An inflammatory pathway to apnea and autonomic dysregulation

Respiratory Physiology & Neurobiology 178 (2011) 449–457

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Review

An inflammatory pathway to apnea and autonomic dysregulation夽 Eric Herlenius ∗ Neonatal Research Unit, Q2:07, Department of Women’s and Children’s Health, Astrid Lindgren Children’s Hospital, Karolinska Institutet, Karolinska University Hospital at Solna, S-171 76 Stockholm, Sweden

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Article history: Accepted 29 June 2011 Keywords: Brainstem Inflammation Hypoxia Neonatal apnea Prostaglandin Dysautonomia

a b s t r a c t Infection in infancy may dramatically aggravate an underlying cardiorespiratory dysfunction during a susceptible postnatal period. Children with immature brainstem respiratory control, as well as infants, may have periodic irregular breathing with potential detrimental apneas that are increased during sleep as well as during infectious episodes. Data now indicate that the proinflammatory cytokine interleukin (IL)-1␤ impairs respiration during infection via prostaglandin E2 (PGE2 ) and that infection, with associated eicosanoid release, is one of the main causes of respiratory disorders in preterm infants. Moreover, brainstem microsomal prostaglandin E synthase-1 (mPGES-1) is rapidly activated during transient hypoxia. An inflammatory mediated activation of the mPGES-1 pathway, e.g., by viral or bacterial infection, rapidly induces release of PGE2 in the vicinity of brainstem cardio-respiratory-related centers. This will depress the autonomic control networks, including the central drive to breathe. Hypoxia may then further reduce the activity of vital brainstem centers and the ability to autoresuscitate. This might have fatal consequences in vulnerable infants during a susceptible time frame. Here the evidence from human, animal and molecular studies to support this hypothesis is reviewed and how the pathogenesis of apnea and the response to hypoxia is associated with inflammatory pathways is discussed. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Apnea and Sudden Infant Death Syndrome (SIDS) represent major medical concerns in the neonatal population. Ventilatory responses to hypoxia and hypercarbia are impaired and inhibitory reflexes are exaggerated in the neonate. These vulnerabilities predispose the neonate to the development of apnea (for review see e.g., Mathew, 2010). SIDS is regarded to be multifactorial but the so-called triple hypothesis involves an impairment of brainstemmediated homeostatic (respiratory and autonomic) control (Moon et al., 2007). This potential brainstem dysfunctional control, postulated as a part of the pathogenesis underlying SIDS, could be due to preterm birth, exogenous factors such as nicotine or other drug exposures during fetal development that render the infant vulnerable to exogenous stresses such as infections (Paterson et al., 2009). Notably, infection and inflammation may play a crucial role in the pathogenesis of neonatal apnea and SIDS. Apnea is a common presenting sign of infection in neonates, and mild viral or bacterial

夽 This paper is part of a special issue entitled “Inflammation and CardioRespiratory Control”, guest-edited by Frank L. Powell and Yu Ru Kou. ∗ Tel.: +46 8 517 706 47; fax: +46 8 517 773 53. E-mail address: [email protected] 1569-9048/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2011.06.026

infection precedes death in the majority of SIDS victims (Fanaroff et al., 1998; Hofstetter, 2006; Prandota, 2004; Weber et al., 2008). The instability of breathing and its sensitivity to infection is well known among preterm infants, even if the role of individual and additive risk factors as well as exact mechanisms has not been fully understood, particularly the role of inflammatory molecules, e.g., cytokines and eicosanoids. Pro-inflammatory cytokines such as interleukin-l␤ (IL-l␤) may serve as key mediators in apnea and SIDS (Guntheroth, 1989). IL-l␤ is produced during an acute phase immune response to infection or inflammation and evokes a variety of sickness behaviors (for review, e.g., Dantzer, 2001; Dinarello, 1996, 2009). IL-1␤ has emerged as a major culprit in an expanding number of systemic and local inflammatory conditions termed “auto-inflammatory” diseases, e.g., osteoarthritis. In such diseases therapeutic neutralization of IL-1␤ results in a rapid and sustained reduction in disease severity (Dinarello, 2011). Several studies indicate that this pro-inflammatory cytokine also alters respiration and autoresuscitation (Froen et al., 2000; Hofstetter and Herlenius, 2005; Lindgren and Grogaard, 1996; Olsson et al., 2003; Stoltenberg et al., 1994). IL-l␤ induces expression of the immediate-early gene c-fos in respiration-related regions of the brainstem such as the nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla oblongata (RVLM) (Ericsson et al., 1997). However, IL-l␤ is a large lipophobic protein that does not readily diffuse across

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the blood–brain barrier. Moreover, the NTS and the RVLM do not appear to express IL-I receptor mRNA (Engblom et al., 2002), and IL-l␤ does not alter brainstem respiration-related neuronal activity in vitro (Olsson et al., 2003). Other molecules or pathways are thus involved in the effects that infection and inflation have on breathing and the response to hypoxia. In the present paper I will review and discuss probable pathways for the sometimes beneficial but in susceptible individuals possibly fatal interaction between the immune and the brainstem cardiorespiratory systems. The evidence and hypotheses as to why preterm infants and also older children are susceptible to infection will be presented. Finally, evidence is presented supporting the hypothesis that a rapid release of PGE2 in the vicinity of brainstem respirationrelated centers, induced by inflammation as well as hypoxia, could have a vital role in the potential deleterious dysfunctional response to hypoxia.

