Hepatic stellate cell activation promotes alcohol-induced steatohepatitis through Igfbp3 and SerpinA12

Abstract

Background & aims: Steatohepatitis drives fibrogenesis in alcohol-related liver disease. Recent studies have suggested that hepatic stellate cells (HSCs) may regulate the parenchymal cell injury and inflammation that precedes liver fibrosis, although the mechanism remains incompletely defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation. In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on the development of steatohepatitis in response to alcohol feeding in mice.

Methods: Mice with HSC-selective deletion of NRP (Col^Cre/Nrp1^loxP) or synectin (Col^Cre/synectin^loxP) vs. paired Nrp1^loxP or synectin^loxP mice were fed a control diet or the chronic/binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated.

Results: Col^Cre/Nrp1^loxP mice showed less fibrosis, as expected, but also less inflammation and steatosis, with lower hepatic triglyceride content. Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSCs from Col^Cre/Nrp1^loxP mice compared to supernatant from Nrp1^loxP mice were protected against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array from the supernatant of HSCs with NRP-1 knockdown showed a significant reduction in Igfbp3 (a major insulin-like growth factor-binding protein with multiple metabolic functions) and an increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wild-type HSCs. Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally, Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with alcoholic hepatitis.

Conclusion: Selective deletion of NRP-1 from HSCs attenuates alcohol-induced steatohepatitis through regulation of Igfbp3 and SerpinA12 signaling.

Lay summary: Hepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we describe their role in the modulation of fat deposition and inflammation in the liver, which occurs secondary to alcohol damage.

Keywords: Alcohol; Alcoholic hepatitis; Alcoholic liver disease; Hepatic stellate cell; Igfbp3; Insulin-like growth factor-binding protein 3; Integrin; Neuropilin-1; SerpinA12; Src-kinase; Steatohepatitis; Steatosis; Vaspin.

Fig. 1.. Impaired HSC activation by selective NRP-1 deletion reduces EtOH-induced steatosis and inflammation in vivo.

Panel (A), Hepatic stellate cells isolated from Col^Cre/NRP-1^loxP mice showed the absence of signal for NRP-1 by western blot compared to NRP-1^loxP mice (n=3–6; p<0.05). Panels (B) and (C), represent α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (Col1α1) mRNA expression respectively, in response to Transforming Growth Factor-β (TGF-β) and Cre recombinase containing adenovirus (AdCRE) in NRP-1^loxP and Col^Cre/NRP-1^loxP mice (n=3–6; p<0.05). (D) Alcohol fed Col^Cre/NRP-1^loxP mice exhibit less steatosis compared to matched NRP-1^loxP mice as assayed by Oil-Red O staining (intensity quantification in the adjacent panel; n=6–9; 5x magnification; p<0.0001; one-way ANOVA). (E) HSC selective deletion of neuropilin-1 inhibits triglyceride accumulation in the liver in response to chronic/binge alcohol feeding (n=6–9; p<0.05; one-way ANOVA). (F-H) Col^Cre/NRP-1^loxP mice exhibit lower expression of inflammatory markers in response to alcohol as evidenced by reduced TNFα, IL1β, and MCP-1 levels (n=6–9; p<0.05; one-way ANOVA). (I) Alcohol fed Col^Cre/NRP-1^loxP mouse livers exhibit less infiltrative macrophages (arrowheads) compared to matched NRP-1^loxP mice based on CD68 staining (intensity quantification adjacent to representative images; n=6–9; 20X magnification; area 1.3mm², p<0.05, one-way ANOVA). (J) Alcohol fed Col^Cre/NRP-1^loxP mouse livers exhibit less number of inflammatory foci (arrowhead) per field compared to matched NRP-1^loxP mice (representative images; n=6–9; 10X magnification, p<0.05, one-way ANOVA).

Fig. 2.. Supernatant from HSC with selective deletion or knockdown of NRP-1 prevents lipid droplets formation in primary hepatocytes treated with ethanol.

