Mechanisms of microglial activation in models of inflammation and hypoxia: Implications for chronic intermittent hypoxia.

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J Physiol 594.6 (2016) pp 1563–1577

Neuroscience

SYMPOSIUM REVIEW

Mechanisms of microglial activation in models of inflammation and hypoxia: Implications for chronic intermittent hypoxia Elizabeth A. Kiernan1 , Stephanie M. C. Smith1 , Gordon S. Mitchell2 and Jyoti J. Watters1 1 2

Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA Department of Physical Therapy, University of Florida, Gainesville, FL 32610, USA

The Journal of Physiology

Peripheral inflammation

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B

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Direct diffusion BBB breakdown Vagal transmission

Direct diffusion BBB breakdown Vagal transmission

Reactive oxygen species Intermittent hypoxia Reactive oxygen species

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C Inflammation Microglial activation

Neuron death

D DAMPs

Abstract Chronic intermittent hypoxia (CIH) is a hallmark of sleep apnoea, a condition associated with diverse clinical disorders. CIH and sleep apnoea are characterized by increased reactive oxygen species formation, peripheral and CNS inflammation, neuronal death and neurocognitive deficits. Few studies have examined the role of microglia, the resident CNS immune cells, in models of CIH. Thus, little is known concerning their direct contributions to neuropathology or the cellular mechanisms regulating their activities during or following pathological CIH. In this review, we identify gaps in knowledge regarding CIH-induced microglial activation, and propose mechanisms based on data from related models of hypoxia and/or hypoxia–reoxygenation. CIH

Gordon Mitchell is a Professor of Neuroscience in the Department of Physical Therapy and Director of the Center for Respiratory Research and Rehabilitation at the University of Florida in Gainesville. His research interests focus on fundamental mechanisms of spinal respiratory plasticity induced by acute exposures to intermittent hypoxia, and translation of those findings to treat devastating clinical disorders that compromise breathing and non-respiratory movements. Jyoti Watters is a Professor of Pharmacology in the Department of Comparative Biosciences at the University of Wisconsin-Madison. She studies cellular signalling and epigenetic mechanisms that regulate transitions between microglial inflammatory and beneficial activities to harness and manipulate them for therapeutic benefit. She uses rodent models of neuroinflammation induced by exposure to chronic intermittent hypoxia.

E. A. Kiernan and S. M. C. Smith contributed equally to this work. This was presented at the 1st PanAmerican Congress of Physiological Sciences ‘Physiology without Boarders’, Iguassu Falls, Brazil, August 2–4 2014. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

DOI: 10.1113/JP271502

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may directly affect microglia, or may have indirect effects via the periphery or other CNS cells. Peripheral inflammation may indirectly activate microglia via entry of pro-inflammatory molecules into the CNS, and/or activation of vagal afferents that trigger CNS inflammation. CIH-induced release of damage-associated molecular patterns from injured CNS cells may also activate microglia via interactions with pattern recognition receptors expressed on microglia. For example, Toll-like receptors activate mitogen-activated protein kinase/transcription factor pathways required for microglial inflammatory gene expression. Although epigenetic effects from CIH have not yet been studied in microglia, potential epigenetic mechanisms in microglial regulation are discussed, including microRNAs, histone modifications and DNA methylation. Epigenetic effects can occur during CIH, or long after it has ended. A better understanding of CIH effects on microglial activities may be important to reverse CIH-induced neuropathology in patients with sleep disordered breathing. (Received 30 August 2015; accepted after revision 16 January 2016; first published online 18 February 2016) Correspondiing author J. J. Watters: 2015 Linden Drive, Madison, WI 53706, USA. Email: [email protected] Abstract figure legend Putative mechanisms whereby CIH may contribute to CNS inflammation. Schematic diagram depicting three independent, but interacting, mechanisms of CIH-induced inflammation and neuropathology, and potential interactions between them. CIH increases reactive oxygen species with subsequent peripheral inflammation (1), neuronal injury (2) and microglial activation (3). Peripheral inflammation can cause CNS inflammation via diffusion of inflammatory molecules across an intact or compromised blood–brain barrier (BBB), and/or via vagal afferent neuron activation leading to secondary inflammation in the CNS, and pro-inflammatory microglial activities (A). Peripheral inflammation can induce neuronal injury or cell death by similar mechanisms (B), and/or by microglial production of pro-inflammatory or neurotoxic molecules (C). Neuron–microglial communication propagates neuroinflammation with damaged neurons or glia releasing damage-associated molecular patterns (DAMPs) into the extracellular space where they elicit microglial inflammatory activities via activation of pattern recognition receptors (e.g. Toll-like receptors) or scavenger receptors (D). Abbreviations CIH, chronic intermittent hypoxia; CSA, central sleep apnoea; DAMP, damage-associated molecular pattern; HRO, hypoxia–reoxygenation; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; OSA, obstructive sleep apnoea; PRR, pattern recognition receptor; TLR, Toll-like receptor.

