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Purinergic Signalling

, Volume 15, Issue 3, pp 375–385 | Cite as

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

  • Sonja Rothweiler
  • Linda Feldbrügge
  • Zhenghui Gordon Jiang
  • Eva Csizmadia
  • Maria Serena Longhi
  • Kahini Vaid
  • Keiichi Enjyoji
  • Yury V. PopovEmail author
  • Simon C. RobsonEmail author
Original Article

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.

Keywords

CD39 Liver fibrosis Primary sclerosing cholangitis Kupffer cells Purinergic signaling 

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

Notes

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.

Compliance with ethical standards

Ethical approval

“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.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11302_2019_9664_Fig6_ESM.png (32 kb)
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)

11302_2019_9664_MOESM1_ESM.eps (984 kb)
High Resolution Image (EPS 984 kb)
11302_2019_9664_Fig7_ESM.png (395 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)

11302_2019_9664_MOESM2_ESM.tif (86.2 mb)
High Resolution Image (TIF 88261 kb)
11302_2019_9664_MOESM3_ESM.doc (38 kb)
ESM 1 (DOC 38 kb)

References

  1. 1.
    Bataller R, Brenner DA (2005) Liver fibrosis. J Clin Invest 115:209–218.  https://doi.org/10.1172/JCI24282 CrossRefGoogle Scholar
  2. 2.
    O’Leary JG, Lepe R, Davis GL (2008) Indications for liver transplantation. Gastroenterology 134:1764–1776.  https://doi.org/10.1053/j.gastro.2008.02.028 CrossRefGoogle Scholar
  3. 3.
    Fickert P, Stöger U, Fuchsbichler A, Moustafa T, Marschall HU, Weiglein AH, Tsybrovskyy O, Jaeschke H, Zatloukal K, Denk H, Trauner M (2007) A new xenobiotic-induced mouse model of Sclerosing cholangitis and biliary fibrosis. Am J Pathol 171:525–536.  https://doi.org/10.2353/ajpath.2007.061133 CrossRefGoogle Scholar
  4. 4.
    Lazaridis KN, Strazzabosco M, LaRusso NF (2004) The cholangiopathies: disorders of biliary epithelia. Gastroenterology 127:1565–1577.  https://doi.org/10.1053/j.gastro.2004.08.006 CrossRefGoogle Scholar
  5. 5.
    Glaser SS, Gaudio E, Miller T, Alvaro D, Alpini G (2009) Cholangiocyte proliferation and liver fibrosis. Expert Rev Mol Med 11:e7.  https://doi.org/10.1017/S1462399409000994 CrossRefGoogle Scholar
  6. 6.
    Popov Y, Schuppan D (2009) Targeting liver fibrosis: strategies for development and validation of antifibrotic therapies. Hepatology 50:1294–1306.  https://doi.org/10.1002/hep.23123 CrossRefGoogle Scholar
  7. 7.
    Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11:723–737.  https://doi.org/10.1038/nri3073 CrossRefGoogle Scholar
  8. 8.
    Gordon S, Martinez FO (2010) Alternative activation of macrophages: mechanism and functions. Immunity 32:593–604.  https://doi.org/10.1016/j.immuni.2010.05.007 CrossRefGoogle Scholar
  9. 9.
    Martinez FO, Sica A, Mantovani A, Locati M (2008) Macrophage activation and polarization. Front Biosci 13:453–461  https://doi.org/10.2741/2692 CrossRefGoogle Scholar
  10. 10.
    Tacke F, Zimmermann HW (2014) Macrophage heterogeneity in liver injury and fibrosis. J Hepatol 60:1090–1096.  https://doi.org/10.1016/j.jhep.2013.12.025 CrossRefGoogle Scholar
  11. 11.
    Wynn TA, Barron L (2010) Macrophages: master regulators of inflammation and fibrosis. Semin Liver Dis 30:245–257.  https://doi.org/10.1055/s-0030-1255354 CrossRefGoogle Scholar
  12. 12.
    Braga TT, Agudelo JSH, Camara NOS (2015) Macrophages during the fibrotic process: M2 as friend and foe. Front Immunol 6.  https://doi.org/10.3389/fimmu.2015.00602
  13. 13.
    Rivera CA, Bradford BU, Hunt KJ, Adachi Y, Schrum LW, Koop DR, Burchardt ER, Rippe RA, Thurman RG (2001 Jul) (2001) attenuation of CCl4-induced hepatic fibrosis by GdCl3 treatment or dietary glycine. Am J Physiol Gastrointest Liver Physiol 281(1):G200–G207.  https://doi.org/10.1152/ajpgi.2001.281.1.G200 CrossRefGoogle Scholar