(A) Supernatant from HSC from NRP-1loxP and Col1Cre/NRP-1loxP mice prevent lipid droplets formation in primary hepatocytes treated with ethanol. Representative images of primary mouse hepatocytes treated with supernatant of HSC from Col^Cre/NRP-1^loxP or NRP-1^loxP and different concentrations of ethanol (EtOH) (0, 25 and 50mM). (n=5–9, 63x). Quantification of lipid droplets by BODIPY® 493/503 intensity evaluation. (n=6–9, *p<0.05, one-way ANOVA). Hepatocytes treated with supernatant of Col^Cre/NRP-1^loxP HSC are prevented from the development of lipid droplets in response to EtOH. (B) Supernatant from HSC with knockdown of NRP-1 by siRNA prevents lipid droplets formation in primary hepatocytes treated with ethanol. Representative images of hepatocytes treated with supernatant from NRP-1 KD HSC and different concentrations of ethanol (EtOH) (0, 25 and 50mM). (n=5–9, 40x). Quantification of lipid droplets by BODIPY® 493/503 intensity evaluation. (n=6–9, *p<0.05, one-way ANOVA).

Fig. 3.. NRP-1 knockdown in human-derived primary HSC increases Igfbp3 while decreases SerpinA12 levels.

(A) Representative BODIPY® staining images and quantification from HepG2^Cyp2E1 cells treated with (I) medium, (II) control HSC supernatant, (III) siRNA mediated NRP-1 knockdown HSC supernatant. (III) shows decreased lipid droplet formation in vitro (n=6–9, 63X, one-way ANOVA). (B) Top ten down/upregulated molecules from adipokine and inflammatory protein array from NRP-1 KD HSC supernatant compared to controls. IGFBP3 and SerpinA12 were the most significantly decreased and increased proteins respectively (n=3, p<0.05, unpaired t-test). (C) Igfbp3 mRNA and protein expression in NRP-1 KD HSC is lower than human WT HSC on qPCR and WB (n=6–9, p<0.05, unpaired t-test). (D) SerpinA12 mRNA expression in NRP-1 KD HSC is higher than human WT HSC (n=6–9, p<0.05, unpaired t-test).

Fig. 4.. Recombinant murine-Igfbp3 and ethanol (EtOH) increase lipid droplet formation, triglycerides, and lipogenic gene expression in primary mouse hepatocytes.

(A) Representative images and BODIPY staining from hepatocytes treated with basal medium ± EtOH in different concentrations ± murine recombinant Igfbp3 (BODIPY® + DAPI staining, n=6, 63x, one-way ANOVA). Treatment of primary mouse hepatocytes with murine recombinant Igfbp3 and ethanol significantly increases lipid droplets formation in vitro. (B) Quantification of triglycerides. Cultured primary mouse hepatocytes were treated with Igfbp3, EtOH 50mM, Igfbp3 plus EtOH, or vehicle. All treated groups showed higher cellular triglyceride content compared to control (n=6, *p<0.05 compared to control, one way ANOVA). (C) Expression of lipogenic genes in response to recombinant Igfbp3 administration in primary mouse hepatocytes. Igfbp3 administration induces key lipogenic genes involved in hepatic lipid homeostasis (SREBP-1c, ACC, FASN, SCD1, and DGAT1) (n=6, *p<0.05 compared to control, one way ANOVA). (D) Protein expression of SREBP-1c and FASN by WB in response to Igfbp3. Igfbp3 administration increases both protein expression (n=6, *p<0.05 compared to control, unpaired t-test). (E) In vitro human recombinant IGFBP3 treatment to human hepatocytes induces the main lipogenic genes involved in triglyceride de novo synthesis viz. SREBP-1c, ACC, FASN, SCD1, DGAT1 (n=6–9, *p<0.05, one-way ANOVA). (F) Supernatant from siRNA-mediated NRP-1 knockdown HSC reduces the protein expression of SREBP-1c by WB (n=6, *p<0.05, unpaired t-test). (G) In vitro reversal of protection against alcohol of NRP-1 siRNA-mediated knockdown in HSC. Hepatocytes treated with supernatant and alcohol +/− recombinant Igfbp3. Hepatocytes treated with supernatant from NRP-1 KD HSC developed fewer lipid droplets, and this protective effect was reverted in the presence of Igfbp3 (n=6, *p<0.05 compared to control siRNA + Igfbp3, one way ANOVA).

Fig. 5.. Igfbp3 increases p-Akt through integrin receptor/Src-kinase signaling in primary hepatocytes.

