Introduction:: Limb girdle muscular dystrophy 2B (LGMD2B) is an untreatable progressive skeletal muscle disease caused by the loss of a membrane-repair protein dysferlin and eventual replacement of muscle by fat [1-2]. LGMD2B disease progression is initially characterized by extensive immune cell infiltration before onset of muscle weakness, suggesting immune cell contribution to LGMD2B pathogenesis [1-2]. Specifically, macrophages are the most abundant cell type in LGMD2B immune cell infiltrates with a preferential M1 pro-inflammatory phenotype [3]. Macrophages express dysferlin and LGMD2B macrophages lacking dysferlin have altered phagocytic ability [4]. It is unknown if this functional change in LGMD2B macrophages stems from altered muscle microenvironment or a cell-autonomous lack of dysferlin, and is difficult to study in vivo. Thus in this study, we for the first time utilized human induced pluripotent stem cell (hiPSC) derived macrophages (iMP) from healthy individuals and LGMD2B patients to study the role of dysferlin-deficiency on macrophage function and polarization.
Materials and Methods:: hiPSCs from three healthy and three LGMD2B donors were differentiated into monocytes and magnetically sorted for the CD14 monocyte marker. Monocytes were differentiated into non-polarized (M0) macrophages via treatment with M-CSF. Similarly, primary monocytes were isolated from healthy peripheral blood mononuclear cells (PBMCs) via CD14 magnetic sorting and differentiated into M0 macrophages. Primary and hiPSC-derived macrophages were polarized into an M1 or M2 phenotype via pro-inflammatory (LPS and IFN-λ) or anti-inflammatory (IL-4 and IL-10) cytokine stimulation. Macrophage polarization was compared using immunofluorescent staining for CD80 and CD206 as markers of M1 and M2 phenotype, respectively. Macrophage polarization was also studied in 2D experiments in co-cultures of healthy or diseased macrophages with hiPSC-derived muscle progenitor cells, which were then further differentiated into myotubes. Cytokine release profiles for healthy and LGMD2B iMP were compared for each type of polarization using a 36-panel human cytokine proteome profiler assay (R&D Systems). Macrophage phagocytic ability was measured using the well-established fluorescent E. coli bioparticle uptake assay and receptor-mediated endocytosis was assessed via fluorescent acetylated low-density lipoprotein (AcLDL) uptake. For a more physiologically relevant assessment of macrophage function, we also developed an efferocytosis assay to measure uptake of fluorescent myoblast debris in macrophages.
Results, Conclusions, and Discussions:: Results: No significant differences in yield or morphology were observed between healthy and LGMD2B monocytes. Additionally, immunofluorescent staining revealed similar levels of macrophage polarization in the presence of classic pro/anti-inflammatory cues (Fig 1A), suggesting similar differentiation and polarization in both healthy and LGMD2B iMPs. More than 15 differentially secreted cytokines were identified between healthy and LGMD2B iMPs polarized to M0, M1, or M2 phenotypes (data not shown). Bacterial phagocytosis (Fig 1B) and AcLDL uptake (Fig 1C) were not significantly different between healthy PBMC-derived macrophages and hiPSC-derived macrophages, suggesting similar functionality of primary vs. hiPSC-derived cells. Bacterial phagocytosis was significantly impaired in both M1 and M2 polarized LGMD2B vs. healthy macrophages (Fig 1B). Additionally, decreased myoblast debris uptake suggests defective efferocytosis ability in M0, M1, and M2 LGMD2B iMPs (data not shown). Receptor-mediated endocytosis of AcLDL was significantly higher for M2 polarized LGMD2B vs. healthy macrophages (Fig 1C). Macrophage polarization in the presence of healthy and LGMD2B muscle progenitor cells is currently being investigated in a 2D co-culture transwell assay and in 3D tissue engineered skeletal muscle tissues.
Discussion and Conclusion: LGMD2B iMPs display decreased phagocytosis and increased endocytosis, indicating a cell-autonomous role of dysferlin in regulating macrophage function. Future work will further characterize the functional differences between healthy and LGMD2B iMPs and study their transcriptomic differences by RNA sequencing. Additionally, we will incorporate iMPs into a 3D model of tissue-engineered skeletal muscle to determine the macrophage roles in LGMD2B muscle pathogenesis.
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