/kg/day) RT-PCR following 2–4 weeks BDL or 6 weeks CCl-4 treatment. BDL and CCl-4 groups were (n = 10 and n = 13). Sham treated mice were used as control for the BDL and CCl-4 groups (n = 8 and respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 and n = 4) and IL-1Ra treated mice (50 n = 4, histology not shown). (A,D) Liver sections immunostained for -SMA (green) and stained with mg/kg/day) (n = 10 and n = 13). Sham treated mice were used as control for the BDL and CCl-4 groups evans blue (red). Activated hepatic stellate cells appear in green and hepatic parenchyma areas appear (n = 8 and n = 4, histology not shown). (A,D) Liver sections immunostained for α-SMA (green) and in red. Morphometric quantification of stellate cell activation was performed on multiple liver sections stained with evans blue (red). Activated hepatic stellate cells appear in green and hepatic parenchyma and expressed as percentage of -SMA+ area for the BDL (B) and CCl-4 (E) groups. Liver -SMA areas appear in red. Morphometric quantification of stellate cell activation was performed on multiple mRNA levels were measured by RT-PCR in mice following BDL (C) or CCl-4 treatment (F). Scale bars, liver sections and expressed as percentage of α-SMA+ area for the BDL (B) and CCl-4 (E) groups. 400 m. * p < 0.05, ** p < 0.01, *** p < 0.001, comparing two groups as indicated. Liver α-SMA mRNA levels were measured by RT-PCR in mice following BDL (C) or CCl-4 treatment 2.1.6. Liver Enzymes (F). Scale bars, 400 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, comparing two groups as indicated. Hepatocyte death and liver injury were assessed by measuring alanine transaminase (ALT) in mice 2.1.5. Kupffer Cell Activation serum in the two groups (Figure 6). In the BDL model, WT mice had higher level of ALT compared to We then investigated the activation of Kupffer cells during liver fibrosis in the two models by IL-1Ra KO mice or IL-1Ra treated mice (Figure 6A). In the CCl-4 model, WT mice treated with IL-1Ra quantifying the ionized calcium binding adaptor molecule 1 (IBA-1) [27,28] on liver sections (Figure had higher ALT levels compared to WT mice or IL-1Ra KO mice (Figure 6B). IL-1Ra KO mice had the 5). The results varied along with fibrosis intensity in each model, respectively. In the BDL model, the lowest ALT level in the later model. numbers of liver IBA-1 expressing cells were significantly increased in IL-1Ra KO mice compared to WT mice or IL-1Ra treated mice (Figure 5A,B). The opposite situation was observed in the CCl-4 model; a lower level of activation was observed in the IL-1Ra KO group compared to the two other groups (Figure 5C,D). Overall, these results are consistent with those observed in fibrosis level variations and further highlight model specific effects during IL-1Ra modulation. aSMA mRNA (fold change) aSMA mRNA (fold change) aSMA+ cell area (%) aSMA+ cell area (%) Int. J. Mol. Sci. 2019, 20, x 8 of 18 BDL IBA-1+ BDL in IL-1Ra KO BDL +IL-1Ra *** BDL Int. J. Mol. Sci. 2019, 20, 1295 8 of 18 Int. J. Mol. Sci. 2019, 20, x 8 of 18 CCl4 CCl4in IL-1Ra KO CCl4+IL-1Ra 15 ** BDL IBA-1+ BDL in IL-1Ra KO BDL +IL-1Ra *** CCl Sham WT IL-1Ra KO WT, IL-1Ra t 1 reated BDL CCl4 CCl4in IL-1Ra KO CCl4+IL-1Ra D ** Figure 5. Differential and model-dependent variation in Kupffer cell number in fibrotic mice following IL-1Ra knockout or treatment. (A–D) Mice livers were fixed in formalin and embedded in paraffin following 2–4 weeks BDL or 6 weeks CCl-4 treatment. BDL and CCl-4 groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 and n = 5) and IL-1Ra treated mice (50 mg/kg/day) (n = 8 and n = 14). Sham treated mice were used as control for the BDL and CCl-4 groups (n = 8 and n = 6, histology not shown). (A,C) Liver sections immunostained for IBA-1 (green) and CCl stained with evans blue (red). Activated Kupffer cells appear in green and hepatic parenchyma areas Sham WT IL-1Ra KO WT, IL-1Ra treated appear in red. Morphometric quantification of activated Kupffer cell numbers was performed on Figure mu5. ltiple l Differ iver ential secti and ons and model-dependent expressed avariation s percentag in e Kupf of IB fer A-1 + cell number area for inthe BDL fibrotic ( mice B) an following d CCl-4 (D) Figure 5. Differential and model-dependent variation in Kupffer cell number in fibrotic mice IL-1Ra grou knockout ps. Scale b or ars, 400 treatment. µm. * (A * p – < 0. D) Mice 01, *** livers p < 0.001, comparing two groups as indicate were fixed in formalin and embedded d. in paraffin following following IL-1 2–4 weeks Ra knockout or treatment. ( BDL or 6 weeks CCl-4A tr –eatment. D) Mice liver BDLs and were fix CCl-4 ed gr in f oups ormwer alin and e e respectively mbedded asin 2. follows: 1.paraffin follo 6. Live WT r E( nnzym = wing 2–4 wee 7 and es n = 8), IL-1Ra ks BDL or KO 6 (n =weeks CCl-4 t 7 and n = 5) and reatment. BDL and CCl-4 IL-1Ra treated mice (50 mg/kg/day) groups were (nrespectiv = 8 and n ely = as 14). follows Sham : WT treated (n = 7 and mice wer n = 8) e used , IL-1Ra KO ( as control n for = 7 and the BDL n = 5) an and CCl-4 d IL-1Ra treate groups (nd mice = 8 and (50 Hepatocyte death and liver injury were assessed by measuring alanine transaminase (ALT) in n = mg/k 6, histology g/day) (nnot = 8 and shown). n = 14). (A,C Sham treated mice were use ) Liver sections immunostained d as control for for IBA-1 the BDL and C (green) and stained Cl-4 group with s mice serum in the two groups (Figure 6). In the BDL model, WT mice had higher level of ALT evans (n = 8 and blue (red). n = 6, Activated histology not shown). ( Kupffer cells appear A,C) Liver sections immunostained for in green and hepatic parenchyma Iar BA-1 (green) and eas appear in compared to IL-1Ra KO mice or IL-1Ra treated mice (Figure 6A). In the CCl-4 model, WT mice treated red. Morphometric quantification of activated Kupffer cell numbers was performed on multiple liver stained with evans blue (red). Activated Kupffer cells appear in green and hepatic parenchyma areas with IL-1Ra had higher ALT levels compared to WT mice or IL-1Ra KO mice (Figure 6B). IL-1Ra KO sections and expressed as percentage of IBA-1 + area for the BDL (B) and CCl-4 (D) groups. Scale bars, appear in red. Morphometric quantification of activated Kupffer cell numbers was performed on mice had the lowest ALT level in the later model. 400 m. ** p < 0.01, *** p < 0.001, comparing two groups as indicated. multiple liver sections and expressed as percentage of IBA-1 + area for the BDL (B) and CCl-4 (D) groups. Scale bars, 400 µm. ** p < 0.01, *** p < 0.001, comparing two groups as indicated. A B 2.1.6. Liver Enzymes Hepatocyte death and liver injury were assessed by measuring alanine transaminase (ALT) in mice serum in the two groups (Figure 6). In the BDL model, WT mice had higher level of ALT compared to IL-1Ra KO mice or IL-1Ra treated mice (Figure 6A). In the CCl-4 model, WT mice treated with IL-1Ra had higher ALT levels compared to WT mice or IL-1Ra KO mice (Figure 6B). IL-1Ra KO mice had the lowest ALT level in the later model. BDL CCl Sham WT IL-1Ra KO WT, IL-1Ra treated Figure 6. Serum alanine aminotransferase levels (ALT). Serum ALT levels were measured in sham miceFigure 6. and miceSerum alanine aminotransferase levels (ALT). following 2–4 weeks BDL (A) or 6 weeks CCl-4 Setr ru eatment m ALT l (B ev ).eBDL ls weand re m CCl-4 easure gr doups in sham were mice and mice respectively as following 2–4 follows: WT (weeks BD n = 7 and L ( n = A8), ) or 6 IL-1Ra weeks CCl-4 treatment KO (n = 8 and n = 5) (B or ). IL-1Ra BDL and CCl treated-4 groups mice (50 mg/kg/day) were respectiv (n ely = as 8 and follow n =s13). : WT ( Sham n = 7 a treated nd n = mice 8), IL-1Ra were used KO (n as = 8 a contr nd ol n = for 5)the or IL-1Ra BDL and treate CCl-4 d mice groups (50 mg/kg/d (n = 8 and ayn) ( =n6). = 8 and * p < 0.05, n = 13). Sham tre ** p < 0.01, comparing ated mice wer two gr eoups usedas as control for indicated. the BDL and CCl-4 BDL CCl groups (n = 8 and n = 6). * p < 0.05, ** p < 0.01, comparing two groups as indicated. 2.1.7. Matrix Metalloproteinases We further analyzed MMP-2, MMP-9, MMP-13 and tissue inhibitor of matrix metalloproteinases 1 Sham WT IL-1Ra KO WT, IL-1Ra treated (TIMP-1) hepatic gene expressions after BDL- and CCl-4-induced liver fibrosis using RT-PCR (Figure 7). In the BDL model, MMP-2, 9,13 and TIMP-1 levels were all higher in the WT mice group compared Figure 6. Serum alanine aminotransferase levels (ALT). Serum ALT levels were measured in sham to the IL-1Ra KO mice group or WT mice treated with IL-1Ra (Figure 7A–D). In the CCl-4 model, mice and mice following 2–4 weeks BDL (A) or 6 weeks CCl-4 treatment (B). BDL and CCl-4 groups MMP-2 and MMP-13 were higher in the IL-1Ra KO mice