Abstract As the major cellular component of the innate immune system in the central nervous system (CNS) and the first line of defense whenever injury or disease occurs, microglia play a critical role in neuroinflammation following a traumatic brain injury (TBI). In the injured brain microglia can produce neuroprotective factors, clear cellular debris and orchestrate neurorestorative processes that are beneficial for neurological recovery after TBI. However, microglia can also become dysregulated and can produce high levels of pro-inflammatory and cytotoxic mediators that hinder CNS repair and contribute to neuronal dysfunction and cell death. The dual role of microglial activation in promoting beneficial and detrimental effects on neurons may be accounted for by their polarization state and functional responses after injury. In this review article we discuss emerging research on microglial activation phenotypes in the context of acute brain injury, and the potential role of microglia in phenotype-specific neurotrestorative processes such as neurogenesis, angiogenesis, olgogendrogenesis and regeneration. We also describe some of the known molecular mechanisms that regulate phenotype switching, and highlight new therapeutic approaches that alter microglial activation state balance to enhance long-term functional recovery after TBI. An improved understanding of the regulatory mechanisms that control microglial phenotypic shifts may advance our knowledge of post-injury recovery and repair, and provide opportunities for the development of novel therapeutic strategies for TBI. Keywords: Traumatic brain injury, neuroinflammation, microglia, macrophage, phenotype, M1-like, M2-like, polarization, neurodegeneration, repair

Introduction Neuroinflammation is a prominent feature of many neurodegenerative diseases (Eikelenboom et al., 2010; Perry et al., 2010), and is increasingly being recognized as an important pathophysiological mechanism underlying chronic neurodegeneration following traumatic brain injury (TBI) (Faden and Loane, 2015; Johnson et al., 2013; Smith et al., 2013; Smith et al., 2012). As the primary mediators of the innate immune response in the central nervous system (CNS), microglia play a critical role in neuroinflammation and secondary injury after TBI. The understanding of the functional role of microglia in the injured brain and spinal cord has developed significantly in recent years, with strong support for dual beneficial and detrimental roles for microglia, resulting in tissue repair or neurodegeneration respectively (David and Kroner, 2011; Kumar and Loane, 2012). By removing cellular debris by phagocytosis and releasing neurotrophic factors and anti-inflammatory cytokines microglia can prevent neuronal injury and restore tissue integrity in the injured brain. However, it is clear that the development of a disinhibited and highly reactive microglial activation state results in the release of high levels of pro-inflammatory and cytotoxic mediators that contribute to neuronal dysfunction and cell death (David and Kroner, 2011; Kumar and Loane, 2012). Immune cells within the CNS milieu such as microglia and infiltrating macrophages appear to be heterogeneous with diverse functional phenotypes that range from pro-inflammatory (M1-like) phenotypes to immunosuppressive (M2-like) phenotypes. The “M1/M2” paradigm has been increasingly studied in neurodegenerative diseases in an attempt to uncover mechanisms of immunopathogenesis, and advances in understanding of molecular and functional states of microglia and macrophages may provide a framework to dissect out and interrogate the dual beneficial and destructive roles of these cells after TBI. In this review, we focus on microglia and macrophage phenotypes in the context of acute CNS injury and post-traumatic repair. We discuss their role in phenotype-specific neurotrestorative processes such as neurogenesis, angiogenesis, oligodendrogenesis and regeneration, and the extracellular and molecular signals that regulate phenotype switching. We also highlight new therapeutic approaches that alter M1-/M2-like balance following TBI and enhance long-term functional recovery via promotion of tissue repair processes and suppression of neurodegeneration.

The immune system response to TBI Within minutes of TBI there is a robust neuroinflammatory response that is mediated by complex molecular and cellular inflammatory events. The temporal profile of these events have been studied using animal models of TBI, as well as human surgical and post-mortem tissue samples and analysis of cerebrospinal fluid (CSF) and plasma from TBI patients (Ziebell and Morganti-Kossmann, 2010). Depending on the intensity of the injury and whether it is penetrating or not, TBI induces instantaneous cell death at the site of impact (primary injury). Damaged cells release DAMPs that signal to other resident and infiltrating immune cells via pattern recognition receptors, and astrocytes, microglia and damaged neurons at the site of injury secrete cytokines and chemokines. These potent immune mediators activate microglia and astrocytes at the site of injury and recruit peripheral immune cells that traffic through the damaged blood-brain barrier during the acute post-traumatic period. Neutrophils are the first peripheral cells to accumulate in the brain after TBI (Clark et al., 1994), and they attempt to clear cell debris by phagocytosis. However, neutrophils also contribute to ongoing tissue damage by releasing toxic mediators such as ROS (Rhodes, 2011). Experimental studies indicate that neutrophil infiltration in the TBI brain is maximal at 1 day post-injury, and is followed by accumulation of leukocyte subsets that peak at about 3 days post-injury (Soares et al., 1995). Monocytes are recruited to the damage brain in response to local chemokine signals (e.g. CCL2, CXCL10, CCL5 etc.), and once in the brain they differentiate into macrophages. Two subpopulations of monocytes have recently been defined based on their relative cell-surface expression of the chemokine receptors CCR2 and CX3CR1, with CCR2+ cells representing ‘inflammatory’ (CD11b+CD45hiCCR2+Ly6Chi) monocytes and the CX3CR1+ cells representing ‘patrolling’ (CD11b+CD45hiCX3CR1+) monocytes (Auffray et al., 2007). It is the inflammatory monocytes that are preferentially recruited to the TBI brain (Hsieh et al., 2013), and they predominate the lesion site at 3 days post-injury. Dendritic cells (DCs), T lymphocytes and natural killer (NK) cells are also recruited to the TBI brain during this period (Jin et al., 2012), but at much lower numbers than infiltrating monocytes. The exact functional role of DCs and T cells in TBI pathology has yet to be established, but distinct T cell subsets may modulate local inflammatory responses to be either harmful or protective (Walsh et al., 2014). Within this time frame brain resident glial cells become highly activated. Astrocytes surrounding the lesion are reactive and up-regulate GFAP and produce cytokines and chemokines that contribute to additional recruitment and activation of resident microglia and peripheral immune cells. Microglia transform from a ramified to an amoeboid morphology and when activated they are morphologically indistinguishable from recruited blood-derived macrophages. They secrete pro-inflammatory cytokines and free radicals that are cytotoxic to neurons and can contribute to neurodegeneration after TBI. Thus, the inflammatory response to TBI is highly complex with several interrelated molecular and cellular events initiated after injury to activate resident microglia and astrocytes, recruit additional peripheral immune cells, and damage neurons. Determining the relative contribution and functional role of M1- and M2-like polarized microglia and infiltrating macrophages in this complex tissue injury environment is challenging, but experimental studies are beginning to shed light on these phenotypic responses.

