No abstract

Gene delivery from biomaterials can create an environment that promotes and guides tissue formation. However, the immune response induced upon biomaterial implantation can be detrimental to tissue regeneration. Macrophages play a central role in mediating early phases of this response, and functional “polarization” of macrophages towards M1 (inflammatory) or M2 (anti-inflammatory) phenotypes may bias the local immune state at the implant site. Since gene delivery from biomaterial scaffolds can confer transgene expression in macrophages in vivo, we investigated whether transduction of macrophages with an IL-10 encoding lentivirus can (1) induce macrophage polarization toward an M2 phenotype even in an pro-inflammatory environment, and (2) prevent a shift in polarization from M2 to M1 following exposure to pro-inflammatory stimuli. IL-10 lentivirus delivery to pre-polarized M1 macrophages reduced TNF-α production 1.5-fold when compared to cells treated with either a control virus or a bolus delivery of recombinant IL-10 protein. IL-10 lentivirus delivery to naïve macrophages reduced the amount of TNF-α produced following an inflammatory challenge by 2.5-fold compared to cells treated with both the control virus and recombinant IL-10. At a mechanistic level, IL-10 lentivirus delivery mediated sustained reduction in NF-κB activation and, accordingly, reduced transcription of TNF-α. In sum, lentiviral delivery of IL-10 to macrophages represents a promising strategy for directing and sustaining macrophage polarization towards an M2 phenotype in order to promote local immune responses that facilitate tissue engineering.

Targeted approaches to treat autoimmune diseases would improve upon current therapies that broadly suppress the immune system and lead to detrimental side effects. Antigen-specific tolerance was induced using poly(lactide-co-glycolide) nanoparticles conjugated with disease-relevant antigen to treat a model of multiple sclerosis. Increasing the nanoparticle dose and amount of conjugated antigen both resulted in more durable immune tolerance. To identify active tolerance mechanisms, we investigated downstream cellular and molecular events following nanoparticle internalization by antigen-presenting cells. The initial cell response to nanoparticles indicated suppression of inflammatory signaling pathways. Direct and functional measurement of surface MHC-restricted antigen showed positive correlation with both increasing particle dose from 1 to 100 μg/mL and increasing peptide conjugation by 2-fold. Co-stimulatory analysis of cells expressing MHC-restricted antigen revealed most significant decreases in positive co-stimulatory molecules (CD86, CD80, and CD40) following high doses of nanoparticles with higher peptide conjugation, whereas expression of a negative co-stimulatory molecule (PD-L1) remained high. T cells isolated from mice immunized against myelin proteolipid protein (PLP) were co-cultured with antigen-presenting cells administered PLP-conjugated nanoparticles, which resulted in reduced T cell proliferation, increased T cell apoptosis, and a stronger anti-inflammatory response. These findings indicate several potential mechanisms used by peptide-conjugated nanoparticles to induce antigen-specific tolerance.

Inflammation associated with autoimmune diseases and chronic injury is an initiating event that leads to tissue degeneration and dysfunction. Inflammatory monocytes and neutrophils systemically circulate and enter inflamed tissue, and pharmaceutical based targeting of these cells has not substantially improved outcomes and have had side effects. Herein, we investigated the design of drug-free biodegradable nanoparticles, notably without any active pharmaceutical ingredient or targeting ligand, that target circulating inflammatory monocytes and neutrophils in the vasculature to inhibit them from migrating into inflamed tissue. Nanoparticles were formed from 50:50 poly(DL-lactide-co-glycolide) (PLG) with two molecular weights (Low, High) and poly(DL-lactide) (PLA) formed (termed PLG-L, PLG-H, and PDLA, respectively) and were analyzed for their association with monocytes and neutrophils and their impact on disease course along with immune cell trafficking,. For particles injected intravenously for 6 consecutive days to

Current therapeutic options for autoimmune diseases, such as multiple sclerosis (MS), often require lifelong treatment with immunosuppressive drugs, yet strategies for antigen-specific immunomodulation are emerging. Biodegradable particles loaded with disease-specific antigen, either alone or with immunomodulators, have been reported to ameliorate disease. Herein, we hypothesized that the carrier could impact polarization of the immune cells that associate with particles and the subsequent disease progression. Single injection of three polymeric carriers, 50:50 poly (DL-lactide-co-glycolide) (PLG) with two molecular weights (Low, High) and poly (DL-lactide) (PLA), loaded with the disease-specific antigen, proteolipid protein (PLP139–151), were investigated for the ability to attenuate clinical scores in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. At a low particle dose, mice treated with PLA-based particles had significantly lower clinical scores at the chronic stage of the disease over 200 days post immunization, while neither PLG-based particles nor OVA control particles reduced the clinical scores. Compared to PLG-based particles, PLA-based particles were largely associated with Kupffer cells and liver sinusoidal endothelial cells, which had a reduced co-stimulatory molecule expression that correlated with a reduction of CD4+ T-cell populations in the central nervous system. Delivery of PLA-based particles encapsulated with higher levels of PLP139–151 at a reduced dose were able to completely ameliorate EAE over 200 days along with inhibition of Th1 and Th17 polarization. Collectively, our study demonstrates that the carrier properties and antigen loading determine phenotypes of immune cells in the peripheral organs, influencing the amelioration of both acute and chronic stages of autoimmunity.