2. How do cytokines communicate with the autonomic nervous system? Infection by microbes, their products or injured endogenous cells will induce an inflammatory reaction, initially by interacting with pattern recognition receptors expressed on monocytes, macrophages and other cells of the innate immune system. These toll-like receptors (TLR) and NOD-like receptors transduce intracellular signals leading to the production and release of cytokines, eicosanoids and other inflammatory molecules that directly mediate cellular responses causing inflammation (Huston and Tracey, 2011). Inflammatory stimuli will rapidly increase circulating levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-␣), interleukin-1 beta (IL-l␤) and interleukin-6 (IL6). Cytokines synthesized in the periphery may act by crossing the blood–brain barrier and acting directly via neuronal cytokine receptors. As cytokines are large lipophobic molecules direct passage across the barrier protecting the brain seems to have a minor part in immune-to-brain communication. At least three parallel and coherent pathways for immune-to-brain communication to activate host defenses against infection exist. Putative pathways for cytokine signaling across the blood–brain barrier (BBB) include the binding of cytokines to: (a) circum-ventricular organs without BBB; (b) afferent neuronal pathways, such as the vagus and the carotid bodies; and (c) cytokine-mediated induction of the synthesis of a non-cytokine pyrogen, i.e. PGE2 , in endothelial cells constituting the BBB (Conti et al., 2004). All of these pathways will affect the control of breathing, either indirectly (e.g., fever) or via direct action on brainstem respiratory control centers. One possible pathway involves the area postrema, a circumventricular organ with incomplete endothelial blood–brain barrier (BBB) and therefore regarded as ‘gateway to the brain’ allowing blood-borne action of circulating IL-l␤. It also receives direct primary viscerosensory signals via the vagus nerve. Lipopolysaccharide (LPS) treatment induces IL-l␤ immunoreactivity in perivascular cells as well as dendritic cells of unknown origin (Goehler et al., 2006). Thus it may be a direct entry point for the immune system to communicate with the brain. However, it does not seem to be critical for IL-l␤- or viral-induced fever (Conti et al., 2004; Ericsson et al., 1994). The second pathway mentioned use sensory afferents in the immune-to-brain communication. The vagus nerve expresses IL-l␤ receptors and is important in conveying a central, CNS-mediated, response to immune stimuli outside the blood–brain barrier (Ek et al., 1998; Goehler et al., 1997, 2000). Vagal signaling has been shown to down-regulate inflammation via a cholinergic antiinflammatory pathway (Huston and Tracey, 2011). Vagus nerve

Fig. 1. COX and mPGES mediated biosynthesis of PGE2 . Availability of arachidonic acid (AA) is determined by the active regulation of phospholipases. AA can be metabolized to prostaglandin H2 via the cyclooxygenase COX pathway. COX-1 is constitutively expressed while COX-2 can be induced by a variety of inflammatory stimuli. One of the prostaglandin-E2 synthases, mPGES-1, is inducible and up-regulated in concert with COX-2 by inflammatory stimuli. mPGES-1 rapidly converts PGH2 to PGE2 and is crucial for inflammation-induced fever, pain, sickness behavior, anorexia and alterations in breathing behavior.

activity and the subsequent release of acetylcholine block cytokine production through the nicotinic alpha 7 acetylcholine receptor subunit, a regulator of the intracellular signals that control cytokine transcription and translation (Huston and Tracey, 2011; Liu et al., 2009). Thus the vagus is important in bi-directional communication between the nervous and the immune systems. The nucleus tractus solitarius (NTS) of the brainstem is a key nucleus for immune-to-brain signaling as the main site for termination of vagal afferents (Marty et al., 2008). Part of the fever response induced by pro-inflammatory cytokines seems to be mediated via vagal afferents. Nonetheless when all vagal input to the central nervous system is eliminated (i.e. by electrolytic lesion of the NTS) a febrile response, although reduced, remains. Mechanisms independent of vagal afferent projections to the NTS are thus involved in immune-to-brain signaling during inflammatory responses (Gordon, 2000). The third pathway is the one that has emerged as the major one during inflammation-induced fever, pain and sickness behavior and involves the production of prostaglandin, an immunomodulator that also has direct effects on respiration (Fig. 1). Prostaglandin E2 (PGE2 ) acts as a major inflammatory messenger across the blood–brain barrier (Engblom et al., 2002). Ericsson et al. (1997) presented evidence that an intramedullary pathway is involved in the stimulatory effect that circulating IL-l␤ has on neurons constituting the central limb of the hypothalamo-pituitary-adrenal axis. In a series of elegant studies Engblom et al. (2003) then demonstrated that activation of mPGES-1 and subsequent production of PGE2 is the rate-limiting steps in systemic inflammation and immune induced fever. This cytokine-induced pathway across the BBB using PGE2 is schematically illustrated in Fig. 2. Briefly, circulating IL-1␤ binds to IL-1 receptors on vascular endothelial cells of the blood–brain barrier and induces cyclooxygenase-2 (COX-2) and microsomal

