**6.1 Classical role of macrophages as pro-inflammatory cells in TBI (M1 phenotype): time course**

The classically activated or M1 phenotype macrophages are known to function as pro-inflammatory cells. Early studies indicated that these cells become activated by a combination of IFNγ signaling and either direct TNF signaling or Toll-like receptor-induced production of TNF, usually triggered by lipopolysaccharide (LPS) [47]. In the typical response to wound healing outside the CNS, these cells are important in protection against bacterial infection. M1-polarized macrophages generate reactive oxygen species and also activate inducible nitric oxide synthase (iNOS) to generate nitric oxide as well as an array of pro-inflammatory cytokines including IL-12, TNFα, IL-6, IL-1β, and nitric oxide [47, 48]. Identification of M1 macrophages is typically done by measuring gene expression of characteristic markers including IL-12, IL-1β, iNOS, TNFα, and IL-6 [46]. High levels of CCR2 with low CX3CR1 expression have also been used as an indicator of pro-inflammatory status in macrophages [49]. Bystander tissue damage from M1 macrophages can be catastrophic in the normally immune-privileged setting of the CNS.

Peripheral-derived macrophages have been shown to rapidly infiltrate the injured brain within the first 1–3 days postinjury [46]. Although both M1 and M2 macrophages are likely present at this stage, early studies of macrophage polarization following TBI indicated that the M1 phenotype predominates in the *initial response* to brain trauma [48]. CCI-induced increase in expression of pro-inflammatory markers has been demonstrated as early as 6 hours following injury, suggesting that macrophages expressing M1 traits are a key part of the acute response to TBI [46]. One study found that increases in the number of IL-12-expressing macrophages/microglia were evident by 24 hours following CCI injury, and the number remained increased compared to sham controls out to 7 days postinjury [46]. Other work demonstrated that either macrophages polarized toward the M1 phenotype or a transitional phenotype between M1/2 (to be discussed later) become predominant over M2 phenotype by 7 days following CCI injury. This phenomenon correlates with neurodegeneration [46].

Based on these reports, macrophages seem to be skewed toward the M1 phenotype for an extended period following CNS injury, with corresponding negative effects on recovery. This contrasts with the typical immune response outside neural tissue, where an early increase in M1 macrophages gives way to pro-recovery M2 macrophages [46]. The neurotoxic effect of M1 macrophages is most likely mediated by pro-inflammatory cytokines. Levels of M1-associated pro-inflammatory cytokines transiently increase in brain tissue during the acute response to injury. Specifically, IL-1β, IL-6, and TNFα levels have been shown to significantly increase in brain tissue by 12 hours postinjury in a mouse model. These cytokines return to sham levels by 7 days postinjury [50]. Support for the importance of these cytokines as mediators of M1-induced secondary neural damage following TBI is provided by a study targeting

**23**

macrophages in TBI.

*Peripheral Immune Response Following Traumatic Brain Injury*

these cytokines as a potential TBI therapeutic. For example, treatment with Minozac, an inhibitor of pro-inflammatory cytokines, ameliorated the TBI-induced increase of pro-inflammatory cytokines in cortex and hippocampus and resulted in decreased

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

macrophages following weight-drop TBI

neuronal damage and improved neurocognitive function following TBI [50].

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

Macrophage polarization toward the alternatively activated or M2 phenotype

expressing M2 phenotypic traits are a significant factor in TBI recovery.

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

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

*DOI: http://dx.doi.org/10.5772/intechopen.93597*

showed increased expression of CD163+

these cytokines as a potential TBI therapeutic. For example, treatment with Minozac, an inhibitor of pro-inflammatory cytokines, ameliorated the TBI-induced increase of pro-inflammatory cytokines in cortex and hippocampus and resulted in decreased neuronal damage and improved neurocognitive function following TBI [50].
