Mills in 2000 applied the TH1/ TH2 nomenclature to define macrophage activation, hence proposing the concept of M1/M2 macrophage activation [121]. The M1/M2 model stemmed from the main following in vitro data: (i) macrophages from C57BL/6 and Balb/c mice, described to be prototypical Th1 and Th2 strains respectively, respond from a metabolic point of view, i.e. in terms of NO and ornithine production, differently to the same Th1-like stimuli, namely IFN-γ, LPS, or IFN-γ + LPS. In particular, while stimulated macrophages from C57BL/6 produce predominantly NO (iNOS or kill pathway), macrophages from Balb/c produce predominantly ornithine/urea (arginase or Heal pathway). (ii) The same observation was made for macrophages isolated from SCID or NUDE C57BL/6 or Balb/c mice, in which lymphocytes are reduced or absent. This led Mills to hypothesize that macrophages have an inherent capability to display a classical M1 or alternative M2 phenotype, and do not necessarily need “instruction” from lymphocytes. He further proposed that macrophages are in fact the first line of defense cells that can then direct the nature of the adaptive immune response of the T lymphocytes, i.e. TH1, TH2 or other [122–124]. This latter view remains nevertheless contested by immunologists who consider the lymphocytes and the cytokines they release as the orchestrators of macrophage activation [125]. A notable difference between the Th1/Th2 and the M1/M2 hypotheses is that M1/M2 polarized macrophages do not constitute separate clones, like Th1/Th2 cells, but rather represent the extremes of a spectrum of different phenotypes depending on stimulus/environment.

The M1/M2 nomenclature was then enlarged, with the introduction of M2a, M2b, M2c [126] and M2d subgroups [127, 128], defined by partially overlapping or distinct transcriptional profiles/signaling cascades induced by specific stimuli. Immunologists now suggest to use a more precise polarization nomenclature that includes the source of macrophage, the stimulus used and corresponding set of activation markers [125]. Thus, while the value of the M1/M2 framework is acknowledged, it is also recognized that it does not fully capture the complexity of macrophage responses especially in in vivo settings, as now revealed by the numerous analyses of macrophage transcriptomes via high-throughput RNA sequencing-bulk, single cell or single nucleus—methodologies in diverse pathological conditions (for reviews, see [129, 130]).

Even though it may only seem natural that the M1/M2 concept was also applied to microglia, the brain resident macrophages, neuro/immunologists are reconsidering the use and the validity of such concept [104, 105, 131]. As for macrophages, microglial gene expression profiles typically cannot be fitted into M1- or M2-like subphenotypes, as observed in rat models of adult ischemic stroke [132] or neonatal HI [98], in rodent models of neurodegenerative disorders [100, 107, 133, 134] and in human microglia isolated from Alzheimer’s patients [135, 136]. There again, the complexity of microglia cannot be reflected into the M1/M2 concept.