Introduction

Chronic intermittent hypoxia (CIH) is a hallmark of sleep-disordered breathing. Although sleep apnoea (and CIH) is highly prevalent in otherwise normal individuals (Peppard et al. 2013), it is even more prevalent in clinical disorders of the central nervous system, including traumatic brain and spinal cord injury (Tran et al. 2010), ischaemic injury (stroke; Park et al. 2011), and genetic (e.g. Down’s syndrome; Marcus et al. 1991) or neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s diseases, multiple sclerosis, amyotrophic lateral sclerosis; Hoch et al. 1986; Manon-Espaillat et al. 1989; Atalaia et al. 2007; Bombois et al. 2010). Obstructive (OSA) and central (CSA) sleep apnoea cause morbidities such as systemic and pulmonary hypertension, metabolic syndrome, increased cancer risk and neuropathology (Beebe & Gozal, 2002; Drager et al. 2007; Gozal et al. 2015). CIH and/or OSA-induced neuropathologies include hippocampal cell death (Gozal et al. 2001), cognitive deficits (Engleman et al. 2000) and affective disorders (Barnes et al. 2004; Peppard et al. 2006). These morbidities are often

attributed to CIH/OSA-induced CNS inflammation (neuroinflammation) (Prabhakar et al. 2007; Gozal & Kheirandish-Gozal, 2008; Gozal, 2009; McNicholas, 2009; de Lima et al. 2016), although mechanisms of CIH-induced inflammation have scarcely been studied. In this brief review, we consider recent advances in our understanding of causes and consequences of neuroinflammation induced by multiple paradigms of hypoxia (including intermittent hypoxia and sustained hypoxia with or without rexoygenation) in the hope of gaining valuable insights concerning mechanisms of neuropathology in patients with OSA and other forms of sleep disordered breathing. Since resident CNS immune cells, microglia, are often major contributors to CNS inflammation, we focus on mechanisms regulating microglial activities, including their direct activation by hypoxia–reoxygenation (including CIH), and their indirect activation secondary to peripheral inflammation (the presence of inflammatory molecules and activated immune cells in the blood) (Gozal, 2009; Nadeem et al. 2013; Chen et al. 2015). Although certain doses of intermittent hypoxia are associated with plasticity and have functional benefits C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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Mechanisms of microglial activation in chronic intermittent hypoxia

(Dale et al. 2014; Navarrete-Opazo & Mitchell, 2014; Mateika et al. 2015; Peters et al. 2015), we focus on chronic intermittent hypoxia (CIH) protocols that simulate pathological aspects of sleep disordered breathing, inducing neuroinflammation and neuropathology (Dale et al. 2014; Navarrete-Opazo & Mitchell, 2014). In each section, we discuss data from OSA patients and/or experimental models of pathological CIH when available, but also present evidence from other models of hypoxia–reoxygenation and/or inflammation that may suggest neuroinflammatory mechanisms during CIH. Thus, our intent is to focus on gaps in knowledge and/or hypotheses that can be tested in the context of CIH. CIH-induced inflammation and cell death

Inflammation is a complex biological response initiated by pathogens and damaged tissue. Its primary function is to eliminate foreign bodies and repair injured tissues. A normal immune response involves the release of multiple inflammatory molecules, combined with growth/trophic (reparative) factors to heal any associated wounds. Improper control of either the inflammatory or reparative aspects of the normal response leads to chronic inflammation that exacerbates pathology (Medzhitov, 2008; Steinman, 2013). Inflammatory molecules produced include cytokines, such as tumour necrosis factor α (TNFα), interleukins (eg. IL-1β and IL-6), chemokines and adhesion molecules. The transcriptional regulation of many inflammatory cytokines, chemokines and enzymes responsible for inflammation is often controlled by pro-inflammatory transcription factors, particularly nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) (Lawrence, 2009). Molecules released from dead or dying cells (called damage-associated molecular patterns; DAMPs; Erridge, 2010) can trigger sterile inflammation (inflammation in the absence of a pathogen (Lehnardt et al. 2008)). Known DAMPs include proteins such as heat-shock proteins (e.g. HSP60) and high mobility group protein B1 (HMGB1). DAMPs may underlie immune responses observed during and following CIH. Inflammation can also be induced by production of reactive oxygen species (ROS), reactive nitrogen species and prostaglandins, produced by enzymes such as NADPH oxidase, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. These same enzymes are key players in normal inflammatory responses (Medzhitov, 2008). Rodent models of CIH and sleep disordered breathing (i.e. sleep apnoea) are associated with peripheral and CNS inflammation. OSA patients with severe sleep apnoea (greater apnoea/hypopnoea index (AHI), and/or greater oxygen desaturation independent of the AHI) exhibit increased levels of circulating adhesion molecules (Ursavas et al. 2007) as well as the cytokines TNFα and IL-6 (Ciftci C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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et al. 2004; Svensson et al. 2012). Similarly, rodents exposed to CIH exhibit increased NF-κB activity in cardiovascular tissues (Greenberg et al. 2006), upregulated carotid body cytokines and chemokines (Lam et al. 2012) and liver inflammation, including increased lipid peroxidation and cytokine production (Savransky et al. 2007). CNS Inflammation is also evident in rodents exosed to CIH, and it is thus predicted in OSA patients, where it is harder to establish CNS inflammation. CIH-exposed rats exhibit increased hippocampal pro-inflammatory proteins, such as heat shock proteins 60 and 70 (Gozal et al. 2002). After multiple days of CIH, rat microglia increase their expression of IL-1β, IL-6, TNFα, COX-2 and Toll-like receptor (TLR) 4 (Smith et al. 2013a), a receptor that responds to DAMPs such as HSP60 and HMGB1. CIH-induced ROS formation also increases inflammatory processes (Gao et al. 2012) and regulates pro-inflammatory enzyme expression, including iNOS and COX-2 (Zhan et al. 2005b; Block et al. 2007; Amor et al. 2010; Nair et al. 2011; Gao et al. 2012). Both iNOS and COX-2 are implicated in CIH-induced neuronal injury (Li et al. 2003, 2004) via their production of neurotoxic reactive nitrogen species and prostaglandins, respectively. Consequently, CIH increases net lipid peroxidation in the rodent CNS (Row et al. 2003). Similar ROS-dependent effects are observed in cortical neurons in vitro after even a single hypoxia–reoxygenation event; antioxidants (Burckhardt et al. 2008; Hui-guo et al. 2010) and inhibition of ROS producing enzymes (Hui-guo et al. 2010) reverse these effects, underscoring the potential for ROS to elicit CIH-induced neuronal toxicity. CIH-induced neuronal injury and death exhibit regional susceptibility. For example, there is greater injury in the cortex (Gozal et al. 2001; Row et al. 2003; Li et al. 2004; Xu et al. 2004) and hippocampus (Gozal et al. 2001; Row et al. 2003), key areas for learning and memory. CIH also promotes neural injury in CNS regions important in cardiorespiratory control such as cerebellar Purkinje neurons and the astigial nucleus (Pae et al. 2005). OSA patients and CIH-exposed rodents are reported to have reduced white matter in multiple CNS regions, suggesting demyelination and axon loss beyond neuronal apoptosis (Macey et al. 2008; Cai et al. 2012; Chen et al. 2015). Importantly, neuronal death and cognitive impairments in rodents exposed to CIH are minimized by iNOS, COX-2 and NADH oxidase inhibition (Li et al. 2003, 2004; Zhan et al. 2005a,b; Burckhardt et al. 2008; Hui-guo et al. 2010; Nair et al. 2011), suggesting that these pro-inflammatory molecules are key to CIH-induced neural injury. Peripheral inflammation, neuronal death and increased expression of pro-inflammatory molecules are consistent with a role for microglia in OSA and CIH neuropathology. In healthy brains, microglia have a dynamic, ramified morphology, involved in continuous surveillance of brain function, including synaptic activity and neuronal health,