group compared to the two other groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 8 and n = 5) or IL-1Ra treated mice (Figure 7E,G). Regarding MMP-9 and TIMP-1, expression was decreased in the IL-1Ra KO mice group (50 mg/kg/day) (n = 8 and n = 13). Sham treated mice were used as control for the BDL and CCl-4 compared to the two other groups (Figure 7F,H). Comparison between WT and KO baseline -SMA groups (n = 8 and n = 6). * p < 0.05, ** p < 0.01, comparing two groups as indicated. IBA-1+ cell area (%) IBA-1+ cell area (%) IBA-1+ cell area (%) IBA-1+ cell area (%) Int. J. Mol. Sci. 2019, 20, x 9 of 18 2.1.7. Matrix Metalloproteinases We further analyzed MMP-2, MMP-9, MMP-13 and tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) hepatic gene expressions after BDL- and CCl-4-induced liver fibrosis using RT-PCR (Figure 7). In the BDL model, MMP-2, 9,13 and TIMP-1 levels were all higher in the WT mice group compared to the IL-1Ra KO mice group or WT mice treated with IL-1Ra (Figure 7A–D). In the CCl-4 model, MMP-2 and MMP-13 were higher in the IL-1Ra KO mice group compared to the two other groups (Figure 7E,G). Regarding MMP-9 and TIMP-1, expression was decreased in the IL-1Ra KO Int. J. Mol. Sci. 2019, 20, 1295 9 of 18 mice group compared to the two other groups (Figure 7F,H). Comparison between WT and KO baseline α-SMA mRNA levels in sham animals without fibrosis induction is provided in mRNA levels in sham animals without fibrosis induction is provided in Supplementary Figure S3. Supplementary Figure S3. These results confirm that IL-1Ra regulates genes in a model specific- These manner during liver results confirm that fibroIL-1Ra sis. regulates genes in a model specific-manner during liver fibrosis. AB 0 0 BDL BDL C D 0 0 BDL BDL Int. J. Mol. Sci. 2019, 20, x 10 of 18 Sham WT IL-1Ra KO WT, IL-1Ra treated E F 8 2 4 1 0 0 CCl CCl 4 4 G H *** ** 2 6 CCl CCl 4 4 Sham WT IL-1Ra KO WT, IL-1Ra treated Figure 7. Matrix metalloproteinase (MMP) 2, 9 and 13 and tissue inhibitor of metalloproteinase 1 (TIMP-1) expression. MMP-2, 9 and 13 and TIMP-1 liver mRNA levels were measured by RT-PCR in Figure 7. Matrix metalloproteinase (MMP) 2, 9 and 13 and tissue inhibitor of metalloproteinase 1 sham mice and mice following 2–4 weeks BDL (A–D) or 6 weeks CCl-4 treatment (E–H). BDL and (TIMP-1) expression. MMP-2, 9 and 13 and TIMP-1 liver mRNA levels were measured by RT-PCR in CCl-4 groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 and n = 4) or IL-1Ra sham mice and mice following 2–4 weeks BDL (A–D) or 6 weeks CCl-4 treatment (E–H). BDL and treated mice (50 mg/kg/day) (n = 10 and n = 13). Sham treated mice were used as control for the CCl-4 groups were respectively as follows: WT (n = 7 and n = 8), IL-1Ra KO (n = 7 and n = 4) or IL-1Ra BDL treated and mic CCl-4 e (50 mg/kg/d groups (n = ay) ( 8 and n = 10 n and = 4).n* =p 13). < 0.05, Sham treat ** p < 0.01, ed mice *** were u p < 0.001, sed as control f comparingotwo r the gr BDL oups as indicated. and CCl-4 groups (n = 8 and n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, comparing two groups as indicated. 3. Discussion Acute and chronic liver injuries are accompanied by a major inflammatory response, including an increased expression of IL-1β and its natural antagonist IL-1Ra [8,10–12,16,22,29–39]. We previously demonstrated that the absence of IL-1Ra caused an increased inflammatory state and a delayed liver regeneration in a mouse model of partial hepatectomy [16] and that IL-1Ra might be an important factor in MSC antifibrotic effect in the liver [19]. In the present study, we analyzed the effect of IL-1β alone or blocked with IL-1Ra on primary human HSCs, as well as the effect of IL-1Ra deficiency or supplementation in two models of in vivo experimental liver fibrosis. Our in vitro experiments showed that the treatment of primary human HSCs with IL-1β resulted in an increase of its own transcription but reduced α-SMA expression, with the effects being prevented by recombinant IL-1Ra. In vivo, IL-1β and IL-1Ra expressions were strongly upregulated in WT mice livers following BDL and CCl-4 injuries, confirming the implication of IL-1 signaling in this process and its regulation by the liver [40,41]. The knockout of IL-1Ra resulted in opposite patterns in the two liver fibrosis models; IL-1Ra KO mice had increased fibrosis in the BDL model while fibrosis