  14. 14.
    Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP (2005) Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Investig 115:56–65.  https://doi.org/10.1172/JCI22675 CrossRefGoogle Scholar
  15. 15.
    Best J, Verhulst S, Syn W-K, Lagaisse K, van Hul N, Heindryckx F, Sowa JP, Peeters L, van Vlierberghe H, Leclercq IA, Canbay A, Dollé L, van Grunsven LA (2016) Macrophage depletion attenuates extracellular matrix deposition and Ductular reaction in a mouse model of chronic Cholangiopathies. PLoS One 11:e0162286.  https://doi.org/10.1371/journal.pone.0162286 CrossRefGoogle Scholar
  16. 16.
    Idzko M, Ferrari D, Eltzschig HK (2014) Nucleotide signalling during inflammation. Nature 509:310–317.  https://doi.org/10.1038/nature13085 CrossRefGoogle Scholar
  17. 17.
    Cohen HB, Briggs KT, Marino JP, Ravid K, Robson SC, Mosser DM (2013) TLR stimulation initiates a CD39-based autoregulatory mechanism that limits macrophage inflammatory responses. Blood 122:1935–1945.  https://doi.org/10.1182/blood-2013-04-496216 CrossRefGoogle Scholar
  18. 18.
    Haskó G, Cronstein B (2013) Regulation of inflammation by adenosine. Front Immunol 4(85).  https://doi.org/10.3389/fimmu.2013.00085
  19. 19.
    Cekic C, Linden J (2016) Purinergic regulation of the immune system. Nat Rev Immunol 16:177–192.  https://doi.org/10.1038/nri.2016.4 CrossRefGoogle Scholar
  20. 20.
    Clausen BE, Burkhardt C, Reith W, Renkawitz R, Förster I (1999) Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 8:265–277CrossRefGoogle Scholar
  21. 21.
    Enjyoji K, Sévigny J, Lin Y, Frenette PS, Christie PD, Esch JSA, Imai M, Edelberg JM, Rayburn H, Lech M, Beeler DL, Csizmadia E, Wagner DD, Robson SC, Rosenberg RD (1999) Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. Nat Med 5:1010–1017.  https://doi.org/10.1038/12447 CrossRefGoogle Scholar
  22. 22.
    Peng Z, Rothweiler S, Wei G et al (2017) The ectonucleotidase ENTPD1/CD39 limits biliary injury and fibrosis in mouse models of sclerosing cholangitis. Hepatol Commun 1:957–972.  https://doi.org/10.1002/hep4.1084 CrossRefGoogle Scholar
  23. 23.
    Peng Z-W, Ikenaga N, Liu SB, Sverdlov DY, Vaid KA, Dixit R, Weinreb PH, Violette S, Sheppard D, Schuppan D, Popov Y (2016) Integrin αvβ6 critically regulates hepatic progenitor cell function and promotes Ductular reaction, fibrosis, and tumorigenesis. Hepatology 63:217–232.  https://doi.org/10.1002/hep.28274 CrossRefGoogle Scholar
  24. 24.
    Popov Y, Sverdlov DY, Sharma AK, Bhaskar KR, Li S, Freitag TL, Lee J, Dieterich W, Melino G, Schuppan D (2011) Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice. Gastroenterology 140:1642–1652.  https://doi.org/10.1053/j.gastro.2011.01.040 CrossRefGoogle Scholar
  25. 25.
    Jakubzick C, Bogunovic M, Bonito AJ, Kuan EL, Merad M, Randolph GJ (2008) Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes. J Exp Med 205:2839–2850.  https://doi.org/10.1084/jem.20081430 CrossRefGoogle Scholar
  26. 26.
    Pinsky DJ, Broekman MJ, Peschon JJ, Stocking KL, Fujita T, Ramasamy R, Connolly ES Jr, Huang J, Kiss S, Zhang Y, Choudhri TF, McTaggart RA, Liao H, Drosopoulos JHF, Price VL, Marcus AJ, Maliszewski CR (2002) Elucidation of the thromboregulatory role of CD39/ectoapyrase in the ischemic brain. J Clin Invest 109:1031–1040.  https://doi.org/10.1172/JCI10649 CrossRefGoogle Scholar
  27. 27.
    Gouw ASH, Clouston AD, Theise ND (2011) Ductular reactions in human liver: diversity at the interface. Hepatology 54:1853–1863.  https://doi.org/10.1002/hep.24613 CrossRefGoogle Scholar
  28. 28.
    Robson SC, Wu Y, Sun X, Knosalla C, Dwyer K, Enjyoji K (2005) Ectonucleotidases of CD39 family modulate vascular inflammation and thrombosis in transplantation. Semin Thromb Hemost 31:217–233.  https://doi.org/10.1055/s-2005-869527 CrossRefGoogle Scholar
  29. 29.