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facilitating synaptic pruning or remodelling (Kettenmann et al. 2011). When cellular homeostasis is disturbed, microglia undergo complex activation processes, enabling them to migrate to the site of injury, proliferate, phagocytose dying cells or debris, and produce large quantities of neurotoxic and/or neuroprotective molecules (Kettenmann et al. 2011). Microglial activation and inflammatory molecule production contribute to neuroinflammation in almost all known neurodegenerative, traumatic and ischaemic neural disorders (Hanisch & Kettenmann, 2007). However, the specific involvement of microglia in CIH-induced neuropathology has scarcely been investigated. In the following sections, we will examine microglial activation in multiple models of hypoxia (including CIH), and explore potential mechanisms whereby CIH-induced peripheral and CNS pathology may activate microglia. Mechanisms of hypoxia-induced microglial activation: implications for CIH

Although little is known concerning mechanisms of CIH-induced neuroinflammation, even a single hypoxia–reoxygenation (HRO) event promotes inflammation in cultured microglia (Wang, 2007), and activates microglia in vivo (Smith et al. 2013b). Molecules regulating HRO-induced microglial inflammatory responses include mitogen-activated protein kinases (MAPKs), transcription factors and oxidative enzymes (Spranger et al. 1998; Vogt et al. 1998; Wang et al. 2000; Kim et al. 2003; Suk, 2004; Lai & Todd, 2006; Lu et al. 2006), many of which also play a role in peripheral CIH pathology (Yuan et al. 2011; Makarenko et al. 2014; Nanduri et al. 2015). Given the profound response of microglia to even a single HRO event, we suggest that cumulative effects from recurrent HRO may contribute to CIH-induced neuroinflammation, using many of the same molecular pathways identified for actions of CIH in peripheral tissues. Microglia may be activated by ROS from CIH per se, by factors released from injured neurons, and/or by the indirect effects of peripheral inflammation (Pineau & Lacroix, 2009). Thus, based on observations in models of hypoxia and reoxygenation, at least three mechanisms could regulate microglial activities during CIH: (1) peripheral inflammation, indirectly activating microglia via neural transmission or direct passage of pro-inflammatory molecules across the blood–brain barrier; (2) indirect microglial activation due to DAMPs released from nearby injured cells; and (3) direct microglial activation by CIH. Each potential mechanism is interrelated, and shares some common signalling mechanisms (Abstract Figure). Indirect microglial activation due to peripheral inflammation. Although the CNS is partially protected from

