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Derivation of injury-responsive dendritic cells for acute brain targeting and therapeutic protein delivery in the stroke-injured rat - PubMed

  • ️Tue Jan 01 2013

Derivation of injury-responsive dendritic cells for acute brain targeting and therapeutic protein delivery in the stroke-injured rat

Nathan C Manley et al. PLoS One. 2013.

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.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflict: The techniques described in this manuscript are part of patent number 11/713,218 filed with the U.S. Patent and Trademark Office on Feb. 28, 2007. The name of the patent in question is "Trojan Horse Immunotherapy." This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Morphological and surface marker profile of 7-day-old, lentivirus-transduced, rat bone marrow-derived DC cultures.

(A) Large clusters of round cells predominate by culture day 7, forming a semi-adherent layer over stromal-type cells (top left and center panels). Fluorescent micrograph of a harvested, GFP-transgenic DC with representative spherical, mononuclear morphology (top right panel). Scale bars: (left) 50 µm, (center) 5 µm, (right) 0.5 µm. (B) Flow cytometric analysis of DC cultures showing forward- and side-scatter dot plot with the gated population prior to singlet gating (upper left panel), and histograms of mean fluorescent intensity (x-axis) versus percentage of gated population (y-axis) for CD11b/c (OX42), CD11c, MHC class II (OX6), CD80, CD68, CCR2, and VLA4. Results shown in (B) are representative of at least 3 independent experiments, assaying 20,000 cells per experiment.

Figure 2
Figure 2. In vivo migration profile of cultured DCs.

(A)–(C) Bioluminescent imaging of rats positioned dorsally (left panels) and ventrally (right panels) 6 h post-tMCAO and 3 h post-infusion of: (A) vehicle, (B) 2×106 luciferase-DCs, or (C) 4×106 luciferase-DCs (D) High resolution (low-binning) image of a luciferase-DC-infused rat at the same time point as (A)–(C). (E) In vivo tracking of radiolabeled DCs with SPECT. Representative images of 3 rats infused with radiolabeled DCs 3 h post-tMCAO and imaged by SPECT at 5–20 min and again at 2.5–6 h post-DC infusion. Inset in top left panel indicates the orientation of rats during imaging. Rat number (1–3) and imaging time post-DC infusion are indicated at the bottom of each image panel.

Figure 3
Figure 3. Immunofluorescent staining of GFP-positive cells at the tMCAO lesion site.

(A) Graphical representation of GFP-positive cells in the rat brain 6 h post-tMCAO and 3 h post-DC infusion based on reconstructed whole brain section fluorescent images from a single animal. Overlying numbers indicate position relative to bregma. (B) Time course of GFP-positive cells in the brain following tMCAO and DC infusion. Values are expressed as the total number of GFP-positive cells per hemisphere extrapolated from counting 6 coronal sections/brain (shown in (A)). Error bars denote SEM, n = 9, 11, 5, 3 for 10 min, 3 h, 6 h, and 12 h, respectively. (C) Confocal images of GFP-positive cells (green) inside RECA-stained blood vessels (red). Left panel: ortho image of GFP-positive cell in the cerebrovasculature at 10 min post-cell infusion with nuclei DAPI-counterstained (blue); middle/right panel: z-stack images of GFP-positive cells in the cerebrovasculature at 3 h post cell-infusion with co-staining of GFAP-positive astrocytes (white). Scale bar: (C) 10 µm.

Figure 4
Figure 4. In vitro characterization of protein cargo.

(A) Representative micrographs of harvested LV-hBDNF-transduced DCs. (B) Quantification of hBDNF protein in transduced-DC lysate (intracellular) and conditioned medium (secreted) by ELISA. Values reflect the combined average of 3 independent experiments, with each derived from separate lentiviral preps and DC culture preps. ** P<0.01, *** P<0.001 versus LV-GFP-transduced cultures by one-way ANOVA and Tukey post-hoc analysis. (C) Quantification of cortical neuron loss following OGD and DC treatment. Results were combined from at least 3 independent experiments. Error bars denote SEM. n = 46–60 wells/treatment group. * P<0.05, *** P<0.001 by one-way ANOVA and Tukey post-hoc analysis. (D) Representative fluorescent micrographs of LV-tBH4-transduced and LV-GFP-transduced-DC cultures. (E) Representative western blot showing anti-HisG immunoreactivity at 14 kDa for GFP-DC lysate or tBH4-DC lysate and conditioned medium. Time post-LV transduction is indicated for each sample. (F) Quantification of hippocampal neuron loss following glutamate exposure and DC treatment. Average neuron loss was calculated from at least 3 independent experiments. Error bars denote SEM. n = 24–48 wells/treatment group. * indicates significance by one-way ANOVA and Tukey post-hoc analysis with respect to each no insult control (vehicle or DC treatment alone, P<0.05). Scale bars: (A) and (D) 100 µm.

Figure 5
Figure 5. Infarct quantification following treatment with protein-loaded DCs.

(A) Representative cresyl violet stain for each treatment group showing the 6 coronal sections used to quantify lesion size at 24 h post-tMCAO. (B) Quantification of lesion size at 24 h post-tMCAO by coronal brain section and treatment group. * indicates significance by one-way ANOVA plus Student-Newman-Keuls post-hoc analysis of tBH4-DCs and GFP-DCs for the sum infarct area of coronal sections 2–4, P = 0.040.

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