A Pilot Study Identifying Brain-Targeting Adaptive Immunity in Pediatric Extracorporeal Membrane Oxygenation Patients With Acquired Brain Injury

Abstract

Objectives: Extracorporeal membrane oxygenation provides short-term cardiopulmonary life support, but is associated with peripheral innate inflammation, disruptions in cerebral autoregulation, and acquired brain injury. We tested the hypothesis that extracorporeal membrane oxygenation also induces CNS-directed adaptive immune responses which may exacerbate extracorporeal membrane oxygenation-associated brain injury.

Design: A single center prospective observational study.

Setting: Pediatric and cardiac ICUs at a single tertiary care, academic center.

Patients: Twenty pediatric extracorporeal membrane oxygenation patients (0-14 yr; 13 females, 7 males) and five nonextracorporeal membrane oxygenation Pediatric Logistic Organ Dysfunction score matched patients INTERVENTIONS:: None.

Measurements and main results: Venous blood samples were collected from the extracorporeal membrane oxygenation circuit at day 1 (10-23 hr), day 3, and day 7 of extracorporeal membrane oxygenation. Flow cytometry quantified circulating innate and adaptive immune cells, and CNS-directed autoreactivity was detected using an in vitro recall response assay. Disruption of cerebral autoregulation was determined using continuous bedside near-infrared spectroscopy and acquired brain injury confirmed by MRI. Extracorporeal membrane oxygenation patients with acquired brain injury (n = 9) presented with a 10-fold increase in interleukin-8 over extracorporeal membrane oxygenation patients without brain injury (p < 0.01). Furthermore, brain injury within extracorporeal membrane oxygenation patients potentiated an inflammatory phenotype in adaptive immune cells and selective autoreactivity to brain peptides in circulating B cell and cytotoxic T cell populations. Correlation analysis revealed a significant relationship between adaptive immune responses of extracorporeal membrane oxygenation patients with acquired brain injury and loss of cerebral autoregulation.

Conclusions: We show that pediatric extracorporeal membrane oxygenation patients with acquired brain injury exhibit an induction of pro-inflammatory cell signaling, a robust activation of adaptive immune cells, and CNS-targeting adaptive immune responses. As these patients experience developmental delays for years after extracorporeal membrane oxygenation, it is critical to identify and characterize adaptive immune cell mechanisms that target the developing CNS.

Figure 1.

Enrollment diagram. This diagram shows all enrollment for this pilot study, including status/post (s/p) exclusion criteria. Patient recruitment was determined a prior to be completed after 20 patients. CPR = cardiopulmonary resuscitation, ECMO = extracorporeal membrane oxygenation, PI = principal investigator, VA = venoarterial, VV = venovenous.

Figure 2.

Cytokine levels are elevated in extracorporeal membrane oxygenation (ECMO) patients with acquired brain injury. ECMO induces early cytokine up-regulation for (A) interleukin (IL)–6 and (B) IL-8 at day 1 of ECMO (n = 20) compared with disease-control patients without ECMO (n = 5). Circle shapes are nonbrain injury patients, and triangles represent brain injury patients. C, Heat map representation of all mean ± sd values (text shown, pg/mL) of cytokines separated by brain injury status. Significance is defined as *p < 0.05, **p < 0.01 by nonparametric Kruskal-Wallis analysis of variance versus sick control shown in the far left column of the heat map.

Figure 3.

Plasma from brain-injured patients supported with extracorporeal membrane oxygenation (ECMO) drives healthy adaptive immune cells toward a pro-inflammatory phenotype. Black bar graphs show nonparametric Kruskal-Wallis analysis of variance results for triplicate experiments for activation status (CD25⁺, n = 6) of (A) CD4⁺ helper T cells, (B) CD8⁺ cytotoxic T cells, and (C) CD19⁺ B cells after exposure to healthy plasma-containing media (left columns, squares, n = 5), or ECMO patient-derived plasma-containing media from patients without brain injury (middle columns, circles, n = 2), or with brain injury (right columns, triangles, n = 4). Permutations of test conditions resulted in 10 test conditions for control plasma, six for nonbrain injury ECMO, and 13 for brain injury ECMO. Plasma from ECMO patients with brain injury also elevated intracellular interleukin (IL)–17 production in (D) helper T cells and (F) B cells, but not (E) cytotoxic T cells. Values are mean ± sd and significance between groups on an individual day is shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus healthy plasma unless otherwise indicated by brackets.

Figure 4.

Brain injury associated with cytotoxic T cell autoreactivity to CNS antigens. Autoreactivity data separated by brain injury were analyzed by two-way analysis of variance, Fisher Least Significant Difference. Positive responses (change in proliferation fraction [ΔPF]) from all patients tested (#/#) and corresponding % indicated below graph. A, CD4 helper T cell responses did not differ between extracorporeal membrane oxygenation (ECMO) patients without brain injury (circles) and those with brain injury (triangles). B, Autoreactive CD8 T cell responses were, however, more abundant in brain injury ECMO patients, and (C) B cell autoreactivity was increased ECMO patients without brain injury at day 1, which was reversed by day 3 as brain-injured ECMO patients exhibited increased autoreactivity. Significance between groups on an individual day is shown as *p < 0.05, **p < 0.01, ***p < 0.001 or between groups, as indicated by brackets.