Neuroinflammation

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Chapter 15 Neuroinflammation

Pierre Gressens, Zinaida S. Vexler

Introduction

Inflammation and the developing brain

Until recently, the central nervous system (CNS) has been thought to be an immune privileged organ. However, neuronal injury or a potent immune stimulation can induce a cascade of immune responses within the CNS. It is now clear that neuroinflammation is linked with the development of several diseases affecting the adult and developing CNS, including infectious, autoimmune, degenerative, ischemic, and traumatic disorders. The impact and mechanisms of action of neuroinflammation might be different in the developing brain, as compared to the adult CNS. In addition, there is growing evidence that, in the context of inflammation, there is important crosstalk between the CNS and the periphery, and that systemic inflammation can have deleterious effects on the developing brain, further extending the concept of neuroinflammation. In the developing brain, these different aspects of neuroinflammation have been particularly well characterized in the context of perinatal brain damage; they will be further described in the present chapter in models of developmental neuroinflammation.

Perinatal Brain Damage

The consequences of perinatal injury include a spectrum of disorders, such as mental retardation, cerebral palsy, epilepsy, vision and hearing loss, learning difficulties, and school failure. Periventricular white matter damage, including periventricular leukomalacia, is most frequently observed in human preterm neonates [Volpe, 2009]. Full-term neonates with perinatal encephalopathy generally develop gray matter damage that most frequently affects the neocortex, basal ganglia, and hippocampus [Volpe, 2001]. Cerebrovascular occlusion leading to perinatal stroke may be arterial or venous, but excludes global injuries due to hypoxic-ischemic injury. Neurodevelopmental disability, including cerebral palsy, epilepsy, and behavior disorders, as well as impaired vision and language, is common after perinatal stroke [Kirton and deVeber, 2009].

The pathophysiology of perinatal brain damage (see Chapters 17 and 18) has proven to be multifactorial, with sensitizing factors occurring in utero that make the brain more vulnerable to secondary insults occurring around birth, such as hypoxia-ischemia (HI), and excess release of glutamate leading to excitotoxicity [Dammann et al., 2002; Mesples et al., 2005b; Nelson and Chang, 2008; Degos et al., 2008]. Systemic inflammation linked to chorioamnionitis has been recognized as a key sensitizing factor, while CNS inflammatory responses have been shown to play a key modulatory role in the amplitude of brain damage and subsequent adverse neurological outcome.

Systemic Inflammation and Perinatal Brain Damage

Epidemiological studies have shown a strong association between fetal infection/inflammation (chorioamnionitis) and brain damage in the newborn and/or neurological handicap in survivors [Dammann and Leviton, 2007]. Experimental studies have confirmed a sensitizing effect of systemic inflammation on perinatal brain lesions induced by hypoxic-ischemic or excitotoxic insults [Dommergues et al., 2000; Eklind et al., 2005]. In addition, some experimental data also suggest that perinatal exposure to infectious/inflammatory factors can alter, in a more or less subtle manner, the programs of brain development that will result in lasting neurological deficits. The relationship between this latter observation and human diseases remains to be fully demonstrated, although clinical evidence is supportive of this hypothesis [Volpe, 2009].

Sensitizing Effect

As mentioned above, perinatal brain damage may be caused by a combination of several insults. In this so-called multiple hit hypothesis, systemic infection/inflammation linked to chorioamnionitis can act as predisposing factors, making the brain more susceptible to a second stress (sensitization process). Indeed, systemic injection of low doses of lipopolysaccharide (LPS) to developing rats makes the newborn brain significantly more susceptible to hypoxic-ischemic insult [Eklind et al., 2005]. Similarly, systemic injection of interleukin-1-beta (IL-1β) or LPS to newborn mice or rats makes the brain much more sensitive to an excitotoxic insult [Dommergues et al., 2000]. The mechanisms by which sensitization is working are not yet fully understood, but could include changes in gene transcription and modifications of glutamate receptor activity.

