Cathepsin D and G expression correlates with human fracture healing phases and specific neutrophil N1 and N2 phenotypes

BACKGROUND: The role and involvement of immune cells in the fracture healing cascade, specifically neutrophils, is not yet fully understood. During tissue regeneration, cathepsin (CTS) D and cathepsin G have been identified as being involved in various cellular processes and associated with neutrophil function. This study aimed to determine the expression of these 2 cathepsins in fracture hematoma. Firstly, these two cathepsins were identified and scrutinized using a bioinformatics approach. Secondly, these two cathepsins were investigated for their expression in the human fracture hematoma (FH) as well as in N1 (pro-inflammatory) and N2 (regenerative) neutrophil phenotypes. METHODS: To review the latest research on CTSD and G gene expressions in fracture healing, bioinformatics analysis was firstly performed. Subsequently, to identify CTSD and G genes, the expression of them was assessed in human FH samples throughout different phases of the fracture healing cascade, and potential correlations with patient characteristics were explored. To confirm that gene expression translated to protein production, their corresponding protein levels in FH were evaluated via immunofluorescence. Finally, human neutrophils were harvested and polarized into N0, N1, or N2 phenotypes, after which their expression of CTSD and CTSG was analyzed. RESULTS: Bioinformatics analysis revealed distinct expression patterns of CTSD and CTSG in the fracture healing cascade in one earlier rodent study. In human, 58 FHs (0-19 days post-trauma) were harvested. The expression of CTSD significantly increased over fracture healing time, while the expression of CTSG remained constant throughout the early phases of fracture healing. Both proteins were found to be expressed throughout the FH. In neutrophils from five human donors, the expression of CTSD was higher in N2 neutrophils compared to N1, while CTSG was expressed more in N1 compared to N2 neutrophils. CONCLUSION: This study was the first to investigate the association of CTSD and CTSG in the ...