**7. Continuum of expression between M1/2 and influence on TBI**

As previously mentioned, macrophages *in vivo* do not always show a sharply demarcated M1 or M2 phenotype. Several studies have shown expression of both M1 and M2 traits in macrophages following TBI, while others have demonstrated that macrophages can switch between phenotypes [54, 55]. One study using the CCI model demonstrated co-expression of iNOS, a classical M1 marker, with Arg1, an M2 marker, in perilesional macrophages/microglia following injury [46]. Another study assessed expression of a wide array of pro-inflammatory (associated with M1) and anti-inflammatory (associated with M2) genes in mouse cortical tissue following CCI injury and found that both sets of genes are co-expressed at 1, 2, and 7 days postinjury. This study also showed that perilesional microglia/macrophages co-labeled with both M1 and M2 markers at all three time points [56]. A different study using flow cytometry to sort Arg1-positive and Arg1-negative brain macrophages following TBI demonstrated that neither Arg1+ or Arg1− cells displayed gene expression profiles consistent with the M1 or M2 patterns defined by *in vitro* studies, although two distinct populations of macrophages did seem to exist in this context [48]. These findings suggest that the classic M1 and M2 traits may actually coexist in the same macrophages following TBI. To confirm this at the level of the individual macrophage, one study employed single-cell RNA sequencing to assess the expression of classical and alternative markers in individual macrophages 1 day following TBI. This work demonstrated that traditional M1/2 markers are frequently co-expressed at high levels in the same cell [55]. This study also demonstrated that high expression of well-known M1 or M2 markers did not seem to down-regulate expression of markers of the opposite class. Some macrophages with high expression of Arg1, an established M2 marker, also displayed high expression of TNF and/or IL-1β, known M1 markers [55]. This type of M1/2 combination profile was displayed in a variety of genes, demonstrating that macrophage polarization *in vivo* can widely differ from the traditional M1/2 paradigm established primarily by *in vitro* studies [55]. Surprisingly, this study actually failed to find any macrophages that fit entirely within the M1 or M2 category, suggesting that all macrophages responding to TBI respond to injury stimuli along a continuum of expression [55]. Intermediate macrophage phenotypes with traits of both M1 and M2 have also been found in studies of spinal cord injury and Alzheimer's disease [46]. The results of these studies indicate the existence of a continuum between M1 and M2 macrophages in the setting of brain injury and disease.

The specific stimuli and mechanisms involved in the continuum of M1/2 expression are currently areas of active research. Some authors have suggested that dual expression of M1 and M2 characteristics is a necessary part of the macrophage response to TBI, as these cells must respond to both pro- and anti-inflammatory environmental signals simultaneously in the setting of brain trauma [54]. This concept is supported by the results of the previously mentioned study demonstrating concurrent expression of both pro- and anti-inflammatory gene signatures [56]. The function of infiltrating monocyte/macrophages, therefore, appears to depend more on the specific gene expression and cytokine profile than on overall classification as M1 or M2. These findings underscore the importance of improving our understanding of the pathways involved in regulation of expression on the M1/2 continuum. Data from multiple studies have indicated that the Tie2/Angiopoietin pathway is an important factor in the continuum of expression between M1 and M2 macrophages. In addition, data from our project, to be discussed in the following chapters, have specifically implicated this pathway in the context of M1/2 polarization after TBI.

**25**

*Peripheral Immune Response Following Traumatic Brain Injury*

**8. Tie2/Angiopoietin signaling in immune cells**

The Tie2/Angiopoietin signaling axis was first identified for its key role in the regulation of angiogenic pathways, but this receptor complex is also gaining increasing recognition for its importance in peripheral immune cells. The receptor tyrosine kinase Tie2 (also known as Tek) interacts with its ligands, the angiopoietin family of proteins, to influence vascular development [57]. Studies in endothelial cells have shown that Tie2 is differentially regulated by its ligands Angiopoietin 1 (Angpt1) and Angiopoietin 2 (Angpt2). Angpt1 typically acts as an agonist for Tie2, while Angpt2 serves as an antagonist with several exceptions [58, 59]. Although Tie2/Angiopoietin signaling has been most studied for its role in regulation of vascular function, Tie2 is also expressed in a subpopulation of monocyte/macrophages called Tie2-expressing monocytes (TEMs) implicated in tumor formation and inflammation [60]. This review will discuss the mechanisms involved in the Tie2/Angiopoietin signaling axis and investigate the function of

Tie2 is a receptor tyrosine kinase first identified on vascular endothelial cells [61]. There are multiple components to the Tie2 signaling pathway where the angiopoietin ligands serve as binding partners [59]. In addition to its expression on endothelial cells, Tie2 is expressed in TEMs, hematopoietic stem cells, neutrophils, eosinophils, and some muscle satellite cells [59, 62]. Angiopoietin 1 (Angpt1) is primarily expressed in platelets and perivascular cells, while Angiopoietin 2 (Angpt2) is expressed in endothelial cells [63]. Expression of both Angiopoietins has also been demonstrated in hematopoietic stem cells and some immune cell types including monocyte/macrophages [64, 65]. Angpt1 serves as a Tie2 agonist, activating this receptor and increasing endothelial vessel stability [59]. However, the function of Angiopoietin 2 (Angpt2) is more variable. Some studies have shown that Angpt2 can act as either an agonist or antagonist of Tie2 depending on cellular context, and increased expression of Angpt2 has been demonstrated in multiple disease states [59]. Angpt2 has been found to act as a Tie2 agonist in the context of decreased Angpt1 signaling, absence of Tie1/Tie2 heterocomplexes, or inhibition of vascular endothelial protein tyrosine phosphatase (VE-PTP) in the endothelium [59, 66, 67]. However, the dominant role of Angpt2 and/or these cocomplexes in TBI has not been established. This ligand has repeatedly been shown to act as an antagonist in the setting of inflammation [68]. Although less studied than its counterpart, Tie1 has also been found to interact with Tie2 to promote Tie2/Angiopoietin interactions in vascular remodeling [59, 69]. The interactions between Tie2, Tie1, Angpt1, and Angpt2 have a profound influence on cell survival

The downstream cellular effects of Tie2 binding with an Angiopoietin ligand can vary widely with cellular context. This is partially due to the differing effects of Angpt1 vs. Angpt2—Angpt1 binding has been shown to oppose the effects of inflammatory cytokines and decrease vascular permeability, while Angpt2 has been found to increase vascular permeability in a number of inflammatory models [59]. Binding patterns of these two ligands with Tie2 are distinct from each other, which may contribute to their differing effects. The fibrinogen-like domain of Angpt1 binds an immunoglobulin domain of Tie2, which may help Angpt1 increase cluster formation and cross-phosphorylation of Tie2 upon binding [68]. In contrast, Angpt2 has a slightly different amino acid sequence in the fibrinogen-like domain

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

TEMs in various cellular contexts.

and vascular permeability [59, 61].

**8.1 Overview of the Tie2/Angiopoietin axis**