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peripheral immune responses by the blood–brain barrier, peripheral inflammation nevertheless elicits CNS immune responses (Abstract Figure, A and B). Some circulating inflammatory molecules cross the blood–brain barrier into the CNS via active transport (e.g. IL-1, interleukin-1 receptor antagonist and TNFα; Wilson et al. 2002) or via circumventricular regions that lack an effective blood–brain barrier (Konsman et al. 2002). Circulating cytokines also bind to brain endothelial cells and induce proinflammatory molecules such as nitric oxide (NO) or prostaglandins that readily cross the blood–brain barrier or increase blood–brain barrier permeability (Ek et al. 2001), thus enabling CNS entry of cytokines, chemokines and peripheral immune cells. CIH may compromise the blood–brain barrier (Lim & Pack, 2014), although peripheral immune cell accumulation in the CNS of rodent CIH models has not been reported. Vagal afferent neurons may also transmit inflammatory signals to the CNS by reacting to circulating proinflammatory cytokines and rapidly inducing CNS cytokine production (Balan et al. 2011). For example, peripheral IL-1 or lipopolysaccharide (LPS; a TLR4 agonist that does not cross the blood–brain barrier) elicits brainstem inflammation within minutes, an effect abolished by bilateral vagotomy (Laye et al. 1995; Bluthe et al. 1996). Thus, increased serum levels of pro-inflammatory molecules like C-reactive protein, TNFα and IL-6 in OSA patients (Ursavas et al. 2007; Goldbart & Tal, 2008; McNicholas, 2009; Ryan et al. 2009; Svensson et al. 2012) may activate microglia. Indirect microglial activation by damage-associated molecular patterns. Microglia express TLRs, a class

of pattern recognition receptors (PRRs) activated by molecules released from stressed or dying cells (Hanisch & Kettenmann, 2007; Pineau & Lacroix, 2009). Thus, injured or stressed neurons and glia may release DAMPs into the extracellular space where they are expected to activate microglial TLRs (Pineau & Lacroix, 2009) and initiate ‘sterile’ immune responses. PRRs bind highly conserved motifs on microbial cells (pattern-associated molecular patterns; PAMPs) (Kawai & Akira, 2009). Of the four described PRR families, TLRs are most relevant in microglia; however, CNS TLRs are not uniquely expressed by microglia. TLRs are also expressed by neurons, astrocytes and cerebrovascular endothelial cells (Carty & Bowie, 2011). While TLR involvement in CIH-induced microglial activation and neuronal injury has not yet been directly investigated, CIH upregulates TLR4 in rat microglia (Smith et al. 2013a) and TLR4 contributes to OSA-induced peripheral inflammation and HRO-induced CNS inflammation (Kim et al. 2010b; Akinnusi et al. 2013). Monocytes isolated from OSA patients have elevated TLR2 and TLR4 expression (Akinnusi et al. 2013), C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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suggesting increased pro-inflammatory responses in these cells. In agreement, TLR2, TLR4 and pro-inflammatory cytokine expression in OSA patients are normalized by Continuous Positive Airway Pressure (CPAP), suggesting a causal role for CIH and/or sleep fractionation (Akinnusi et al. 2013). Further, TLR4 (Kim et al. 2010b) and TLR2/6 (Kuhlicke et al. 2007) are upregulated by hypoxia in cultured macrophages through a hypoxia-inducible transcription factor 1α (HIF-1α)-dependent pathway, potentiating LPS-induced inflammation in hypoxia (Kim et al. 2010b). DAMPs associated with OSA include circulating TLR2/TLR4 ligands, such as HMGB1, monocyte responsive protein (MRP)-8/14 (formerly S100A8/9), HSP60, fibrinogen and oxidized low density lipoprotein (Chin et al. 1996; Wessendorf et al. 2000; Saletu et al. 2006; Bhattacharjee et al. 2009; Kim et al. 2009, 2010a; Kizawa et al. 2009; Lavie et al. 2010; Wu et al. 2010; Cholidou et al. 2013). Although source(s) of DAMPs in CIH are unknown, they may mediate peripheral inflammation in OSA, and CIH-induced neuroinflammation (Lehnardt et al. 2008; Ziegler et al. 2009; Gao et al. 2011). Microglia are the CNS cells with the highest TLR expression levels, and TLR-mediated microglial inflammation is linked with neurotoxicity in neurodegenerative, infectious, traumatic and ischaemic CNS pathologies (Carty & Bowie, 2011; Okun et al. 2011; Hanamsagar et al. 2012). Microglial TLR activation by PAMPs and DAMPs elicits production of cytokines, chemokines and enzymes promoting neurotoxicity. Although TLR-mediated inflammation induces neurotoxicity with bacterial, viral, fungal and prion infection, TLR activation by endogenous ligands during sterile inflammation, such as in CIH/OSA, remains poorly understood (Okun et al. 2011). In other models of CNS injury, DAMPs released from damaged or stressed cells activate microglial TLRs (Abstract Figure, D) causing inflammation, neurotoxicity, stress or death of surrounding cells and a ‘vicious cycle’ of inflammation and cell death (Abstract Figure C; Lehnardt et al. 2003, 2008; Ziegler et al. 2009; Lehnardt, 2010). HSP60, HMGB1 and MRP8/14 are the primary DAMPs known to induce microglial inflammation (Lehnardt et al. 2008; Ziegler et al. 2009; Gao et al. 2011), and these same molecules are elevated in OSA-patient blood (Wu et al. 2010; Kim et al. 2010a) and/or the hippocampus of CIH-exposed rodents (Gozal et al. 2002). For example, in the rodent brain, CIH increases endogenous TLR4 ligands in the CNS in a region-specific manner. Proteomic analyses of hippocampal CA1 and CA3 subfields revealed increases in HSP60 and α-synuclein in the CA1 but not CA3 region of the hippocampus (Gozal et al. 2002), the primary site of hippocampal cell death (Gozal et al. 2001). Since cell death increases extracellular HSP60 release (Lehnardt et al. 2008), CIH-induced DAMPs may contribute to CA1 neuron cell death, although this remains to be tested. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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Serum HMGB1 and MRP8/14 levels are increased in OSA patients (Wu et al. 2010; Kim et al. 2010a), and both have pro-inflammatory effects (Glass et al. 2010). Although HMGB1 is a nuclear protein, it is released into the extracellular space during cellular stress and inflammasome activation (Kim et al. 2006). In neurons, hypoxic/ischaemic injury leads to HMGB1 translocating from the nucleus to the cytosol where it is secreted from the cell (Faraco et al. 2007). HMGB1 activates microglial TLR2, TLR4 and/or the scavenger receptor MAC-1 (CD11b), thereby inducing inflammation (Yu et al. 2006; Gao et al. 2011). Macrophage HMGB1 release has autocrine/paracrine effects, enhancing macrophage inflammation (Kim et al. 2006; Campana et al. 2008); similar effects may also occur in microglia. MRP8/14 released from activated phagocytes enhances LPS-induced TLR4 signalling (Vogl et al. 2007) and increases MRP8/14 expression in the CNS, an indication of neuroinflammation (Vogl et al. 2007; Ziegler et al. 2009). Although HMGB1 and MRP8/14 induce TLR4-dependent inflammation following ischaemic injury (Kim et al. 2006; Faraco et al. 2007; Vogl et al. 2007; Ziegler et al. 2009), little is known concerning their roles in CIH. Since MRP8/14 oxidation reduces its pro-inflammatory activities (Lim et al. 2009), its neuroinflammatory effects on microglia may actually be constrained during CIH. Hypoxia may also directly enhance TLR4 signalling pathways, changing DAMP-induced inflammation. For example, HRO increases LPS effects on cultured microglia and potentiates iNOS, TNFα and NF-κB expression (Guo & Bhat, 2006), suggesting that HRO ‘primes’ TLR4-mediated inflammation. TLR signalling (Fig. 1) often involves recruitment of the adaptor protein myeloid differentiation factor-88 (MyD88), which initiates MAP kinase and relevant transcription factor activation, including NF-κB and HIF-1α (Hellwig-Burgel et al. 2005; Zughaier et al. 2005; Doyle & O’Neill, 2006; Lu et al. 2008). Interestingly, sustained hypoxia increases TLR4 expression in cultured microglia, and shifts TLR4 signalling from a MyD88 to a TRIF-dominant pathway (Ock et al. 2007); if a similar shift in TLR4 signalling occurs during CIH, it may attenuate CIH-induced microglial pro-inflammatory activities. Recent studies indicate that CIH increases mRNA and protein levels of TLR4 in the myocardium and the hippocampus (Yuan et al. 2014; Deng et al. 2015). It will be important to determine the hippocampal cell types in which TLR4 increases occur following CIH since cells other than microglia also express these proteins. Direct effects of CIH on microglial activities. Antioxidants

