Abstract
The increased incidence of opioid use during pregnancy warrants investigation to reveal the impact of opioid exposure on the developing fetus. Exposure during critical periods of development could have enduring consequences for affected individuals. Particularly, evidence is mounting that developmental injury can result in immune priming, whereby subsequent immune activation elicits an exaggerated immune response. This maladaptive hypersensitivity to immune challenge perpetuates dysregulated inflammatory signaling and poor health outcomes. Utilizing an established preclinical rat model of perinatal methadone exposure, we sought to investigate the consequences of developmental opioid exposure on in vitro activation of peripheral blood mononuclear cells (PBMCs). We hypothesize that PBMCs from methadone-exposed rats would exhibit abnormal chemokine and cytokine expression at baseline, with exaggerated chemokine and cytokine production following immune stimulation compared to saline-exposed controls. On postnatal day (P) 7, pup PMBCs were isolated and cultured, pooling three pups per n. Following 3 and 24 h, the supernatant from cultured PMBCs was collected and assessed for inflammatory cytokine and chemokine expression at baseline or lipopolysaccharide (LPS) stimulation using multiplex electrochemiluminescence. Following 3 and 24 h, baseline production of proinflammatory chemokine and cytokine levels were significantly increased in methadone PBMCs (p < 0.0001). Stimulation with LPS for 3 h resulted in increased tumor necrosis factor (TNF-α) and C-X-C motif chemokine ligand 1 (CXCL1) expression by 3.5-fold in PBMCs from methadone-exposed PBMCs compared to PBMCs from saline-exposed controls (p < 0.0001). Peripheral blood mononuclear cell hyperreactivity was still apparent at 24 h of LPS stimulation, evidenced by significantly increased TNF-α, CXCL1, interleukin 6 (IL-6), and IL-10 production by methadone PMBCs compared to saline control PBMCs (p < 0.0001). Together, we provide evidence of increased production of proinflammatory molecules from methadone PBMCs at baseline, in addition to sustained hyperreactivity relative to saline-exposed controls. Exaggerated peripheral immune responses exacerbate inflammatory signaling, with subsequent consequences on many organ systems throughout the body, such as the developing nervous system. Enhanced understanding of these inflammatory mechanisms will allow for appropriate therapeutic development for infants who were exposed to opioids during development. Furthermore, these data highlight the utility of this in vitro PBMC assay technique for future biomarker development to guide specific treatment for patients exposed to opioids during gestation.
Highlights
The incidence of opioid abuse in the United States has steadily increased since 2000, today reaching epidemic proportions [1, 2]
After 24 h, the level of tumor necrosis factor α (TNF-α) secreted by saline PBMCs was 41% increased from levels measured at 3 h, whereas methadone PBMCs demonstrated a 152% increase from levels measured at 3 h
Consistent with our previous findings that PBMCs derived from methadone-exposed rats secreted increased levels of TNF-α at 3 and 24 h after collection [42], here, we demonstrate that methadone PBMCs secrete elevated levels of CXCL1 at 3 h
Summary
The incidence of opioid abuse in the United States has steadily increased since 2000, today reaching epidemic proportions [1, 2]. Opioid use by pregnant women has escalated to alarming rates [5]. Opioid use in this population increased 5-fold from 2000 to 2009 [6], and the prevalence of women with opioid use disorders at delivery hospitalizations quadrupled between 1999 and 2014 [7]. The growing rate of women using opioids during pregnancy has led to an increase in adverse neonatal outcomes [11, 12]. Recent studies showing an association between opioid use during pregnancy and poor health outcomes for both pregnant women and infants highlight prenatal opioid exposure as a serious public health concern [13, 14]. The devastating consequences of opioid exposure on the physical health and developmental outcomes of exposed children strengthen the need to advance scientific understanding of the underpinnings of opioid-induced neural injury and to advance biomarker development in this patient population
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