The Induced Prostaglandin E2 Pathway Is a Key Regulator of the Respiratory Response to Infection and Hypoxia in Neonates

The induced prostaglandin E2 pathway is a key regulator of the respiratory response to infection and hypoxia in neonates

Annika O. Hofstetter*, Sipra Saha*†, Veronica Siljehav*, Per-Johan Jakobsson‡, and Eric Herlenius*§

*Department of Woman and Child Health, Karolinska Institutet, 171 76 Stockholm, Sweden; †Centre for Structural Biochemistry, Karolinska Institutet, Novum, 141 57 Huddinge, Sweden; and ‡Department of Medicine, Karolinska Proteonic Center, Karolinska University Hospital, S-171 76, Stockholm, Sweden

Edited by Tamas Bartfai, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board April 24, 2007 (received for review January 2, 2007).

Infection during the neonatal period commonly induces apnea epi- induced by IL-1␤ (5). PGE2 itself depresses breathing in fetal and sodes, and the proinflammatory cytokine IL-1␤ may serve as a critical newborn sheep in vivo (17–19) and inhibits respiration-related mediator between these events. To determine the mechanism by neurons in vitro (5). Furthermore, EP3 receptors (EP3R) for PGE2 which IL-1␤ depresses respiration, we examined a prostaglandin E2 are located in the NTS and RVLM (20, 21). (PGE2)-dependent pathway in newborn mice and human neonates. The present study provides evidence that IL-1␤ adversely affects ␤ IL-1 and transient anoxia rapidly induced brainstem-specific micro- central respiration via mPGES-1 activation and PGE2 binding to somal prostaglandin E synthase-1 (mPGES-1) activity in neonatal mice. brainstem EP3R, resulting in increased apnea frequency and failure Furthermore, IL-1␤ reduced respiratory frequency during hyperoxia to autoresuscitate after a hypoxic event. and depressed hypoxic gasping and autoresuscitation in mPGES-1 wild-type mice, but not in mPGES-1 knockout mice. In wild-type mice, Results PGE2 induced apnea and irregular breathing patterns in vivo and Endogenous Brainstem mPGES-1 Activity and Tonic Respiratory Effect. inhibited brainstem respiratory rhythm generation in vitro. Mice We first examined endogenous PGE2 production and its effects on ϩ/ϩ Ϫ/Ϫ lacking the EP3 receptor (EP3R) for PGE2 exhibited fewer apneas and ventilation in 9-d-old mPGES-1 and mPGES-1 mice. Wild- sustained brainstem respiratory activity, demonstrating that PGE2 type mice exhibited basal mPGES-1 activity that was higher in the exerts its respiratory effects via EP3R. In human neonates, the infec- homogenized brainstem than the homogenized cortex (Fig. 1). tious marker C-reactive protein was correlated with elevated PGE2 in Breathing during normoxia was similar between genotypes, al- ϩ/ϩ the cerebrospinal fluid, and elevated central PGE2 was associated though respitory frequency (fR) tended to be lower in mPGES-1 with an increased apnea frequency. We conclude that IL-1␤ adversely mice than mPGES-1Ϫ/Ϫ mice (Kruskal–Wallis test, P ϭ 0.03; affects breathing and its control by mPGES-1 activation and PGE2 Student’s t post hoc test, P ϭ 0.18) [supporting information (SI) binding to brainstem EP3 receptors, resulting in increased apnea Table 2]. The central respiratory drive was examined by a 1-min frequency and hypoxia-induced mortality. hyperoxic challenge (100% O2, 1 min). Mice from both genotypes responded to hyperoxia with a reduction in fR (Fig. 2). However, the pnea and sudden infant death syndrome (SIDS) represent respiratory depression was greater in mPGES-1ϩ/ϩ mice than in Amajor medical concerns in the neonatal population, and mPGES-1Ϫ/Ϫ mice (27 Ϯ 2% vs. 19 Ϯ 3%, respectively). infection may play a crucial role in their pathogenesis. Apnea is a common presenting sign of infection in neonates, and mild viral or IL-1␤ and Anoxia Induced mPGES-1 Activity in the Mouse Brainstem. bacterial infection precedes death in the majority of SIDS victims We also measured the effect of IL-1␤ and short anoxic exposure (1, 2). Proinflammatory cytokines such as IL-1␤ may serve as key (100% N2, 5 min) on mPGES-1 activity in the homogenized mediators between these events (3). IL-1␤ is produced during an brainstem and cortex of 9-d-old mPGES-1ϩ/ϩ, mPGES-1Ϫ/Ϫ, and acute phase immune response to infection and inflammation and EP3Rϩ/ϩ mice (Fig. 1). IL-1␤ induced a time-dependent increase evokes a variety of sickness behaviors (for a review, see ref. 4). in mPGES-1 activity, particularly in the brainstem. Specifically, Previous studies indicate that this immunomodulator also alters there was a 2- and 4-fold increase in brainstem mPGES-1 activity respiration and autoresuscitation (5–10). IL-1␤ induces expression at 90 and 180 min, respectively, after IL-1␤ administration, whereas of the immediate-early gene c-fos in respiration-related regions of cortex activity remained unchanged between 90 and 180 min. the brainstem such as the nucleus tractus solitarius (NTS) and Anoxic exposure also induced mPGES-1 activity in both brainstem rostral ventrolateral medulla (RVLM) (11). However, IL-1␤ is a and cortex. Notably, there was an additive effect of IL-␤ and short large lipophobic protein that does not readily diffuse across the anoxic exposure on mPGES-1 activity, which was more pronounced blood–brain barrier (BBB). Furthermore, the NTS and RVLM do in the brainstem. EP3R wild-type mice displayed similar mPGES-1 not appear to express IL-1 receptor mRNA (12), and IL-1␤ does not alter brainstem respiration-related neuronal activity in vitro (5). Thus, it is likely that an indirect mechanism underlies the central Author contributions: A.O.H. and S.S. contributed equally to this work; A.O.H., S.S., and E.H. ␤ designed research; A.O.H., S.S., V.S., and E.H. performed research; A.O.H., S.S., P.-J.J., and respiratory effects of IL-1 . E.H. analyzed data; and A.O.H., S.S., and E.H. wrote the paper. IL-1␤ binds to IL-1 receptors on vascular endothelial cells of the The authors declare no conflict of interest. BBB and induces cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) activity (for a review, see ref. This article is a PNAS Direct Submission. T.B. is a guest editor invited by the Editorial Board. Freely available online through the PNAS open access option. 13). COX-2 catalyzes the formation of prostaglandin H2 (PGH2) from arachidonic acid, and mPGES-1 subsequently catalyzes the Abbreviations: aCSF, artificial CSF; BBB, blood–brain barrier; COX, cyclooxygenase; CRP, C-reactive protein; CSF, cerebrospinal fluid; EP3R, EP3 receptor; fR, respiratory frequency; synthesis of prostaglandin E2 (PGE2)fromPGH2. PGE2 is then i.c.v., intracerebroventricular; mPGES-1, microsomal prostaglandin E synthase-1; NTS, nu- released into the brain parenchyma where it recently has been cleus tractus solitarius; PGE2, prostaglandin E2; PGH2, prostaglandin H2; preBo¨tC, pre- shown to mediate several central effects of IL-1␤, e.g., fever Bo¨tzinger complex; RVLM, rostral ventrolateral medulla. induction (14), behavioral responses (15), and neuroendocrine §To whom correspondence should be addressed. E-mail: [email protected]. changes (16). Prostaglandin also appears to mediate the ventilatory This article contains supporting information online at www.pnas.org/cgi/content/full/ effects of IL-1␤. We previously showed that indomethacin, a 0611468104/DC1. nonspecific COX inhibitor, attenuates the respiratory depression © 2007 by The National Academy of Sciences of the USA

