IMMUNOPROTEOMIC CHARACTERIZATION OF INNATE IMMUNITY PROCESSES IN A TRANSLATIONAL PORCINE OSTEOMYELITIS MODEL

Aim Diagnosing musculoskeletal related infections with molecular biomarkers remains a clinical challenge. This study aims to characterize a translational porcine model of osteomyelitis using mass spectrometry (MS) based quantitative proteomics to identify novel and differentially expressed proteins of soft tissues associated with infection. The aim was to identify biologically relevant proteins of the innate immune system that may serve as diagnostic biomarkers for osteomyelitis. Method Historic subcutaneous tissue samples were collected from a porcine osteomyelitis model based on implanting a k-wire inoculated with saline (controls) or 10 4 CFU of Staphylococcus aureus S54F9 spa-type t1333 into the right proximal tibia. Animals were euthanized five days post-operatively, and all animals inoculated with bacteria were confirmed infected by microbiology, whereas all animals inoculated with saline were found sterile. In total, eight infected and six sterile animals were included. Subcutaneous samples were exposed to liquid nitrogen and stored at -80°C until processing. Three biological replicates per sample were homogenized by bead blending in SDS and TEAB lysis buffer with protease inhibitors. Proteomic profiling was done using standard protein aggregation capture (PAC) MS methods and acquired in a data independent acquisition (DIA) manner. Raw data was processed for label free quantification. Results In total, 312 significantly upregulated (FDR<0.001, s0=0.1) proteins were identified in the infected versus the sterile subcutaneous tissue. Cytoscape-STRING protein network analysis of upregulated proteins revealed the top two pathways were ‘Reactome pathways’ in innate immune system and neutrophil degranulation, with 55 and 43 constituent proteins, respectively. The top 5 most significant proteins, in order of highest to lowest significance, present in the Innate Immune System pathway are listed here: Cathepsin S, Resistin, Lipocalin 2, Elastase (neutrophil expressed), and Azurocidin. In the upregulated fraction, we detected several proteases, which may further modulate the proteome by creating cleavage fragments of proteins susceptible to the protease's respective cleavage activity. Conclusions We show that proteins upregulated in the soft tissue of osteomyelitis lesions are functionally interconnected and enriched in innate immune responses, including several proteases with potential roles in shaping the local proteome. Ongoing analysis aims to explore the diagnostic potential of selected proteins and enzymatic activities associated with infection-related tissue remodeling. Acknowledgements Funding via Innovation Fund Denmark, grant no. 3194-00017B, and Nordic Bioscience.