**6.2 Pro-resolving macrophages in TBI (M2 phenotype): time course**

The alternatively activated or M2 phenotypic macrophages are known to serve as pro-recovery or anti-inflammatory cells. These cells are activated by IL4 and serve an immunoregulatory function, in contrast to the microbe-killing function of their M1 counterparts [47]. Like M1 macrophages, identification of M2 macrophages employs gene expression levels of a wide array of characteristic markers. Markers commonly used for this purpose include CD206, Fizz1, Ym1, IL1-RN, Arg1, TGFβ, SOCS3, and IL4-RA [46]. Low levels of CCR2 with high CX3CR1 expression have also been used as a marker for pro-repair macrophages [49]. Studies using these markers have demonstrated an important role for M2-polarized macrophages at multiple time points following TBI. Increases in M2 markers have been shown as early as 6 hours following CCI injury [46]. The reported timeline of M2 influence varies depending on the specific markers assessed. For example, the number of TGFβ-expressing macrophages/microglia has been demonstrated to increase by 24 hours post-CCI injury and remain elevated compared to sham out to 7 days following injury [46]. Increase in expression of Arg1 has also been demonstrated in macrophages/microglia following CCI. Interestingly, the increase in Arg1 expression in macrophages/microglia, which first becomes evident at 24 hours post-CCI, continues to rise out to 7 days postinjury rather than decreasing back toward normal levels as was observed for TGFβ [46]. Expression of CD163, another marker of the pro-resolving M2 phenotype, has also been investigated following TBI. One study showed increased expression of CD163+ macrophages following weight-drop TBI in a rat model. This may have anti-inflammatory effects following TBI through suppression of the pro-inflammatory macrophage phenotype [51]. While timing of expression of specific markers can vary, these studies indicate that macrophages expressing M2 phenotypic traits are a significant factor in TBI recovery.

Macrophage polarization toward the alternatively activated or M2 phenotype has beneficial effects on recovery following TBI through a variety of mechanisms. M2-polarized macrophages are characterized by expression of multiple markers including arginase 1 (Arg1), CD206, CD301, resistin-like α, and PDL2 [48]. Alternatively activated macrophages have been shown to decrease T-cell proliferation, promote angiogenesis, assist in generation of extracellular matrix components, and benefit wound healing and tissue repair [47]. In addition, the anti-inflammatory cytokines TGFβ and IL10 secreted by alternatively activated macrophages help to decrease activation of classical macrophages, reducing bystander tissue damage [47]. One study demonstrated that experimentally altering macrophage/microglia phenotype to favor M2 polarization by inhibition of NOX2 results in decreased oxidative damage [46]. In another report, inhibition of high-mobility group box 1 (HMGB1) decreased M1 and increased M2 polarization of macrophages/microglia, which correlated with decreased lesion volume and improved recovery [52]. Moreover, activation of the cannabinoid receptor CB2R decreased M1 and promoted M2 macrophage polarization, accompanied by decreased edema and improved blood flow and behavioral recovery [53]. These studies employed different methods to influence macrophages toward the M2 phenotype with similar results—*increased expression of M2 traits has a positive impact on TBI recovery.* Additional studies are needed to confirm this beneficial role of M2 macrophages in TBI.
