Chorioamnionitis in Rats Precipitates Extended Postnatal Inflammatory Lymphocyte Hyperreactivity

Abstract

Preterm birth is an important cause of perinatal brain injury (PBI). Neurological injury in extremely preterm infants often begins in utero with chorioamnionitis (CHORIO) or inflammation/infection of the placenta and concomitant placental insufficiency. Studies in humans have shown dysregulated inflammatory signaling throughout the placental-fetal brain axis and altered peripheral immune responses in children born preterm with cerebral palsy (CP). We hypothesized that peripheral immune responses would be altered in our well-established rat model of CP. Specifically, we proposed that isolated peripheral blood mononuclear cells (PBMCs) would be hyperresponsive to a second hit of inflammation throughout an extended postnatal time course. Pregnant Sprague-Dawley dams underwent a laparotomy on embryonic day 18 (E18) with occlusion of the uterine arteries (for 60 min) followed by intra-amniotic injection of lipopolysaccharide (LPS, 4 μg/sac) to induce injury in utero. Shams underwent laparotomy only, with equivalent duration of anesthesia. Laparotomies were then closed, and the rat pups were born at E22. PBMCs were isolated from pups on postnatal day 7 (P7) and P21, and subsequently stimulated in vitro with LPS for 3 or 24 h. A secreted inflammatory profile analysis of conditioned media was performed using multiplex electrochemiluminescent immunoassays, and the composition of inflammatory cells was assayed with flow cytometry (FC). Results indicate that CHORIO PBMCs challenged with LPS are hyperreactive and secrete significantly more tumor necrosis factor α (TNFα) and C-X-C chemokine ligand 1 at P7. FC confirmed increased intracellular TNFα in CHORIO pups at P7 following LPS stimulation, in addition to increased numbers of CD11b/c immunopositive myeloid cells. Notably, TNFα secretion was sustained until P21, with increased interleukin 6, concomitant with increased expression of integrin β1, suggesting both sustained peripheral immune hyperreactivity and a heightened activation state. Taken together, these data indicate that in utero injury primes the immune system and augments enhanced inflammatory signaling. The insidious effects of primed peripheral immune cells may compound PBI secondary to CHORIO and/or placental insufficiency, and thereby render the brain susceptible to future chronic neurological disease. Further understanding of inflammatory mechanisms in PBI may yield clinically important biomarkers and facilitate individualized repair strategies and treatments.

Perinatal Opioid Exposure Primes the Peripheral Immune System Toward Hyperreactivity

“…Based on TNF-α secretion levels at 3 h, we determined that stimulation with 100 ng of LPS was ideal to produce a robust PBMC secretory response in both treatment groups. Therefore, consistent with previous studies, 100 ng of LPS was used for LPS challenge experiments ( 41 , 45 , 73 ). Supernatant samples were collected at 3 and 24 h in sterile 2-mL Eppendorf tubes, snap frozen on dry ice, and stored at −80°C until biochemical analysis.…”

“…To further characterize altered immune function, we stimulated cultured PBMCs with 100 ng/mL of LPS to represent an immune challenge and then quantified the chemokine and cytokine profile signature. While the addition of 100 ng/mL of LPS is a supraphysiological dose, it was formulated in consideration of our previous work ( 41 , 42 ) and based on the dose response measured in TNF-α secretion to 10, 50, and 100 ng/mL of LPS that we performed in this study. We demonstrate that 100 ng/mL produces a robust PBMC response in the in vitro PBMC culture assay we performed, allowing us to examine chemokine and cytokine production by PBMCs from both saline and methadone treatment groups.…”

“…The in vitro LPS challenge we perform here is a method that has been used clinically in children with developmental disorders and brain injury ( 35 , 37 ). For instance, in a clinical study of children born preterm with cerebral palsy ( 37 ) and preclinical model reminiscent of preterm CNS injury ( 41 ), PBMCs were assayed consistent with the in vitro approach employed in the current study. Interestingly, in these studies, baseline secretion of PBMCs did not differ between treatment groups ( 37 , 41 ).…”

“…During this same developmental period, consolidation of the immune system in humans and rats occurs, whereby the functional capacity of immune cells evolves, and the number of circulating leukocytes, neutrophils, and monocytes increases ( 26 – 28 , 60 , 70 – 72 ). At P7, PBMC isolation was performed as previously published ( 41 ). Venous blood was collected from the right atrium of deeply anesthetized P7 pups and pooled across three animals in pyrogen-free K2 EDTA Vacutainer tubes (366643; Becton Dickson, Franklin Lakes, NJ, USA).…”

“…Moreover, these advances highlight the neurodevelopmental consequences of perinatal immune activation following perinatal insult ( 29 – 33 ). The findings from both clinical and preclinical studies implicate perinatal immune activation in the pathophysiology of numerous neurodevelopmental disorders, such as cerebral palsy, autism spectrum disorders, Down syndrome, and fetal alcohol spectrum disorders ( 33 – 41 ).…”