Cathepsin D is essential for the correct collagenolytic activity of macrophages during cholestatic-induced liver fibrosis
| Autor: | Ruiz-Blázquez, Paloma, Fernández-Fernández, María, Pistorio, Valeria, Nuñez, Susana, García-Ruiz, Carmen, Pavone, Luigi Michele, Fernández-Checa, José C., Moles, Anna |
|---|---|
| Rok vydání: | 2022 |
| Popis: | Resumen del trabajo presentado en el 47º Congreso AEEH (Asociación Española para el Estudio del Hígado), celebrado en Madrid (España), del 9 al 11 de marzo de 2022 Background and Aims: Liver fibrosis is caused by an excessive accumulation of extracellular matrix (ECM) proteins. Macrophages are important effectors for ECM remodelling through phagocytosis and processing of the ECM within acidic compartments and can contribute to liver fibrosis resolution. Proteases, such as cathepsins, are essential for lysosomal proteolytic activity however, their contribution to ECM remodelling within the macrophages is unknown. Thus, the aim of this study was to investigate the proteolytic and degradative signalling pathways associated to macrophages during liver fibrosis. Method: To study the proteolytic and degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D. We generated and validated a novel macrophage-CtsD knock-out mouse strain by breeding LysMCre (macrophages) with CtsD floxed mice. Fibrosis was established by bile duct ligation at 7 and 14 days in CtsDF/F or CtsDΔMac mice and determined by hydroxyproline, α-SMA, Col1A1 and TGF-β RT-PCR in total liver. Collagen degradation and endocytosis was studied using DQ™ Collagen, type I and 10KDa Dextran probes, respectively and WB for Endo180 and UPAR in peritoneal macrophages. Lysosomal colocalization was demonstrated using LAMP2. Protease secretome was determined using Protease Array from R&D. Results: First, CtsD deletion in macrophages from CtsDΔ Mac mouse was confirmed by CtsD WB and gene expression in macrophages and dual IHP (F4/80-CtsD) in liver tissue. Of note, cathepsin B expression remained unaffected despite deletion of CtsD. CtsD deletion in macrophages, increased liver fibrosis after BDL as shown by an increase in liver hydroxyproline, and hepatic α-SMA, Col1A1 and TGF-β gene expression. No differences of liver damage were detected between CtsDF/F and CtsDΔMac after BDL. As expected CtsD activity increased only in CtsDF/F mice after BDL. Isolated CtsDΔ Mac macrophages showed a significant decrease in DQ™ Collagen, type I degradation versus CtsDF/F ones. Collagen degradative profile colocalized partially with LAMP2, indicating that collagen was degraded within the lysosome. Furthermore, Dextran endocytosis and Endo180 and UPAR collagen internalization receptors remained unaffected in both CtsDF/F and CtsDΔ Mac macrophages. Protease array in cell culture medium from CtsDF/F and CtsDΔ Ma c macrophages revealed CtsA, MMP7, MMP8 and MMP13 as part of CtsD proteolytic secretome. Conclusion: Lysosomal cathepsin D is essential for a correct collagenolytic activity displayed by macrophages during liver fibrosis and its absence contributes to the development of cholestatic-induced liver fibrosis |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|