was reduced in the CCl-4 model. It is known that BDL ligation causes a periportal fibrosis [42] while CCl-4 intoxication leads to an initial centrilobular matrix deposition [43]. Considering our in vitro results and the known predominance of HSCs in the pericentral area [44], we hypothesized that IL-1β signaling modulates liver fibrosis in an HSC-dependent manner. A first question that we aimed to address was to understand the effect of increased IL-1 signaling on HSC, the key player in liver fibrosis. We knew from previous works that HSCs respond to IL-1 stimulation in vitro with various effects on proliferation and α-SMA expression [24,26,45,46]. We MMP-2 mRNA MMP-13 mRNA MMP-2 mRNA MMP-13 mRNA (Fold change) (fold change) (Fold change) (fold change) TIMP-1 mRNA MMP-9 mRNA TIMP-1 mRNA MMP-9 mRNA (Fold change) (Fold change) (fold change) (Fold change) Int. J. Mol. Sci. 2019, 20, 1295 10 of 18 3. Discussion Acute and chronic liver injuries are accompanied by a major inflammatory response, including an increased expression of IL-1 and its natural antagonist IL-1Ra [8,10–12,16,22,29–39]. We previously demonstrated that the absence of IL-1Ra caused an increased inflammatory state and a delayed liver regeneration in a mouse model of partial hepatectomy [16] and that IL-1Ra might be an important factor in MSC antifibrotic effect in the liver [19]. In the present study, we analyzed the effect of IL-1 alone or blocked with IL-1Ra on primary human HSCs, as well as the effect of IL-1Ra deficiency or supplementation in two models of in vivo experimental liver fibrosis. Our in vitro experiments showed that the treatment of primary human HSCs with IL-1 resulted in an increase of its own transcription but reduced -SMA expression, with the effects being prevented by recombinant IL-1Ra. In vivo, IL-1 and IL-1Ra expressions were strongly upregulated in WT mice livers following BDL and CCl-4 injuries, confirming the implication of IL-1 signaling in this process and its regulation by the liver [40,41]. The knockout of IL-1Ra resulted in opposite patterns in the two liver fibrosis models; IL-1Ra KO mice had increased fibrosis in the BDL model while fibrosis was reduced in the CCl-4 model. It is known that BDL ligation causes a periportal fibrosis [42] while CCl-4 intoxication leads to an initial centrilobular matrix deposition [43]. Considering our in vitro results and the known predominance of HSCs in the pericentral area [44], we hypothesized that IL-1 signaling modulates liver fibrosis in an HSC-dependent manner. A first question that we aimed to address was to understand the effect of increased IL-1 signaling on HSC, the key player in liver fibrosis. We knew from previous works that HSCs respond to IL-1 stimulation in vitro with various effects on proliferation and -SMA expression [24,26,45,46]. We made the hypothesis that IL-1 , which is a key cytokine during inflammation, may increase HSC activation. However, our results indicate that, on the contrary, upon IL-1 treatment, HSC have a reduced activation. These findings are consistent with those reported previously, where HSC activation was shown to be inhibited after treatment with IL-1 [45] or IL-1 [24]. As suggested previously, IL-1 might mobilize and increase proliferation of HSC rather than directly promoting fibrosis production [24]. Knowing that HSCs are the major source of proteases that degrade basement membrane collagens such as MMP-9 [25], we analyzed its expression in vitro. We found that IL-1 strongly increased MMP-9 gene transcription in HSCs and this effect was prevented by IL-1Ra. These findings are consistent with those of others showing that, under 3D HSC cell culture conditions, stimulation with IL-1 caused robust induction of pro-MMP-9 [25,26]. Of note, enhanced HSC activation was observed in the latter experiments, however IL-1 was used instead of IL-1 and this may explain the difference with our findings. These results add further evidence that IL-1 is a key regulator of MMP-9 expression by HSC during liver fibrosis [8,25,26]. In addition, we [19] and others [47] previously demonstrated that liver MMP-9 expression increases via mesenchymal stem cells’ paracrine effect and might be a protective factor during liver fibrosis. Overall, whether it is clear from numerous previous works that prolonged inflammation can lead to fibrosis [48], our findings along with