and superoxide dismutase mimetics block CIH-induced neuron death and cognitive impairment (Xu et al. 2004; Zhan et al. 2005b; Burckhardt et al. 2008; Hui-guo et al. 2010; Nair et al. 2011), but it is not yet clear if the beneficial effects of diminished ROS levels result from decreased

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neuronal versus microglial ROS formation. Although the direct impact of CIH on microglial ROS production has not yet been studied, it is possible that CIH directly affects microglia via ROS generation (Abstract Figure, 3). Indeed, ROS are essential for microglial inflammatory responses (Tschopp & Schroder, 2010). For example, HRO induces calcium influx through voltage-gated calcium channels and increases microglial NADPH oxidase activity, the major source of microglial extracellular ROS generation (Spranger et al. 1998). Further, HRO-induced ROS formation promotes microglial pro-inflammatory cytokine production (Spranger et al. 1998; Vogt et al. 1998; Chronic intermittent hypoxia

TLR4 DAMPs

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Figure 1. Model of CIH-induced inflammation via microglial TLR4 activation Damage-associated molecular patterns (DAMPs) are endogenous TLR4 ligands that are among the factors released from stressed and dying cells. Chronic intermittent hypoxia increases several DAMPs that can bind TLR4 in microglia and activate canonical inflammatory signalling cascades. TLR4 signalling utilizes two different pathways: MyD88-dependent and TRIF-dependent. The MyD88-dependent pathway increases pro-inflammatory gene expression through activation of the MAP kinases ERK, p38 and JNK, and transcription factors including NF-κB and AP-1. The TRIF-dependent pathway induces phosphorylation of IRF3, which then translocates to the nucleus and induces expression of type I interferons (IFNs) such as IFNβ and anti-inflammatory cytokines. Both MyD88 and TRIF proteins are increased peripherally and centrally after CIH.