Considerations for future development of therapeutic strategies that alter microglial/macrophage phenotypes Historically, broad-spectrum anti-inflammatory therapies for TBI have translated poorly to the clinic for human head injury (Kumar and Loane, 2012; Maas et al., 2010), which may in part reflect loss of beneficial M2-like effects in microglia. It is likely that efficient tissue repair after TBI requires both M1-like and M2-like functional responses, including coordinated transitions between phenotypes that fine-tune the sequential inflammatory, proliferative and remodeling phases of active repair (Gurtner et al., 2008; Novak and Koh, 2013). Therefore caution is advised against initiating a poorly timed M1- to M2-like phenotypic shift because the M1-like response plays a critical role in early repair processes, and additional beneficial roles for M1-like microglia following TBI may still be discovered. Now that microglia are known to play an active role in sculpting neuronal circuits and synapse remodeling (Salter and Beggs, 2014), it is not inconceivable that M1-like microglia may also drive the clearance of cellular debris from damaged synapses early after brain injury. Therefore, when developing therapeutic strategies to modulate M1-/M2-like phenotypes, timing the transitions between polarization states to facilitate efficient tissue repair needs to be carefully considered, as may tapering M2-like augmenting strategies to avoid chronic immunosuppressive effects and maladaptive repair processes. A long-term repair phase after a rapid pro-inflammatory response that is driven by M2-like macrophages has been demonstrated to result in fibrosis and other aberrant repair (Hesse et al., 2001), because elevated arginase activity shifts the metabolism of L-arginine significantly to produce increased ornithine and proline that simulate cell division leading to hyperplasia and fibrosis. In addition, a long-term increase in arginase activity leads to an uncoupled decrease in NO production, which has been shown to precipitate endothelial dysfunction (Horowitz et al., 2007). Thus, it is critical to boost, and diminish, the correct microglial and/or macrophage phenotypes at the right time in order to drive endogenous repair pathways following acute brain injury and to avoid maladaptive responses. Finally, a note of caution is needed with regard to the translation of M1-/M2-like polarization states developed in rodents to humans because several commonly used markers of the M2-like phenotype, such as arginase 1 and Ym1, are not expressed in human myeloid cells (Raes et al., 2005). However, others such as CD206 and CD163, appear to be consistent (Vogel et al., 2013). Therefore, if markers for M2-like polarization are carefully selected then it may be possible to identify M2-like microglia and macrophages in human brain injury and translate promising therapeutic strategies to the clinic.

Conclusion The latest research implicates M1/M2-like polarization of microglia and/or macrophages in acute brain injury, and activated microglia/macrophages with distinct molecular phenotypes can either promote neurorestorative processes, or hinder CNS repair and expand tissue damage leading to chronic neurodegeneration after TBI. In the context of CNS remodeling, the M2-like phenotype can support enhanced functional recovery after TBI via the induction of key neurorestorative processes, such as neurogenesis, angiogenesis, oligodendrogenesis and remyelination. Future therapeutic approaches that target post-traumatic neuroinflammation should avoid broad suppression of microglial activation, and instead should alter the balance between different phenotypes to promote maximal CNS remodeling and repair. This concept is supported by recent experimental studies that modulated M1/M2-like polarization to protect against post-traumatic neurodegeneration and enhance CNS repair. Therefore, future research is needed to determine key regulatory mechanisms that control phenotype switching in microglia in order to boost their “good” and suppress their “bad” activation states, and promote maximal functional recovery following TBI.

Microglia play a critical role in neuroinflammation following TBI

Microglial activation has beneficial and detrimental effects on neurons after TBI

Microglial phenotypic and functional responses may account for these dual effects

Treatments that alter M1/M2-like balance may enhance CNS repair after TBI

Acknowledgments This work was supported by NIH grant R01NS082308 (D.J. Loane), and The National Institute on Aging (NIA) Claude D. Pepper Older Americans Independence Center P30-AG028747 (D.J. Loane).