BACKGROUND AND AIM Congenital Hepatic Fibrosis (CHF) is a genetic liver disease caused by mutations in PKHD1, a gene encoding for Fibrocystin, a ciliary protein expressed by cholangiocytes. CHF is characterized by biliary dysgenesia associated with progressive portal fibrosis and portal hypertension. In CHF mechanisms of portal fibrosis are unknown. We have recently reported that Pkhd1-/- mice present: 1)activation of b-catenin signaling in cystic cholangiocytes; 2)increased pericystic infiltrate of CD45+/Collagen type1 (Col1)+ cells, reminiscent of fibrocytes. Fibrocytes are monocyte-derived cells, involved in several fibrosing conditions, but their role in liver scarring is controversial. b-catenin is emerging as a regulator of inflammation, therefore we hypothesized that a b-catenin-dependent chemokine production by cholangiocytes drives recruitment of fibrocytes in Pkhd1-/- mice. METHODS In Pkhd1-/- mice we investigated: a)a panel of 32 cyto/chemokines in both apical and basolateral medium of cultured polarized cholangiocytes (Luminex); b)the effects of two different b-catenin inhibitors (ICG-001, 25uM, or quercetin, 50uM) on the expression of CXCL1 and CXCL10 (RT-PCR); c)the immunohistochemical expression of CD45/Col1 (fibrocyte markers) and aSMA (myofibroblast marker) in the portal inflammatory cells, and their correlation with portal fibrosis (Sirius-red) in liver samples at 1-12 months; d)the effects of cholangiocyte conditioned media (CM) and CXCL1+CXCL10 on WEHI265.1-monocyte chemoattraction (Boyden chamber) and transdifferentiation into fibrocytes (RT-PCR for COL1(A1)). WT mice served as controls. RESULTS Pkhd1-/- cholangiocytes secreted significantly higher basolateral levels of CXCL1 and CXCL10 as compared to WT. Expression of CXCL1 and CXCL10 was significantly inhibited in cells treated with ICG-001 and quercetin. Pkhd1-/- mice showed progressive fibrosis, but portal accumulation of aSMA+ cells was evident only after 9 months. In contrast, we observed an early peribiliary recruitment of CD45+/Col1+ cells, whose number strongly correlated with the Sirius-red area (r=0.89,p<0.01). CM from Pkhd1-/- cholangiocytes, as well as CXCL1+CXCL10 stimulated both migration and expression of COL1(A1) mRNA in WEHI265.1 cells, consistent with transdifferentiation of fibrocytes. CONCLUSIONS In Pkhd1-/- mice progressive portal accumulation of CD45+/COL1+ cells (fibrocytes) correlates with portal fibrosis and cholangiocyte secretion of increased levels of CXCL1 and CXCL10 in a b-catenin-dependent way. This novel mechanism may underlay the recruitment of monocytes and their transdifferentiation into fibrocytes and consequently promote fibrosis deposition.
Fabris, L., Locatelli, L., Viganò, D., De Matteis, M., Fiorotto, R., Scirpo, R., et al. (2013). Β-Catenin-Mediated Cxcl1 and Cxcl10 Secretion by Fibrocystin-Defective Cholangiocytes Regulates Peribiliary Recruitment of Fibrocytes in Congenital Hepatic Fibrosis. In AASLD annual meeting 2013 (pp.1-1). Wiley.
Β-Catenin-Mediated Cxcl1 and Cxcl10 Secretion by Fibrocystin-Defective Cholangiocytes Regulates Peribiliary Recruitment of Fibrocytes in Congenital Hepatic Fibrosis
LOCATELLI, LUIGI;SCIRPO, ROBERTO;CADAMURO, MASSIMILIANO;STRAZZABOSCO, MARIO
2013
Abstract
BACKGROUND AND AIM Congenital Hepatic Fibrosis (CHF) is a genetic liver disease caused by mutations in PKHD1, a gene encoding for Fibrocystin, a ciliary protein expressed by cholangiocytes. CHF is characterized by biliary dysgenesia associated with progressive portal fibrosis and portal hypertension. In CHF mechanisms of portal fibrosis are unknown. We have recently reported that Pkhd1-/- mice present: 1)activation of b-catenin signaling in cystic cholangiocytes; 2)increased pericystic infiltrate of CD45+/Collagen type1 (Col1)+ cells, reminiscent of fibrocytes. Fibrocytes are monocyte-derived cells, involved in several fibrosing conditions, but their role in liver scarring is controversial. b-catenin is emerging as a regulator of inflammation, therefore we hypothesized that a b-catenin-dependent chemokine production by cholangiocytes drives recruitment of fibrocytes in Pkhd1-/- mice. METHODS In Pkhd1-/- mice we investigated: a)a panel of 32 cyto/chemokines in both apical and basolateral medium of cultured polarized cholangiocytes (Luminex); b)the effects of two different b-catenin inhibitors (ICG-001, 25uM, or quercetin, 50uM) on the expression of CXCL1 and CXCL10 (RT-PCR); c)the immunohistochemical expression of CD45/Col1 (fibrocyte markers) and aSMA (myofibroblast marker) in the portal inflammatory cells, and their correlation with portal fibrosis (Sirius-red) in liver samples at 1-12 months; d)the effects of cholangiocyte conditioned media (CM) and CXCL1+CXCL10 on WEHI265.1-monocyte chemoattraction (Boyden chamber) and transdifferentiation into fibrocytes (RT-PCR for COL1(A1)). WT mice served as controls. RESULTS Pkhd1-/- cholangiocytes secreted significantly higher basolateral levels of CXCL1 and CXCL10 as compared to WT. Expression of CXCL1 and CXCL10 was significantly inhibited in cells treated with ICG-001 and quercetin. Pkhd1-/- mice showed progressive fibrosis, but portal accumulation of aSMA+ cells was evident only after 9 months. In contrast, we observed an early peribiliary recruitment of CD45+/Col1+ cells, whose number strongly correlated with the Sirius-red area (r=0.89,p<0.01). CM from Pkhd1-/- cholangiocytes, as well as CXCL1+CXCL10 stimulated both migration and expression of COL1(A1) mRNA in WEHI265.1 cells, consistent with transdifferentiation of fibrocytes. CONCLUSIONS In Pkhd1-/- mice progressive portal accumulation of CD45+/COL1+ cells (fibrocytes) correlates with portal fibrosis and cholangiocyte secretion of increased levels of CXCL1 and CXCL10 in a b-catenin-dependent way. This novel mechanism may underlay the recruitment of monocytes and their transdifferentiation into fibrocytes and consequently promote fibrosis deposition.
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