Full-length ArticleNOX2 drives M1-like microglial/macrophage activation and neurodegeneration following experimental traumatic brain injury
Introduction
Recent experimental and clinical studies implicate neuroinflammation as a major contributor to chronic neurodegeneration and related neuropsychiatric dysfunction following single moderate-to-severe traumatic brain injury (TBI) or repetitive mild TBI (Aungst et al., 2014, Coughlin et al., 2015, Johnson et al., 2013, Loane et al., 2014, Mouzon et al., 2014, Ramlackhansingh et al., 2011, Shitaka et al., 2011). TBI-induced inflammation involves both glial cells and infiltrating blood leukocytes (Ziebell and Morganti-Kossmann, 2010). Resident microglia and peripherally derived inflammatory macrophages migrate to the site of injury and release immunomodulatory factors. Microglial and macrophage activation involves multiple phenotypes with different physiological roles (Boche et al., 2013, Cherry et al., 2014, Gordon and Martinez, 2010); the differential functions reflect their composition of polarization markers ranging from M1-like to M2-like (Cherry et al., 2014, Perego et al., 2011). M1-like activation is characterized by up-regulation of pro-inflammatory mediators (e.g. TNFα, IL-1β, NOS2) and the production of reactive oxygen species (ROS) that are essential for host defense (Gordon and Martinez, 2010), but when highly activated or prolonged can contribute to injury and cause progressive tissue loss (Loane et al., 2014, Wang et al., 2013). In contrast, M2-like activation is important for wound healing and resolving inflammation; it is marked by expression of factors such as arginase 1 (Arg1), Ym1, and Fizz 1, and release of neurotrophic factors that can promote tissue repair (Cherry et al., 2014). Although the mechanisms of the dysregulated M1-like neuroinflammatory response after TBI are not well understood, recent evidence has linked ROS from activated microglia to neuroinflammation-mediated oxidative stress and chronic neurodegeneration (Gao et al., 2012, Liao et al., 2012, Qin et al., 2013).
NADPH oxidase (NOX2) is a multi-subunit enzyme complex responsible for the production of both extracellular and intracellular ROS by phagocytes, including microglia and macrophages. Microglial NOX2 has been implicated in the pathogenesis of multiple chronic neurodegenerative disorders, such as Alzheimer’s disease (Choi et al., 2012, Shimohama et al., 2000), Parkinson’s disease (Wu et al., 2003), amyotrophic lateral sclerosis (ALS) (Wu et al., 2006), and multiple sclerosis (Fischer et al., 2012, van Horssen et al., 2012). Microglial NOX2 activation can cause neurotoxicity, both through the production of extracellular ROS (Qin et al., 2004), as well as through initiation of redox signaling that amplifies the pro-inflammatory response (Mander and Brown, 2005, Pawate et al., 2004). NOX2 inhibition limits microglial ROS production and attenuates M1-like activation in response to lipopolysaccharide (LPS), reducing microglial-mediated neurotoxicity (Gao et al., 2003, Qin et al., 2013, Qin et al., 2004).
NOX2 is transiently up-regulated in injured neurons and hypertrophic astrocytes in the contused cortex after TBI (Dohi et al., 2010, Kumar et al., 2015), and is highly expressed in reactive microglia/macrophages surrounding the expanding lesion for weeks and even months post-injury (Byrnes et al., 2012, Loane et al., 2014). Chronic microglial/macrophage NOX2 expression is associated with increased oxidative stress, progressive cortical and hippocampal neurodegeneration, and long-term cognitive impairments following TBI (Loane et al., 2014). Microglia/macrophages can change their activation state depending on the redox state of the microenvironment (Brune et al., 2013), and there is an age-related, NOX2-dependent shift in redox state towards an oxidizing environment that results in exaggerated M1-like activation and suppressed M2-like activation following TBI (Kumar et al., 2013). In injured cortex NOX2 is highly up-regulated in microglia/macrophages that co-express M1-like, or mixed M1-/M2-like activation markers, but not with single M2-like activation markers (Kumar et al., 2015). Increasingly, NOX2 is being recognized as an important therapeutic target for CNS injury (von Leden et al., 2016), and pharmacological or genetic intervention studies demonstrate that NOX2 inhibition after TBI is neuroprotective, mediated, in part, by targeting secondary neuroinflammation and microglial activation (Dohi et al., 2010, Loane et al., 2013, Lu et al., 2014, Zhang et al., 2012).
Here we set out to investigate cellular mechanisms that drive M1-like neuroinflammation and secondary neurodegeneration after TBI, and to establish the role of microglial/macrophage NOX2 in TBI pathology. The aims of the current study were to: (1) test the hypothesis that NOX2 drives M1-like neuroinflammation that contributes to neurodegeneration and loss of neurological function after TBI, and determine the relative role of NOX2 in peripheral macrophages versus resident microglia; (2) investigate cellular mechanisms that promote the alternative M2-like activation response after TBI in the absence of NOX2; and (3) determine if delayed systemic administration of a selective NOX2 peptide inhibitor improves outcomes after TBI by altering the M1-/M2-like neuroinflammatory responses.
Section snippets
Animals
Studies were performed using either NOX2-deficient (NOX2−/−; B6.129S-Cybbtm1Din/J, stock 002365; Jackson Laboratories, Bar Harbor, ME,) adult male mice (10–12 week old, 22–26 g), or age-matched C57Bl/6J (WT) male mice. Mice were housed in the Animal Care facility at the University of Maryland School of Medicine under a 12 h light-dark cycle, with ad libitum access to food and water. All surgical procedures were carried out in accordance with protocols approved by the Institutional Animal Care and
NOX2 is robustly increased in CD68+ macrophages/microglia after TBI and contributes to chronic neurodegeneration and loss of neurological function
Oxidative stress and ROS production represent important secondary injury responses after TBI (Hall et al., 2012) that are mechanistically linked with microglia/macrophage activation and post-traumatic neuroinflammation. TBI rapidly up-regulates NOX2 enzyme subunits in injured neurons and astrocytes during the acute phase after injury, followed by high NOX2 expression in amoeboid-like microgila/macrophages during the sub-acute and chronic periods (Dohi et al., 2010, Kumar et al., 2015, Loane et
Discussion
NOX2 is a key mechanism of microglial-mediated neurotoxicity in neurodegenerative disease (Lull and Block, 2010). NOX2 activation has been shown to contribute to secondary injury after ischemic brain injury (Chen et al., 2011, Chen et al., 2009), spinal cord injury (Khayrullina et al., 2015), and TBI (Byrnes et al., 2012, Dohi et al., 2010, Loane et al., 2014, Zhang et al., 2012). After experimental TBI, NOX2 is chronically up-regulated in CD68+ microglia/macrophages in the peri-lesional cortex
Acknowledgments
We thank Ashley Singh for expert technical assistance. This work was supported by NIH grant R01NS082308 (D.J. Loane), R01NS037313 (A.I. Faden), The National Institute on Aging (NIA) Claude D. Pepper Older Americans Independence Center P30-AG028747 (D.J. Loane), and a CONACYT Scholarship 249772/389071 (DM Alvarez-Croda).
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