Early activation of the inflammatory response in the liver of brain-dead non-human primates

Abstract

Background: Donor brain death (BD) triggers a systemic inflammatory response that reduces organ quality and increases immunogenicity of the graft. We characterized the early innate immune response induced by BD in the liver and peripheral blood of hemodinamically stable non-human primates (NHP).

Methods: Rhesus macaques were assigned to either brain death or control group. BD was induced by inflation of a subdurally placed catheter and confirmed clinically and by cerebral angiography. Animals were monitored for 6 h after BD and managed to maintain hemodynamic stability.

Results: Cortisol, epinephrine, nor-epinephrine, and IL-6 levels were elevated immediately after BD induction. Neutrophils and monocytes significantly increased in circulation following BD induction, while dendritic cells were decreased at 6 h post-induction. Flow cytometry revealed increased expression of chemokine receptors CxCR1, CxCR2, CCR2, and CCR5 in peripheral blood leukocytes from NHP subjected to BD. Microarray analysis demonstrated a significant up-regulation of genes related to innate inflammatory responses, toll-like receptor signaling, stress pathways, and apoptosis/cell death in BD subjects. Conversely, pathways related to glucose, lipid, and protein metabolism were down-regulated. In addition, increased expression of SOCS3, S100A8/A9, ICAM-1, MHC class II, neutrophil accumulation, and oxidative stress markers (carboxy-methyl-lysine and hydroxynonenal) were detected by immunoblot and immunohistochemistry.

Conclusions: Activation of the innate immune response after BD in association with a down-regulation of genes associated with cell metabolism pathways in the liver. These findings may provide a potential explanation for the reduced post-transplant function of organs from brain dead donors. In addition, this work suggests potential novel targets to improve donor management strategies.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

FIG. 1.

Brain death induction in NHP induces changes in physiologic parameters that model the human clinical scenario. Hemodynamic instability characterized by a sharp increase in arterial blood pressure after catheter inflation followed by hypotension (A), sustained tachycardia (B), and progressive increase in urinary output (C) was observed in all NHP subjected to BD induction (n = 5). A subset of NHP were anesthetized and mechanically ventilated without brain death induction to determine control physiologic values over the 6 h period (n = 6).

FIG. 2.

Epinephrine and Nor-epinephrine changes in brain-dead (n = 5) and Non-Brain Dead control (NBD) NHP (n = 6) (A). Bars represent fold change above baseline. Cortisol levels in NBD (n = 5) and brain-dead primates (n = 6) before and after brain death (B). Data are means ± SEM. ns = not-significant. Significance calculated by non-parametric, two-sided t-test.

FIG. 3.

Brain death leads to changes in innate immune components of the peripheral blood leukocyte pool. Peripheral blood was drawn from control non-brain dead (NBD) and brain dead (BD) monkeys at T0 (prior to BD induction) and T6 (6 h post-BD induction) and leukocytes isolated by gradient centrifugation. Neutrophils, monocytes, and myeloid dendritic cells were quantified and changes in population frequency compared at T6 relative to T0 (A), chemokine receptor expression by neutrophils (B), monocytes (C), and myeloid dendritic cells (D). Data shown are the mean ± SEM and P values were calculated by two-way ANOVA.

FIG. 4.

Brain death induces increases in level of proteins involved in cells adhesion, inflammatory processes, and accumulation of oxidative stress products in the liver. Liver protein from NBD donor “Control” and “Brain Dead” NHP were analyzed by Western blotting for the expression of SOCS3 and S100A9 proteins (A). Immunohistochemical assessment of liver biopsies taken from NBD donor controls and brain dead NHP stained for ICAM-1 (B), MPO (C), MHC Class II DR (D), CML (E), and HNE (F). Immunoblot quantification of the identified bands was performed by densitometry and normalized to GAPDH protein levels with p values determined by Student’s t-test. Quantification of staining was performed by image analysis (see the Methods section) and data shown are the individual values and means (line). P values were calculated by Student’s t-test.

FIG. 5.

Model of systemic and localized inflammation affecting the liver in a non-human primate model of brain death. The initial process of BD results in the Cushing response including dysfunction of the hypothalamic-pituitary-adrenal axis and central autonomous dysregulation. The resulting hypoperfusion leads to endothelial cell activation, chemokine secretion and release of DAMPs from necrotic cells and activated innate immune cells. DAMPs bind to and activate TLR and result in NFKB activation followed by production and release of pro-inflammatory cytokines (PIC). PIC release results in further activation of endothelial cells and up-regulation of adhesion molecules including VCAM, ICAM, and P-selectin. PIC also induce systemic release of IL-6 leading to STAT3 activation and production of acute phase proteins by hepatocytes. These events culminate in the recruitment and migration of PMN and monocytes from bone marrow into peripheral circulation and then into the ischemic tissue. Once in the liver, innate immune cells prime and amplify the immune response, produce reactive oxygen species, and induce secondary tissue damage that ultimately leads to decreased organ function after recovery and an increased immunogenic potential.