The dystrophic extracellular matrix promotes distinct macrophage behavior in muscular dystrophy subtypes

Topic:

Pre-Clinical Research

Poster Number: 179 M

Author(s):

Ashlee Long, PhD, Northwestern University, GaHyun Lee, Northwestern University, Lauren Vaught, Northwestern University, Michele Hadhazy, Northwestern University, Rachelle Crosbie, PhD, UCLA, Alexis Demonbreun, PhD, Northwestern University, Elizabeth McNally, MD PhD, Northwestern Feinberg School of Medicine

Introduction. Pathological remodeling of the extracellular matrix (ECM) occurs in all forms of muscular dystrophy (MD) and abnormal ECM protein deposition correlates with disease severity in MD. In dystrophic muscle, ECM structural proteins, as well as the smaller proteins embedded within the ECM, can exert detrimental effects on many cellular components within muscle. Macrophage accumulation accompanies pathological remodeling in MD, where the diversity of macrophage subtypes contributes to disease progression. Structural ECM components and their associated proteins differ between genetically distinct forms of MD, and these matrix components may drive distinct pathological mechanisms involving the ECM-macrophage axis.

Methods. To interrogate how the dystrophic-ECM influences macrophage behavior, we decellularized muscle sections on slides to generate myoscaffolds. Detergent treatment was used to remove cellular components, leaving myoscaffolds with intact ECM and retained spatial information. Decellularized myoscaffolds were generated from dysferlin-deficient (Dysf), dystrophin-deficient mdx (mild-C57), mdx (severe-D2) and WT (C57) mouse muscles. Bone marrow derived macrophages (macrophages) were isolated from WT mice and placed onto myoscaffolds where differential cellular localization and morphology were examined. Macrophage polarization was studied.

Results. WT macrophages placed onto Dysf, mdxC57 and mdxD2 myoscaffolds demonstrated unique localization patterns compared to WT macrophages seeded onto WT myoscaffolds. Macrophages seeded onto dystrophic matrices demonstrated increased circularity compared to those seeded onto WT matrices. WT macrophages seeded onto Dysf scaffolds acquired the “fried egg” (pro-inflammatory) morphology compared to WT macrophages seeded onto mdx and WT myoscaffolds. Cytokine production differed between WT macrophages placed onto WT and mdx myoscaffolds. In polarization assays, WT and mdx macrophages responded similarly, consistent with an ECM- driven mechanism driving macrophage behavior.

Conclusions. Proteomic profiling, coupled with immunofluorescence microscopy and myoscaffolds studies, are positioned to define the ECM components and spatial requirements that drive pathological macrophage differentiation in dystrophic muscle.