Disruption of Brain Programming

Systemic infection/inflammatory factors can also alter brain development by themselves, even if they do not induce major clastic lesions. Accordingly, injection of Escherichia coli to pregnant rabbits induces diffuse white matter cell death [Debillon et al., 2000], and injection of Ureaplasma parvum, a pathogen frequently observed in chorioamnionitis, to pregnant mice induces myelin defects and loss of interneurons in the offspring [Normann et al., 2009]. Similarly, injection of LPS to pregnant rats induces transient central inflammation and myelination defects in the offspring [Rousset et al., 2006]. Of major concern, exposure of newborn mice to low doses of systemic IL-1β induces a moderate and transient inflammatory response during the neonatal period, that may be sufficient to disrupt oligodendrocyte maturation, myelin formation, and axonal development [Favrais and Gressens, personal communication]. These white matter abnormalities are moderate during the developmental period but persist until adulthood. They lead to permanent deficiencies in cognitive testing without detectable effects on motor function. The relevance of these abnormal behaviors to human pathology remains to be confirmed. The underlying molecular mechanisms include alterations of the transcription of genes implicated in oligodendrogenesis, myelin formation and axonal maturation.

Blood–Brain Barrier

At birth, the blood–brain barrier (BBB) in the neonate is substantially more mature than is commonly thought. The tight junctions are present early in embryonic development [Kniesel et al., 1996], restricting entrance of proteins into the brain in a controllable fashion, and by birth the BBB is functional with no fenestrations [Engelhardt, 2003]. The presence of the barrier substantially affects leukocyte passage but does not guarantee minimal leukocyte transmigration. The use of direct inflammatory challenge, such as intrastriatal injections of IL-1β or tumor necrosis factor alpha (TNFα) in rats of different ages, does not show a linear decline of leukocyte transmigration with age [Anthony et al., 1997], but rather that the newborn CNS is more resistant to inflammatory stress than the juvenile brain. The reported magnitude of BBB disturbance following HI or focal stroke in neonatal rodents varies and depends on the aspect of barrier studied [Svedin et al., 2007; Faustino et al., 2009]. Degradation of the extracellular matrix plays a role in neonatal ischemic injury. Excessive activation of matrix metalloproteinase-9 (MMP-9) early after HI is deleterious to the immature brain, as demonstrated by smaller injury size in MMP-9 knockout mice [Svedin et al., 2007] and following pharmacological inhibition of this protease [Leonardo et al., 2008].

Crosstalk between the Periphery and the CNS

The precise molecular mechanisms by which circulating mediators of inflammation have a deleterious effect on perinatal brain lesions remain a matter for debate [Hagberg and Mallard, 2005]. Circulating cytokines do not seem to cross the intact BBB easily, although this issue is contested. Different alternative pathways have been proposed to link serum cytokines with brain damage [Malaeb and Dammann, 2009]. Several major crosstalk mechanisms are outlined in Figure 15-1. Circulating cytokines could initially alter the permeability of the BBB to inflammatory mediators and cells. They could also act directly on parts of the brain lacking the BBB, such as the circumventricular organs, meninges, and choroid plexus, or, as demonstrated in the adult brain, indirectly through activation of the vagal nerve. Cytokine effects also could be mediated by cyclo-oxygenases (Cox) located in the BBB. In particular, cytokines could activate the inducible isoform Cox-2 to enhance the local production of prostaglandin E₂ (PGE₂), which could have deleterious effects on the developing brain. This latter mechanism has been demonstrated in a mouse model of perinatal excitotoxic brain damage [Favrais et al., 2007]. Some of these deleterious effects could involve an autocrine/paracrine loop, leading to excess production of inflammatory cytokines by brain cells.

Fig. 15-1 The relationship between systemic and local inflammation after neonatal brain injury, and the key inflammatory mechanisms that contribute to neonatal brain injury and modulate the role of sensitization.

