Inclusion Body Myositis (IBM) is a chronic, progressive inflammatory disease characterised by skeletal muscle inflammation, leading to muscle weakness. Notably, the presence of immune cells within IBM muscle does not confer responsiveness to conventional immunotherapy, a phenomenon that remains poorly understood. Here we undertake a detailed proteomic analysis of affected muscle tissue and infiltrating immune cells to elucidate the immune pathways involved in IBM. Our goal is to provide novel insights into disease pathogenesis that could lead to more effective treatments.
Formalin-fixed paraffin-embedded samples from four IBM patients and six sex- and age-matched controls were analysed. Sections of 2 µm thickness were prepared on polyethylene naphthalate membranes and stained with hematoxylin and eosin. 250,000 µm2 of muscle tissue and 350,000 µm2 of immune infiltrate were isolated using a Leica LMD7 laser microdissection microscope and prepared for proteomic analysis using an acetonitrile-based sample preparation. Cleaned, tryptic peptides were analysed via LC-MS using a Thermo Astral mass spectrometer in data independent acquisition mode.
Of the roughly 1200 protein groups identified in muscle fibers of IBM and control groups, 65 differentially expressed proteins (DEPs) were found. GO and KEGG analysis found that the DEPs were mainly enriched in antigen processing and presentation, response to type I interferon and MHC class I protein complex, pointing towards a critical role for interferon in IBM.
The more than 4000 protein groups identified in immune cell infiltrate of IBM samples were analysed using CIBERSORTx to infer immune cell identity and abundance. This analysis revealed that mast cells, rather than cytotoxic T cells, were the predominant infiltrating immune cells in the IBM samples.
This study demonstrates the power of LMD and high-sensitivity proteomics to uncover immune cell populations and signalling pathways driving inflammation.