Fig 1: Significant Expression of CCL24 and its receptor observed in the livers and blood of patients with NAFLD/NASH.(A) Representative pictures of liver histological sections stained for CCL24 (magnification ×40, biopsies were taken from NASH and healthy livers n = 10 per group). (B) Representative pictures from double staining of CCL24 and CD68 (macrophage marker) in patients with NASH (magnification ×63, n = 10, staining done on TMA). (C) Representative pictures of liver histological samples from TMA stained for CCR3 and a-SMA (total of n = 7 samples from NASH, n = 4 from healthy livers, magnification ×10). (D) CCR3 expression in epithelial cells and hepatocytes (magnification ×40). (E) Co-staining of CCR3 and a-SMA in patients with NASH, liver biopsies (magnification ×100). (F) Quantitative analysis of CCL24 in sera from healthy donors (n = 20) and patients with NAFLD (FIB-4 =1.45 n = 27, FIB-4 =1.45 n = 23). (G) Quantitative analysis of CCR3 in PBMCs from healthy donors (n = 22) and patients with NAFLD (n = 35); Results are presented as average ± SE; Student's t test; *p =0.05; ***p =0.001. FIB-4, Fibrosis-4 score; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PBMCs, peripheral blood mononuclear cells.
Fig 2: Ccl24 knockout mice exhibited an attenuated response to the MCD diet-induced liver damage.(A) Representative H&E stained histological liver samples. Healthy liver from WT control mice (upper panels), MCD-fed WT mice (Middle panels) and MCD-fed Ccl24 knockout (bottom panels). (B) Histological scoring, comparisons of the 2 MCD-fed groups (WT n = 11 and Ccl24 knockout n = 8). (C-F) NAS, ALT, AST and bilirubin levels in MCD-induced Ccl24 knockout mice compared to WT mice. (G) Average percentage weight change in normal, MCD diet-fed WT and Ccl24 knockout mice. Results are presented as average ± SE; Student's t test; *p =0.05, **p =0.01, ***p =0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; MCD, methionine-choline deficient; NAS, non-alcoholic fatty liver disease activity score; WT, wild-type.
Fig 3: Anti-CCL24 antibody (CM-101 (D8)) reduced NASH-related pathologies in the MCD-diet mouse model (treatment mode).(A) Representative H&E stained histological liver samples. Healthy liver from chow diet-fed mice (upper panels), MCD-fed WT mice (middle panels) and MCD + 5 mg/kg CM-101 (D8) treated group (bottom panels). (B) Histological scoring, comparisons of the 2 groups fed with the MCD diet (MCD diet and MCD diet + 5 mg/kg CM-101 (D8), n = 8). (C-E) AST, ALT and bilirubin levels in the MCD-induced NASH model compared to MCD + 5 mg/kg CM-101 (D8) treated group. (F) CCL24 levels in the liver measured by ELISA using total protein liver lysates from chow diet-fed mice, MCD-fed WT mice and MCD + 5 mg/kg CM-101 (D8) treated mice. Results are presented as average ±SE; Student's t test; *p =0.05, **p =0.01, ***p =0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; MCD, methionine-choline deficient; NAS, non-alcoholic fatty liver disease activity score; WT, wild-type.
Fig 4: CM-101 suppressed CCL24-induced activation of HSCs in vitro.(A) Representative images and quantification of HSC (LX-2 cell line) scratch assay. Images of scratched wells were taken at time 0 h, 24 h and 48 h. Percentage closure of scratched area was evaluated for control group or cells treated with CCL24 (25 ng/ml) with or without CM-101 (5 µg/ml). (B) a-SMA expression measured by FACS following treatment with CCL24, with or without CM-101 (C) Pro-collagen I alpha secretion was quantified with a commercial ELISA kit, following activation of HSCs with CCL24 (100 ng/ml, 48 h incubation) with or without CM-101. Student's t test; *p =0.01, **p =0.005, ***p =0.001, each graph represents an average of 3 different experiments. HSCs, hepatic stellate cells; MFI, median fluorescence intensity.
Fig 5: CCL24 and CCR3 are highly expressed in the liver and on PBMCs of patients with PSC and correlate with fibrotic biomarkers.(A and B) CCL24 staining in PSC sections (×40 original magnification). Positive staining is detected in inflammatory mononuclear cells (red circle) and cholangiocytes (yellow circle). (C) CCR3 staining (×40 original magnification) observed in inflammatory cells (black arrowheads) or fibroblasts (blue circle) surrounding the bile duct. (D) CCR3 staining of PBMCs from PSC patients (n = 10) and healthy individuals (n = 22). Data are mean ± SEM. ****P = 0.0001, t test.
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