1, Direct effect of the systemic inflammatory mediators on brain endothelium. 2, The effect of the systemic inflammatory mediators on leukocyte-mediated activation of local inflammation. 3, Microglia activation. 4, Propagation of neuroinflammation by release of toxic species, including cytokines and chemokines from macrophages, both endogenous and invading. 5, Direct injurious effects of microglial cells. 6, Effects of locally produced inflammatory mediators to propagate injury and directly affect neurons. 7, Protection by microglia-produced growth factors and cytokines. 8, Astrocyte-mediated effects. BBB, blood–brain barrier.

Glial Cells

Macrophages are seen in abundance following neonatal HI [McRae et al., 1995; Ivacko et al., 1996] and focal stroke [Dingman et al., 2006; Denker et al., 2007], producing inflammatory cytokines, high levels of nitric oxide, MMPs, and complement molecules. The early postinjury macrophage population is predominantly comprised of resident microglia rather than invading monocytes [Denker et al., 2007]. The notion that microglia contribute to, rather than limit, acute ischemic injury in the immature brain comes from findings that reduction in injury is associated with diminished microglial activation/monocyte infiltration [Arvin et al., 2002; Dommergues et al., 2003]. At the same time, several studies have shown that anti-inflammatory drugs thought to protect adult brain by reducing macrophage accumulation after stroke protect neonatal brain without directly affecting inflammatory mechanisms associated with microglial activation [Tikka et al., 2001; Fox et al., 2005; van den Tweel et al., 2005; Dingman, et al. 2006]. Distinct steps of microglial maturation and differentiation (such as expression of class II histocompatibility complex [MHC], cathepsin, and other molecules), and the propensity of neurons to undergo apoptosis in the developing brain, may account for this age-dependence of the microglial response. Complement activation – C3 and C1q deposition in particular – is deleterious after HI, whereas sensitizing of neonatal rodents with cobra venom factor or deletion of the C1q gene confers protection [Cowell et al., 2003; Ten et al., 2005]. The mechanisms of protection are not completely understood but may include decreased C3 deposition and reduction in neutrophil activation [Ten et al., 2005].

The relative contribution of proinflammatory mechanisms in astrocytes, as opposed to other roles of these cells in ischemic injury, is not well understood but astrocytes express MHC and can upregulate inducible nitric oxide synthase (iNOS) and increase cytokine production. Mast cells have been shown to play an injurious role in neonatal HI [Jin et al., 2007] and focal stroke [Biran et al., 2008]. The injurious effects of these cells are thought to depend on TGF-β and IL-9 [Mesples et al., 2005a]. Less is known about the contribution of the T and B cell infiltration that is seen at later time points after injury.

Individual Inflammatory and Cell Signaling Molecules

Cytokines and Chemokines

Clinical data on the pathophysiological role of inflammatory cytokines in perinatal brain damage, including in term babies, continue to emerge [Grether and Nelson, 1997; Foster-Barber and Ferriero, 2002; Bartha et al., 2004].

Overall, it appears that IL-1 potentiates ischemic brain injury. Following HI or transient middle cerebral artery occlusion (MCAO) in neonatal rats, brain IL-1β expression is increased rapidly [Hagberg et al., 1996; Denker et al., 2007] following a major rapid systemic increase of IL-1β protein [Denker et al., 2007]. Brain IL-1β levels can be further amplified by concomitant infection or manipulations within the oxidant pathways [Doverhag et al., 2008; Girard et al., 2008]. The pro-inflammatory shift in balance between IL-1β and IL-1ra following HI when combined with infection (modeled by endotoxin or LPS exposure) can play a role in the initiation of perinatal brain damage [Girard et al., 2008]. IL-1β-induced local inflammatory reaction and chemokine expression, with the consequent leukocyte attraction and BBB disruption, is age-dependent [Anthony et al., 1997]. Studies that use genetic deletion of IL-1β or IL-1α alone, or in combination (IL-1αβ knockout), however, show no protection after HI injury [Hedtjarn et al., 2005], indicating the presence of multiple or desynchronized pro- and anti-injurious effects of IL-1, which are all abrogated in knockout mice. IL-18, a pro-inflammatory cytokine from the IL-1 family, also contributes to HI injury [Hedtjarn et al., 2002].