9894–9899 ͉ PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611468104 Downloaded by guest on September 30, 2021 Table 2). All mice, irrespective of treatment, responded to hyperoxic challenge with a reduction in fR, but IL-1␤-treated wild-type mice exhibited a greater respiratory depression than vehicle-treated wild-type mice. IL-1␤ also tended to reduce basal fR in mPGES- 1ϩ/ϩ mice (Kruskal–Wallis test, P ϭ 0.03; Student’s t post hoc test, P ϭ 0.17). Conversely, IL-1␤ did not alter ventilation during normoxia or hyperoxia in mPGES-1Ϫ/Ϫ or EP3RϪ/Ϫ mice.

IL-1␤ Worsened Anoxic Survival in Wild-Type Mice, but Not in Mice Lacking mPGES-1 or EP3R. Next, we investigated whether IL-1␤ affects the hypoxic ventilatory response and autoresuscitation after hypoxic apnea via of a PGE2-mediated mechanism. Using flow plethysmography, respiration during anoxia (100% N2,5min) followed by hyperoxia (100% O2, 8 min) was examined beginning at 80 min after i.p. injection of IL-1␤ or vehicle in mPGES-1ϩ/ϩ, mPGES-1Ϫ/Ϫ, and EP3RϪ/Ϫ mice (Fig. 3 and SI Table 3). All mice Fig. 1. IL-1␤ and anoxia rapidly induce brainstem mPGES-1. mPGES-1 activity exhibited a biphasic response to anoxia with an initial increase in in the microsomal fraction of cortex and brainstem, including endothelial cells ventilation (i.e., hyperpnea) followed by a hypoxic ventilatory of the BBB, was analyzed in 9-d-old mice (n ϭ 33) treated with IL-1␤ or vehicle depression (i.e., primary apnea, gasping, secondary apnea). IL-1␤ ϩ/ϩ and subjected to normoxia or normoxia plus anoxia (100% N2, 5 min). (A)In reduced the number of gasps in mPGES-1 mice, but not in wild-type mice, mPGES-1 activity was measured at 90 min after NaCl (control) mPGES-1Ϫ/Ϫ mice. IL-1␤-treated mPGES-1ϩ/ϩ mice also tended or 90 min and 180 min after IL-1␤ treatment. Higher endogenous mPGES-1 to have a shorter gasping duration compared with IL-1␤-treated activity was observed in the brainstem compared with cortex in control mPGES-1Ϫ/Ϫ mice (Kruskal–Wallis test, P ϭ 0.19; Student’s t post ϩ/ϩ ␤ mPGES-1 mice. In addition, IL-1 induced mPGES-1 activity in a time- hoc test, P ϭ 0.003). Fewer gasps and a shorter gasping duration dependent manner. (B) At 90 min, IL-1␤-treated mice exhibited approximately were correlated with decreased anoxic survival. IL-1␤ significantly 2-fold higher activity in the brainstem compared with saline-treated mice. ϩ/ϩ Anoxia also signiﬁcantly induced mPGES-1 activity. Moreover, the effects of reduced anoxic survival in mPGES-1 mice, but did not decrease IL-1␤ and transient anoxic exposure were additive. When IL-1␤-treated mice survival in mice lacking the mPGES-1 or EP3R genes. IL-1␤ was Ϫ Ϫ were exposed to anoxia, 4-fold higher activity was observed in the brainstem unable to affect the hypoxic ventilatory response of EP3R / mice. compared with control mice. However, mice with genetic deletion of mPGES-1 ␤ gene displayed negligible activity in response to IL-1 and anoxia. Data are PGE2 Decreased Brainstem Respiration-Related Activity and Induced presented as mean Ϯ SEM. , P Ͻ 0.05; , P Ͻ 0.01; , P Ͻ 0.001. * ** *** Apnea via of EP3R. To better determine whether PGE2 depresses respiration by binding specifically to brainstem EP3 receptors, central respiratory activity was measured using the en bloc brain- activity compared with the mPGES-1 wild-type mice at 90 min after stem–spinal cord preparation of 2- to 3-d-old EP3Rϩ/ϩ and IL-1␤. Moreover, the EP3R mice also had higher mPGES-1 activity Ϫ/Ϫ

