Characterizing the Roles of Dysferlin Deficiency in Function, Polarization, and Secretome of Human iPSC-Derived Macrophages 2251399

Abstract

Abstract Introduction Limb-girdle muscular dystrophy R2/2B (LGMDR2/2B) is an untreatable and progressive late-onset skeletal muscle disease caused by the loss of a membrane-repair protein dysferlin. Even before disease symptom onset, LGMDR2 muscles are infiltrated by pro-inflammatory macrophages (MP), implicating immune cells in disease pathogenesis. While MPs express dysferlin, defining the cell-autonomous roles of dysferlin in MP function has been challenging in vivo due to complex multicellular interactions and altered microenvironment in LGMDR2 muscle. Methods To address this, we generated human induced pluripotent stem cell (hiPSC)-derived macrophages (iMPs) from three healthy and three LGMDR2 donors to delineate cell-autonomous roles of dysferlin in macrophage: 1) polarization, 2) transcriptional profile, 3) secretome, and 4) phagocytotic and endocytic function. Results Despite exhibiting comparable polarization under well-characterized pro- and anti-inflammatory cues, RNAseq analyses revealed downregulation of Gene Ontology terms related to cytokine secretion, phagocytosis, and receptor-mediated endocytosis in LGMDR2 iMPs. Proteomic analysis of iMP conditioned media revealed significant differences in 72 secreted proteins, including numerous chemokines, cytokines, and growth factors, suggesting an altered secretory phenotype. Functional assays found no significant differences in the phagocytosis of E. coli bioparticles or fluorescent myotube debris. However, receptor-mediated endocytosis of AcLDL was significantly lower in both M0 and M2 LGMDR2 vs. healthy iMPs. Pharmacological screens identified clathrin-dependent endocytosis as the primary pathway for AcLDL uptake in both genotypes, with altered clathrin trafficking and reduced scavenger receptor expression likely underlying LGMDR2 endocytic deficits. Conclusion Overall, dysferlin loss in iMPs results in cell-autonomously altered transcriptome, secretome, and endocytic function, which may contribute to LGMDR2 muscle pathology and disease progression. Funding Source Jain Foundation grant, NIH grant 1R01AR082979-01, National Science Foundation Graduate Research Fellowship Topic Categories Immune Mechanisms of Human Disease (HUM)