TNFα can exhibit pleiotropic functions in the ischemic adult brain. It may lead to apoptosis by reacting with Fas-associated death domain (FADD) and caspase-8, whereas signaling through TNFR2 can lead to anti-inflammatory and anti-apoptotic functions, depending on the cell origin of TNFα production and the type of receptor involved. In adult, TNFα-induced sensitization prior to cerebral ischemia is shown to be associated with a preconditioning response, whereas TNFα sensitization post stroke is detrimental [Hallenbeck, 2002]. In neonatal brain injury the pathophysiological role of TNFα may also be complex. A key role of TNFα in injury in an excitotoxic model combined with prior inflammatory challenge (IL-1β) has been recently demonstrated [Aden et al., 2009]. A TNFα blocker, etanercept, did not affect brain damage when given before injury, but substantially ameliorated injury when given after the combined inflammatory and excitotoxic insult, suggesting that TNFα may act by producing an imbalance between pro- and anti-inflammatory cytokines in injured brain [Aden et al., 2009].

Type 2 T helper (Th2) cytokines play multiple roles in neonatal brain injury. The IL-9/IL-9 receptor pathway, which is most active in the newborn brain, has a direct anti-apoptotic action in the newborn neocortex [Fontaine et al., 2008], and contributes to HI and excitotoxic injury in the developing brain, presumably by activating mast cells [Dommergues et al., 2000; Patkai et al., 2001]. IL-10, a Th2 cytokine that is synthesized in the CNS and can act on hematopoietic and nonhematopoietic cells, can reverse injury caused by IL-1β, TNFα, and IL-6. In a white matter lesion model in P5 rats, protection is seen when IL-10 is administered post insult, whereas treatment prior to or at the time of insult is ineffective [Mesples et al., 2003].

Chemokines play a key role in crosstalk between peripheral and local responses. The pathophysiological role of the CC-class chemokine, monocyte chemoattractant protein 1 (MCP-1), in neonatal brain injury is evident from protection by functional inactivation of MCP-1 post insult [Galasso et al., 2000] or in mice with depleted IL-1 converting enzyme [Xu et al., 2001]. The role of the CXC-(Cys-X-Cys chemokine) family chemokines after neonatal brain injury is less understood but may be crucial for BBB modulation after inflammatory challenge of injured immature brain [Anthony et al., 1998]. Information on SDF-1 (CXCL12), which has been suggested to play a role in homing stem cells to regions of ischemic injury and repair in the adult, is rather scarce in the neonate but the timing for SDF-1-mediated chemotaxis and recruitment of reparative cells in the neonate may be narrow [Miller and Tran, 2005].

Intracellular Reactive Oxidant Metabolism

Oxidative stress and inflammation are tightly linked. Once activated, the inflammatory cells generate reactive oxygen species (ROS), which, in turn, trigger an inflammatory response. Superoxide anion, which is generated via Cox, xanthine oxidase, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and is utilized via superoxide dismutase (SOD) in the cytosol and mitochondria, plays an important role in ischemic injury. Copper Zinc (CuZn) SOD overexpression exacerbates brain injury after HI by increased superoxide utilization and hydrogen peroxide (H₂O₂) accumulation in injured developing brain [Fullerton et al., 1998; Sheldon et al., 2004]. Enhanced activity of anti-oxidative metabolism is protective [Sheldon et al., 2008], supporting the notion of the pro-injurious role of oxidative stress in neonatal ischemic brain injury.

The role of NADPH oxidase in neonatal brain injury is now better understood. Genetic deletion of gp91-phox (the catalytic subunit of nicotinamide adenine dinucleotide phosphate) increases the extent of brain injury in two neonatal models, the HI model in P9 mice and an excitotoxic model in P5 mice [Doverhag et al., 2008], rather than decreasing injury, as seen in adult stroke. More severe HI injury parallels reduced NADPH oxidase activity and enhanced local neuroinflammation.

