The crisis of opioid use in the United States continues to grow and has a significant impact on many populations including pregnant women and children. Opioid use disorder during pregnancy has risen at alarming rates in the past decade with a 131% increase from 2010 to 2017 in women with a maternal opioid-related diagnosis at time of delivery (1). Paralleling this epidemic has been a sharp increase in the number of infants exposed in utero to opioid medications each year. Neonatal opioid withdrawal syndrome (NOWS) is a well-recognized consequence of prenatal opioid exposure (POE) in the first few weeks of life leading to an extended length of hospitalization for many newborn infants. However, the long-term adverse outcomes associated with POE are just beginning to be understood (2). As the epidemic grows, understanding the mechanisms underlying POE and the long-term consequence of this exposure is paramount to supporting the health and development of this vulnerable population.

Prenatal opioid exposure occurs as a result of maternal use or misuse of prescription opioid medications including oxycodone, hydrocodone, morphine, codeine and fentanyl, or illicit opioids, such as heroin. While it is known that POE is associated with an increased risk of fetal growth restriction and preterm birth, the effects of POE on the developing central nervous system remain poorly understood (3–5). Clinical data shows evidence of abnormal brain development with decreased brain volumes and aberrant structural connectivity in children exposed to opioid medications prenatally (6–12). Others have shown significant cognitive and motor dysfunction in school-age children with POE when compared to age-matched peers without prenatal opioid exposure (13). Larger cohort studies are limited by social, economic, and environmental confounding factors, but demonstrate higher risk of attention deficit hyperactivity disorder and symptoms in school-age children with prenatal opioid exposure as well (3, 14). Elegant preclinical studies using animals show similar findings and elicit concern for long-term neurobehavioral consequences in this patient population. Specifically, prenatal methadone exposure changes open field activity, and impairs sensorimotor developmental milestone acquisition concomitant with reduced neuronal density in motor cortex and aberrant circuit connectivity (15). Prenatal Methadone and buprenorphine cause impaired recognition memory, and nonspatial reference learning in young adult rats (16) corroborating reports of impaired cognitive flexibility and learning acquisition older adults after POE using a touchscreen platform (17). Similarly, buprenorphine and methadone impair social interaction and novel object recognition, diminish elevated plus maze performance, and induce anxiety (18). Studies using other opioids yield similar results. Prenatal fentanyl exposure alters sensory processing defined by multiple changes in synapses, regional changes in excitatory and inhibitory tone, and diminished dendritic arbor (19). Prescription opioids, including oxycodone, disrupt afferent regulation of dopamine activity in the ventral tegmental area during development suggesting disruption of the trajectory of mesolimbic circuity maturation and providing connection to common neuropsychological outcomes such as anxiety, attention, and depression (20).

Understanding the mechanism of opioid-induced neural injury and the lasting impact of this injury across the lifespan is key to identifying therapeutic targets and tailoring intervention to high-risk patients at crucial times in development. The ability to recognize patients at higher risk of long-term adverse outcomes from POE with precise biomarkers is also essential in guiding clinical management for diagnosis and treatment (21–23). Here, we defined alterations in the immune system following POE to test whether adults with POE had persistent brain inflammation. Specifically, we conducted investigations of serum inflammatory cytokine and chemokine profiles and evaluated peripheral immune hyper-reactivity from toddler age equivalent at P21 to an adult human age equivalent at P60. We also used multiparameter flow cytometry to interrogate cerebral and peripheral blood immune cell population dynamics in adulthood. We hypothesized that POE would induce neuroinflammation that could be detected in adulthood, defined by changes in infiltrating immune cells. We also predicted there would be sustained inflammation alongside disruption in the function and composition of the peripheral immune system.