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Hwang et al. 2008), and extracellular superoxide interacts synergistically with pro-inflammatory cytokines to induce neurotoxicity (Block et al. 2007). Common mechanisms of microglial activation

Mechanisms of CIH-induced microglial activation are likely to be complex; evidence from OSA patients and rodent CIH models suggests multiple activators converging on common downstream signalling pathways. In the next section, we discuss potential mechanisms of hypoxia and CIH-induced microglial activation that include (1) TLR4 activation eliciting downstream inflammasome activation; (2) MAPK activation; (3) increased expression and activity of pro-inflammatory transcription factors; and (4) possible long-lasting epigenetic modifications, including DNA methylation, hypoxia-sensitive microRNAs (miRNAs), and post-translational histone modifications. Inflammasome. The inflammasome is a high molecular mass multi-protein complex activated by ROS and TLR ligands (Lamkanfi et al. 2010; Kayagaki et al. 2011). The inflammasome complex (1) activates the enzyme caspase-1, cleaving pro-IL-1β and pro-IL-18 into mature cytokines, enabling their release through a non-canonical secretory pathway (Schroder & Tschopp, 2010) and (2) facilitates HMGB1 release (Lamkanfi et al. 2010; Kayagaki et al. 2011). Inflammasome-related molecules, including IL-18, IL-1β and HMGB1, are increased in the serum of OSA patients (Dyugovskaya et al. 2005; Minoguchi et al. 2005; Lavie & Polotsky, 2009; Wu et al. 2010). Because hypoxia upregulates caspase-11, inducing subsequent caspase-1 activity and IL-1β processing in microglial cultures (Kim et al. 2003), and HRO upregulates caspase-1 and IL-1β in the amygdala and impairs memory formation in rodents (Chiu et al. 2012), CIH may also activate the inflammasome and contribute to microglial IL-1β production (Smith et al. 2013a) and subsequent neuropathology and memory impairment (Gozal et al. 2001). Mitogen-activated protein kinases. MAPKs in immune cells upregulate both pro-inflammatory and cell survival pathway gene expression. MAPKs are activated by TLR4 ligands through a MyD88-dependent pathway (see Fig. 1), and they regulate microglial activities during sterile inflammation. Microglial p38 and ERK-1/2 MAPKs are differentially modulated by HRO. In cultured microglia, HRO activates p38, but not other MAPKs (i.e. ERK or JNK); increased p38 MAPK activity increases microglial TNFα and iNOS expression (Park et al. 2002). On the other hand, p38 MAPK inhibition blocks hypoxiaand HRO-induced iNOS activity and cytokine production (Park et al. 2002). Since HRO-induced p38 MAPK C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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activation increases caspase-11 expression, caspase-1 activation and IL-1β and IL-18 secretion (Kim et al. 2003), pro-inflammatory effects of p38 MAPK activation could be mediated in part via the inflammasome. CIH-induced inflammation in monocytes is associated with p38 MAPK activation and NF-κB activity (Ryan et al. 2007). In the cervical spinal cord, 8 h of a CIH protocol increases p38 MAPK activation in both microglia and motor neurons, and suppresses spinal phrenic motor plasticity (Huxtable et al. 2015). This effect is reversed by spinal p38 MAPK inhibition, confirming that CIH regulates microglial inflammation and its effects on neural function at least in part by MAPKs. Transcription factors. Multiple transcription factors mediate hypoxia-induced microglial inflammatory responses, including HIF-1α, NF-κB, CREB and AP-1. HIF-1α and NF-κB are well-studied and interrelated transcription factors that orchestrate complex inflammatory cascades. During normoxia, HIF-1α is hydroxylated by prolyl hydroxylases, targeting it for poly-ubiquitination, which keeps HIF-1α protein levels low (Sharp & Bernaudin, 2004; Hellwig-Burgel et al. 2005). In hypoxia, prolyl hydroxylases are inhibited, enabling HIF-1α protein accumulation, nuclear translocation and the increase of pro-inflammatory and pro-survival gene transcription. HIF-1α may also regulate innate immunity during normoxia since it is necessary for proper inflammasome function and NF-κB activation (Nizet & Johnson, 2009). TLR4 signalling activates HIF-1α independently of hypoxia, upregulating iNOS and COX-2 (Peyssonnaux et al. 2007), both of which contribute to CIH-induced neuronal death (Li et al. 2003, 2004). NF-κB, a prominent regulator of inflammatory gene transcription, is also hypoxia sensitive. During normoxia, NF-κB is sequestered in the cytosol by IκB. Activated IκB kinase-β (IKKβ) phosphorylates IκB, causing its degradation, freeing NF-κB to translocate into the nucleus (Blackwell & Christman, 1997). Prolyl hydroxylases negatively regulate IKKβ, targeting it for degradation. Thus, hypoxia permits IKKβ activation, enabling NF-κB nuclear translocation and inflammatory gene expression (Rius et al. 2008). Although sustained hypoxia preferentially increases HIF-1α (versus NF-κB) transcriptional activity, CIH preferentially increases NF-κB transcriptional activity (Ryan et al. 2005; Greenberg et al. 2006). For example, cultured monocytes from OSA patients exhibit increased NF-κB (not HIF-1α) activity in association with pro-inflammatory gene expression (Ryan et al. 2005; Greenberg et al. 2006; Htoo et al. 2006). However, these transcription factors work in concert since NF-κB is necessary for HIF-1α protein accumulation in hypoxia (Rius et al. 2008). These observations C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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may be cell-specific since HRO preferentially increases NF-κB activities in microglia versus astrocytes (Guo & Bhat, 2006). Although further evidence is needed, these studies suggest that CIH-induced microglial inflammatory gene expression may be at least partially regulated by NF-κB. Epigenetic regulation. Epigenetic modifications including DNA methylation, microRNAs and histone modifications underlie long-lasting and sometimes heritable changes (Portela & Esteller, 2010; Handy et al. 2011). These modifications prime cells to change transcriptional profiles in response to microenvironmental stimuli, such as hypoxia and inflammation (Muscari et al. 2013; Qiao et al. 2013; Tang et al. 2014; Cronk et al. 2015; Das et al. 2015). Epigenetic modifications may underlie long-lasting physiological or pathophysiological changes well after early life apnoeas (Bates et al. 2014) and/or CIH (Nanduri et al. 2012; Nanduri & Prabhakar, 2013; Bates et al. 2014; Prabhakar & Semenza, 2016). Although few studies directly verify that epigenetic modifications underlie long-lasting behavioural effects (i.e. breathing) in CIH or OSA, CIH-exposed neonatal rats exhibit increased DNA methylation at the superoxide dismutase 2 promoter, a modification that has lasting impact on chemoreflexes in adults (Nanduri et al. 2012). Even less is known concerning possible CIH-induced epigenetic modifications in microglia. Since hypoxia-induced microglial epigenetic modifications may regulate microglial activities long after the initiating stimulus (see below), this is an area in great need of additional research. DNA alterations may underlie epigenetic changes in peripheral cells and tissues in childhood OSA or neonatal CIH (Kim et al. 2012; Du et al. 2014; Khalyfa et al. 2014; Cortese et al. 2015), such as DNA methylation at immune/inflammatory-related genes (Kim et al. 2012). However, it is unclear if these changes persist into adulthood. Children with OSA have increased high-sensitivity C-reactive protein (hs-CRP) levels in their blood, and increased methylation of the promoter of FOXP3, a gene involved in T-cell regulation (Kim et al. 2012). Blood from children with OSA with additional abnormal postocclusive hyperaemic responses also exhibit increased DNA methylation upstream from the endothelial NOS promoter (Kheirandish-Gozal et al. 2013). Together, these observations demonstrate the potential for epigenetic modifications in peripheral immune cells following CIH, establishing a precedent for similar changes in microglia. Changes in DNA methylation regulate macrophage differentiation in tumour microenvironments (Kulshreshtha et al. 2007; Toffoli & Michiels, 2008; Garzon et al. 2010), effects that may be influenced by tumour hypoxia. This observation may help explain links between

