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

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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 (LysMCreCd39fl/fl) were generated. Global CD39 null (Cd39−/−), wild-type (WT), LysMCreCd39fl/fl, and Cd39fl/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 LysMCreCd39fl/fl mice recapitulated the phenotype of global Cd39−/−, after exposure to DDC, and resulted in similar worsening of liver fibrosis when compared to Cd39fl/fl control animals. Further, DDC-treated LysMCreCd39fl/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 LysMCreCd39fl/fl and Cd39fl/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.

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Abbreviations

ALP:

alkaline phosphatase

ALT:

alanine aminotransferase

α-Sma:

α-smooth muscle actin

Col1a1:

Collagen type 1 alpha 1

DDC:

3,5-diethoxycarbonyl-1,4-dihydrocollidine

ECM:

extracellular matrix

HSCs:

hepatic stellate cells

KO:

knockout

Mdr2:

multidrug resistance protein 2

PSC:

primary sclerosing cholangitis

qRT-PCR:

real-time quantitative polymerase chain reaction

TBIL:

total bilirubin

Tgf-β:

transforming growth factor β

Tnf-α:

tumor necrosis factor α

WT:

wild-type

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Acknowledgements

This study was supported by a research award from the Dept. of the Army USAMRAA (W81XWH-15-PRMRP-FPA; DoD) and a NIH grant (5R01DK108894-02) to S.C.R.

S.R. was a recipient of career development award from the Swiss National Science Foundation (P300PB_161098).

A research grant from PSC Partners Seeking a Cure Canada to Y.P. supported this work.

Funding

This study was supported by a research award from the Dept. of the Army USAMRAA (W81XWH-15-PRMRP-FPA; DoD) and a NIH grant (5R01DK108894-02) to S.C.R.

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Correspondence to Yury V. Popov or Simon C. Robson.

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“All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.”

S.R. was a recipient of career development award from the Swiss National Science Foundation (P300PB_161098).

A research grant from PSC Partners Seeking a Cure Canada to Y.P. supported this work.

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The authors declare that they have no conflict of interest.

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Supplementary Fig. 1

CD39 disruption enhances expression of fibrotic markers upon DDC treatment. Gene expression analysis using quantitative RT-PCR in whole livers of control Cd39−/− (n = 3) and DDC-fed WT (n = 5) and Cd39−/− (n = 8) mice. Values are shown as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01 (PNG 32 kb)

High Resolution Image (EPS 984 kb)

Supplementary Fig. 2

CD39 deficiency on macrophages does not affect macrophage numbers under normal conditions or in response to DDC feeding. a Hepatic mRNA expression of the macrophage marker Cd68 was quantified by qPCR in control LysMCreCd39fl/fl (n = 4) and Cd39fl/fl (n = 3) mice. Values are shown as mean ± SEM. b Representative immunohistochemistry images for macrophage marker F4/80 stained in livers from 3 weeks DDC-fed LysMCreCd39fl/fl and Cd39fl/fl mice (original magnification, ×200). Brown pigments indicate porphyrin accumulation. (PNG 395 kb)

High Resolution Image (TIF 88261 kb)

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Rothweiler, S., Feldbrügge, L., Jiang, Z.G. et al. Selective deletion of ENTPD1/CD39 in macrophages exacerbates biliary fibrosis in a mouse model of sclerosing cholangitis. Purinergic Signalling 15, 375–385 (2019). https://doi.org/10.1007/s11302-019-09664-3

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