Therapeutic targeting of adipose tissue macrophages ameliorates liver fibrosis in non-alcoholic fatty liver disease

Abstract

Background & aims: : The accumulation of adipose tissue macrophages (ATMs) in obesity has been associated with hepatic injury. However, the contribution of ATMs to hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD) remains to be elucidated. Herein, we investigate the relationship between ATMs and liver fibrosis in patients with patients with NAFLD and evaluate the impact of modulation of ATMs over hepatic fibrosis in an experimental non-alcoholic steatohepatitis (NASH) model.

Methods: Adipose tissue and liver biopsies from 42 patients with NAFLD with different fibrosis stages were collected. ATMs were characterised by immunohistochemistry and flow cytometry and the correlation between ATMs and liver fibrosis stages was assessed. Selective modulation of the ATM phenotype was achieved by i.p. administration of dextran coupled with dexamethasone in diet-induced obesity and NASH murine models. Chronic administration effects were evaluated by histology and gene expression analysis in adipose tissue and liver samples. In vitro crosstalk between human ATMs and hepatic stellate cells (HSCs) and liver spheroids was performed.

Results: Patients with NAFLD presented an increased accumulation of pro-inflammatory ATMs that correlated with hepatic fibrosis. Long-term modulation of ATMs significantly reduced pro-inflammatory phenotype and ameliorated adipose tissue inflammation. Moreover, ATMs modulation was associated with an improvement in steatosis and hepatic inflammation and significantly reduced fibrosis progression in an experimental NASH model. In vitro, the reduction of the pro-inflammatory phenotype of human ATMs with dextran-dexamethasone treatment reduced the secretion of inflammatory chemokines and directly attenuated the pro-fibrogenic response in HSCs and liver spheroids.

Conclusions: Pro-inflammatory ATMs increase in parallel with fibrosis degree in patients with NAFLD and their modulation in an experimental NASH model improves liver fibrosis, uncovering the potential of ATMs as a therapeutic target to mitigate liver fibrosis in NAFLD.

Impact and implications: We report that human adipose tissue pro-inflammatory macrophages correlate with hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD). Furthermore, the modulation of adipose tissue macrophages (ATMs) by dextran-nanocarrier conjugated with dexamethasone shifts the pro-inflammatory phenotype of ATMs to an anti-inflammatory phenotype in an experimental murine model of non-alcoholic steatohepatitis. This shift ameliorates adipose tissue inflammation, hepatic inflammation, and fibrosis. Our results highlight the relevance of adipose tissue in NAFLD pathophysiology and unveil ATMs as a potential target for NAFLD.

Keywords: Adipose tissue inflammation; Dextran dexamethasone conjugates; Drug delivery; Liver injury; Nanomedicine; Nanoparticle; Non-alcoholic steatohepatitis; Targeted therapy.

Graphical abstract

Fig. 1

Pro-inflammatory ATMs increase with hepatic fibrosis in patients with NAFLD. (A) Immunohistochemical analysis of ATMs in the adipose tissue and hepatic fibrosis assessment by trichrome or α-SMA staining in liver biopsies. Scale bar: 200 μm. (B) Correlation of percentage of ATMs with the BMI, body weight, and waist circumference of patients with NAFLD: F0 (n = 20); F1–F2 (n = 6) and F3–F4 (n = 12). (C) Quantification of the percentage of pro-inflammatory (CD14⁺CD11c⁺); anti-inflammatory (CD14⁺CD163⁺) and intermediate (CD14⁺CD11c⁺CD163⁺) ATMs in patients with NAFLD with different fibrosis stages: F0 (n = 7) and F3–F4 (n = 7). (D) Immunohistochemical analysis of CD163 and CD11c staining in adipose tissue F0 (n = 15) and F≥2 (n = 16). Data represented as mean ± SD. Scale bar: 100 μm. Correlations were performed with Spearman’s correlation. The Mann–Whitney test was used to compare patients at different stages. ∗p <0.05, ∗∗p <0.01 by ANOVA (A), Mann–Whitney test (C, D). α-SMA, α-smooth muscle actin; AT, adipose tissue; ATM, adipose tissue macrophages; FOV, field of view; NAFLD, non-alcoholic fatty liver disease.

Fig. 2

Dextran nanocarriers are captured by macrophages and predominantly retained in the adipose tissue of the NASH animals. (A) Representative immunofluorescence of dextran 500 (green) phagocytised by macrophages (F4/80, red) in the liver and adipose tissue and in isolated macrophages in obese and NASH models. Scale bar: 20 μm. (B) Flow cytometry analysis of the dextran (F4/80⁺CD11b⁺dextran⁺) captured by isolated macrophages from liver and adipose tissue of obese and NASH model animals (n = 5, each group). Histogram of mean intensity fluorescence of the dextran signal captured by macrophages is also shown. Data represented as mean ± SD. (C) Representative immunofluorescence of dextran 500 (green) and anti-inflammatory ATMs (CD163, red); pro-inflammatory ATMs (CD11c, red) or lipid associated macrophages (CD9, red) in adipose tissue in NASH mice. Scale bar: 100 μm. ANOVA test. ∗∗p <0.01. AT, adipose tissue; ATMs, adipose tissue macrophages; D500, Dextran 500 kDa; HepMs, hepatic macrophages; NASH, non-alcoholic steatohepatitis.