EP3R mice after administration of artificial cerebrospinal fluid PHYSIOLOGY in the brainstem than the cortex (PGE2: 1,111 Ϯ 49 and 710 Ϯ 44 (aCSF) or PGE2. During control conditions, similar respiratory ⅐ ⅐ ϩ ϩ Ϫ Ϫ pmol min mg protein, respectively). activity was recorded in preparations from EP3R / and EP3R / mice. However, PGE reversibly inhibited respiration-related fre- ؉ ؉ 2 IL-1␤ Depressed Respiration in mPGES-1 / Mice, but Not in mPGES- quency in EP3Rϩ/ϩ preparations but had no affect on EP3RϪ/Ϫ ؊/؊ ؊/؊ 1 or EP3R Mice. To examine the role of PGE2 in mediating the preparations (Fig. 4). The ability of PGE2 to alter breathing via ventilatory effects of IL-1␤, we analyzed respiration during nor- EP3R was further assessed using flow plethysmography. After moxia and hyperoxia (100% O2, 1 min) by using flow plethysmog- intracerebroventricular (i.c.v.) injection of PGE2 or vehicle in raphy after i.p. administration of IL-1␤ or vehicle in 9 d-old EP3Rϩ/ϩ and EP3RϪ/Ϫ mice, respiration during normoxia and ϩ/ϩ Ϫ/Ϫ Ϫ/Ϫ mPGES-1 , mPGES-1 , and EP3R mice (Fig. 2 and SI hyperoxia was analyzed (Fig. 4 and Table 1). PGE2 induced a

Fig. 2. IL-1␤ depresses respiration through mPGES-1 activation. Using whole-body ﬂow plethysmography, basal respiration and the ventilatory response to hyperoxia were examined in 9-d-old mPGES-1 wild-type mice (n ϭ 66) and mPGES-1 knockout mice (n ϭ 34) after i.p. administration of either IL-1␤ (n ϭ 52) or NaCl (n ϭ 48). (A) Plethysmograph recordings illustrate breathing during normoxia and hyperoxia in wild-type mice given NaCl or IL-1␤ (5-s period, breath amplitude 1 ␮l/s). (B and C) All mice responded to hyperoxia with a reduction in fR (breaths per min). IL-1␤ depressed fR to a greater extent than NaCl in mPGES-1 miceϩ/ϩ, whereas IL-1␤ did not alter respiration during normoxia or hyperoxia in mPGES-1Ϫ/Ϫ mice. mPGES-1ϩ/ϩ mice exhibited a greater respiratory depression Ϫ Ϫ ϩ ϩ during hyperoxia compared with mPGES-1 / mice. Data are presented as mean Ϯ SEM. *, P Ͻ 0.05 compared with mPGES-1 / mice given NaCl.

Hofstetter et al. PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 ͉ 9895 Downloaded by guest on September 30, 2021 tion and PGE2 binding to EP3 receptors in respiration-related regions of the brainstem (Fig. 6). Additionally, severe hypoxia rapidly induces mPGES-1 activity, indicating that endogenous PGE2 may modulate brainstem respiratory neurons during hypoxia in the newborn period. Lastly, we reveal a correlation between infection, central PGE2, and apnea events in human neonates.