Background and Purpose Bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng+/−) mice. We hypothesized that Eng+/− BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. Methods BM transplantation was performed using adult wild-type (WT) and Eng+/− mice as donors/recipients. An adeno-associated viral vector expressing VEGF (AAV-VEGF) was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 μm/100 vessels), and BMDCs in the angiogenic foci were analyzed. Results The dysplasia index of WT/Eng+/− BM mice was higher than WT/WT BM mice (p<0.001) and was similar to Eng+/−/Eng+/− BM mice (p=0.2). Dysplasia in Eng+/− mice was partially rescued by WT BM (p<0.001). WT/WT BM and WT/Eng+/− BM mice had similar numbers of BMDCs in the angiogenic foci (p=0.4), most of which were CD68+. Eng+/− monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1. Conclusions ENG-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations.

Background and Purpose Adeno-associated viral vector (AAV) is a powerful tool for delivering genes to treat brain diseases. Intravenous delivery of a self-complementary, but not single-stranded, AAV9 vector (ssAAV9) mediates robust gene expression in the adult brain. We tested if ssAAV9 effectively mediates gene expression in the ischemic stroke lesion and angiogenic foci. Methods Focal ischemic stroke was induced by permanent occlusion of the left middle cerebral artery (MCAO), and focal angiogenesis, by injecting an AAV vector expressing vascular endothelial growth factor (AAV-VEGF) into the basal ganglia. ssAAV vectors that have CMV promoter driving (AAV-CMVLacZ) or hypoxia response elements controlling (AAV-H9LacZ) LacZ expression were packaged in AAV9 or AAV1 capsid, and injected into mice through the jugular vein one hour after MCAO or four weeks after the induction of angiogenesis. LacZ gene expression was analyzed in the brain and other organs five days post LacZ vector-injection. Results LacZ expression was detected in the peri-infarct region of AAV9-CMVLacZ and AAV9-H9LacZ-injected MCAO mice, and the brain angiogenic foci of AAV9-CMVLacZ-injected mice. Minimum LacZ expression was detected in the brain of AAV1-CMVLacZ-injected mice. Robust LacZ expression was found in the liver and heart of AAV-CMVLacZ-injected mice, but not AAV9-H9LacZ-injected mice. Conclusion ssAAV9 vector could be a useful tool to deliver therapeutic genes to the ischemic stroke lesion or brain angiogenic foci.

Background and Purpose: Stroke occurs mostly in patients with advanced age and older patients have a less favorable prognosis than younger patients. To understand the underlying mechanisms involved in this phenomenon, we tested the hypothesis that an increased inflammatory response to acute ischemic injury in the aged mice leads to more severe brain damage and functional deficits. Methods: An ischemic stroke model was created in two- (young) and twelve-month-old (aged) C57BL/6 mice (n=6) through permanent occlusion of the left distal middle cerebral artery (pMCAO). Infarct volumes in the brain sections were quantified at one day after pMCAO by Cresyl Violet staining. Sensorimotor function was assessed using corner test and adhesive removal test before, 3, and 14 days after pMCAO. Macrophage/microglia (CD68+ cells) and apoptotic neurons (TUNEL+ and NeuN+) in the ischemic region were quantified one day after pMCAO. The interleukin-6 (IL-6) and interleukin-1 β (IL-1β) levels in the ischemic brain tissue were measured using ELISA at one and three days after pMCAO. Results: At one day post pMCAO, infarct volume was larger in aged mice than in young mice (31±3/mm3 vs 27±5, p= 0.05), Comparing aged mice to young mice, more TUNEL+ neurons (22±5 vs 15±5, p=0.04) were detected in the infarct region. Both young and aged mice showed significant sensorimotor dysfunction on day 3 and 14 post pMCAO; turning more frequently to the left on the corner test and taking longer time to remove tape on left paws. More severe functional deficits were detected on aged mice by adhesive removal test at day 14 post pMCAO (12±5 seconds vs 6±3, p=0.01, aged vs young). There were more CD68+ cells in the peri-infarct region in aged mice compared to young mice (79±22/ 40X objective field vs 58±18, p=0.01). Infarct volume was positively correlated with both the numbers of CD68+ cells (R2= 0.80) and TUNEL+ neurons (R2= 0.86). Compared to young, cytokine levels also increased in aged mice at one day (IL-6: 308 ±152 pg/mg vs 147±44, p=0.03; IL-1β: 96±35 vs 55±8, p=0.02) and three days (IL-6: 149 ±53 vs 90±27, p=0.03; IL-1β: 55 ±14 vs 38± 5, p= 0.02) after pMCAO. Conclusion: Our data suggest that a higher inflammatory response at the acute stage of ischemic stroke in aged mice is associated with more severe neuronal injury (larger infarct volume and more apoptotic neurons) and long-term functional deficits. The upstream mechanisms responsible for the enhanced response will require additional studies. However, modulating inflammatory response in the aged population at the acute stage might be a strategy to reduce neuronal injury and improve functional recovery in older stroke patients with monitoring of brain inflammation a feasible means of risk stratification.