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Fig. 2. The induced PGE2 pathway is implicated in causing increased apnea frequency and failure to autoresuscitate after a hypoxic event. During a systemic immune response the pro-inflammatory cytokine IL-1␤ is released into the peripheral bloodstream. It binds to its receptor (IL-1R) located on endothelial cells of the blood–brain barrier. Activation of IL-1R induces the synthesis of PGH2 from arachidonic acid via COX-2 and the synthesis of PGE2 from PGH2 via the rate-limiting enzyme mPGES-1. PGE2 is subsequently released into the brain parenchyma and binds to EP3R located in respiratory control regions of the brainstem, e.g., nucleus of the solitary tract (NTS) and the rostral ventrolateral medulla (RVLM). Moreover, as mPGES-1 is activated by hypoxia per se this may have an additive effect on PGE2 release in brainstem areas. This action of PGE2 may induce apneas and a reduced ability to autoresuscitate during a hypoxic event.

prostaglandin E synthase-1 (mPGES-1) activity (for review, see Engblom et al., 2002, 2003). COX-2 catalyzes the formation of prostaglandin H2 (PGH2 ) from arachidonic acid and mPGES-1 subsequently catalyzes the synthesis of PGE2 from PGH2 . PGE2 is then released into the brain parenchyma where it mediates several central effects of IL-1␤, e.g., fever induction (Coceani and Akarsu, 1998), behavioral responses (Crestani et al., 1991) and neuroendocrine changes (Ericsson et al., 1994). PGE2 signaling is thus a major pathway acting as a lipid mediator and messenger in immune-to-brain communication. The question then arises as to what role PGE2 plays in the brain and brainstem control of breathing? Let us first consider its role in the fetus and the perinatal transition that we call birth. 3. Cytokines and PGE2 effects on the perinatal brainstem and the fetal hypoxic response Together with adenosine and endorphins, prostaglandins play an active role in the regulation of fetal breathing (Herlenius et al., 2002; Kitterman et al., 1983; Takita et al., 1998). Prostaglandin released from the placenta, especially PGE2 , is a major regulator of the fetus. PGE2 acts as a circulatory regulator of the physiological function of several fetal organs including the brain. Prostaglandin contributes to maintenance of a patent ductus arteriosus, suppresses non-shivering thermogenesis, stimulates fetal insulin secretion and suppresses the fetal hepatic gluconeogenic pathway. Moreover, it contributes to the inhibition of fetal movements, including breathing movements, thus decreasing oxygen consumption (Dawes, 1984; Thorburn, 1995). PGE2 also possibly suppresses the activity of peripheral chemoreceptors during fetal life (Thorburn, 1992). If a fetus is challenged by severe hypoxia it does not respond with a ‘flight-or-fight reaction’ as an adult would, but rather becomes immobilized, stops breathing and becomes bradycardia (Lagercrantz, 1996). This paralytic state of the fetus can be caused by inhibition of chemical neurotransmission. When PO2 (oxygen

partial pressure) and glucose might be temporarily reduced to levels that would be associated with severe neuronal damage in an unprotected baby, adenosine and PGE2 levels are increased (Walker et al., 2000). Adenosine is a neuromodulator that is involved in suppression in the fetal brain. The low oxygen level in the womb, sometimes referred to as the “Mount Everest in utero” (PO2 is <30 mm Hg), contributes to higher concentrations of adenosine compared to those after birth. Adenosine inhibits central brainstem respiration-related neurons in fetal rats (Herlenius et al., 1997, 2002) thereby reducing their activity. Furthermore, it protects the brain and neurons by decreasing synaptic transmission and neuronal activity via adenosine-A1 receptors (Herlenius and Lagercrantz, 1999; Johansson et al., 2001). Adenosine concentration increases during energy failure and hypoxia, and it can act as a modulator to cope with the hypoxic situation (Berne, 1986; Fredholm, 2010). Possibly the same applies for prostaglandins (Walker et al., 2000). PGE2 released during birth and hypoxia may have acute neuroprotective effects during fetal hypoxia and ‘the stress of being born’ (Lagercrantz and Slotkin, 1986). The action is through stimulating E-prostanoid receptor subtype 3 (EP3R)-Gi-activation and subsequent lowering of cAMP and reduction of neuronal activity leading to increased brain resistance to acute hypoxia. This neuroprotective effect of PGE2 is supported by data indicating that PGE2 decreases neocortical network activity through postsynaptic reduction of excitatory synaptic transmission (Koch et al., 2010). Although a decrease of the placental inhibitor PGE2 is not crucial for establishing continuous breathing movements at birth, its removal allows the newborn baby to be vigilant after birth (Alvaro et al., 2004). Post-birth the decreased inhibitory ‘tone’ exhibited by adenosine and prostaglandins originating from the placenta on the newborn infant and the newborn brain contributes to resetting of the peripheral chemoreceptors and increased central respiratory pattern generation. Together with cooling at birth which initiates the first breaths, this contributes to the transition from fetal intermittent breathing movements to the continuous breathing necessary for life outside of the womb (Blanco et al., 1987).