PGE2 Exerts a Tonic Effect on Brainstem Respiratory-Related Activity via mPGES-1. Our study clearly demonstrates an endogenous expression of mPGES-1 activity, particularly in the brainstem. mPGES-1 is expressed mainly by endothelial cells along the BBB (25). A constitutive and rapidly inducible expression of mPGES-1 at endothelial cells overlying the brainstem, near crucial respiration- related centers, suggests an important role of PGE2 in control of breathing. The significant respiratory depression in wild-type mice compared with mice lacking mPGES-1 during hyperoxia also provides evidence that endogenous PGE2 has a tonic effect on respiratory rhythmogenesis during the perinatal period. This finding is consistent with evidence that prostaglandin synthesis inhib- itors, which block endogenous prostaglandin production, increase fetal breathing movements and central respiration during early postnatal life (26–28). Developmental changes occur in the modulatory effects of prostaglandin with an initial inhibition of ventilation during the perinatal period (18, 26, 27, 29) followed by smaller changes in respiration with increasing age (19). However, PGE may still disrupt regular breathing with induction of apnea at Fig. 3. IL-1␤ reduces anoxic survival via mPGES-1. Nine-day-old mPGES-1ϩ/ϩ 2 mice (n ϭ 37) and mPGES-1Ϫ/Ϫ mice (n ϭ 20) were exposed to 5 min of anoxia older ages (19). Developmental changes could be secondary to (100% N2) at 80 min after peripheral administration of IL-1␤ (n ϭ 29) or vehicle alterations in brainstem PGE2 receptor expression beyond the (n ϭ 28). (A) Plethysmograph recording of mPGES-1ϩ/ϩ mouse given NaCl depict- perinatal period, although EP3R gene and protein are expressed in ing the initial hyperpnea and subsequent gasping response to anoxia. The mouse adult rodent RVLM (20, 21, 30). In addition, even though prosta- autoresuscitated after 100% O2 was administered. (B) Plethysmograph recording glandin binding density may decrease, it is located in the same of mPGES-1ϩ/ϩ mouse given IL-1␤ showing the brief hyperpnea period and brainstem regions at all ages (31). Further investigation of the subsequent gasping response to anoxia. The mouse failed to autoresuscitate ontogenesis of EP3R expression and mechanisms underlying po- after 100% O2 was administered. (C) The number of gasps tended to differ tential developmental changes in the respiratory effects of PGE ␹2 ϭ 2 between groups (Wilcoxon , P 0.06). When comparing treatment effects (e.g., posttranslational EP3R modification, suprapontine influ- within each genotype, IL-1␤ decreased the number of gasps in wild-type mice, whereas this effect was not observed in mice lacking mPGES-1. (D) IL-1␤ reduced ences) is warranted but outside the scope of the present study. the survival rate anoxic compared with NaCl in mPGES-1ϩ/ϩ mice, but not in Ϫ Ϫ mPGES-1 / mice. Data are presented as mean Ϯ SEM. *, P Ͻ 0.05; **, P Ͻ 0.01. Transient Anoxia Induces mPGES-1 Activity in the Mouse Brain. PGE2 also appears to play a crucial role in the respiratory response to anoxia. A short anoxic exposure increased mPGES-1 activity in the significantly greater apnea frequency and irregular breathing pat- homogenized mouse brain. This rapid increase in mPGES-1 activity tern during normoxia and hyperoxia in EP3Rϩ/ϩ mice, but not in in vivo is a new finding but is consistent with evidence that anoxia Ϫ/Ϫ EP3R mice. The mice were subsequently exposed to anoxia induces PGE2 production in mice cortical sections ex vivo and followed by hyperoxia, which enabled them to autoresuscitate. All prostaglandin H synthase-2 mRNA expression in the piglet brain mice continued gasping beyond the 5-min anoxic exposure, and (32, 33). Transient asphyxia similarly increases PGE2 concentra- only one of 38 mice failed to autoresuscitate (PGE2-treated tions in the newborn guinea pig brain, and this effect is inhibited by Ϫ/Ϫ EP3R mouse). PGE2 did not alter the gasping response or pretreatment with indomethacin (34). anoxic survival of EP3Rϩ/ϩ or EP3RϪ/Ϫ mice compared with No known mechanisms of mPGES-1 enzyme regulation may vehicle. explain the rapid changes in mPGES-1 activity revealed here. Finally, we investigated whether respiration-related neurons in Induced gene expression is unlikely to occur during such a short the RVLM express EP3R. Specifically, NK1R immunolabeling was anoxic event. However, posttranscriptional regulation of constitu- used as a tool to identify respiration-related neurons located in the tively expressed mPGES-1, e.g., phosphorylation, is a potential RVLM ventral to the nucleus ambiguous and including the pre- etiology. Stabilization of mPGES-1 mRNA is another possibility, as Bo¨tzinger Complex (preBo¨tC) (22–24). We show that these neu- previously shown with COX-2 mRNA in a human cell system (35) rons coexpressed NK1R and EP3R (Fig. 4). and recently in cardiac myocytes (36). Further investigation is required to clarify the underlying mechanism. Central PGE2 Concentration Correlated with Increased Apnea Fre- quency in Human Infants. To further elucidate the mechanism IL-1␤ Alters Respiration via mPGES-1 Activation and PGE2 Binding to underlying the association between infection and apnea in human Brainstem EP3 Receptors. We reveal that mPGES-1 activation is newborns, we examined the association between the infectious necessary for IL-1␤ to depress central respiration. First, IL-1␤ marker C-reactive protein (CRP), CSF PGE2 levels, and apnea increased brainstem mPGES-1 activity in a time-dependent man- events in newborn infants. CRP was positively correlated with ner. Second, IL-1␤ depressed respiration in mPGES-1؉/؉ mice, but Ϫ/Ϫ central PGE2, and there was a positive association between PGE2 not in mPGES-1 mice. Indomethacin, by blocking prostaglandin concentrations in the CSF and apnea frequency (Fig. 5). synthesis, has been shown to similarly attenuate the effects of IL-1␤ on basal respiration (5). In addition, we provide evidence that after Discussion mPGES-1 activation, newly synthesized PGE2 exerts the respiratory In the current investigation, we demonstrate that systemic IL-1␤ actions of IL-1␤ centrally. We show here that PGE2 hindered depresses breathing and autoresuscitation via of mPGES-1 activa- breathing in wild-type mice, consistent with studies demonstrating

9896 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611468104 Hofstetter et al. Downloaded by guest on September 30, 2021 that PGE2 depresses respiration in fetal and newborn animals (18, 29, 37). Moreover, these effects occur centrally because PGE2 did not alter peripheral chemosensitivity in vivo and directly inhibited brainstem respiratory activity in vitro. These findings are in accordance with data showing that PGE2 inhibits respiration-related neurons in neonatal rats (5) and similarly inhibits fetal breathing movements in sheep after sham-operation or denervation of the carotid sinus and vagus nerve (38). Furthermore, the modulatory effects of PGE2 occur via binding to brainstem EP3 receptors. IL-1␤ was unable to alter respiration Ϫ/Ϫ in EP3R mice. PGE2 induced apnea and irregular breathing in vivo in EP3R؉/؉ mice, but not in EP3RϪ/Ϫ mice. Finally, the presence of EP3 receptors was required to inhibit brainstem respiration-related rhythmic activity in vitro. Although the specific prostaglandin receptor subtype EP3R has been localized to the NTS and RVLM (20, 21), no prior studies have shown that the respiratory effects of prostaglandin occur via action at these receptors and that they are expressed in respiration-related neurons.

