Defective proteolytic processing of collagen in macrophages enhances liver fibrosis

Trabajo presentado en el International Workshop on Liver and Gut Fibrosis, celebrado en Valencia (España), los días 7 y 8 de octubre de 2021
Background and Aims: Changes in proteolytic activity are essential to liver fibrosis development. Previous reports suggest that macrophages are important effectors for ECM remodelling through phagocytosis and processing of ECM within acidic compartments. However, the role of macrophages in ECM remodelling during liver fibrosis is unknown. Methods: To study the degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D (CtsD). We generated and validated a novel macrophage-CtsD knock-out mouse by breeding LysMCre (macrophages) with CtsD floxed mice. Fibrosis was established chronically by CCl4 (0.5μl/g) or bile duct ligation in CtsDF/F or CtsDΔMac mice and determined by SR staining, hydroxyproline and fibrogenic genes by RT-PCR. Collagen degradation and endocytosis was studied using DQ™ Collagen type I and 10KDa Dextran probes respectively in peritoneal macrophages. Lysosomal colocalization was determined by IF using LAMP2. WB for Endo180 and UPAR and dual IF (Collagen- F4/80) was performed in fibrotic livers. Results: First, CtsD deletion in macrophages from CtsDΔMac mouse was confirmed by WB and RT PCR in macrophages and dual IF (F4/80-CtsD) in liver tissue. Of note, cathepsin B expression remained unaffected despite deletion of CtsD. CtsDΔMac mice demonstrated enhanced liver fibrosis in both CCl4 and BDL models as shown by an increase in Sirius red staining, hydroxyproline and hepatic Col1A1 and TGF-β mRNA. CtsDΔMac macrophages showed a significant decrease in DQ™ Collagen type I degradation versus CtsDF/F ones. DQTM collagen profile partially colocalized within the lysosomes (LAMP2). No affectation of the endocytosis process in macrophages or the expression of the collagen receptors UPAR and Endo180 was detected in livers from CtsDF/F or CtsDΔMac after CCl4 challenge. Finally, F4/80+ Collagen loaded macrophages were detected in CCl4 livers. Conclusions: Lysosomal cathepsin D is essential for the correct collagenolytic activity displayed by macrophages during liver fibrosis.