Chorioamnionitis disrupts erythropoietin and melatonin homeostasis through the placental-fetal-brain axis during critical developmental periods.

Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins (opioids, other drug exposures, environmental exposure), hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury-induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors (HIF1α, HIF2α). Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI. Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry. Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels. Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptor-mediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms.

Corrigendum: Methadone alters the peripheral inflammatory and central immune landscape following prenatal exposure in rats.

[This corrects the article DOI: 10.3389/adar.2022.10792.].

Methadone alters the peripheral inflammatory and central immune landscape following prenatal exposure in rats.

Opioid use during pregnancy continues to rise at alarming rates with a parallel trend in the number of infants and children exposed to opioid medications each year. Prenatal opioid exposure (POE) occurs at a critical timepoint in neurodevelopment disrupting intricate pathways essential for neural-immune maturation with the potential for devastating long-term consequences. Understanding the mechanisms underlying injury associated with POE is essential to address long-term outcomes and identify diagnostic and therapeutic biomarkers in this vulnerable patient population. Using an established preclinical model of POE, we investigated changes in cerebral and peripheral inflammation and peripheral blood mononuclear cell (PBMC) activity. We hypothesized that neuroinflammation, as defined by changes in specific cerebral immune cell populations, would exist in adult rats following POE concomitant with sustained peripheral immune hyperreactivity (SPIHR). Our data demonstrated alterations in cerebral immune cells at postnatal day 60 (P60) typified by increased regulatory T cells (p < 0.01) and neutrophils (p < 0.05) in rats with POE compared to controls. Evaluation of serum revealed increased levels of IL-6 (p < 0.05) and CXCL1 (p < 0.05) at P21 in rats with POE compared to controls with no significant difference in cytokine or chemokine levels between the two groups at P60. Additionally, PBMCs isolated from rats with POE at P21 demonstrated baseline hypersecretion of IL-6 (p < 0.01) and SPIHR with increased levels of TNF-α (p < 0.05) and CXCL1 (p < 0.05) following stimulation with LPS. At P60, however, there was no significant difference found in cytokine or chemokine levels secreted by PBMCs isolated from rats with POE at baseline or with LPS stimulation when compared to controls. Taken together, these data demonstrate cerebral inflammation months after prenatal opioid exposure and long after the resolution of systemic inflammation and SPIHR seen at toddler age equivalent. Chronic alterations in the cerebral immune cell populations secondary to prenatal opioid exposure may underly long-term consequences of developmental brain injury including deficits in cognition and attention. These findings may be invaluable to further investigations of precise biomarkers of injury and targeted therapeutics for this vulnerable population.

Sustained peripheral immune hyper-reactivity (SPIHR): an enduring biomarker of altered inflammatory responses in adult rats after perinatal brain injury.

BACKGROUND: Chorioamnionitis (CHORIO) is a principal risk factor for preterm birth and is the most common pathological abnormality found in the placentae of preterm infants. CHORIO has a multitude of effects on the maternal-placental-fetal axis including profound inflammation. Cumulatively, these changes trigger injury in the developing immune and central nervous systems, thereby increasing susceptibility to chronic sequelae later in life. Despite this and reports of neural-immune changes in children with cerebral palsy, the extent and chronicity of the peripheral immune and neuroinflammatory changes secondary to CHORIO has not been fully characterized. METHODS: We examined the persistence and time course of peripheral immune hyper-reactivity in an established and translational model of perinatal brain injury (PBI) secondary to CHORIO. Pregnant Sprague-Dawley rats underwent laparotomy on embryonic day 18 (E18, preterm equivalent). Uterine arteries were occluded for 60 min, followed by intra-amniotic injection of lipopolysaccharide (LPS). Serum and peripheral blood mononuclear cells (PBMCs) were collected at young adult (postnatal day P60) and middle-aged equivalents (P120). Serum and PBMCs secretome chemokines and cytokines were assayed using multiplex electrochemiluminescent immunoassay. Multiparameter flow cytometry was performed to interrogate immune cell populations. RESULTS: Serum levels of interleukin-1ß (IL-1ß), IL-5, IL-6, C-X-C Motif Chemokine Ligand 1 (CXCL1), tumor necrosis factor-α (TNF-α), and C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) were significantly higher in CHORIO animals compared to sham controls at P60. Notably, CHORIO PBMCs were primed. Specifically, they were hyper-reactive and secreted more inflammatory mediators both at baseline and when stimulated in vitro. While serum levels of cytokines normalized by P120, PBMCs remained primed, and hyper-reactive with a robust pro-inflammatory secretome concomitant with a persistent change in multiple T cell populations in CHORIO animals. CONCLUSIONS: The data indicate that an in utero inflammatory insult leads to neural-immune changes that persist through adulthood, thereby conferring vulnerability to brain and immune system injury throughout the lifespan. This unique molecular and cellular immune signature including sustained peripheral immune hyper-reactivity (SPIHR) and immune cell priming may be a viable biomarker of altered inflammatory responses following in utero insults and advances our understanding of the neuroinflammatory cascade that leads to perinatal brain injury and later neurodevelopmental disorders, including cerebral palsy.