    Bono MR, Fernández D, Flores-Santibáñez F et al (2015) CD73 and CD39 ectonucleotidases in T cell differentiation: beyond immunosuppression. FEBS Lett 589:3454–3460.  https://doi.org/10.1016/j.febslet.2015.07.027 CrossRefGoogle Scholar
  30. 30.
    Lévesque SA, Kukulski F, Enjyoji K, Robson SC, Sévigny J (2010) NTPDase1 governs P2X7-dependent functions in murine macrophages. Eur J Immunol 40:1473–1485.  https://doi.org/10.1002/eji.200939741 CrossRefGoogle Scholar
  31. 31.
    Pellicoro A, Ramachandran P, Iredale JP, Fallowfield JA (2014) Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 14:181–194.  https://doi.org/10.1038/nri3623 CrossRefGoogle Scholar
  32. 32.
    Pradere J-P, Kluwe J, De Minicis S et al (2013) Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 58:1461–1473.  https://doi.org/10.1002/hep.26429 CrossRefGoogle Scholar
  33. 33.
    Thomas JA, Pope C, Wojtacha D, Robson AJ, Gordon-Walker TT, Hartland S, Ramachandran P, van Deemter M, Hume DA, Iredale JP, Forbes SJ (2011) Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function. Hepatology 53:2003–2015.  https://doi.org/10.1002/hep.24315 CrossRefGoogle Scholar
  34. 34.
    Bansal R, Nagórniewicz B, Prakash J (2016) Clinical advancements in the targeted therapies against liver fibrosis. In: Mediators of inflammation. https://www.hindawi.com/journals/mi/2016/7629724/. Accessed 14 Jan 2018
  35. 35.
    Lefebvre E, Moyle G, Reshef R, Richman LP, Thompson M, Hong F, Chou HL, Hashiguchi T, Plato C, Poulin D, Richards T, Yoneyama H, Jenkins H, Wolfgang G, Friedman SL (2016) Antifibrotic effects of the dual CCR2/CCR5 antagonist Cenicriviroc in animal models of liver and kidney fibrosis. PLoS One 11:e0158156.  https://doi.org/10.1371/journal.pone.0158156 CrossRefGoogle Scholar
  36. 36.
    Friedman SL, Ratziu V, Harrison SA et al (2018) A randomized, placebo-controlled trial of Cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis. Hepatology n/a-n/a.  https://doi.org/10.1002/hep.29477
  37. 37.
    Guicciardi ME, Trussoni CE, Krishnan A, Bronk SF, Lorenzo Pisarello MJ, O'Hara SP, Splinter PL, Gao Y, Vig P, Revzin A, LaRusso NF, Gores GJ (2018) Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice. J Hepatol 69:676–686.  https://doi.org/10.1016/j.jhep.2018.05.018 CrossRefGoogle Scholar
  38. 38.
    Krenkel O, Tacke F (2017) Liver macrophages in tissue homeostasis and disease. Nat Rev Immunol 17:306–321.  https://doi.org/10.1038/nri.2017.11 CrossRefGoogle Scholar
  39. 39.
    Bartneck M, Warzecha KT, Tacke F (2014) Therapeutic targeting of liver inflammation and fibrosis by nanomedicine. Hepatobiliary Surg Nutr 3:364–376.  https://doi.org/10.3978/j.issn.2304-3881.2014.11.02 Google Scholar
  40. 40.
    Wang N, Liang H, Zen K (2014) Molecular mechanisms that influence the macrophage M1–M2 polarization balance. Front Immunol 5.  https://doi.org/10.3389/fimmu.2014.00614
  41. 41.
    Eltzschig HK, Sitkovsky MV, Robson SC (2012) Purinergic signaling during inflammation. N Engl J Med 367:2322–2333.  https://doi.org/10.1056/NEJMra1205750 CrossRefGoogle Scholar
  42. 42.
    Morganti JM, Riparip L-K, Rosi S (2016) Call off the dog(ma): M1/M2 polarization is concurrent following traumatic brain injury. PLoS One 11:e0148001.  https://doi.org/10.1371/journal.pone.0148001 CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Sonja Rothweiler
    • 1
  • Linda Feldbrügge
    • 1
    • 2
  • Zhenghui Gordon Jiang
    • 1
  • Eva Csizmadia
    • 1
  • Maria Serena Longhi
    • 1
  • Kahini Vaid
    • 1
  • Keiichi Enjyoji
    • 1
  • Yury V. Popov
    • 1
    Email author
  • Simon C. Robson
    • 1
    Email author
  1. 1.Division of Gastroenterology and HepatologyBeth Israel Deaconess Medical Center and Harvard Medical SchoolBostonUSA
  2. 2.Department of Surgery, Charité Universitätsmedizin, Freie Universität BerlinHumboldt-Universität zu Berlin and Berlin Institute of HealthBerlinGermany

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