NO generation can have opposing roles in the process of ischemic injury, depending on the NOS isoform and cell type. iNOS inhibition is neuroprotective in neonatal HI models [Peeters-Scholte et al., 2002; Dingman et al., 2006], but inhibition is not necessarily associated with reduced proliferation of microglial cells.

Intracellular Inflammatory Signaling Pathways

The transcription factor nuclear factor-κβ (NF-κβ) is involved in the regulation of inflammation and neuronal death after stroke. It is normally located in the cytoplasm as a heterodimer composed of p65 and p50 subunits, bound to the endogenous inhibitor protein Iκβ (Nfκβ subunit). Dissociation of this complex by phosphorylation of Iκβ allows it to translocate to the nucleus, bind to functional κβ sites, and induce an array of genes involved in inflammation. NF-κβ plays a dual role in HI [Nijboer et al., 2008a, b]. NF-κβ inhibition during the early peak of activation prevents upregulation and accumulation of p53 in the nucleus and caspase-3 activation, and results in major neuroprotection weeks after injury [Nijboer et al., 2008a], whereas prolongation of inhibition abolishes the effect on neuronal apoptosis and even exacerbates injury [Nijboer et al., 2008a]. Taken together, these results demonstrate the crucial role of timing in the effects of potentially neuroprotective strategies.

Data on the contribution of mitogen-activated protein kinases (MAPK) to neonatal ischemic injury continue to accumulate. The extracellular signal-regulated kinase (ERK1/2) activation is generally neuroprotective in brain injury, whereas MAPK p38 activation is deleterious after HI [Hee Han et al., 2002]. The contribution of p38 to injury may depend on the model used, however [Fox et al., 2005]. Targeted deletion of the brain-specific c-Jun N-terminal kinase (JNK) isoform has been shown to reduce the overall JNK activity and protect mice against HI [Pirianov et al., 2007].

Anti-Inflammatory Strategies and Neuroprotection

The progressive elucidation of the pathophysiological mechanisms by which inflammation can harm the developing brain allows the design of new neuroprotective strategies specifically targeting mediators of neuroinflammation. Different strategies have been tested in relevant animal models; some of them, especially those for which clinically relevant drugs are available [Wolfberg et al., 2007], are summarized in Table 15-1.

Table 15-1 Potential Targets and Drugs for Protection Against Neuroinflammation

Target	Examples of Drugs with Potential Clinical Use
Inflammation	Glucocorticoids, hypothermia
BBB integrity	?
MMP-9	?
Cox	Nonsteroidal anti-inflammatory drugs (Cox-2 inhibitors)
Microglia	Minocycline, melatonin, cannabinoids, hypothermia
Mast cells	Cromolyn, histamine receptor blockers
Cytokines	Etanercept (soluble TNFα receptor), IL-1 receptor antagonist, IL-10, tianeptine
Chemokines	Broad-spectrum chemokine inhibitors
ROS	N-acetyl-cysteine, melatonin, hypothermia
iNOS	Aminoguanidine, tilarginine acetate
NF-κβ	?
JNK	JIP peptide
Mitochondria	Caspase antagonists, minocycline
Multiple known targets	Hypothermia, erythropoietin, topiramate

BBB, blood–brain barrier; IL, interleukin; iNOS, inducible nitric oxide synthase; JIP,jnk interacting protein; JNK, Jun N-terminal kinase; MMP, matrix metalloproteinase; NF, nuclear factor; ROS, reactive oxygen species; TNF, tumor necrosis factor.

Although some of these strategies offer the potential to limit brain damage in the newborn and other neuroinflammatory diseases affecting the developing brain, several points need to be considered before moving towards clinical trials. The developing brain is very different from the adult brain and therefore data from the adult literature cannot be extrapolated without confirmatory developmental studies. Some inflammatory mediators, such as microglia, have deleterious or beneficial roles, depending on the timing after the insult and/or the cellular/molecular context in which they are administered. Therefore, the timing of intervention might be critical for outcome. Use of some of these agents also has to be evaluated in the context of their systemic and nervous system side effects during maturation, and balanced with their potential benefits.