(A) The phosphorylated form of Akt is induced by the administration of murine recombinant Igfbp3 in vitro in primary hepatocytes (n=6, p<0.05, one way ANOVA). (B) Transfection with either WT Igfbp3 and Igfbp3 GGG mutant adenovirus (I56G, L80G and L81G, mutant IGF binding sites), which expresses full-length Igfbp3 but has no binding affinity to IGF1 and does not bind to IGF1R) induced key lipogenic genes expression (n=3, p<0.05, one way ANOVA). (C) Igfbp3-induced phosphorylation of p-Akt is blunted by Akt inhibitor pretreatment (triplicates, n=9, p<0.05, one way ANOVA). (D) Igfbp3-induced phosphorylation of p-Akt is blunted by Cytochalasin D (Cyt D) pretreatment (n=6, p<0.05, one way ANOVA). (E) Igfbp3-induced phosphorylation of p-Akt is blunted by RGD peptide pretreatment (n=6, p<0.05, one way ANOVA). (F) Igfbp3-induced phosphorylation of p-Akt is blunted by PP2 pretreatment (duplicates, n=6, p<0.05, one way ANOVA). (G) Igfbp3-induced phosphorylation of p-Akt is not affected by IGF-1R inhibitor (PQ401) pretreatment (n=6, p<0.05, one way ANOVA). (H) Representative microscopy images of lipid droplets and Bodipy staining quantification from primary mouse hepatocytes treated with vehicle or Igfbp3 ± PP2 or Akt inhibitor. Pretreatment with PP2 or Akt inhibitor protects against lipid droplet formation induced by Igfbp3 (n=6, p<0.05, 63x, one way ANOVA). (I) mRNA expression of lipogenic genes by qPCR. Igfbp3 administration to in vitro primary mouse hepatocytes induces key lipogenic genes involved in hepatic lipid homeostasis and this effect is blunted by Cytochalasin D, Akt inhibitor, and PP2 (SREBP-1c, ACC, FASN, SCD1, and DGAT1) (n=6, *p<0.05 compared to control/vehicle, #p<0.05 compared to Igfbp3 alone, one way ANOVA).

Fig. 6.. SerpinA12 treatment protects against ethanol-induced steatosis in primary mouse hepatocytes and correlates with the p-AMPK pathway.

(A) Representative images and BODIPY® staining quantification show pretreatment with SerpinA12 protects against lipid droplet formation induced by EtOH (n=6, 63x, p<0.05, one way ANOVA). (B) WB and densitometry quantification shows that EtOH induces a reduction in the phosphorylation of AMPK compared to control. SerpinA12 induces phosphorylation of AMPK, even in the presence of EtOH, and this effect is blunted by the presence of AMPK inhibitor (duplicates, n=6, *p<0.05, one way ANOVA). (C) Representative images and BODIPY staining quantification show protection of SerpinA12 against EtOH-induced lipid droplet formation on treatment with SerpinA12 ± EtOH. This effect was blunted by using an AMPK inhibitor (n=6, 63x, *p<0.05, one way ANOVA). (D) EtOH induces key lipogenic genes involved in hepatic lipid homeostasis, and this effect is reduced by SerpinA12. The protective effect of SerpinA12 is attenuated by an AMPK inhibitor (n=6, *p<0.05 compared to control, #p<0.05 compared to EtOH alone, one way ANOVA). (E) Human hepatocytes treated with supernatant from human HSC with a siRNA-mediated knockdown of SerpinA12 ± EtOH. EtOH ± supernatant from siRNA-mediated knockdown of SerpinA12 increases expression of SREBP-1c, FASN, and SCD1 compared to EtOH in siControl cells (n=9, *p<0.05 compared to control, #p<0.05 compared to EtOH alone, one way ANOVA).

Fig. 7.. Igfbp3 is increased while SerpinA12 is decreased in serum and liver tissue from patients with alcoholic liver disease.

(A) Representative WB and densitometry quantification for Igfbp3 and SerpinA12 from plasma from control subjects and with alcoholic hepatitis. Subjects with alcoholic hepatitis have significative higher levels of Igfbp3 and significative lower levels of SerpinA12 compared to controls (n=9, p<0.05, unpaired t-test). (B) Schematic representation of the proposed mechanism of hepatic stellate cells (HSC)-activation-induced steatosis. Activated HSC increase Insulin-like growth factor-binding protein 3 (Igfbp3) and reduces SerpinA12 secretion, which have a paracrine effect over hepatocytes. Igfbp3 increases p-Akt signaling through integrin receptor leading to lipid droplets formation, triglyceride content, and lipogenic gene expression; EtOH reduces p-AMPK, increasing lipogenesis. SerpinA12 protects against ethanol-induced steatosis through increasing p-AMPK.