Optimized Flow Cytometry Strategy for Phenotyping Intramuscular Leukocytes: Application to the Evaluation of
Tassadit Saidj1, Yasmine Baba Amer1, Anne Plonquet2
1From the Université Paris Est Créteil, INSERM, IMRB, Equipe Relaix, Creteil, France.
Insights
Flow cytometry (FC) effectively analyzes immune cells in muscle tissue, aiding diagnosis of inflammatory myopathies. This method preserves cell surface markers, crucial for identifying immune cell types and their activation states in both mouse models and human patients.
Area of Science:
- Immunology
- Cell Biology
- Pathology
Background:
- Characterizing intramuscular immune cells is vital for understanding dysimmune/inflammatory myopathies (DIM).
- Flow cytometry (FC) is a reliable method for analyzing leukocyte subpopulations and their activation.
- A mechanical cell extraction protocol preserves cell surface epitopes for accurate analysis.
Purpose of the Study:
- To develop and validate a mechanical protocol for muscle cell extraction for flow cytometry analysis.
- To assess the utility of this protocol in experimental mouse models and human myopathological diagnosis.
- To characterize intramuscular immune cell populations and activation states in DIM.
Main Methods:
- Developed a purely mechanical protocol for extracting cells from muscle tissue.
- Applied the protocol to mouse models of muscle regeneration and inherited muscle dystrophies.
- Analyzed 16 human muscle samples (9 DIM, 7 non-immune conditions) using FC.
Main Results:
- Skeletal muscle regeneration in mice showed transient increases in macrophages, myeloid dendritic cells (MDCs), CD8+ T cells, and NK cells.
- In murine muscular dystrophies, leukocytes were abundant, with CD8+ T cells mirroring CD45+ cell percentages.
- Human DIM samples revealed intramuscular recruitment of CD8+ T cells, with elevated CD4+ and CD8+ T cell activation markers (HLA-DR, CD25, CD38, CD57).
Conclusions:
- The mechanical cell extraction protocol is applicable to experimental pathology and human myopathological diagnosis.
- Flow cytometry is suitable for quantifying intramuscular leukocyte subpopulations and their activation states in DIM.
- CD8+ T cell infiltration and activation are key features of human DIM.
Abstract:
Phenotyping intramuscular immune cells is essential for the characterization of dysimmune/inflammatory myopathies (DIM). Flow cytometry (FC) is the most reliable technique for analyzing leukocyte subpopulations and evaluating their activation levels. We developed a purely mechanical protocol for extracting cells from muscle tissue allowing us to preserve cell surface epitopes and determined its applicability to experimental pathology in mice and myopathological diagnosis in human. Skeletal muscle regeneration in mice was associated with a transient enrichment of macrophages (CD11bhighGr-1+), myeloid dendritic cells (CD3-C8+CD11bhigh), CD8+ T cells (CD3+C8+), and NK cells (CD3- CD11bhighNKp46+). In murine models of inherited muscle dystrophies, leukocytes represented 23%-84% of intramuscular mononuclear cells, with a percentage of CD8+ T cells (4%-17%) mirroring that of all CD45+ cells, while MDCs remained a minority. In human 16 samples (DIM: n = 9; nonimmune conditions: n = 7), DIM was associated with intramuscular recruitment of CD8+ T cells, but not CD4+ T cells and NK cells. FC allowed concomitant quantification of HLA-DR, CD25, CD38, and CD57 activation/differentiation biomarkers and showed increased activation levels of CD4+ and CD8+ T cells in DIM. In conclusion, FC is an appropriate method for quantifying intramuscular leukocyte subpopulations and analyzing their activation states.