IL-1␤ Inhibits Autorescuscitation and Anoxic Survival via mPGES-1 and EP3R. This study demonstrates that PGE2 also plays a crucial role in mediating the anoxic ventilatory effects of IL-1␤. IL-1␤ inhibited autoresuscitation after hypoxic apnea in wild-type mice, but not in mice lacking mPGES-1 or EP3R. These findings are consistent with our previous investigation showing that indomethacin attenuates the adverse effects of IL-1␤ on hypoxic gasping and anoxic survival in neonatal rats (5). Our data suggests that PGE2 induced by IL-1␤ as well as hypoxia selectively modulates respiration-related neurons in the RVLM, including the preBo¨tC, via EP3R. Other neuromodulators, including PGE1, have been shown to inhibit preBo¨tC neurons and slow respiration-related rhythm (22, 23), and preBo¨tC lesions may disrupt anoxic gasping and evoke central apneas and ataxic breath-

ing (39, 40). Moreover, these respiration-related neurons were PHYSIOLOGY recently shown to be critical for adequate response to hypoxia, maintaining brainstem homeostasis with gasping and autoresuscitation and thus restoring oxygen levels (41). PGE2-induced depression of this vital brainstem neuronal network, e.g., during an infectious response, could result in gasping and autoresuscitation failure and ultimately death.

Correlation Between Infectious Parameters, PGE2, and Apnea in Hu- man Neonates. Apnea is a common presenting sign of sepsis in the neonatal population (1), yet the mechanism underlying this association remains unclear. Here, we show that the infectious marker CRP is correlated with elevated PGE2 levels in the CSF of human neonates. Importantly, we also demonstrate that PGE2 is associated with an increased apnea frequency. These findings suggest that infection depresses respiration in human neonates through systemic release of cytokines followed by the biosynthesis and central action of PGE2. The mechanism described here could explain previous Fig. 4. PGE2 depresses brainstem respiratory activity and induces apnea via brainstem EP3Rs. Respiration was examined in EP3Rϩ/ϩ (n ϭ 13) and EP3RϪ/Ϫ reports showing an independent association between CRP levels (n ϭ 25) neonatal mice after administration of PGE (n ϭ 19) or NaCl (n ϭ 19). and the apnea/hypopnea index in children with sleep apnea (42) as 2 ␤ (A) PGE2 was injected i.c.v. at 0 min followed by normoxia and a 1-min well as a positive correlation between IL-1 concentrations in hyperoxic challenge in newborn EP3Rϩ/ϩ (■) and EP3RϪ/Ϫ (ᮀ) mice. The pharyngeal secretions of human infants and clinical severity of ϩ/ϩ EP3R mouse exhibited a lower fR (breaths per min) and an irregular respi- apnea (8). Transient apneas are also a common side effect of ratory rhythm with elevated coefficient of variation (C.V.) during normoxia prostaglandin treatment in human neonates (43), which may be due Ϫ/Ϫ and hyperoxia due to apneic breathing. In the EP3R mouse, basal fR did not decrease after the postanesthesia period, and there was less variability in the respiratory pattern. No temperature difference or dependency was observed during the first 20 min after i.c.v. administration of PGE2.(B) Plethysmograph (fR) (ANOVA repeated measures design, P Ͻ 0.01). PGE2 did not affect respi- recordings (10-s periods with breath amplitude of 1 ␮l/s) demonstrate apnea ratory activity in preparations from EP3RϪ/Ϫ mice (ᮀ, n ϭ 6). (E) In transverse ϩ/ϩ episodes in response to PGE2 during normoxia in an EP3R mouse, but not medullary sections, respiration-related neurons within the RVLM ventral to Ϫ/Ϫ ϩ/ϩ in an EP3R mouse. (C) In EP3R mice, PGE2 induced more apneas during the nucleus ambiguus (NA) and including the preBo¨tC coexpress NK1R (red) normoxia and hyperoxia compared with vehicle. This effect of PGE2 was not and EP3R (green). The arrows indicate EP3R and NK1R colocalization (yellow) in observed in EP3RϪ/Ϫ mice. (D)Inen bloc brainstem spinal–cord preparations some RVLM respiration-related neurons. (F) NK1R, but no EP3R, expression was ϩ/ϩ Ϫ/Ϫ from 2- to 3-d-old EP3R pups (■, n ϭ 5), PGE2 (20␮g/liter) reversibly identified in an EP3R mouse. (Scale bar, 100 ␮m.) Data are presented as ϩ ϩ depressed respiratory rhythm generation to 64 Ϯ 5% of control frequency mean Ϯ SEM. *, P Ͻ 0.05 compared with EP3R / mice given NaCl.

Hofstetter et al. PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 ͉ 9897 Downloaded by guest on September 30, 2021 Table 1. Respiration during normoxia, hyperoxia, and anoxia in EP3R mice after central PGE2 administration Normoxia Hyperoxia Hyperpnea

Genotype Treatment fR VT VE fR VT VE fR

EP3Rϩ/ϩ NaCl (nϭ 7) 281 Ϯ 17 3.8 Ϯ 0.4 1,065 Ϯ 75 234 Ϯ 19 7.0 Ϯ 3.0 1,598 Ϯ 642 327 Ϯ 13

PGE2 (nϭ 6) 247 Ϯ 13* 3.7 Ϯ 0.4 901 Ϯ 154 190 Ϯ 16 4.4 Ϯ 1.1 745 Ϯ 102 267 Ϯ 11** EP3RϪ/Ϫ NaCl (nϭ12) 247 Ϯ 15 5.3 Ϯ 0.6 1,322 Ϯ 157 200 Ϯ 23 5.4 Ϯ 0.9 1,057 Ϯ 213 288 Ϯ 11