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OSA and cancer where the risk of dying from cancer is higher in patients with OSA (Almendros et al. 2012; Campos-Rodriguez et al. 2013). Further research is needed to identify epigenetic mechanisms involved in CIH pathology and their relationship to cancer. There is at least one ongoing 5-year study evaluating DNA methylation and RNA transcriptome changes in the blood of OSA patients (Marin et al. 2014); results of this study will be of considerable interest. Many microRNAs (miRNAs), small non-coding RNAs that repress target gene translation, are induced by hypoxia (Nallamshetty et al. 2013). However, to date, only a few hypoxia-sensitive miRNAs have been implicated in macrophage or microglial inflammation (Taganov et al. 2006; Zhang et al. 2012; Kong et al. 2013). In primary microglia, oxygen and glucose deprivation upregulates miR-146a, miR-21, miR181a/c and miR-221 (Zhang et al. 2012; Kong et al. 2013), miRNAs with gene targets that include tumour suppressors, pro-inflammatory signalling molecules and cell cycle/survival genes. For example, miR-181c decreases TNFα production and, as a result, protects neurons from microglia-induced death (Zhang et al. 2012). Although the roles (if any) of these miRNAs in the inflammatory response to hypoxic injuries involving reoxygenation are not known, some clues may be provided by studies of miRNA regulation of TLR4 signalling, given the increase in DAMPs during CIH and OSA. The miRNAs miR-203, miR-146 and miR-155 regulate different components of TLR4 signalling (Taganov et al. 2006; Chassin et al. 2012; Qi et al. 2012; Schulte et al. 2013; Jiang et al. 2014; Yang et al. 2015). miR-203 negatively regulates microglial activation by targeting the MyD88 pathway (Yang et al. 2015), and miR-146a decreases IRAK1 and TRAF6 downstream of MyD88 in macrophages (Jiang et al. 2014). miR-155 underlies negative feedback control of macrophage TLR4 signalling (Schulte et al. 2013), and can additionally target the TRIF pathway (O’Connell et al. 2007). Thus, multiple miRNAs can constrain TLR4-induced inflammation. Whether these miRNAs or others are regulated by CIH is not yet known; however, hypoxia-dependent, epigenetic regulation of microglial inflammatory responses may occur via miRNAs. Hypoxia and pro-inflammatory molecules may also alter microglial gene transcription via histone modifications (e.g. methylation, acetylation, sumoylation and phosphorylation) (Portela & Esteller, 2010; Watson et al. 2010; Tang et al. 2014; Cho et al. 2015). The best-studied histone modification in response to hypoxia is lysine 27 methylation on histone H3 (H3K27me) via the Jumonji C (JmjC) family of histone demethylases (Shmakova et al. 2014). Unfortunately, to our knowledge, no studies of histone methylation have been done in microglia in response to any paradigm of hypoxia–reoxygenation. Although the expression of many

