Heme oxygenase-1 mitigates liver injury and fibrosis via modulation of LNX1/Notch1 pathway in myeloid cells

Abstract

Activation of resident macrophages (Mϕ) and hepatic stellate cells is a key event in chronic liver injury. Mice with heme oxygenase-1 (HO-1; Hmox1)-deficient Mϕ (LysM-Cre:Hmox1 ^flfl ) exhibit increased inflammation, periportal ductular reaction, and liver fibrosis following bile duct ligation (BDL)-induced liver injury and increased pericellular fibrosis in NASH model. RiboTag-based RNA-sequencing profiling of hepatic HO-1-deficient Mϕ revealed dysregulation of multiple genes involved in lipid and amino acid metabolism, regulation of oxidative stress, and extracellular matrix turnover. Among these genes, ligand of numb-protein X1 (LNX1) expression is strongly suppressed in HO-1-deficient Mϕ. Importantly, HO-1 and LNX1 were expressed by hepatic Mϕ in human biliary and nonbiliary end-stage cirrhosis. We found that Notch1 expression, a downstream target of LNX1, was increased in LysM-Cre:Hmox1 ^flfl mice. In HO-1-deficient Mϕ treated with heme, transient overexpression of LNX1 drives M2-like Mϕ polarization. In summary, we identified LNX1/Notch1 pathway as a downstream target of HO-1 in liver fibrosis.

Keywords: Biological sciences; Cell biology; Human physiology; Stem cells research.

Graphical abstract

Figure 1

The role of myeloid-cell-expressed HO-1 in the BDL murine model of liver fibrosis (A) Immunohistochemical (IHC) staining with antibodies against HO-1 in the liver samples from control and BDL-treated mice showing staining in sinusoidal macrophages. Scale bar: 100 μm. (B) Western blot analysis of HO-1 and E-cadherin expression in liver lysates from mice untreated (naive) or subjected to BDL surgery (17 days post-surgery, BDL). (C) Percentage of liver weights change per body weights (BW) of Hmox1^flfl (WT) or LysM-Cre:Hmox1^flfl (KO) mice, which were untreated or subjected to BDL surgery, 11 or 17 days post-surgery. ANOVA, p < 0.0001, Student’s t test: ∗∗∗p < 0.001, ∗p < 0.05. Data are represented as mean ± SEM n = 5–18 female and male mice per group. (D) Hydroxyproline levels in the livers exposed to BDL surgery as described in (A) and harvested 17 days after BDL. ANOVA, p = 0.0376, Student’s t test: ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SEM. (E–H) Lobular necrosis (H &E, E) and fibrosis (Sirius red, G) in the livers from mice treated as in (A). Representative sections are shown from BDL-treated mice in (E) and (G) (100x magnification) and quantification of the data is presented in (F) and (H). Data are represented as mean ± SEM. ANOVA, p < 0.0001, Tukey’s multiple comparison test: ∗∗p < 0.01, ∗∗∗p < 0.001. Scale bar: 200 μm.

Figure 2

Deletion of HO-1 in macrophages leads to enhanced ductal reaction (A–C) RT-PCR using the total liver mRNA and primers for COL1 (A), TGFβ (B), and ACTA2 (C). Data are represented as mean ± SEM. ANOVA, p < 0.0001, Tukey’s multiple comparison test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D–G) IHC staining with antibodies against E-cadherin (D) or CK-19 (F) in the liver samples from control and BDL-treated mice. Magnification of 200× (upper panels) or 400x (lower panels). Quantification of the data is shown in (E) and (G). Data are represented as mean ± SEM. ANOVA, p < 0.0001, Tukey’s multiple comparison test: ∗∗∗p < 0.001. Scale bar: 50 μm.

Figure 3

Deletion of HO-1 in myeloid cells impacts Mφ phenotype as assessed by RiboTag approach (A and B) Liver lysates from LysM-Cre:RiboTag mice were subjected to immunoprecipitation (IP) with anti-HA antibody. A scheme of the technology is shown in (A). Myeloid markers were assessed by RT-PCR and are shown in (B). Data are represented as mean ± SEM. (C–J) LysM-Cre:RiboTag or LysMCre:Hmox1^flfl: RiboTag mice were subjected to BDL surgery as described in Figure 1. RT-PCR was performed on the RNA from the IP with anti-HA antibody. Data are represented as mean ± SEM. ANOVA, p < 0.0001, Tukey’s multiple comparison test: ∗∗∗p < 0.001, ∗∗p < 0.01. n = 5–18 female and male mice per group.

