Selective deletion of ENTPD1/CD39 in macrophages exacerbates biliary fibrosis in a mouse model of sclerosing cholangitis

Abstract

Purinergic signaling is important in the activation and differentiation of macrophages, which play divergent roles in the pathophysiology of liver fibrosis. The ectonucleotidase CD39 is known to modulate the immunoregulatory phenotype of macrophages, but whether this specifically impacts cholestatic liver injury is unknown. Here, we investigated the role of macrophage-expressed CD39 on the development of biliary injury and fibrosis in a mouse model of sclerosing cholangitis. Myeloid-specific CD39-deficient mice (LysMCreCd39^fl/fl) were generated. Global CD39 null (Cd39^-/-), wild-type (WT), LysMCreCd39^fl/fl, and Cd39^fl/fl control mice were exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce biliary fibrosis. Hepatic hydroxyproline levels, liver histology, immunohistochemistry, mRNA expression levels, and serum biochemistry were then assessed. Following 3 weeks of DDC-feeding, Cd39^-/- mice exhibited more severe fibrosis, when compared to WT mice as reflected by morphology and increased liver collagen content. Myeloid-specific CD39 deletion in LysMCreCd39^fl/fl mice recapitulated the phenotype of global Cd39^-/-, after exposure to DDC, and resulted in similar worsening of liver fibrosis when compared to Cd39^fl/fl control animals. Further, DDC-treated LysMCreCd39^fl/fl mice exhibited elevated serum levels of transaminases and total bilirubin, as well as increased hepatic expression of the profibrogenic genes Tgf-β1, Tnf-α, and α-Sma. However, no clear differences were observed in the expression of macrophage-elaborated specific cytokines between LysMCreCd39^fl/fl and Cd39^fl/fl animals subjected to biliary injury. Our results in the DDC-induced biliary type liver fibrosis model suggest that loss of CD39 expression on myeloid cells largely accounts for the exacerbated sclerosing cholangitis in global CD39 knockouts. These findings indicate that macrophage expressed CD39 protects from biliary liver injury and fibrosis and support a potential therapeutic target for human hepatobiliary diseases.

Keywords: CD39; Kupffer cells; Liver fibrosis; Primary sclerosing cholangitis; Purinergic signaling.

Fig. 1

CD39 deficiency worsens liver fibrosis in the DDC-induced model of biliary fibrosis. a Representative images of Sirius Red stained liver sections from WT and CD39 KO mice after 3 weeks of DDC feeding (original magnification × 40 in the upper and × 100 in the lower panel). b, c Relative hepatic collagen content (μg/g liver), serum ALT, ALP, and TBIL in age-matched WT and CD39 null mice after 3 weeks DDC administration or standard chow diet. Data are shown as mean ± SEM (n = 5–8 animals per bar). **P ≤ 0,001. d Liver sections from WT, Mdr2^−/−, and DDC-fed WT mice were subjected to immunohistochemistry for CD39 (upper panel) and F4/80 (lower panel) (original magnification × 20)

Fig. 2

Generation and characterization of macrophage specific CD39 KO mice (LysMCreCd39^fl/fl). a Schematic illustration of gene deletion strategy. Mice carrying the Cd39 gene construct with 2 loxP sites flanking exon 5 and 6 were crossed with LysMCre mice to generate LysMCreCd39^fl/fl mice. b LysMCreCd39^fl/fl mice show expression of both, LysMCre and CD39 floxed fragments, monitored by polymerase chain reaction. c Flow cytometry analysis of peritoneal macrophages (F4/80⁺CD11b⁺) confirms effective Cre-mediated CD39 deletion in LysMCreCd39^fl/fl mice. Cd39^fl/fl and Cd39^−/− mice were included as positive and negative controls. Histogram shows CD39 expression in gated macrophages. Blue: Cd39^fl/fl, red: Cd39^−/−, green: LysMCreCd39^fl/fl. d Flow cytometry analysis of splenocytes from LysMCreCd39^fl/fl mice shows CD39 expression in splenic B cells (B220⁺CD19⁺) from Cd39^fl/fl (positive control) and Cd39^−/− (negative control) mice. Representative histogram showing expression of CD39 in B cells of Cd39^fl/fl (blue), Cd39^−/− (red), and LysMCreCd39^fl/fl (green) mice. e Immunofluorescence staining for F4/80 (green) and CD39 (red) in frozen liver sections shows specific CD39 deletion in Kupffer cells from LysMCreCd39^fl/fl mice, but not in endothelial cells. Kupffer cells and endothelial cells in Cd39^fl/fl livers show CD39 expression (original magnification, × 200)

Fig. 3

Loss of CD39 on macrophages increases biliary fibrosis and liver injury in the DDC-fed liver fibrosis model. a Representative images of Sirius Red stained liver sections from Cd39^fl/fl and LysMCreCd39^fl/fl mice after 3 weeks of DDC feeding (original magnification × 40 in the upper panel and ×200 in the lower panel). b, c Relative hepatic collagen content (μg/g liver), serum ALT, ALP, and TBIL in age-matched WT and CD39 null mice after 3 weeks DDC administration or standard chow diet. Data are shown as mean ± SEM (n = 5–8 animals per bar). *P ≤ 0.05, ***P ≤ 0.001

Fig. 4

Loss of CD39 on macrophages increases biliary fibrosis and causes more liver injury in the DDC-fed liver fibrosis model. a qRT-PCR was performed to test the expression of the pro-fibrogenic genes Tgf-β1, Tnf-α, α-Sma, and Col1a1 in whole liver tissue. b Representative pan-cytokeratin (p-CK) immunohistochemistry of liver sections from DDC fed Cd39^fl/fl and LysMCreCd39^fl/fl mice and quantification of pan-CK staining (10 random portal fields, n = 3, × 40). Statistical significance was assessed using unpaired t-test with *P ≤ 0.05

Fig. 5

Profile expression patterns of macrophage cytokines are comparable in livers of LysMCreCd39^fl/fl and control mice. Expression of Ifng, Il1b, Il6, iNos, Arg1, and Il10 mRNA levels was measured in DDC-fed LysMCreCd39^fl/fl and Cd39^fl/fl mice by qRT-PCR. Values are presented as mean ± SEM. ns, not significant (t test)