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4. Preterm infants have unstable breathing – purinergic antagonists may help Preterm infants have periodic unstable breathing that is sensitive to the environment (Barrington and Finer, 1991). A decreased respiratory drive and impaired pulmonary function predispose the premature infant to apnea and hypoventilation that in turn may precipitate desaturation and/or bradycardia (Martin and Fanaroff, 1998). Peripheral chemoreceptors are only active at very low oxygen levels during fetal life and become essentially silent in the immediate postnatal period due to the sudden increase in PO2 from <30 mm Hg to 50 to 70 mm Hg range or higher (Mathew, 2010). Resetting of the peripheral chemoreceptors gradually allows them to participate in postnatal control of breathing (Holgert et al., 1995; Roux et al., 2005). The peripheral chemoreceptor response is reduced in preterm infants with bronchopulmonary dysplasia (BPD) (Katz-Salamon, 1995). This likely contributes to the instability and inadequate response to environmental stresses such as hypoxia that children with BPD (bronchopulmonary dysplasia) exhibit. Preterm infants with apnea, regardless of its type and severity, have depressed and delayed ventilatory responses to hypercapnia, and appropriate but somewhat delayed responses to hyperoxia (Katz-Salamon, 2004). Development of the intrinsic properties and functional organization of the central respiratory network continues after birth. This includes maturation of dendritic morphology and increase in synaptic connections and myelination with progressive maturation of the brainstem. Thus immature central control of respiration is an important mechanism in the pathogenesis of ‘irregular breathing’ and apnea of infancy. It is still unclear whether this early instability of respiratory control is associated with longer-term morbidity in this high-risk population, but the pressure to provide treatment for prevention of these episodes is huge. As the irregular breathing and apneas prolong the stay in hospital this may have so adverse an effect that mechanical ventilation is required and can in worst-case scenarios be fatal. Management of premature infants with apnea involves pharmacological and nonpharmacological therapies. Non-pharmacological treatment includes altered body position, supplemental oxygen and non-invasive ventilator support such as CPAP. When non-pharmacological measures are ineffective, which they often are, drugs such as methylxanthines (theophylline and caffeine) are used (Martin and Wilson, 2009). Thus some 90% of infants who are born before 34 weeks of development receive methylxanthine derivatives such as caffeine during their stay in neonatal wards. Daily caffeine doses up to an equivalent of 25 cups of coffee a day might be given to individual infants. Caffeine acts on adenosine-A1 and adenosine A2 receptors (A1R and A2R) (Yang et al., 2009). The A1R expressed on neuronal cells has a direct effect on the rostral ventrolateral medulla respirationrelated neurons, central pattern generation and breathing output (Herlenius and Lagercrantz, 1999; Johansson et al., 2001). Caffeine treatment in very preterm infants <1250 g seems to yield respiratory and neurodevelopmental benefits compared with placebo according to the Caffeine for Apnea of Prematurity (CAP) trial (Schmidt et al., 2007). This is encouraging, since deleterious effects have been described in several animal models (Thurston et al., 1978). Nonetheless there are still lingering concerns about manipulating adenosinergic and related neurotransmitter function during early life (Martin and Wilson, 2009; Schmidt, 1999). Animal studies indicate that long-term effects of caffeine during pregnancy and postnatal life may alter behavior including respiratory control in the offspring. For example; fetal and postnatal exposure to caffeine (equivalent to 3 cups of coffee per day) in rats induces longterm changes in the regulation of breathing without detectable