Fig. 3

Chronic dextran-dexamethasone treatment modulates pro-inflammatory ATMs and adipose tissue inflammation in obese and NASH models. (A) Experimental design of the model protocol. (B) Body weight measurements during the experimental protocol in the obese and NASH models. The arrow indicates the beginning of the treatment. Body weight and peritoneal fat pads were weighed at the time of the euthanisation of all experimental groups. (C) Relative number of adipocytes and the average area of adipocytes per field of view (FOV). (D) Relative gene expression of key lipid metabolic and inflammatory markers in the adipose tissue of obese and NASH models treated with FD or DD. Expression values are represented as fold change related to FD group (E) Representative immunohistochemical staining of F4/80 in the adipose tissue of treated NASH and obese animals (FD or DD) and its quantification. (F) Representative flow cytometry plots and histograms showing the pro-inflammatory ATMs subset (F4/80⁺CD11b⁺CD11c⁺) of the obese and NASH animals after treatment (FD or DD) and their quantification. (G) Representative immunohistochemical staining of CD9 in the AT of NASH animals (FD or DD). Quantification of %CD9 positive area in all experimental animal groups: obese FD (n = 10), obese DD (n = 7); NASH FD (n = 10), and NASH DD (n = 9). Data represented as mean ± SD. Scale bar: 100 μm ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 by the Mann–Whitney test. DD, dextran-dexamethasone; FD, free dextran; FOV, field of view; NASH, non-alcoholic steatohepatitis.

Fig. 4

Long-term dextran-dexamethasone administration reduces hepatic inflammation without affecting the phenotype of liver macrophages. (A) Representative haematoxylin and eosin staining of liver sections of obese or NASH mice after DD treatment. Scale bar: 200 μm. Semi quantitative histological analysis of the steatosis grade, ballooning degree, lobular inflammation grade, and NAFLD activity score in each experimental group. (B) Gene expression levels measured by quantitative PCR of inflammatory markers in liver tissue. (C) Representative immunohistochemical staining of iNOS and immune cell populations such as macrophages (F4/80), neutrophils (MPO), and lymphocytes (CD3) are shown. Scale bar: 50 μm. (D) Determination of liver pro-inflammatory macrophages by flow cytometry determined by the F4/80⁺CD11b⁺CD11c⁺ population. (E) Expression analysis of pro-inflammatory genes in isolated liver macrophages. Experimental groups are: Obese FD (n = 10), Obese DD (n = 7); NASH FD (n = 10), and NASH DD (n = 9). Data represented as mean ± SD. Groups were compared using the Mann–Whitney test. ∗p <0.05; ∗∗p <0.01. DD, dextran-dexamethasone; FD, Free dextran; FOV, field of view; HepMs, hepatic macrophages; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase.

Fig. 5

Chronic dextran-dexamethasone conjugate treatment reduces hepatic fibrosis. (A) Representative images of the Sirius Red staining of liver sections of NASH and obese mice after treatment. Semi-quantitative analysis of pericellular fibrosis and quantification of positively stained area in all experimental groups are shown. (B) Immunohistochemical analysis of αSMA staining and quantification of the stained area in liver sections of obese and NASH mice treated with FD or DD. (C) Relative gene expression analysis of a panel of fibrogenic markers in liver tissue of animals in all experimental groups. Expression values are represented relative to the obese FD group. Animals were grouped in obese FD (n = 10), obese DD (n = 7); NASH FD (n = 10), and NASH DD (n = 9). Data are represented as mean ± SD. Scale bar: 100 μm. The Mann–Whitney test was used to compare experimental groups. ∗p <0.05 were consider significant; ∗∗p <0.01. DD, dextran-dexamethasone; FD, free dextran; NASH, non-alcoholic steatohepatitis.

Fig. 6

Human ATMs treated with dextran-dexamethasone conjugates decrease their pro-inflammatory phenotype and reduce hepatic stellate cell activation in vitro. (A) Flow cytometry analysis of isolated human ATMs after a 24-h treatment with FD or DD (500 nM). Percentages of CD11c and CD206 cells relative to all macrophages. Obese patients (n = 11) were used for this study. (B) Quantitative analysis of TNFα and IFNγ secretion by ATMs treated with FD or DD (500 nM) measured by ELISA. Replicates per group, n = 7. (C) Relative gene expression analysis of pro-inflammatory markers in human ATMs after FD or DD (500 nM) treatment under basal or inflammatory conditions (IFNγ + LPS). Replicates per group, n = 7. (D) Gene expression analysis of inflammatory and activation markers in iPSC-HSCs after culture with secretome of ATMs derived from patients without (F0) or with (F>2) fibrosis. Replicates per group n = 10. (E) Gene expression analyses of inflammatory and activation markers in iPSC-HSCs after incubation with conditioned medium (CM) of ATMs treated with FD or DD for 24 h. Replicates per group, n = 10. (F) Representative images of human liver spheroids, containing HepG₂ and iPSC-HSCs at a 2:1 ratio, cultured for 48 h with conditioned medium derived from ATMs treated with FD or DD. Scale bar: 100 μm. (G) Gene expression analyses of activation and inflammatory markers in liver spheroids cultured with conditioned medium obtained from human FD- or DD-treated ATMs. Replicates per group, n = 10. ATMs replicates are represented as single values whereas iPSC-HSCs data are represented as mean ± SD. The Wilcoxon test was performed for paired samples comparison (A, B, C) and the Mann–Whitney test was performed for unpaired samples analysis (D, E, G). ∗p <0.05; ∗∗p <0.01. ATMs, adipose tissue macrophages; CM, conditioned medium; DD, dextran–dexamethasone; FD, free dextran; hATMs, human adipose tissue macrophages; IFNγ, interferon gamma; iPSC-HSCs, hepatic stellate cells derived from induced pluripotent stem cells; TNFα, tumour necrosis factor alpha.