Macrophage activation and polarization: nomenclature and experimental guidelines

Abstract

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.

Figure 1. Framework for describing activated macrophages

A. Examples of widely used macrophage preparations. CSF-1 grown mouse adherent macrophages from the bone marrow or CD14⁺ monocytes were used as the exemplars for marker evaluation and standardized activation conditions. Macrophages can also be generated with GMCSF, where a CD11c⁺ DC population is also present depending on the culture conditions. In the mouse, thioglycollate injection followed by peritoneal lavages are used to generate macrophage populations with differing yields and properties, while many organ systems in the mouse and human are sources of tissue infiltrating macrophages. B. Marker systems for activated macrophages. Shown are functional subdivisions according to stimulation of mouse CSF-1 macrophages or human monocyte-derived CSF-1 macrophages with the existing M1–M2 spectrum concept (Martinez and Gordon, 2014; Mosser and Edwards, 2008; Stout and Suttles, 2004). Stimulation conditions are IL-4, immune complexes (Ic), IL-10, glucocorticoids with TGFβ, glucocorticoids alone, LPS, LPS and IFN-γ and IFN-γ alone. Marker data was drawn from a wide range of published and unpublished data from the authors’ laboratories and represents a starting consensus (Edwards et al., 2006; Fleetwood et al., 2009; Gratchev et al., 2008; Gundra et al., 2014; Krausgruber et al., 2011; Lang et al., 2002; Shirey et al., 2008; Shirey et al., 2014; Shirey et al., 2010; Xue et al., 2014). Asterisk indicates corroboration of human IL-4 genes by deep sequencing (KAS and SNV, unpublished). C. Use of genetics to aid in macrophage activation studies. Mutations in Akt1 and Klf4 cause a ‘switch’ to M(LPS) and M(IFNγ) associated gene expression while mutations in Akt2 and Klf6 show the reverse phenotype. Mutations in Stat6, Ppard, Pparg, Irf4 and IRF5 depletion are involved in the maintenance and/or amplitude of activation.