Perinatal Opioid Exposure Primes the Peripheral Immune System Toward Hyperreactivity

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.

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

“…Indeed, phenotypic plasticity of lymphocytes may be maladaptive and important to injury progression, or at the very least, a hindrance to neural repair in many individuals with CP or other forms of perinatal brain injury. Similar findings have been documented in children with Down Syndrome [ 97 , 98 ] and after exposure to opioids [ 59 , 70 ]. On the other hand, decreased levels of pro-inflammatory cytokines in sera and PBMC supernatants have been found in response to LPS stimulation in critically ill patients in intensive care [ 99 , 100 ], and in children with neonatal encephalopathy who received hypothermia [ 100 , 101 ].…”

“…PBMCs, defined as any blood cell with a round nucleus (i.e., a lymphocyte, a monocyte, or a macrophage), were isolated as previously described [ 58 , 59 , 70 ]. Specifically, on postnatal day 60 (P60, young adult) and P120 (middle-age adult), venous blood was collected from the right atrium.…”

“…After plating, 100 ng/ml of LPS or vehicle was added to each well and PBMCs were allowed to incubate for 3 or 24 h as previously described [ 58 , 59 , 70 ]. Upon conclusion of incubation, media were collected and centrifuged at 500 g for 10 min.…”

“…A separate kit was used for the detection of Monocyte Chemoattractant Protein-1 (MCP-1)/ C–C motif chemokine ligand 2 (CCL2) (R-PLEX Rat F231D, Meso Scale Diagnostics, Rockville, MD, USA). In accordance with the manufacturer's specifications and as previously described [ 62 , 70 – 72 ], serum or culture supernatant was diluted to 1:4 and then loaded in duplicate with the prepared standard on to a blocked and washed multi-spot 96-well plate. After a series of washes and incubations with antibody detection solution, the plates were washed and loaded with read buffer and then read with a Quickplex SQ 120.…”

“…After a series of washes and incubations with antibody detection solution, the plates were washed and loaded with read buffer and then read with a Quickplex SQ 120. Consistent with the standard in the field, samples reading below the detectable limits or with a coefficient of variation greater than 25% in an individual assay were removed from further analyses [ 59 , 62 , 70 , 72 ]. Both the pro-inflammatory and CCL2 panels performed with less than 10% variability between runs.…”