Improved Macrophage Isolation from Mouse Skeletal Muscle

Background

Macrophages were discovered by Metchnikoff and colleagues more than a century ago as ‘professional’ phagocytes (Underhill et al., 2016). Later studies revealed that macrophages constitute a heterogenous class of cells that exert diverse functions in tissues throughout the body (Wynn et al., 2013). Macrophages can be divided into two major types: tissue-resident and non-tissue-resident macrophages (Ginhoux and Guilliams, 2016). Tissue-resident macrophages can be further divided into two distinct populations: embryo-derived self-renewing and bone marrow-derived non-self-renewing macrophages. Typical self-renewing macrophages derived from the embryonic yolk sac or fetal liver include microglia, Kupffer cells, alveolar macrophages, and Langerhans cells. Bone marrow-derived non-self-renewing resident macrophages, which must be replenished by circulating monocytes at tissue-specific levels, include tissue-resident macrophages in the intestine, pancreas, and dermis (Chakarov et al., 2019). Non-resident macrophages are derived from bone marrow progenitor cells and infiltrate tissues following injury or infection (Kratofil et al., 2016).

Macrophages are highly versatile cells that are capable of ingesting and digesting pathogens as well as necrotic and infected cells, activating T and B lymphocytes, and inducing or suppressing inflammation (Shapouri-Moghaddam et al., 2018). The functional diversity of macrophages is well represented by their dynamic polarization abilities. Depending on signals from the local environment, macrophages can be polarized toward functionally opposite roles: pro-inflammatory M1 or anti-inflammatory M2 subtypes. Cytokines produced by Th1 (T helper type 1) lymphocytes, including interferon γ (IFNγ or IFNG) and tumor necrosis factor (TNF), polarize macrophages to the M1 subtype, while cytokines produced by Th2 lymphocytes, such as interleukin (IL) 4 and IL13, promote macrophage M2 polarization (Mills et al., 2000; Martinez et al., 2008). Polarized M1 macrophages induce inflammation, destroy pathogens, and clean up cell debris, partly through upregulation of the nitric oxide synthase (NOS) pathway (Rath et al., 2014). By contrast, M2 macrophages suppress inflammation and promote tissue repair, partially through upregulation of the arginase pathway (Rath et al., 2014). While M1 and M2 are well-known macrophage subtypes, more recent single-cell studies have identified additional subtypes in several mouse tissues (Chakarov et al., 2019; Jaitin et al., 2019). These subtypes share similarities and differences with M1 and M2, which further reveal the heterogeneity and versatility of macrophages.

Macrophages adapt to individual tissues and largely act in a tissue-dependent manner. Macrophages from different tissues possess distinct gene expression profiles and transcriptional regulatory pathways (Gautier et al., 2012). Recent studies suggest that local environmental factors in each tissue contribute to the tissue specificity of resident macrophages (Gosselin et al., 2014; Lavin et al., 2014). For instance, tumor growth factor β (TGFβ or TGFB) promotes the development of microglia by affecting the enhancer/promoter landscape of brain macrophages, while retinoic acid determines peritoneal macrophage specificity (Hoeksema and Glass, 2012). These studies have further uncovered the capacity of macrophages to adapt to local environments and acquire tissue-specific identities.

As the largest organ in mammals, skeletal muscle contains numerous and diverse resident macrophages. In skeletal muscle, macrophages are localized in the perimysium and endomysium (Cui et al., 2019), where they resolve infections and repair injury (Arnold et al., 2007; Tidball, 2011 and 2017). For example, when skeletal muscle is damaged, monocytes from the bloodstream differentiate and polarize into pro-inflammatory M1 macrophages, which eliminate pathogens and clean up tissue debris. Subsequently, M1 macrophages convert to M2 macrophages to suppress inflammation and repair tissues along with resident M2 macrophages (Yang and Hu, 2018; Cui and Ferrucci, 2020). Recently, skeletal muscle macrophages have been associated with physiological adaptations to exercise that differ between young and elderly individuals (Walton et al., 2019; Jensen et al., 2020), although the full spectrum of macrophage subtypes in skeletal muscle and their functions are only partially known. We have recently found that macrophages residing in human and mouse skeletal muscle are mostly of the M2 subtype (Cui et al., 2019); however, recent single-cell analyses from skeletal muscle and other tissues suggest that the identities of skeletal muscle macrophages are likely more complex (Chakarov et al., 2019; Jaitin et al., 2019; Wang et al., 2020). Furthermore, skeletal muscle macrophages have been shown to have mixed origins, including the embryonic yolk sac, fetal liver, and adult bone marrow (Wang et al., 2020). It remains unclear whether macrophages from various origins behave differently, and the function of each macrophage subtype in skeletal muscle physiology and aging is yet to be elucidated. To answer these questions, it is critical to isolate macrophages from skeletal muscle. In a recent report, Liu and coworkers established a protocol for the isolation of muscle stem cells that was also effective in isolating macrophages from human skeletal muscle (Liu et al., 2015; Kosmac et al., 2018). Here, we report modifications to this methodology for the isolation and purification of resident macrophages from young and old mouse skeletal muscle, allowing the characterization of resident macrophages by flow cytometry analysis and single-cell transcriptomics.