Figure 4

LNX1 is the HO-1 target gene in myeloid cells (A) Gene clustering based on the RNA-seq on the RNA obtained from the IP with anti-HA antibody from the liver lysates of LysM-Cre:RiboTag (WT) and LysMCre:Hmox1^flfl:RiboTag (HO-1^KO−M) subjected to BDL surgery as in Figure 1. n = 2 mice per group. (B–G) Confirmatory RT-PCR using the RNA isolated as in (A), assessing the top target genes as identified by RNA-seq (17 days after BDL). Data are represented as mean ± SEM. ANOVA, p < 0.0001, Dunn’s multiple comparison test: ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. n = 5–18 female and male mice per group. (H–J) Immunohistochemical staining of LNX1 in the liver samples from control and BDL-treated mice. Quantification of the data is shown in (I) and (J). Data are represented as mean ± SD. ANOVA, p < 0.01, Student’s t test: ∗∗p < 0.01, ∗p < 0.05. Scale bar: 100 μm. (L–N) Immunohistochemical staining of Aco1/Irp1 in the liver samples from control and BDL-treated mice. Quantification of the data is shown in (I) and (J). Data are represented as mean ± SD. ANOVA, p < 0.01, Student’s t test: ∗p < 0.05. Scale bar: 100 μm.

Figure 5

Detection of HO-1⁺ and LNX1⁺ cells in the human samples with increasing fibrosis stage and assessment of the role of HO-1 in myeloid cells in fibrosis progression in murine NASH model (A–D) IHC staining with antibodies against LNX1 or HO-1 in (A) human liver biopsies from n = 5 patients with various stages of NAFLD/NASH with the clinical characteristic as described in STAR Methods. (B–D) Human liver explants obtained from patients undergoing orthotopic liver transplantation due to end-stage liver disease of various etiologies (n = 3 PBC, n = 5 Alc, n = 5 Crypt) (Peng et al., 2016). Representative staining in myeloid cells/macrophages is shown in (A) and (B). Scale bar: 100 μm (A) and 50 μm (B). Quantification of the HO-1⁺ and LNX1⁺ cells are shown in (C) and (D). Data are represented as mean ± SEM. ANOVA, not significant. (E–G) Hmox1^flfl (Hmox1^flfl) or LysM-Cre:Hmox1^flfl (Cre:Hmox1^flfl) mice were fed with methionine- and choline-deficient (MCD) diet for 3 weeks. Livers were evaluated for fibrosis and fatty scores based on the Sirius red staining and H&E. Data are represented as mean ± SEM. Student’s t test: ∗p < 0.05. n = 10 mice/group. Scale bar: 100 μm.

Figure 6

The role of LNX1 in Mφ (A–C) Bone-marrow-derived Mφ (BMDM) were isolated from Hmox1^flfl or LysM-Cre:Hmox1^flfl and treated with heme (1–50 μM) for 24 h. Protein analyses were performed with antibodies against HO-1, LNX1, and Aco1/IRP1. Quantification of LNX1 expression is shown in (C). Data are represented as mean ± SEM. ANOVA, p = 0.144, Student’s t test: ∗∗p < 0.01. (D–G) LNX1 was overexpressed in RAW264.7 Mφ by transient transfection. Western blot with antibody against LNX1 is shown in (D). RT-PCR analyses with indicated primers in the RAW264.7 Mφ with transient overexpression of LNX1 and treated with heme (50 μM) for 6 h are shown in (E–G). Data are represented as mean ± SEM. ANOVA, p < 0.01, Tukey’s multiple comparison test: ∗p < 0.05, ∗∗p < 0.01.

Figure 7

A crosstalk of HO-1 and Notch1 signaling in Mφ (A and B) Immunostaining with antibody against Notch1 total was performed in the livers isolated from Hmox1^flfl or LysM-Cre:Hmox1^flfl (Cre:Hmox1^flfl) mice after BDL surgery. Scale bar: 100 μm (insets: 400x magnification). The quantification of a number of Notch1+ cells is shown in (B). Data are represented as mean ± SEM. ANOVA, p < 0.001, Tukey’s multiple comparison test: ∗p < 0.05, ∗∗∗p < 0.001. (C–E) LysM-Cre:RiboTag (Hmox1^flfl) or LysMCre:Hmox1^flfl: RiboTag (Cre:Hmox1^flfl) mice were subjected to BDL surgery as in Figure 2. RT-PCR was performed using RNA isolated from the immunoprecipitates with the anti-HA antibody. Data are represented as mean ± SEM. ANOVA, p < 0.0001, Tukey’s multiple comparison test: ∗∗∗p < 0.001, ∗∗p < 0.01. n = 5–18 female and male mice per group. (F) Scheme representing the interaction of LNX1 in HO-1 and regulation of Mφ phenotype during liver injury.