PGE2 (nϭ13) 256 Ϯ 10 5.2 Ϯ 0.5 1,350 Ϯ 129 229 Ϯ 9 6.7 Ϯ 1.3 1,509 Ϯ 299 290 Ϯ 9

fR (breaths per min), tidal volume (VT)(␮l per breath per g), and minute ventilation (VE)(␮l⅐min⅐g) during normoxia, hyperoxia (100% ϩ/ϩ Ϫ/Ϫ O2), and anoxia (100% N2) were examined in 9-d-old EP3R mice (n ϭ 13) and EP3R mice (n ϭ 25) after i.c.v. injection of PGE2 or vehicle. When comparing treatment effects within each genotype, PGE2 signiﬁcantly depressed fR during normoxia and hyperpnea in ϩ/ϩ Ϫ/Ϫ ϩ/ϩ EP3R mice, but not in EP3R mice. PGE2 also tended to reduce fR during hyperoxia in EP3R mice (ANOVA, P ϭ 0.11), but not in Ϫ Ϫ EP3R / mice. Data are presented as mean Ϯ SEM. *, P Ͻ 0.05; **, P Ͻ 0.01.

to activation of EP3 receptors in brainstem respiration-related (mean gestational age: 32 Ϯ 2 weeks) from the neonatal intensive centers. Furthermore, our data provide an explanation for the care unit were included (n ϭ 12). Infants were eligible for inclusion positive correlation between central apneas and urine PGE me- if they underwent a lumbar puncture for clinical indications and if tabolites in newborn infants (44). informed written consent was obtained. These studies were per- Inflammatory mediators have been proposed as important mark- formed in accordance with European Community Guidelines and ers for detecting infection and asphyxia in newborns. The rapid approved by regional ethics committees. See SI Materials and synthesis of PGE2 in response to cytokine and hypoxic stimulation Methods for more information. may make it particularly useful in the diagnosis and surveillance of infants with increased apneas due to suspected infection or as- Impedance Pneumography. Infant cardiorespiratory activity was phyxia. Studies to evaluate the potential diagnostic benefits of measured noninvasively using impedance pneumography and was monitoring PGE2 compared with other infectious markers such as recorded by an event monitoring system (KIDS; Hoffrichter, CRP are necessary. Schwerin, Germany). The monitor recorded baseline respiratory The present discoveries have important treatment implications rates and events defined as Ն85% reduction in breathing move- for neonatal apnea related to infection because the adverse effects ments for Ն10 s. The 60-s periods before and after the event were ␤ of IL-1 were attenuated by selectively deleting the mPGES-1 and also stored in the monitor’s memory. EP3R genes. Indomethacin has been used previously to treat apnea of prematurity (45). However, indomethacin causes multiple ad- Plethysmography After i.p. Injection of IL-1␤ or NaCl. Respiration was verse effects in the newborn population (46), and thus treatment examined using whole-body flow plethysmography in (refs. 5 and 6 modalities selectively targeting mPGES-1 or EP3 receptors could in SI Materials and Methods) 9-d-old DBA/1lacJ mice (n ϭ 143) and be more beneficial. C57BL/6 mice (n ϭ 16) with variable expression of mPGES-1 and Future investigations examining the mechanism described in the EP3R, respectively. Each mouse received an i.p. injection (0.01 present study may improve our understanding of the epidemiolog- ml/g) of recombinant mouse IL-1␤ (10 ␮g/kg; Nordic Biosite, Ta¨by, ical and scientific evidence linking infection, apnea, autoresuscita- Sweden) or vehicle. At 70 min, the mouse was placed unrestrained tion failure, and sudden infant death syndrome (SIDS). into the plethysmograph chamber. Respiration was assessed during Materials and Methods 4 min of normoxia (21% O2) followed by 1 min of hyperoxia (100% O ). After a 5-min recovery period in normoxia, the respiratory Subjects. Neonatal mice of the inbred DBA/1lacJ strain (n ϭ 158) 2 response to anoxia (100% N ) was examined. Finally, 100% O was (Jackson Laboratory, Bar Harbor, ME) and C57BL/6 strain (n ϭ 2 2 75) were used. The mPGES-1 and EP3R genes were selectively deleted in knockout mice as described in refs. 47 and 48. Infants

Fig. 5. PGE2 in CSF is correlated to apnea index in neonates. CSF was collected from infants in the neonatal intensive care unit who had clinical indications for lumbar puncture (n ϭ 12, mean postnatal age 16 Ϯ 4 d; mean gestational age Fig. 6. Model for IL-1␤-induced respiratory depression and autoresuscitation 32 Ϯ 2 week). Infants then underwent a cardiorespiratory recording (duration failure via a PGE2-mediated pathway. During a systemic immune response, the 9.2 Ϯ 2.4 h). PGE2 concentrations in the CSF were analyzed using a standard- proinﬂammatory cytokine IL-1␤ is released into the peripheral blood stream. It ized enzyme immunoassay (EIA) protocol and correlated to the infectious binds to its receptor (IL-1R) located on endothelial cells of the BBB. Activation of marker CRP and apnea index (number of apneas per h). Central PGE2 concen- IL-1R induces the synthesis of PGH2 from arachidonic acid (AA) via COX-2 and the trations were positively correlated to the CRP levels in blood (P ϭ 0.01). synthesis of PGE2 from PGH2 via of the rate-limiting enzyme mPGES-1. PGE2 is Moreover, a striking association was observed between central PGE2 concen- released into the brain parenchyma and binds to its EP3R located in respiratory trations and apnea index (P Ͻ 0.05). Here, we distinguish between undetect- control regions of the brainstem, e.g., NTS and the RVLM. This results in depres- able levels of PGE2 (0 Ϯ 0 pg/ml) compared with high levels of PGE2 (52 Ϯ 22 sion of central respiration-related neurons and breathing, which may fatally pg/ml). Data are presented as mean Ϯ SEM. decrease the ability to gasp and autoresuscitate during hypoxic events.