changes in adenosine receptors (Herlenius et al., 2002). Subtle but long-term changes in the adenosine receptor system (A1 and A2a receptors) might explain the altered respiratory response to hypoxia in adult male rats exposed as neonates to caffeine (Picard et al., 2008). Furthermore, even a single dose of caffeine to pregnant mice (equivalent to two cups of coffee in humans) decreases cardiac development and has long-term adverse effects on postnatal cardiac function in offspring (Wendler et al., 2009). In preterm humans treated for apnea with methylxanthines secondary effects include sleep deprivation (Hayes et al., 2007). Potential long-term effects of this in humans are still unknown. Moreover, the CAP study indicates that the effects of caffeine may vary in subgroups. In contrast to preterms in need of ventilator support that receive caffeine within the first days of life, no beneficial effects of caffeine are evident in preterms treated with caffeine as a prophylaxis for apnea (Davis et al., 2010; Henderson-Smart and De Paoli, 2010). Thus the uncertainties are not fully resolved as to when, if and how to use methylxanthines as a therapeutic tool in preterms. Caffeine modulates both innate and adaptive immune responses (for review see Horrigan et al., 2006). Indeed, two effects of caffeine treatment in very preterm infants seem to be that it reduces the frequency of patent ductus arteriosus and the incidence of BPD, related to prostaglandin and inflammation. Caffeine, in low concentration (<20 mg/kg, roughly 2–3 cups of coffee/24 h in adult humans), may act as an anti-inflammatory modulator as it can suppress the production of the proinflammatory cytokines (TNF-␣, IL-2 and IL-5) as well as chemotaxis, and leukocyte proliferation (Horrigan et al., 2006; Valdez et al., 2011). However, caffeine also has pro-inflammatory effects as shown in human adults and young preterms (Bishop et al., 2005; Valdez et al., 2011). This is likely through the antagonism of A2a receptors that seems to be the most important subtype of adenosine receptors in mediating the immunomodulator effects of adenosine (Bishop et al., 2005; Horrigan et al., 2006). Caffeine may reduce the levels of antinflammatory (IL-10) and increase the pro-inflammatory cytokines, thus reversing the anti-inflammatory effect observed at lower caffeine concentration (Ohta et al., 2007; Valdez et al., 2011). Considering in vitro, in vivo animal and human data the dose dependent immunomodulating effects of caffeine on inflammation could have important, good and bad, roles in the treatment of apnea of prematurity and its long term consequences. Indeed, the non-steroidal anti-inflammatory drug indomethacin has been used to treat apnea of prematurity (Hammerman and Zangen, 1993). However, indomethacin causes multiple adverse effects in the newborn population including drug-induced reductions in renal, intestinal, and cerebral blood flow (Schmidt et al., 2001, 2006). If irregular breathing and apnea occur in all preterm infants should we treat it and if so according to what indications and in which infants? To treat an immature, not yet fully adapted or myelinated respiratory control system by increasing the tone/excitability of neural networks, as caffeine does via Ad-A1R, could indeed stabilize the output, but considering the potential long-term side effects one should be aware of what to treat and why. Part of the effects of caffeine seems to be related to its modulation of inflammation. Apnea can be the presenting sign of sepsis (Fanaroff et al., 1998). Do viral infections or even minor inflammations make the already unstable breathing even more irregular and increase apnea/hypopnea and hypoxemia incidence in human neonates?

5. Preterms have unstable breathing patterns that are sensitive to infection Very preterm infants have a delayed maturation of respiratory control that places them at increased risk of cardiorespiratory

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events beyond term-equivalent gestation and increases their vulnerability to postnatal events such as infection. To characterize the occurrence of cardiorespiratory events in extremely preterm infants a prospective study was performed in 33 extremely preterm infants born between 23 and 28 weeks gestational age (GA) (Hofstetter et al., 2008). Infants were eligible for the study if they were clinically stable with no mechanical ventilation requirements at the time of recruitment. Infants with congenital abnormalities or medical conditions likely to cause secondary apnea (i.e. intraventricular hemorrhage (IVH), grade >2; white matter disease; seizure disorder) were excluded. Weekly overnight cardiorespiratory recordings were performed between 25 and 45 weeks post-conceptional age during and after their discharge from the neonatal intensive care unit. Methylxanthine treatment, which was used by patients during half (49%) of the recordings, had no effect on baseline respiratory or heart rate or saturation. Methylxanthine therapy neither affected the incidence of apneic and hypoventilation episodes nor affected the periodic breathing that the majority of extremely preterm infants displayed (Hofstetter et al., 2008). Notably, in very preterm infants cardiorespiratory events still occurred frequently at termequivalent age and following hospital discharge. This concords with previous studies indicating that asymptomatic preterm infants exhibit frequent and potentially clinically adverse cardiorespiratory events at term-equivalent age (Darnall et al., 2006). Preterm infants born at later gestational ages (>33 weeks) have significantly fewer apnea events at term-equivalent age (Henderson-Smart and Duck-Chong, 1981). Moreover, infants demonstrated a pronounced vulnerability to infection during early postnatal development. Although infection did not affect baseline respiratory rate, heart frequency or O2 saturation, it had a pronounced impact on the occurrence of apneas (apneic index) and hypoxemia incidence in infants ≤30 weeks postconceptional age (PCA) (Hofstetter et al., 2008). The persistence of cardiorespiratory events beyond term-equivalent age as well as the marked impact of infection on cardiorespiratory function indicates that close surveillance after hospitalization is of crucial importance in extremely preterm infants (Hofstetter et al., 2008). In addition, infection was more important than gestational age in determining the occurrence of apneas and hypoxic episodes. Furthermore, this study reveals a correlation between the inflammatory marker CRP and Apneic Index in infants at younger PCA (Hofstetter et al., 2008). Thus regarding the autonomic and cardiorespiratory control systems very preterm neonates are still immature at term equivalent age and are particularly vulnerable to infection. This finding supports previous investigations revealing a similar association between infection and apnea in newborn infants (Bruhn et al., 1977; Fanaroff et al., 1998). The increased susceptibility and vulnerability to infection is reflected in the increased risk for central apneas that preterms have when exposed to respiratory syncytial virus (RSV) compared to children born at term-age (Schiller et al., 2011). While infection is a major contributor to apnea/hypopnea events and frequency of O2 desaturations/hour in very preterm infants, the question remains as to Why? Infection and immediate early inflammatory proteins (IL-l␤, IL-6 and TNF-␣) could all be involved in the altered respiratory behavior of a child. As described in the preceding text the lipid mediator PGE2 is important during fetal development but is also a major and rapid inflammatory messenger responsible for several of the body’s responses to infection/inflammation. So what role does PGE2 play in postnatal life and in relation to breathing and hypoxic responses? PGE2 has long been known to inhibit breathing after birth and causes hypoventilation and apnea in newborns of mammals, e.g., rodents, lambs and humans (Guerra et al., 1988; Hofstetter, 2006). Its role during infection-related auto-