Chorioamnionitis Precipitates Perinatal Alterations of Heme-Oxygenase-1 (HO-1) Homeostasis in the Developing Rat Brain.

Chorioamnionitis (CHORIO), placental insufficiency, and preterm birth are well-known antecedents of perinatal brain injury (PBI). Heme-oxygenase-1 (HO-1) is an important inducible enzyme in oxidative and inflammatory conditions. In the brain, HO-1 and the iron regulatory receptor, transferrin receptor-1 (TfR1), are known to be involved in iron homeostasis, oxidative stress, and cellular adaptive mechanisms. However, the role of HO pathway in the pathophysiology of PBI has not been previously studied. In this study, we set out to define the ontogeny of the HO pathway in the brain and determine if CHORIO changed its normal developmental regulation. We also aimed to determine the role of HO-1/TfR1 in CHORIO-induced neuroinflammation and peripheral inflammation in a clinically relevant rat model of PBI. We show that HO-1, HO-2, and TfR1 expression are developmentally regulated in the brain during the perinatal period. CHORIO elevates HO-1 and TfR1 mRNA expression in utero and in the early postnatal period and results in sustained increase in HO-1/TfR1 ratios in the brain. This is associated with neuroinflammatory and peripheral immune phenotype supported by a significant increase in brain mononuclear cells and peripheral blood double negative T cells suggesting a role of HO-1/TfR1 pathway dysregulation in CHORIO-induced neuroinflammation.

Prenatal opioid exposure: The next neonatal neuroinflammatory disease.

The rates of opioid use disorder during pregnancy have more than quadrupled in the last decade, resulting in numerous infants suffering exposure to opioids during the perinatal period, a critical period of central nervous system (CNS) development. Despite increasing use, the characterization and definition of the molecular and cellular mechanisms of the long-term neurodevelopmental impacts of opioid exposure commencing in utero remains incomplete. Thus, in consideration of the looming public health crisis stemming from the multitude of infants with prenatal opioid exposure entering school age, we undertook an investigation of the effects of perinatal methadone exposure in a novel preclinical model. Specifically, we examined the effects of opioids on the developing brain to elucidate mechanisms of putative neural cell injury, to identify diagnostic biomarkers and to guide clinical studies of outcome and follow-up. We hypothesized that methadone would induce a pronounced inflammatory profile in both dams and their pups, and be associated with immune system dysfunction, sustained CNS injury, and altered cognition and executive function into adulthood. This investigation was conducted using a combination of cellular, molecular, biochemical, and clinically translatable biomarker, imaging and cognitive assessment platforms. Data reveal that perinatal methadone exposure increases inflammatory cytokines in the neonatal peripheral circulation, and reprograms and primes the immune system through sustained peripheral immune hyperreactivity. In the brain, perinatal methadone exposure not only increases chemokines and cytokines throughout a crucial developmental period, but also alters microglia morphology consistent with activation, and upregulates TLR4 and MyD88 mRNA. This increase in neuroinflammation coincides with reduced myelin basic protein and altered neurofilament expression, as well as reduced structural coherence and significantly decreased fractional anisotropy on diffusion tensor imaging. In addition to this microstructural brain injury, adult rats exposed to methadone in the perinatal period have significant impairment in associative learning and executive control as assessed using touchscreen technology. Collectively, these data reveal a distinct systemic and neuroinflammatory signature associated with prenatal methadone exposure, suggestive of an altered CNS microenvironment, dysregulated developmental homeostasis, complex concurrent neural injury, and imaging and cognitive findings consistent with clinical literature. Further investigation is required to define appropriate therapies targeted at the neural injury and improve the long-term outcomes for this exceedingly vulnerable patient population.