Chitinase 1: a novel therapeutic target in metabolic dysfunction-associated steatohepatitis

Abstract

Background: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by persistent inflammatory cascades, with macrophage activation playing a pivotal role. Chitinase 1 (CHIT1), produced by activated macrophages, is a key player in this cascade. In this study, we aimed to explore the role of CHIT1 in MASH with progressive liver fibrosis.

Methods: Fibrotic liver tissue and serum from distinct patient groups were analyzed using nCounter MAX, flow cytometry, immunohistochemistry, and enzyme-linked immunosorbent assay. A MASH mouse model was constructed to evaluate the effectiveness of OATD-01, a chitinase inhibitor. Macrophage profiling was performed using single-nuclei RNA sequencing and flow cytometry.

Results: CHIT1 expression in fibrotic liver tissues was significantly correlated with the extent of liver fibrosis, macrophages, and inflammation. Single-nuclei RNA sequencing demonstrated a notable increase in macrophages numbers, particularly of lipid-associated macrophages, in MASH mice. Treatment with OATD-01 reduced non-alcoholic fatty liver disease activity score and Sirius red-positive area. Additionally, OATD-01-treated mice had lower CHIT1, F4/80, and α-smooth muscle actin positivity, as well as significantly lower levels of inflammatory markers, pro-fibrotic genes, and matrix remodeling-related mRNAs than vehicle-treated mice. Although the population of F4/80⁺CD11b⁺ intrahepatic mononuclear phagocytes remained unchanged, their infiltration and activation (CHIT1⁺MerTK⁺) significantly decreased in OATD-01-treated mice, compared with that observed in vehicle-treated mice.

Conclusions: Our study underscores the pivotal role of CHIT1 in MASH. The observed significant improvement in inflammation and hepatic fibrosis, particularly at higher doses of the CHIT1 inhibitor, strongly suggests the potential of CHIT1 as a therapeutic target in MASH accompanied by progressive liver fibrosis.

Keywords: MASH mouse mod; chitinase 1; inflammation; metabolic dysfunction-associated steatohepatitis; mononuclear phagocyte.

Figure 1

CHIT1 is upregulated in patients with liver fibrosis and MASH with progressive liver fibrosis and correlates with disease severity. (A) Correlation analysis of hepatic CHIT1. (B) Hepatic CHIT1 expression in CD14⁺CD80⁺ macrophages. (C) Liver biopsies stained with hematoxylin and eosin, Masson’s trichrome, CD3, CD68, and CHIT1 (scale bar: 100 µm, 400×). Double IHC for CD68 (Magenta Red) and CHIT1 (DAB Brown) (scale bar: 50 µm). Correlation analysis of hepatic CHIT1 and CD68. (D) Serum CHIT1 and correlation of CHIT1 with NFS and platelets. (A and C-D) Pearson’s correlation test; (B and C-D) Two-tailed independent t-test; data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. CHIT1, chitinase 1; MASH, metabolic dysfunction-associated steatohepatitis; NFS, non-alcoholic fatty liver disease (NAFLD) fibrosis score; SD, standard deviation.

Figure 2

Single-nuclei RNA sequencing (sNuc-Seq) analysis of mice with MASH-related liver fibrosis. (A) Annotation of cell populations and uniform manifold approximation and projection (UMAP) clustering of integrated sNuc-Seq dataset. (B) UMAP plots of the five different macrophage subpopulations. (C) Violin plot of the expression of specific genes across various macrophage subpopulations. (D) UMAP plots of the genes associated with the lipid-associated macrophage (LAM) subtype (Mmp12, Gpnmb, and Cd36) in cluster 3 lineage. MASH, metabolic dysfunction-associated steatohepatitis.

Figure 3

CHIT1 inhibition ameliorates liver injury in mice with MASH-related liver fibrosis. (A) Development of the MASH mouse model using streptozotocin and a high-fat, high-cholesterol diet. (B) Serum alanine aminotransferase and aspartate aminotransferase levels at 6 and 10 weeks. (C) NAS from mouse liver specimens (n = 8–12 per group). (D) Hematoxylin and eosin (scale bar: 200 µm, 200×) and Sirius red (scale bar: 100 µm, 400×) staining of liver tissues. Two-tailed unpaired independent t-test; data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. CHIT1, chitinase 1; MASH, metabolic dysfunction-associated steatohepatitis; NAS, non-alcoholic fatty liver disease activity score; SD, standard deviation.

Figure 4

CHIT1 inhibition attenuates hepatic inflammation and fibrosis in mice with MASH-related liver fibrosis. (A) CHIT1, F4/80, and α-SMA immunohistochemistry (scale bar: 100 µm, 400×). Correlation analysis of hepatic CHIT1 and α-SMA (Pearson’s correlation test). (B-D) Hepatic mRNA levels of genes related to inflammation, fibrosis, and matrix remodeling. (E) Expression of proteins involved in inflammation. (A-E) Two-tailed unpaired independent t-test; data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. CHIT1, chitinase 1; MASH, metabolic dysfunction-associated steatohepatitis; α-SMA, alpha smooth muscle actin; β2M, beta 2 microglobulin; SD, standard deviation.

Figure 5

CHIT1 inhibition alters the phenotype of activated mononuclear phagocytes (MPs) in MASH. (A) Dot plot illustrating the proportions of recruited/resident MPs. (B) Recruited (F4/80^low CD11b^high) and resident (F4/80^high CD11b^low) MP number per g liver weight and cell percentage in vehicle or OATD-01-treated STZ-injected, HFHC diet-fed MASH mice. (C) CHIT1⁺, MerTK⁺, and CHIT1⁺MerTK⁺ cell number per g liver weight. Frequency of CHIT1⁺MerTK⁺ intrahepatic MP cells in STZ-injected, HFHC diet-fed MASH mice with or without OATD-01 treatment. Two-tailed unpaired independent t-test; data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Correlation analysis of hepatic CHIT1⁺ and MerTK⁺ MPs (Pearson’s correlation test). CHIT1, chitinase 1; MASH, metabolic dysfunction-associated steatohepatitis; STZ, streptozocin; HFHC, high-fat and high-cholesterol; MerTK, MER Proto-Oncogene, Tyrosine Kinase; SD, standard deviation.