Derivation of Injury-Responsive Dendritic Cells for Acute Brain Targeting and Therapeutic Protein Delivery in the Stroke-Injured Rat

Abstract

Research with experimental stroke models has identified a wide range of therapeutic proteins that can prevent the brain damage caused by this form of acute neurological injury. Despite this, we do not yet have safe and effective ways to deliver therapeutic proteins to the injured brain, and this remains a major obstacle for clinical translation. Current targeted strategies typically involve invasive neurosurgery, whereas systemic approaches produce the undesirable outcome of non-specific protein delivery to the entire brain, rather than solely to the injury site. As a potential way to address this, we developed a protein delivery system modeled after the endogenous immune cell response to brain injury. Using ex-vivo-engineered dendritic cells (DCs), we find that these cells can transiently home to brain injury in a rat model of stroke with both temporal and spatial selectivity. We present a standardized method to derive injury-responsive DCs from bone marrow and show that injury targeting is dependent on culture conditions that maintain an immature DC phenotype. Further, we find evidence that when loaded with therapeutic cargo, cultured DCs can suppress initial neuron death caused by an ischemic injury. These results demonstrate a non-invasive method to target ischemic brain injury and may ultimately provide a way to selectively deliver therapeutic compounds to the injured brain.

Soluble TNF Receptor 1-Secreting ex Vivo-Derived Dendritic Cells Reduce Injury After Stroke

“…3 To define the broader therapeutic window for this approach, we characterized the time-course of exDC recruitment to the injury site after stroke. ExDCs from the bone marrow of postnatal day 15 to 17 GFP þ / À transgenic rats were isolated and cultured.…”

“…Brain sections were stained with the endothelial marker RECA-1 and all of GFP þ exDCs colocalized with RECA-1 staining, appearing to fill the vessel, indicating the cells did not leave the brain vasculature to cross into the brain parenchyma, however entry into the glia limitans was not ruled out ( Figures 1C-1H); this is in agreement with our prior findings. 3 Ex vivo-Derived Dendritic Cells Migration Correlates with Inflammation Levels in the Brain While exDCs do not enter the brain parenchyma, the production of inflammatory factors such as TNF-a and IL-1b by cells in the infarcted region directly affect vascular environment. For example, TNF-a increases expression of ICAM-1 on the endothelial cell surface 18 and in vitro human endothelial migration models have shown that ICAM-1 directly mediates exDC firm adhesion and migration across endothelial cells.…”

“…3 Upon lipopolysaccharide stimulation, exDCs become mature with increased CD80 expression and decreased retention in the injury site. 3 Previous studies in our lab have also shown that after clearing from the brain vasculature, exDCs delivered at 6 hours poststroke migrate to the lung, liver, spleen, and gut over time. 3 Previously published in vitro studies have shown that exDCs respond to chemoattractants such as MCP-1 (ref.…”

“…As an alternative, we have designed a novel delivery system using leukocytes that allow for protein delivery in a minimally invasive manner. 3 Leukocyte-mediated delivery of proteins shows temporal and spatial selectivity, presenting a promising, new method of targeted protein delivery to reduce stroke damage. 3 Because leukocytes respond to proinflammatory factors and the migration of endogenous immune cells to the injured brain is concomitant with the increase in proinflammatory factors after stroke, leukocytes are an ideal vehicle for delivery of therapeutic proteins.…”

“…3 Leukocyte-mediated delivery of proteins shows temporal and spatial selectivity, presenting a promising, new method of targeted protein delivery to reduce stroke damage. 3 Because leukocytes respond to proinflammatory factors and the migration of endogenous immune cells to the injured brain is concomitant with the increase in proinflammatory factors after stroke, leukocytes are an ideal vehicle for delivery of therapeutic proteins. After ischemia, messenger RNA expression of potent inflammatory cytokines and chemokines TNF-a, IL-1b, and MCP-1 increases in the brain within 1 to 3 hours and peaks between 24 and 36 hours.…”