those of others [24] suggest that this process is not solely driven by a direct effect of IL-1 on HSC. We then thought to investigate the effect of IL-1 signaling downregulation on HSC activation and fibrosis in vivo using IL-1Ra KO mice. In both models, IL-1 was upregulated upon liver fibrosis induction in the liver (2–5-fold) and serum (4–6-fold). IL-1Ra was strongly upregulated in the liver (15–50-fold) and serum (35-fold); knowing that its main production source is the liver [49]. The increase in IL-1 signaling (in IL-1Ra KO mice) resulted in increased fibrosis in the BDL model reduced fibrosis in the CCl-4 model. These results were consistent within their respective models and corresponding changes in collagen type I mRNA expression and Kupffer cells activation were observed. We hypothesized that these conflicting results may represent a model-specific influence. Using IL-1 receptor knockout mice, Pradere et al. found a non-significant decrease in liver fibrosis in both BDL and CCl-4 models [24]. They further showed that antagonizing both IL-1 and TNF- was essential to significantly reduce NF-B in HSCs and reduce liver fibrosis in a BDL model (no data in Int. J. Mol. Sci. 2019, 20, 1295 11 of 18 the CCl-4 model). In their experiments, IL-1 and TNF- did not lead to HSC activation but promoted survival of activated HSCs. Consistent with the work of Pradere et al., Gieling et al. found that IL-1 receptor-deficient mice exhibited reduced fibrosis after thioacetamide treatment for 8 weeks [8]. Overall, these data are in accordance with what we observed in the BDL model (more fibrosis in IL-1Ra KO mice and diminished HSC activation). Altogether, it is likely that both IL-1 and TNF- are both required to successfully translate inflammation into fibrosis; however, increased IL-1 signaling by itself may contribute to an increase in liver fibrosis. Regarding IL-1 signaling in the CCl-4 model, previous evidence is scarce and difficult to compare with our experiments. Based on our consistent findings in the IL-1Ra KO group showing diminished fibrosis, collagen type I expression, activated HSC numbers, -SMA and TIMP-1 expression, we can only hypothesize a direct effect of CCl-4-mediated centrilobular increased inflammation and IL-1 signaling on pericentral HSC. Given our observation that IL-1 diminish HSC activation in vitro, we hypothesize that when inflammation is mainly pericentral (CCl-4 model), IL-1Ra absence (namely, unopposed IL-1 signaling) ultimately partially block the profibrogenic signals. All together our results suggest that blocking IL-1-mediated inflammation may be beneficial mostly in biliary type liver fibrosis. Certain IL-1 gene polymorphisms has been associated with liver fibrosis progression in primary biliary cholangitis [50,51] and further confirm this eventuality. Drug-related injury to the liver may then not be amenable to benefit from IL-1 signaling inhibition since the injury is predominantly centrilobuar. Finally, since IL-1 is implicated in virus clearance, viral hepatitis does not represent good candidates for IL-1 inhibition [52,53]. In our experiments, we also investigated the effect of IL-1Ra treatment (anakinra) and observed that it provided marginal protective effect on liver fibrosis after BDL and increased fibrosis in the CCl-4 model. Using a different model and animal species, Mancini et al. observed a decreased collagen content and fewer activated HSC in WT rats subjected to dimethylnitrosamine-induced liver fibrosis and treated with IL-1Ra [22]; we could marginally reproduce these findings in the BDL model only. Our primary hypothesis to explain this modest improvement is a failure of exogenous IL-1Ra to efficiently increase circulating levels due a possible negative feed-back mechanism regulating its own expression (Figure 2F). Indeed, we observed that IL-1Ra blood levels were not much increased compared to the WT group despite daily anakinra administration. Another argument in favor of this hypothesis is that IL-1Ra expression by the liver was reduced upon IL-1Ra administration. In the clinical setting, IL-1Ra treatment was demonstrated to be successful in situations in which IL-1Ra is absent or nonfunctional [54] while a very limited success was observed with recombinant IL-1Ra to treat human inflammatory disease [2] suggesting that endogenous IL-1Ra production cannot be