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nomic dysregulation and the central response to hypoxia has begun to emerge.

6. IL-l␤ alters respiration and the hypoxic response via mPGES-1 activation and PGE2 binding to brainstem EP3 receptors IL-1␤ prolongs the duration of larynx-stimulation induced apnea and alters resuscitation in piglets (Stoltenberg et al., 1994). Moreover, the concentration of IL-1␤ in pharyngeal secretions is also positively correlated with the clinical severity of apnea in human infants (Lindgren and Grogaard, 1996). It was recently revealed that IL-1␤ exerts these effects via a PGE2 -mediated pathway. Indomethacin, a non-specific COX inhibitor, attenuates the respiratory depression induced by IL-l␤ (Olsson et al., 2003). PGE2 itself depresses breathing in fetal and newborn sheep in vivo (Guerra et al., 1988; Kitterman et al., 1983; Tai and Adamson, 2000) and inhibits respiration-related neurons in vitro (Olsson et al., 2003). To further investigate the effects of IL-l␤ on breathing in neonates we used 9-day-old wildtype mice and examined their response to changes in ambient CO2 and O2 90 min after IL-1␤-administration. IL-1␤ reduced breathing during normoxia but not temperature or peripheral chemoreceptors when examined after 90 min. However, the response to severe hypoxia was decreased and the ability to autoresuscitate and survive was reduced (Hofstetter and Herlenius, 2005). To determine the mechanism underlying these detrimental effects of IL-l␤ on breathing and hypoxic response we used mice lacking the inducible enzyme for PGE2 production mPGES-1. Such mice were unaffected by the detrimental effects of IL-1␤. Consequently, the induced mPGES-1 PGE2 pathway is a key regulator of the respiratory response to infection and hypoxia (Hofstetter et al., 2007). The induced PGE2 pathway is depicted in Fig. 2. Furthermore, E-prostanoid receptor subtype 3 (EP3R) receptors for PGE2 are located in respiration-related regions of the brainstem: the NTS (nucleus tractus solitarius) and RVLM (rostral ventrolateral medulla oblongata) (Ek et al., 2000; Nakamura et al., 2000). Activation of human EP3R causes a decrease in [cAMP]i and a modest increase in [Ca2+ ]i (Yang et al., 1994). Reduction of cAMP decreases the firing amplitude and rate in respiration-related brainstem neurons and duly breathing activity (Ballanyi et al., 1997). Using wildtype and EP3R-knockout mice we determined that EP3R is expressed in the rostral ventrolateral medulla oblongata, including co-expression of in EP3R some Neurokinin-1 receptor (NK1R) expressing neurons in pre-Bötzinger and pFRG regions (Hofstetter et al., 2007). These NK1R+ cells are putative pacemaker neurons in the neural network that generate breathing rhythm and dysfunction of these NK1R+ cells can induce apnea. Furthermore, the apneas and respiratory depression evident following intracerebroventricular injection of PGE2 were absent in EP3R-knockout mice. Wild-type mice compared with mice lacking mPGES-1 exhibited a lower respiratory activity during hyperoxia, indicating that endogenous PGE2 has a tonic effect on respiratory rhythmogenesis during the perinatal period (Hofstetter et al., 2007). This would be consistent with evidence that prostaglandin synthesis inhibitors, which block endogenous prostaglandin production, increase breathing movements and central respiration during early postnatal life (Guerra et al., 1989). Hypoxia, per se, rapidly induces an increase in mPGES-1 activity in vivo subsequent to brainstem-specific release of PGE2 with a synergistic depression of respiratory centers to that induced by IL-1␤, e.g., see Fig. 1B in Hofstetter et al. (2007). This was unexpected but is consistent with evidence that anoxia induces PGE2 production in mice cortical sections ex vivo and prostaglandin H