Given the continued rise in opioid use across the globe, there is an urgent need to address the evolving public health crisis affecting countless pregnant women and children exposed to opioid medications in utero. Alongside growing recognition of the long-term neurologic impact associated with in utero opioid exposure, an improved understanding of the mechanisms underlying the brain injury caused by POE is imperative to establish biomarkers for identification of injury and to determine novel therapeutic targets. POE occurs at a critical timepoint in development disrupting delicate pathways essential for proper maturation of neural-immune function. Opioids readily cross the placenta and blood-brain barrier and lead to direct stimulation of inflammatory pathways via TLR4-mediated signaling (41–43). By shifting these pathways towards a pro-inflammatory state, opioids alter the developing immune system, and this alteration is sustained throughout the lifespan (2, 17, 44).

In this study, we show shifts in cerebral immune cell populations, defined specifically by increased neutrophils and regulatory T-cells, occurring months after prenatal opioid exposure. Furthermore, we demonstrate evidence of peripheral inflammation alongside immune priming and sustained peripheral immune reactivity (SPIHR) following prenatal opioid exposure that extends beyond the neonatal period. Even as markers of serum inflammation and SPIHR normalize into adulthood, elevated cerebral neutrophil and regulatory T-cell levels remain, highlighting the long-term impact of prenatal opioid exposure on the brain. Understanding the precise mechanisms underlying this injury is crucial to identifying those children at high risk of injury and to identifying targets for neuroimmunomodulation.

Previously, using the same model of POE, we identified a robust systemic inflammatory response syndrome and immune system dysfunction during the neonatal period concomitant with microstructural white matter injury and cognitive deficits in adulthood (17). POE led to priming of the immune system in the immediate perinatal period with significant baseline elevation in secretion of pro-inflammatory cytokines and chemokines, as well as an exaggerated inflammatory response from PBMCs acutely after stimulation with LPS (25). Specifically, we found of significant elevation of the inflammatory cytokines IL-1β, TNF-α, IL-6, and CXCL1 in the peripheral circulation at P10, around human term age equivalent, in POE rats suggesting a systemic inflammatory response syndrome (SIRS)-like response. We also showed that PBMCs, at P7, demonstrate significant baseline hypersecretion of TNF-α, CXCL1, and IL-6 with decreased levels of anti-inflammatory interleukin-10 (IL-10) and an exaggerated response to LPS stimulation with increased levels of TNF-α, CXCL1, IL-6, and IL-10 in rats with POE compared to controls (17, 25). Here, we extend those finding to show increased serum CXCL1 and IL-6 at P21, in POE rats concomitant with pro-inflammatory, dynamic, PBMC reactivity at P21, toddler age equivalent. CXCL1 is a potent chemokine responsible for neutrophil chemotaxis that has been implicated in significant intrauterine, placental, and fetal inflammation secondary to chorioamnionitis (26, 27, 33). Dysregulation in peripheral cytokine levels is not unique to POE and has also been identified in children with neonatal encephalopathy, cerebral palsy, and trisomy 21 (45–48). Our PBMC data reveals baseline hypersecretion of IL-6 that persists at P21. Further interrogation of PBMCs in culture with LPS stimulation at P21 demonstrates dysregulation in immune response following POE. After a 3 and 24 h incubation period, we note hypersecretion of TNF-α and CXCL1 in methadone-exposed PBMCs compared to saline-exposed PBMCs. These alterations in the baseline immune system function and immune response are indicative of SPIHR. The implications of inflammation during this timeframe from the neonatal period into toddler age equivalent cannot be understated, as it coincides with the elaborate neurodevelopmental program guiding myelination, oligodendrocyte maturation, and neural circuit formation that remains vulnerable to disruption (49, 50). Taken together with emerging clinical literature supporting priming of the immune system secondary to opioid exposure in utero, these data support a unique inflammatory signature followed by increased sensitivity to future insults. This altered immune landscape may lead to aberrant neural maturation and long-term cognitive and functional impairment that is just starting to be recognized in countless children and adults who have been exposed to opioid medications prenatally and has been demonstrated in elegant preclinical studies (15, 19–23).This suggests important future directions to identify correlations with severity of brain injury with functional magnetic resonance imaging, longitudinal assessment of white matter injury, and assessment of additional domains of cognition, including attention and inhibitory control using the touchscreen platform.