9898 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611468104 Hofstetter et al. Downloaded by guest on September 30, 2021 administered for 8 min, and the ability to autoresuscitate was enzyme immunoassay (EIA) protocol (Cayman Chemicals). In- evaluated. fants underwent a cardiorespiratory recording as soon as possible after the lumbar puncture (mean recording duration: 9.2 Ϯ 2.4 h). Plethysmography After i.c.v. Injection of PGE2 or Vehicle. Respiration Blood concentrations of infectious markers (e.g., C-reactive pro- was examined using flow plethysmography in 9-d-old C57BL/6 mice tein, white blood cells) measured within 12 h before lumbar (n ϭ 38) with variable expression of EP3R. After the administration puncture were also recorded. of sevoflurane anesthesia for Ϸ60 s, PGE2 (4 nmol in 2–4 ␮lof aCSF) or vehicle was slowly injected into the lateral ventricle by Plethysmography Data Analysis. Periods of calm respiration without using a thin pulled glass pipette attached to polyethylene tubing. movement artifact were selected for analysis. Mean fR, tidal volume The mouse was then placed immediately into the plethysmograph (VT), and minute ventilation (VE) values during normoxia and chamber. After a 10-min recovery period in normoxia, the mouse hyperoxia as well as the anoxic response (i.e., hyperpnea, primary was exposed to hyperoxic and anoxic challenge as described above. apnea, gasping, secondary apnea, and autoresuscitation) were In both i.c.v. and i.p. experiments, skin temperature was measured analyzed as described in ref. 6. Survival was recorded for all animals. at baseline time points through experimentation and after removal Apnea was defined as cessation of breathing for more than or equal from the chamber by using thermistor temperature probe. Cham- to three respiratory cycles. ber temperature was maintained at 30.1 Ϯ 0.1°C in accordance with the documented thermoneutral range for neonatal mice by im- Infant Cardiorespiratory Data Analysis. The monitoring software was mersing the chamber in a thermostat-controlled water bath (49). used to report baseline respiratory rates and to visualize all cardiorespiratory events. The apnea index (A.I.) (number apneas per Brainstem Respiratory Activity. Brainstem–spinal cord preparations ϩ/ϩ h recording) was determined. The correlation between cardiore- were rapidly isolated from 2-d-old C57BL/6 mice with EP3R spiratory activity, infection status, and PGE levels in the CSF was Ϫ/Ϫ ϭ 2 and EP3R genotypes as described in refs. 50 and 51 (n 11). evaluated. All movement artifacts were excluded from analysis. Respiratory-related activity corresponding to the inspiratory rhythm was monitored at the C4 ventral root through a glass suction Statistics. One-way ANOVA compared those parameters with electrode, recorded (5 kHz), and analyzed off-line. Control record- normal distribution and equal variance. Multiple comparisons were ings were performed for at least 20 min before perfusion with aCSF performed using the Student’s t post hoc test. Wilcoxon ␹2 test was containing PGE2 followed by an aCSF washout period. used for nonparametric measurements and data with non-Gaussian distributions. Change in variables over time was examined using Measurement of mPGES-1 Activity. Newborn mouse brains (n ϭ 33) multivariate ANOVA (MANOVA) repeated measures design. The were homogenized in 0.1 M KPi (potassium inorganic phosphate) ␳ ϫ Spearman’s correlation test determined correlations between buffer containing 0.25 M sucrose, 1 complete protease inhibitor Ϯ Ͻ (Roche Diagnostics, Indianapolis, IN), and 1 mM reduced gluta- variables. Data are presented as mean SEM. A value of P 0.05 thione followed by sonication. Membrane fraction was isolated by was considered statistically significant. subcellular fractionation. mPGES-1 activity was measured in the membrane fraction as described in ref. 52. We thank Prof. Hugo Lagercrantz for his support and valuable comments; Zachi Horn, Ruth Detlofsson, and Lena Legnevall for technical assistance; and Dr. Beverly Koller (University of North Carolina, Chapel Hill, NC) for PHYSIOLOGY Immunohistochemistry. Transversal sections (14 ␮M) of brainstems ϩ/ϩ Ϫ/Ϫ providing the C57BL/6 EP3R-deficient mice. This research was supported from EP3R and EP3R pups were stained with mouse by Swedish Research Council Grants A0988 and 2004–5259, the Karolinska monoclonal NK1R and EP3R antibodies according to standard Institutet, the Swedish Society of Medicine, the Stockholm County Council, protocols. See SI Materials and Methods for further details. the Swedish Rheumatology Association, the Freemason Children’s House, the Jerring Foundation, the Wiberg Foundation, the Tielmann Foundation, CSF Analysis and Cardiorespiratory Recordings. CSF samples were the Child Care Foundation, the Mayflower Foundation, Swedish Heart– analyzed for PGE2 and PGE2 metabolites by using a standardized Lung Foundation, and King Gustav V’s 80 Years Foundation.