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synthase-2 mRNA expression in the piglet brain (Degi et al., 1998). PGE2 is thus also involved in the acute hypoxic response, Fig. 2. The preceding examples suggest that PGE2 induced by IL-1␤ as well as hypoxia selectively modulates respiration-related neurons in the RVLM, including the pre-Bötzinger complex (preBötC), via EP3R. Other neuromodulators, including PGE1 , inhibit preBötC neurons and slow respiration-related rhythm (Ballanyi et al., 1997), and preBötC lesions may disrupt anoxic gasping and evoke central apneas and ataxic breathing (McKay and Feldman, 2008). These respiration-related neurons might be critical for an adequate response to hypoxia, maintaining brainstem homeostasis with gasping and autoresuscitation and thus restoring oxygen levels (Paton et al., 2006). PGE2 -induced depression of this vital brainstem neuronal network, e.g., during an infectious response, could result in gasping autoresuscitation failure and ultimately death. This could have important treatment implications for neonatal apnea related to infection since the adverse effects of IL-1␤ were attenuated by selectively deleting the mPGES-1 and EP3R genes. Indomethacin has been previously used to treat apnea of prematurity. However, indomethacin causes multiple adverse effects in the newborn population (Schmidt et al., 2001), and thus treatment modalities selectively targeting mPGES-1 or EP3 receptors could possibly be more beneficial. To investigate if the described mechanism had any clinical relevance we next examined cerebrospinal fluid (CSF) from preterm babies (mean post-conceptional age 32 week). There is a significant correlation between prostaglandin E2 concentrations in the CSF, C-reactive protein (CRP) levels and apneic breathing in human neonates (Hofstetter et al., 2007). This concurs with a relationship previously identified between PGE metabolites and apnea in preterm infants (Hoch and Bernhard, 2000). PGE2 thus seems to have a central role of in breathing disorders such as apnea and diminished autoresuscitation following hypoxia in neonates. In particular, increased levels of PGE2 and/or metabolites thereof in CSF are associated with increased apnea frequency and decreased ability to autoresuscitate following hypoxia. A correlation between CRP levels, PGE2 levels and apnea (Hofstetter et al., 2007) indicates that monitoring PGE2 levels and/or metabolites thereof alone or in conjunction with markers of infection, such as CRP, can provide diagnostic benefits in relation to inflammationrelated breathing disorders.

in the diagnosis and surveillance of inflammation-related breathing disorders in mammals (Herlenius et al., 2009). Among infants having an infection and associated apnea the elevation of PGE2 and its metabolite levels in CSF appears to occur at an earlier stage than elevation of CRP levels in blood (<30 min versus >12 h). Assessment of levels of PGE2 and/or metabolites thereof in a biological sample (e.g., urine, blood or CSF) could thus offer advantages for diagnosis, treatment and management of patients having infection-associated inflammation and breathing dysfunction in comparison with assessment of CRP levels. Infection depresses respiration in human neonates via systemic release of cytokines followed by the biosynthesis and central action of PGE2 . The mechanism described herein could explain previous reports describing an independent association between CRP levels and the apnea/hypopnea index in children with sleep apnea (Tauman et al., 2004) as well as a positive correlation between IL1␤ concentrations in pharyngeal secretions of human infants and clinical severity of apnea (Lindgren and Grogaard, 1996). Transient apneas are also a common side-effect of prostaglandin treatment in human neonates, which may be due to activation of EP3 receptors in brainstem respiration-related centers. Furthermore, our data provide an explanation for the positive correlation between central apneas and urine PGE metabolites in newborn infants (Hoch and Bernhard, 2000). Moreover, viral infection (e.g., viral bronchiolitis) may cause severe breathing obstruction and central depression of the ‘breathing pacemaker’ in the brainstem of neonates. As mentioned, such infection typically causes only a mild increase in CRP. The measurement of prostaglandin metabolites (e.g., PGEM levels) is therefore expected to provide an indication of potential inflammation and/or breathing disorders at an earlier stage of the infection. Studies to evaluate the potential diagnostic benefits of monitoring PGE2 compared to other infectious markers such as CRP are necessary. The conceptual change introduced by recent data is that endogenous prostaglandins are central pathogenic factors in cardiorespiratory disorders and the hypoxic response. This may open up an opportunity for new diagnostic and therapeutic ventures that should significantly improve the diagnostics and treatment of newborn, pediatric and adult patients.