To our knowledge, this is the first investigation highlighting changes in cerebral immune cell populations into adulthood following opioid exposure commencing in utero. Our data shows persistently abnormal populations of cerebral lymphocytes following POE, with increased regulatory T cells and neutrophils compared to control rats. This finding offers invaluable insight into long-term immune alterations that coincides with long-term deficits seen in individuals with prenatal opioid exposure (51–53). It also underscores the need for improved biomarker development to better identify those at risk of persistent inflammation into adulthood including imaging or functional assessments of injury. Furthermore, increased regulatory T cells in the brain is consistent with other models of perinatal brain injury that show tissue injury, neuronal loss, and abnormal long-term neurodevelopment associated with increases in T cells (54). Indeed, modulation of regulatory T cells may be neuroprotective under specific conditions of developmental brain injury and offers an important therapeutic avenue for neuroimmunomodulation to treat brain injury and neuroinflammation secondary to POE (54). Serum elevations of IL-6 and CXCL1, as well as hypersecretion of TNF-α and CXCL1 following LPS stimulation of PBMCs in rats with POE at P21 may also serve as valuable markers of earlier dysfunction in the toddler age equivalent that could suggest risk for future persistent immune alterations. This is evident as CXCL1, a potent chemokine in neutrophil chemotaxis, is upregulated after POE and exerts a lasting impact on cerebral inflammation with a persistent increase of neutrophils months after opioid exposure. Promoting homeostasis, rather than antagonism of these unbalanced inflammatory pathways, may be crucial in the treatment of brain injury secondary to POE due to key neurodevelopmental roles of both CXCL1 and TNF-α.

Critically, our data in rats with POE also suggests that the period in which to intervene, from a clinical perspective, extends beyond the neonatal period. While ongoing inflammation, detected even months after cessation of exposure to opioids, can negatively impact brain function into adulthood (44), it also potentially broadens the timeframe for intervention. Targeting persistent inflammation into toddlerhood may still influence long-term developmental outcomes in this high-risk population. Further investigation of changes in immune cell populations in the peripheral circulation using flow cytometric analyses across a similar time course beginning at P21 may offer insight into the specific pattern of inflammation and immune dysregulation associated with POE and provide guidance for discovery of earlier biomarkers, especially in discrete developmental subsets. Additionally, analysis of central immune cell populations in animals, including inflammatory activation and morphology assessments, may help characterize the impact of these alterations on the developing and aging CNS.

There are limitations to the design and scope of our study and future investigations will address these constraints. Firstly, our study was not powered to evaluate differences in outcome measures based on sex. Further investigation into sex as a modifier of inflammation secondary to POE is important to identifying at-risk infants and children and evaluating responsiveness to novel therapeutic approaches including neuroimmunomodulation. Second, opioid exposure in this model occurs from E16 through P21 and does not capture opioid exposure from the onset of pregnancy (E0). Last, a brief period of isoflurane anesthesia was administered during the third trimester of rat gestation to implant osmotic minipumps, and this may have been an additional inflammatory stimulus during pregnancy. Future studies will address the correlation between immune function, functional and structural brain injury, and deficits of cognition and attention in adulthood. Longitudinal assessment of inflammation and the immune system following POE in the same rat, over time would be beneficial.

In conclusion, we provide evidence of peripheral inflammation alongside immune hyper-reactivity following prenatal opioid exposure. The importance of neural-immune communication and crosstalk with the peripheral immune system and central immune system is further highlighted with durable changes in cerebral immune cell populations of regulatory T cells and neutrophils months after POE. Beyond molecular inflammation, this study demonstrates immune cell population changes in adulthood secondary to prenatal exposure that may be critical to understanding the underpinnings of injury associated with opioid exposure. This study adds to a growing body of important literature linking sustained changes in immune reactivity with developmental brain injury (45–48). Furthermore, this study offers insight into potential treatment targets and widens the time course for potential intervention to help countless children with brain injury resulting from prenatal opioid exposure.