surpassed. Of note, in a confirmatory experiment, we could demonstrate that IL-1Ra treatment can rescue IL-1Ra KO mice. However, it is also possible that IL-1Ra given systemically is not fully equivalent to the endogenous natural section of IL-1Ra. An example of that discrepancy is depicted by the difference observed in fasting insulin levels between the different groups of mice after BDL. We observed a significant increase in fasting insulin levels in mice after BDL in WT mice and KO mice (consistent with insulin resistance that is commonly observed during liver fibrosis/dysfunction [55]), however that increase was completely reversed by IL-1Ra treatment in WT mice (and KO mice) (Supplementary Figure S2), consistently with the known anti-diabetic effect of exogenous IL-1Ra [56]. In the CCl-4 model, the profibrogenic effect of IL-1Ra treatment was consistent with all the other findings in this model and further reinforces the hypothesis of a dual and topographical-dependent effect of IL-1 signaling modulation. 4. Materials and Methods 4.1. HSC Isolation and Culture Human HSCs were obtained from biopsies of healthy liver parenchyma from three different patients undergoing partial hepatectomy. The protocol was approved by the local research ethics committee of the Department of Surgery of the Geneva University Hospital. All donors provided Int. J. Mol. Sci. 2019, 20, 1295 12 of 18 their written informed consent for use of the samples in the present study. HSCs were isolated as previously described [57,58]. Cells were cultured in 24-well plates (100,000 cells/well) in IMDM medium (Invitrogen, Basel, Switzerland) containing 10% FCS, penicillin, and streptomycin (Invitrogen, Basel, Switzerland) at 37 C with 5% CO . Cells were used for experiments between passages 3 to 6. HSCs were either left untreated, or treated with recombinant IL-1 (10 ng/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 48 h, or IL-1 (10 ng/mL) and IL-1Ra (15 g/mL, Kineret, Amgen Europe B.V, Breda, The Netherlands) together, or Transforming growth factor- 1 (TGF- 1) (50 ng/mL, PreproTech, UK) for 48h. For -SMA protein detection, HSCs were treated and cultured for 5 days. 4.2. Electrophoretic and Immunoblot Analysis Proteins from lysed cells were separated on polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Milipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in wash buffer (20 mM Tris–HCl, pH 7.4, 140 mM NaCl, 0.1% Tween 20) and incubated with anti-alpha smooth muscle actin ( -SMA) [59] (obtained from C. Chaponnier, Geneva, Switzerland) or anti-vimentin (Dako, Baar, Switzerland) antibodies diluted in blocking solution. Following three washes, membranes were incubated with peroxidase-conjugated goat anti-mouse antibodies (Molecular Probes Inc, Eugene, OR, USA) diluted 1:6000 in wash buffer. Proteins were revealed by chemiluminescence (ECL, Interchim Inc., Montluçon, France) on photographic film (GE healthcare, Chicago, IL, USA) and signals were quantified using the Quantity One software (PDI, Inc., Huntington Station, NY, USA) and normalized by the expression of GAPDH or vimentin. 4.3. Animals DBA-1 mice were purchased from Janvier (Le Genest-St-Isles, France). IL-1Ra knockout (IL-1Ra KO) mice were generated from DBA-1 background and were a kind gift from Prof. Cem Gabay of Geneva University [60,61]. Eight to ten-week-old male mice were used. Liver fibrosis was induced by 2 to 4 weeks of BDL or 6 weeks of CCl-4 treatment, as described here below. All animal studies were approved by the Animal Ethics Committee of the Geneva Veterinarian Office and Geneva University, Geneva, Switzerland (protocol 1043/3603/2, Oct 1, 2010). 4.4. Fibrosis Induction in Mice Liver fibrosis was induced by BDL or CCl-4. BDL: briefly, mice were anesthetized with isoflurane, a midline laparotomy was performed, and the common bile duct was dissected and cut between four ligatures under a dissecting microscope. CCl-4: Three ml per kilogram of CCl-4 50% (v/v) solution in corn oil (Sigma Co., Milan, Italy), containing 1.0 ml/kg of CCl-4, was administered by intraperitoneal injections twice a week for 6 weeks. All mice were maintained under standard conditions at the animal facility of Geneva University. Water and food were provided ad libitum. IL-1Ra treated animals received 50mg/kg/day recombinant IL-1Ra, intraperitoneally, every day from BDL (or CCl-4) to sacrifice (Anakinra/Kineret, Amgen Europe B.V, Breda, The Netherlands). Sham operated and sham injected (NaCl 0.9%) mice were used as controls. In accordance with the 3R principles of animal experimentation in our institution (reduce, refine, replace), the severity of the BDL model in DBA-1 mice prompted us to sacrifice mice as soon as they presented signs of suffering. Accordingly, mice were sacrificed between 2 to 4 weeks. No mortality was observed in the CCl-4 model and all animals were sacrificed at 6 weeks. Blood and liver samples were collected to be analyzed. 