1. Fanaroff AA, Korones SB, Wright LL, Verter J, Poland RL, Bauer CR, Tyson JE, Philips, JB, 28. Long W (1988) J Appl Physiol 64:409–418. III, Edwards W, Lucey JF, et al. (1998) Pediatr Infect Dis J 17:593–598. 29. Guerra FA, Savich RD, Wallen LD, Lee CH, Clyman RI, Mauray FE, Kitterman JA (1988) 2. Prandota J (2004) Am J Ther 11:517–546. J Appl Physiol 64:2160–2166. 3. Guntheroth W (1989) Med Hypotheses 28:121–123. 30. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, 4. Dantzer R (2001) Ann NY Acad Sci 933:222–234. Brockway KS, Byrnes EJ, et al. (2007) Nature 445:168–176. 5. Olsson A, Kayhan G, Lagercrantz H, Herlenius E (2003) Pediatr Res 54:326–331. 31. Tai T, MacLusky NJ, Adamson SL (1994) Brain Res 652:28–39. 6. Hofstetter AO, Herlenius E (2005) Respir Physiol Neurobiol 146:135–146. 32. Degi R, Bari F, Thore C, Beasley T, Thrikawala N, Busija DW (1998) Neurobiology (Bp) 6:467–468. 7. Froen JF, Akre H, Stray-Pedersen B, Saugstad OD (2000) Pediatrics 105:E52. 33. Shohami E, Gross J (1986) J Neurochem 47:1678–1681. 8. Lindgren C, Grogaard J (1996) Acta Paediatr 85:798–803. 34. Allen LG, Louis TM, Kopelman AE (1982) Biol Neonate 42:8–14. 9. Stoltenberg L, Sunder T, Almaas R, Storm H, Rognum TO, Saugstad OD (1994) J Perinat Med 35. Ristimaki A, Garfinkel S, Wessendorf J, Maciag T, Hla T (1994) J Biol Chem 269:11769–11775. 22:421–432. 36. Degousee N, Angoulvant D, Fazel S, Stefanski E, Saha S, Iliescu K, Lindsay TF, Fish JE, 10. Vege Å, Rognum TO, Aasen AO, Saugstad OD (1998) Acta Paediatr 87:819–824. Marsden PA, Li RK, et al. (2006) J Biol Chem 281:16443–16452. 11. Ericsson A, Kovacs KJ, Sawchenko PE (1994) J Neurosci 14:897–913. 37. Savich RD, Guerra FA, Lee CC, Kitterman JA (1995) J Appl Physiol 78:1477–1484. 12. Ericsson A, Liu C, Hart RP, Sawchenko PE (1995) J Comp Neurol 361:681–698. 38. Murai DT, Wallen LD, Lee CC, Clyman RI, Mauray F, Kitterman JA (1987) J Appl Physiol 13. Engblom D, Ek M, Saha S, Ericsson-Dahlstrand A, Jakobsson PJ, Blomqvist A (2002) JMolMed 62:271–277. 80:5–15. 39. Feldman JL, Del Negro CA (2006) 7:232–241. 14. Coceani F, Akarsu ES (1998) Ann NY Acad Sci 856:76–82. 40. McKay LC, Janczewski WA, Feldman JL (2005) Nat Neurosci 8:1142–1144. 15. Crestani F, Seguy F, Dantzer R (1991) Brain Res 542:330–335. 41. Paton JF, Abdala AP, Koizumi H, Smith JC, St-John WM (2006) Nat Neurosci 9:311–313. 16. Ericsson A, Arias C, Sawchenko PE (1997) J Neurosci 17:7166–7179. 42. Tauman R, Ivanenko A, O’Brien LM, Gozal D (2004) Pediatrics 113:e564–e569. 17. Guerra F, Savich RD, Wallen LD, Lee CH, Clyman RI, Mauray FE, Kitterman JA (1988) J Appl 43. Singh G, Fong LV, Salmon AP, Keeton BR (1994) Eur Heart J 15:377–381. Physiology 64:2160–2166. 44. Hoch B, Bernhard M (2000) Acta Paediatr 89:1364–1368. 18. Kitterman J, Liggins GC, Fewell JE, Tooley WH (1983) J Appl Physiol 54:687–692. 45. Hammerman C, Zangen D (1993) Crit Care Med 21:154–155. 19. Tai TC, Adamson SL (2000) Am J Physiol 278:1460–1473. 46. Schmidt B, Davis P, Moddemann D, Ohlsson A, Roberts RS, Saigal S, Solimano A, Vincer M, 20. Ek M, Arias C, Sawchenko P, Ericsson-Dahlstrand A (2000) J Comp Neurol 428:5–20. Wright LL (2001) N Engl J Med 344:1966–1972. 21. Nakamura K, Kaneko T, Yamashita Y, Hasegawa H, Katoh H, Negishi M (2000) J Comp Neurol 47. Trebino CE, Stock JL, Gibbons CP, Naiman BM, Wachtmann TS, Umland JP, Pandher K, 421:543–569. Lapointe JM, Saha S, Roach ML, et al. (2003) Proc Natl Acad Sci USA 100:9044–9049. 22. Gray PA, Rekling JC, Bocchiaro CM, Feldman JL (1999) Science 286:1566–1568. 48. Fleming EF, Athirakul K, Oliverio MI, Key M, Goulet J, Koller BH, Coffman TM (1998) Am J 23. Ballanyi K, Lalley PM, Hoch B, Richter DW (1997) J Physiol 504:127–134. Physiol 275:F955–F961. 24. Pagliardini S, Ren J, Greer JJ (2003) J Neurosci 23:9575–9584. 49. Jacobi MS, Thach BT (1989) J Appl Physiol 66:2384–2390. 25. Yamagata K, Matsumura K, Inoue W, Shiraki T, Suzuki K, Yasuda S, Sugiura H, Cao C, 50. Herlenius E, Lagercrantz H, Yamamoto Y (1997) Pediatr Res 42:46–53. Watanabe Y, Kobayashi S (2001) J Neurosci 21:2669–2677. 51. Suzue T (1984) J Physiol 354:173–183. 26. Kitterman J, Liggins GC, Clements JA, Tooley WH (1979) J Dev Physiol 1:453–466. 52. Engblom D, Saha S, Engstrom L, Westman M, Audoly LP, Jakobsson PJ, Blomqvist A (2003) 27. Guerra FA, Savich RD, Clyman RI, Kitterman JA (1989) J Dev Physiol 11:1–6. Nat Neurosci 6:1137–1138.

Hofstetter et al. PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 ͉ 9899 Downloaded by guest on September 30, 2021