8. Inflammation and breathing dysfunction in older infants and children 7. Biomarkers for inflammation and breathing dysfunction The most used biomarker for infection-related inflammation in clinical use today is the blood level of the acute phase protein CRP, which is synthesized by hepatocytes in response to interleukin-6 (IL-6) during inflammation (Marnell et al., 2005). CRP may prevent autoimmune reactions as well as have an anti-inflammatory role. However, despite raised IL-6 levels and extensive systemic inflammation, serum CRP levels remain low during most viral infections including viral bronchiolitis. Many viral infections are characterized by high blood levels of interferon-alpha (IFN-␣) that activate NK cells and blocks virus replication (Carter and De Clercq, 1974; De Clercq, 2005). IFN-␣ is an inhibitor of CRP promoter activity and CRP secretion. This could explain the big difference in CRP responses between viral and bacterial infections (Enocsson et al., 2009). Thus CRP is an inadequate biomarker for most viral-induced inflammations. The number of white blood cells (WBCs) in plasma is another indicator of inflammation. However, during bacterial infection the WBC count is increased in children but often initially decreased in neonates due to WBC consumption, the kinetics together with an often mild reaction to viral infections make it an unreliable biomarker. Conversely, the rapid synthesis of PGE2 in response to cytokine and hypoxic stimulation could make it particularly useful

Respiratory syncytial virus (RSV) infection is well known to be associated with breathing dysfunction in preterms (Schiller et al., 2011). RSV is a Paramyxo virus, with annual seasonal epidemics and >95% of children have antibodies at 2 years of age. Most infants experience only mild upper respiratory tract symptoms. However, a substantial proportion of patients have serious manifestations, such as respiratory distress, hypoxia or apnea (Bruhn et al., 1977). RSV infections and antibody responses do not render protective immunity, but subsequent infections are milder. One out of four children develops signs of lower respiratory tract infection, e.g., bronchiolitis, 1–2% of infants require hospital care and more (3–5%) in preterms <32 weeks (Henckel et al., 2004). Indeed, premature infants born
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RSV infection increases not only the respiratory frequency but also the number, duration and consequences of apneas in humans as well as in experimental animal models. Age differences and development of the homeostatic control matters as the potential fatal apneic effects of RSV are seen in weaning but not in adult rats (Peng et al., 2007). In humans RSV infections may have apneas as a presenting symptom and induce profound central autonomic dysfunction. This is evident in preterm born infants or term infants before two months of age (Stock et al., 2010). In preterms and infants <6 weeks of age, temperature does not seem to be associated with apneas but rather absence of fever is correlated with risk of apnea (Schiller et al., 2011). Viral bronchiolitis may induce PGE2 synthesis in bronchial epithelial cells. In contrast to its pro-inflammatory role elsewhere, PGE2 in the lung has bronchoprotective effects limiting the immune-inflammatory response as well as tissue repair processes (Asano et al., 1997; Vancheri et al., 2004). We suggest that activation of the PGE2 -induced pathway both in the lungs and in the vicinity of the brainstem respiration-related control centers, could explain apneas in young infants (Fig. 2) (Herlenius et al., 2009; Stock et al., 2010). We hypothesise that the mechanism for inflammation-induced disturbances of brainstem cardiorespiratory control in neonates is also of clinical relevance for some older children beyond the first weeks of life. Children with non-optimal or delayed brainstem respiratory control such as preterm infants, children with Congenital Central Hypoventilation Syndrome (CCHS) (Cutz et al., 1997; Weese-Mayer et al., 2008), Rett Syndrome (Rohdin et al., 2007) and Prader–Willi Syndrome (PWS) (Festen et al., 2006) have periodic irregular breathing with apnea that are increased during sleep as well as during infectious episodes when the resulting apnea can be, and sometimes is, fatal if external- or auto-resuscitation does not occur (Herlenius et al., 2009). The Prader–Willi Syndrome affects approximately 1/10,000–1/15,000 newborns, making it one of the most prevalent congenital syndromes in children after trisomy 21. Nearly all PWS-patients display an absence of parental generegion15q11q13. One of the genes lost is the NECDIN-gene, coding for a key protein regulating polarization of the cytoskeleton during development, reducing cell migration with hypothalamic dysfunction as one consequence (Bush and Wevrick, 2010). Symptoms include hypotonia, hypogonadism and obesity (Festen et al., 2006). Notably, PWS children have a disturbed breathing pattern and autonomic control and are known to die suddenly (2–3% yearly prevalence), often in association with mild upper respiratory infection (Tauber et al., 2008). Studies are currently ongoing to determine if respiratory infections may aggravate the existing breathing dysfunction in PWS children and if prostaglandins are involved. The preliminary results are encouraging and an inflammatory PGE2 -mediated pathway to autonomic instability might be involved in the PWS adolescent sudden death syndrome associated with upper respiratory tract infections. The final pathway to SIDS is widely believed to involve immature cardiorespiratory autonomic control, together with a failure of arousal responsiveness from sleep (Moon et al., 2007). A persistent or delayed maturity of the brainstem autonomic control is likely an important factor in the increased risk for SIDS for children born prematurely (Moon et al., 2007). As described in the preceding paragraphs, apnea is a common presenting sign of infection in neonates, and mild viral or bacterial infection precedes death in the majority of SIDS victims (Fanaroff et al., 1998; Hofstetter, 2006; Prandota, 2004; Weber et al., 2008). We speculate that a PGE2 -mediated depression of a primed vulnerable brainstem cardiorespiratory center could be a trigger event involved in the pathogenesis of SIDS. This hypothesis needs to be examined, e.g., in post-mortem materials from SIDS victims.

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