4.5. Assessment of Hepatic Fibrosis Liver collagen content was determined using sirius red histochemistry as previously described [62]. Tissue sections were observed using an Axiophot microscope (Carl Zeiss AG, Feldbach, Switzerland) and images were acquired with an Axiocam color camera (Zeiss, Feldbach, Switzerland). Hepatic fibrosis extent was determined using morphometric quantification (MetaMorph Software, Universal Imaging, West Chester, PA, USA). Int. J. Mol. Sci. 2019, 20, 1295 13 of 18 4.6. Immunostaining for Alpha-Smooth Muscle Actin (a-SMA) and Ionized Calcium Binding Adaptor Molecule 1 (IBA-1) Paraffin sections were dewaxed, and rehydrated using xylene/ethanol baths and then heated at 95 C in a 10 mM/pH 6.0 sodium citrate bath for 10 min. The detection of activated HSCs was performed using anti -SMA antibody (a gift from Christine Chaponnier, Geneva University) (1:50). The detection of macrophages was performed using an anti IBA-1 antibody (Wako Chemicals, Richmond, VA, USA) (1:500). The liver sections were incubated overnight at 4 C with primary antibody diluted in PBS containing 0.1% BSA, washed in PBS, and incubated for 1 h with a secondary antibody (Alexa Fluor 488 goat-anti mouse) diluted (1:1000) in PBS containing 0.1% BSA. All sections were stained with 0.09% Evans blue solution. Images of immunostained sections were acquired using Axiophot microscope and Axiocam color camera. The percentage of positive cells for -SMA or IBA-1 was determined using MetaMorph, Image J and Definiens Software. Cells area positive for -SMA or IBA-1 staining were counted and normalized to the total liver surface. An average of 3000 cells were counted on 4 histology fields per animal. 4.7. Real-Time Polymerase Chain Reaction (RT-PCR) RT-PCR was used to determine the expression levels of fibrosis-related genes. Total RNA was extracted from liver samples or cultured cells using Qiagen RNeasy Mini kit (Qiagen, San Diego, CA, USA) according to manufacturer ’s instructions. cDNA was synthesized from 1 g of total RNA using SuperScript III reverse transcriptase (Invitrogen, Basel, Switzerland). RT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems Inc, San Diego, CA, USA), with 2 ng cDNA and 300 nM of each primer following the following protocol: two minutes at 50 C, 10 min at 95 C, and for 45 cycles of 15 s at 95 C, and 60 s at 60 C using a SDS 7900 HT machine (Applied Biosystems Inc.). Reactions were performed in three replicates on 384-well plate. Raw C values obtained with SDS 2.2 (Applied Biosystems Inc.) were imported in Excel software (Microsoft, Redmond, WA, USA) and normalization factor and fold changes were calculated using the GeNorm method [63]. Briefly, GeNorm was used to determine which normalization genes are the most stable in the sample population studied and select them for normalization. We computed a normalization factor for each individual sample represented by the geometric mean of the quantity values from the most stable normalization genes. Each sample will therefore have its own normalization factor. Primers used for amplification were designed using Primer3 online software (http://frodo.wi.mit.edu/) unless otherwise specified (Table 1). All primers were tested with Amplifix Software (http://ifrjr.nord.univ-mrs.fr/AmplifX-Home-page) and blasted on http://www.ensembl.org. Human matrix metalloproteinase (MMP) -2 was purchased from Qiagen, San Diego, CA, USA. The efficiency of each primer was tested using positive control cDNA serial dilutions and negative control. Table 1. RT-PCR Primer Sequences. 