Macrophages have well-characterized roles in skeletal muscle repair and regeneration. Relatively little is known regarding the role of resident macrophages in skeletal muscle homeostasis, extracellular matrix remodeling, growth, metabolism and adaptation to various stimuli including exercise and training. Despite speculation into macrophage contributions during these processes, studies characterizing macrophages in non-injured muscle are limited and methods used to identify macrophages vary. A standardized method for the identification of human resident skeletal muscle macrophages will aide in the characterization of these immune cells and allow for the comparison of results across studies. Here, we present an immunohistochemistry (IHC) protocol, validated by flow cytometry, to distinctly identify resident human skeletal muscle macrophage populations. We show that CD11b and CD206 double IHC effectively identifies macrophages in human skeletal muscle. Furthermore, the majority of macrophages in non-injured human skeletal muscle show a ‘mixed’ M1/M2 phenotype, expressing CD11b, CD14, CD68, CD86 and CD206. A relatively small population of CD11b+/CD206- macrophages are present in resting skeletal muscle. Changes in the relative abundance of this population may reflect important changes in the skeletal muscle environment. CD11b and CD206 IHC in muscle also reveals distinct morphological features of macrophages that may be related to the functional status of these cells.
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This work was supported by the National Institute of Aging grant (AG046920) and by the NIH Clinical and Translational Science Award (CTSA) (UL1TR001998) at the University of Kentucky. This work also utilized de-identified samples obtained through Merit Review Award #RX0012030 to Richard A. Dennis from the US Department of Veterans Affairs (VA), Rehabilitation R&D Service. Multi-channel flow cytometry was collected with help from the UK Flow Cytometry & Cell Sorting core facility (www.research.uky.edu/core/flow). The UK Flow Cytometry & Cell Sorting core facility is supported in part by the Office of the Vice President for Research, the Markey Cancer Center and an NCI Center Core Support Grant (P30 CA177558) to the University of Kentucky Markey Cancer Center.
Kosmac, Kate; Peck, Bailey D.; Walton, R. Grace; Mula, Jyothi; Kern, Philip A.; Bamman, Marcas M.; Dennis, Richard A.; Jacobs, Cale A.; Lattermann, Christian; Johnson, Darren L.; and Peterson, Charlotte A., "Immunohistochemical Identification of Human Skeletal Muscle Macrophages" (2018). Physical Therapy Faculty Publications. 99.