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

Bone, vol. 199, pp. 117574

Abstract

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 fracture healing cascade. It was shown that the expression of CTSD enzyme was associated with early fracture healing phases, as well as with specific neutrophil phenotypes (N1 or N2). Furthermore, these expression dynamics of CTSD and CTSG support the increasing N2/N1 phenotype ratio over time during fracture healing in humans, reflecting a shift from inflammation to regeneration. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study determined to investigate the cathepsin D and G expression in human fracture healing after reviewing the latest research progress. Along with characterizing the dynamics of these cathepsins in fracture hematoma, we demonstrate their association with two distinct neutrophil phenotypes, N1 and N2. These findings enhance the understanding of cathepsins and the roles of N1 and N2 neutrophils in fracture healing, providing a theoretical foundation for developing future therapies and biomaterials.

Authors 8

1. Fangzhou Lu corresponding

   Maastricht University · Maastricht University Medical Centre

   Affiliation as printed

   Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht 6229 ER, the Netherlands; Division of Trauma Surgery, Department of Surgery, Maastricht University Medical Center, Maastricht 6229 HX, the Netherlands. Electronic address: fangzhoulu35@163.com

2. Rald V. M. Groven Aachen Department of Orthopaedic, Trauma, and Reconstructive Surgery

   RWTH Aachen University · Universitätsklinikum Aachen · Maastricht University

   Affiliation as printed

   Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht 6229 ER, the Netherlands; Department of Orthopaedic, Trauma- and Reconstructive Surgery, RWTH Aachen University Hospital, Aachen, Germany

3. Dennis M. Meesters

   Maastricht University

   Affiliation as printed

   Department of Genetics & Cell Biology, Maastricht University, 6229ER Maastricht, the Netherlands; NUTRIM, Institute of Nutrition and Translational Research in Metabolism, Maastricht University, 6229ER Maastricht, the Netherlands

4. Albert V. Bitorina

   Maastricht University

   Affiliation as printed

   Department of Genetics & Cell Biology, Maastricht University, 6229ER Maastricht, the Netherlands; NUTRIM, Institute of Nutrition and Translational Research in Metabolism, Maastricht University, 6229ER Maastricht, the Netherlands

5. Martijn Poeze

   Maastricht University · Maastricht University Medical Centre

   Affiliation as printed

   Division of Trauma Surgery, Department of Surgery, Maastricht University Medical Center, Maastricht 6229 HX, the Netherlands; NUTRIM, Institute of Nutrition and Translational Research in Metabolism, Maastricht University, 6229ER Maastricht, the Netherlands

6. Martijn van Griensven

   Maastricht University

   Affiliation as printed

   Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht 6229 ER, the Netherlands

7. Taco Johan Blokhuis

   Maastricht University · Maastricht University Medical Centre · Capital Medical University · Beijing YouAn Hospital

   Affiliation as printed

   Division of Trauma Surgery, Department of Surgery, Maastricht University Medical Center, Maastricht 6229 HX, the Netherlands; NUTRIM, Institute of Nutrition and Translational Research in Metabolism, Maastricht University, 6229ER Maastricht, the Netherlands

   Division of Trauma Surgery, Department of Surgery, Maastricht University Medical Center, Maastricht 6229 HX, the Netherlands; The First Unit, Department of Hepatology, Beijing YouAn Hospital, Capital Medical University, Beijing, China

8. Ronit Shiri‐Sverdlov

   Maastricht University

   Affiliation as printed

   Department of Genetics & Cell Biology, Maastricht University, 6229ER Maastricht, the Netherlands; NUTRIM, Institute of Nutrition and Translational Research in Metabolism, Maastricht University, 6229ER Maastricht, the Netherlands