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JmjC-family members is increased during hypoxia (Beyer et al. 2008; Pollard et al. 2008; Lee et al. 2014), their catalytic activity requires molecular oxygen to repress or activate specific genes (De Santa et al. 2009; Hickok et al. 2013). In macrophages, hypoxia increases H3K27 methylation (Hickok et al. 2013), indicating that hypoxia may regulate macrophage gene transcription by inhibiting this demethylase. By extrapolation from macrophages, hypoxia may have similar effects in microglia, although this remains to be directly tested, particularly in the context of CIH. Net histone methylation during hypoxia can either increase or decrease gene transcription depending on the specific methylated lysine residue. For example, histone 3 lysine 9 (H3K9) methyl marks generally repress gene transcription, whereas H3K4 methylation stimulates transcriptional activation (Bannister & Kouzarides, 2011). Hypoxia can increase both repressive (H3K9) and permissive (H3K4) methylation marks (Tausendschon et al. 2010; Zhou et al. 2010), indicating that the effects of hypoxia are likely to be gene-specific. In addition to the direct effects on histone demethylase enzyme activity via oxygen availability, hypoxia can also exert indirect effects on histone methylation. For example, NO production inhibits demethylase enzyme activity (Hickok et al. 2013); HRO increases NO production in macrophages (Park et al. 2002) and CIH increases iNOS expression in microglia (Smith et al. 2013a). Thus, histone demethylase activity may be controlled both directly and indirectly in the context of hypoxia and inflammation. Summary and concluding remarks

CIH elicits complex effects that can impact microglia in multiple ways. An understanding of the effects of CIH on microglial activities including inflammation may be essential to combat some of the neuropathology that is characteristic of sleep disordered breathing. Despite its potential importance, we know very little about mechanisms of microglial regulation in response to any profile of hypoxia, including sustained hypoxia, hypoxia–reoxygenation, therapeutic (low dose) IH and/or more severe protocols of CIH that simulate IH during sleep disordered breathing. Recent efforts to harness low-dose intermittent hypoxia as a therapeutic tool (Dale et al. 2014; Navarrete-Opazo & Mitchell, 2014) require an understanding of how intermittent hypoxia transitions from a beneficial stimulus promoting neuroplasticity without neuroinflammation, to one that promotes neuroinflammation, neuronal death and cognitive deficits. In particular, we need to know the role microglial cells play in that transition (Dale et al. 2014). Microglial activities can be both beneficial and pathological. For example, after the initial insult of C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

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CIH, with associated neuronal cell death and cognitive decline, a period of reparative neurogenesis and functional recovery ensues (Gozal et al. 2001, 2003; Zhu et al. 2005, 2010; Tsai et al. 2011). We have yet to understand mechanisms underlying such compensation and partial recovery, but microglia are a known source of neurotrophic factors (e.g. brain-derived neurotrophic factor and vascular endothelial growth factor) that underlie neuroplasticity (Parkhurst & Gan, 2010), neuroprotection (Franco & Fernandez-Suarez, 2015) and neurogenesis (Sierra et al. 2014; Ribeiro Xavier et al. 2015). With greater understanding, we may optimize microglial activities to maximize reparative and protective versus deleterious and pathological effects. The CNS is a highly complex organ with extensive regional and cellular heterogeneity, and associated microenvironment differences. Indeed, microglia also exhibit regional hetergeneity (Hanisch & Kettenmann, 2007; Crain et al. 2009; Olah et al. 2011; Crain et al. 2013) that may contribute to region-specific neuronal responses when faced with intermittent hypoxia. We need to understand this heterogeneity and mechanisms that underlie microglial responses to intermittent hypoxia in short and long time domains. In this regard, of particular interest is the potential for epigenetic mechanisms to regulate microglial activities acutely and throughout life. Epigenetic regulation of microglia during intermittent hypoxia is an important area for future research. References Akinnusi M, Jaoude P, Kufel T & El-Solh AA (2013). Toll-like receptor activity in patients with obstructive sleep apnea. Sleep Breath 17, 1009–1016. Almendros I, Montserrat JM, Ramirez J, Torres M, Duran-Cantolla J, Navajas D & Farre R (2012). Intermittent hypoxia enhances cancer progression in a mouse model of sleep apnoea. Eur Respir J 39, 215–217. Amor S, Puentes F, Baker D & van der Valk P (2010). Inflammation in neurodegenerative diseases. Immunology 129, 154–169. Atalaia A, De Carvalho M, Evangelista T & Pinto A (2007). Sleep characteristics of amyotrophic lateral sclerosis in patients with preserved diaphragmatic function. Amyotroph Lateral Scler 8, 101–105. Balan KV, Kc P, Hoxha Z, Mayer CA, Wilson CG & Martin RJ (2011). Vagal afferents modulate cytokine-mediated respiratory control at the neonatal medulla oblongata. Respir Physiol Neurobiol 178, 458–464. Bannister AJ & Kouzarides T (2011). Regulation of chromatin by histone modifications. Cell Res 21, 381–395. Barnes M, McEvoy RD, Banks S, Tarquinio N, Murray CG, Vowles N & Pierce RJ (2004). Efficacy of positive airway pressure and oral appliance in mild to moderate obstructive sleep apnea. Am J Respir Crit Care Med 170, 656–664.

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Additional information Competing interests None declared. Author contributions All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Funding Supported by: NIH training grants GM0075007 (EAK) and HL07694 (SMCS), and NIH R01 HL111598 (GSM and JJW).

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