0 0 Primer-Probe Sequence 5 -3 Name Forward Reverse Mice IL-1 ACT CCT TAG TCC TCG GCC A TGG TTT CTT GTG ACC CTG AGC IL-1Ra CTG CAC TTC CAC AGT CCA GA ATA TGT GAT GCC CTG GTG GT Collagen type I alpha 1 GCA TGG CCA AGA AGA CAT CC CCT CGG GTT TCC ACG TCT C MMP-2 TGG GGG AGA TTC TCA CTT TG CAT CAC TGC GAC CAG TGT CT MMP-9 AGT TGC CCC TAC TGG AAG GT GTG GAT AGC TCG GTG GTG TT MMP-13 AGT TGA CAG GCT CCG AGA AA AGT TCG TTT GGG ACC ATT TG TIMP-1 GCA TCT CTG GCA TCT GGC ATC GAA GGC TGT CTG TGG GTG GG1 [8] Human IL-1 TCC AGG GAC AGG ATA TGG AG TCT TTC AAC ACG CAG GAC AG [64] IL-1Ra AAG ACC AGT CCA TGA GGG AG CTC CCC GAA AGA ACA TAA TCT C -SMA CAT CTA TGA GGG CTA TGC CTT G GTG AAG GAA TAG CCA CGC TC MMP-9 TTG ACA GCG ACA AGA AGT GG GCC ATT CAC GTC GTC CTT AT MMP-2 Ref: QT00088396 Ref: QT00088396 Int. J. Mol. Sci. 2019, 20, 1295 14 of 18 4.8. Serum Assays Serum ALT was measured using UniCel DxC 800 Synchron Clinical Systems (Beckman Coulter, Inc., Brea, CA, USA), following manufacturer ’s instructions. Serum IL-1 , IL-1Ra and fasting insulin contents were measured using mouse IL-1 ELISA set (BD Bioscience, San Jose, CA, USA), mouse IL-1Ra ELISA kit (RayBio, Norcross GA, USA) respectively, and Ultrasensitive Mouse Insulin ELISA (Mercodia, Uppsala, Sweeden) following manufacturer ’s instructions. 4.9. Statistical Analysis Results were expressed as means SEM. Differences between groups were analyzed using the Student t-test or Mann-Whitney U Test and one-way analysis of variance with Kruskal-Wallis testing corrections. A p-value < 0.05 was considered to be statistically significant. 5. Conclusions In conclusion, our study demonstrated that IL-1Ra has opposite effects in two different liver fibrosis models. IL-1Ra was detrimental in the CCl-4 model, whereas it was protective in the BDL model. Since IL-1 can decrease -SMA expression in primary HSC, and given the predominant pericentral distribution of HSC and pericentral inflammation in the CCl-4 model, we postulated that this may represents the cause of decreased fibrosis in the latter model. Altogether these data suggest that blocking IL-1-mediated inflammation with IL-1Ra may only be beneficial in selective liver fibrotic disease. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/6/ 1295/s1. Author Contributions: Conceptualization, R.P.H.M., S.C., F.N. and L.H.B.; Formal analysis, R.P.H.M.; Funding acquisition, P.M. and L.H.B.; Investigation, R.P.H.M., J.M., E.M., S.L., A.B., Y.D.M., S.C. and C.T.; Methodology, R.P.H.M.; Project administration, R.P.H.M. and L.H.B.; Resources, P.M.; Software, R.P.H.M.; Supervision, P.M. and L.H.B.; Writing–original draft, R.P.H.M.; Writing–review & editing, R.P.H.M., J.M., E.M., S.L., A.B., Y.D.M., S.C., F.N., C.T., P.M. and L.H.B. Funding: This work was supported by the Fondation Privée des Hôpitaux Universitaires de Genève (formerly Fondation Artères), the Insuleman Foundation, the University Hospitals of Geneva and Geneva University. Christian Toso was supported by a grant from the Swiss National Science Foundation (PP00P3_165837). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments: We thank Cem Gabay of Geneva University for kindly providing us with IL-1Ra KO mice and Christine Chaponnier for her supply of -SMA antibody. We thank Nadja Perriraz-Mayer, Elodie Perroud, Solange Charvier, Didier Chollet, Sergei Startchik, Danielle Ben Nasr, Marie Ebrahim Malek, Corinne Sinigaglia, Nadine Pernin, Caroline Rouget, and David Matthey-Doret for their technical assistance. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations ALT Alanine transaminase BDL Bile duct ligation CCl-4 Carbon tetrachloride HSC Hepatic stellate cells IL-1Ra Interleukin-1 receptor antagonist IL-1 Interleukin-1 KO Knockout MMP Matrix metalloproteinase MSCs Mesenchymal stem cells TIMP-1 Tissue inhibitor of matrix metalloproteinases 1 TGF- 1 Transforming growth factor- 1 TNF- Tumor necrosis factor- WT Wild type Int. J. Mol. Sci. 2019, 20, 1295 15 of 18 References 1. Dinarello, C.A. Interleukin-1beta and the autoinflammatory diseases. New Engl. J. Med. 2009, 360, 2467–2470. [CrossRef] 2. Gabay, C.; Lamacchia, C.; Palmer, G. IL-1 pathways in inflammation and human diseases. Nat. Rev. Rheumatol. 2010, 6, 232–241. [CrossRef] [PubMed] 3. Seckinger, P.; Williamson, K.; Balavoine, J.F.; Mach, B.; Mazzei, G.; Shaw, A.; Dayer, J.M. A urine inhibitor of interleukin 1 activity affects both interleukin 1 alpha and 1 beta but not tumor necrosis factor alpha. J. Immunol. 1987, 139, 1541–1545. 4. 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International Journal of Molecular Sciences – Unpaywall
Published: Mar 14, 2019