Kae M. Pusic

IFNγ-stimulated dendritic cell exosomes as a potential therapeutic for remyelination.

Dendritic cells (DCs) release exosomes with different characteristics based on stimulus. Here, we showed that DC cultures stimulated with low-level IFNγ released exosomes (IFNγ-DC-Exos) that contained microRNA species that can increase baseline myelination, reduce oxidative stress, and improve remyelination following acute lysolecithin-induced demyelination. Furthermore, nasally administered IFNγ-DC-Exos increased CNS myelination in vivo. IFNγ-DC-Exos were preferentially taken up by oligodendrocytes, suggesting that they directly impact oligodendrocytes to increase myelination. Thus, our results show great potential for use of these IFNγ-DC-Exos as a therapeutic to promote remyelination in multiple sclerosis and dysmyelinating syndromes.

Spreading Depression Requires Microglia and is Decreased by Their M2a Polarization from Environmental Enrichment

Microglia play an important role in fine‐tuning neuronal activity. In part, this involves their production of tumor necrosis factor‐alpha (TNFα), which increases neuronal excitability. Excessive synaptic activity is necessary to initiate spreading depression (SD). Increased microglial production of proinflammatory cytokines promotes initiation of SD, which, when recurrent, may play a role in conversion of episodic to high frequency and chronic migraine. Previous work shows that this potentiation of SD occurs through increased microglial production of TNFα and reactive oxygen species, both of which are associated with an M1‐skewed microglial population. Hence, we explored the role of microglia and their M1 polarization in SD initiation. Selective ablation of microglia from rat hippocampal slice cultures confirmed that microglia are essential for initiation of SD. Application of minocycline to dampen M1 signaling led to increased SD threshold. In addition, we found that SD threshold was increased in rats exposed to environmental enrichment. These rats had increased neocortical levels of interleukin‐11 (IL‐11), which decreases TNFα signaling and polarized microglia to an M2a‐dominant phenotype. M2a microglia reduce proinflammatory signaling and increase production of anti‐inflammatory cytokines, and therefore may protect against SD. Nasal administration of IL‐11 to mimic effects of environmental enrichment likewise increased M2a polarization and increased SD threshold, an effect also seen in vitro. Similarly, application of conditioned medium from M2a polarized primary microglia to slice cultures also increased SD threshold. Thus, microglia and their polarization state play an essential role in SD initiation, and perhaps by extension migraine with aura and migraine. GLIA 2014;62:1176–1194

Environmental Enrichment Stimulates Immune Cell Secretion of Exosomes that Promote CNS Myelination and May Regulate Inflammation

Environmental enrichment (EE) consists of increased physical, intellectual, and social activity, and has wide-ranging effects, including enhancing cognition, learning and memory, and motor coordination. Animal studies have demonstrated that EE improves outcome of brain trauma and neurodegenerative disorders, including demyelinating diseases like multiple sclerosis, making it a promising therapeutic option. However, the complexity of applying a robust EE paradigm makes clinical use difficult. A better understanding of the signaling involved in EE-based neuroprotection may allow for development of effective mimetics as an alternative. In prior work, we found that exosomes isolated from the serum of rats exposed to EE impact CNS myelination. Exosomes are naturally occurring nanovesicles containing mRNA, miRNA, and protein, which play important roles in cell function, disease, and immunomodulation. When applied to hippocampal slice cultures or nasally administered to naïve rats, EE-serum exosomes significantly increase myelin content, oligodendrocyte precursor (OPC) and neural stem cell levels, and reduce oxidative stress (OS). We found that rat EE exosomes were enriched in miR-219, which is necessary and sufficient for OPC differentiation into myelinating cells. Thus, peripherally produced exosomes may be a useful therapy for remyelination. Here, we aim to better characterize the impact of EE on CNS health and to determine the cellular source of nutritive exosomes found in serum. We found that exosomes isolated from various circulating immune cell types all increased slice culture myelin content, contained miR-219, and reduced OS, suggesting that EE globally alters immune function in a way that supports brain health.

Iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine

This study explored the novel use of iron oxide (IO) nanoparticles (<20 nm) as a vaccine delivery platform without additional adjuvants. A recombinant malaria vaccine antigen, the merozoite surface protein 1 (rMSP1), was conjugated to IO nanoparticles (rMSP1‐IO). Immunizations in outbred mice with rMSP1‐IO achieved 100% responsiveness with antibody titers comparable to those obtained with rMSP1 formulated with a clinically acceptable adjuvant, Montanide ISA51 (2.7×10–3 vs. 1.6×10–3; respectively). Only rMSP1‐1O could induce significant levels (80%) of parasite inhibitory antibodies. The rMSP1‐IO was highly stable at 4°C and was amenable to lyophilization, maintaining its antigenicity, immunogenicity, and ability to induce inhibitory antibodies. Further testing in nonhuman primates, Aotus monkeys, also elicited 100% immune responsiveness and high levels of parasite inhibitory antibodies (55–100% inhibition). No apparent local or systemic toxicity was associated with IO immunizations. Murine macrophages and dendritic cells efficiently (>90%) internalized IO nanoparticles, but only the latter were significantly activated, with elevated expression/secretion of CD86, cytokines (IL‐6, TNF‐α, IL1‐b, IFN‐γ, and IL‐12), and chemokines (CXCL1, CXCL2, CCL2, CCL3, CCL4, and CXCL10). Thus, the IO nanoparticles is a novel, safe, and effective vaccine platform, with built‐in adjuvancy, that is highly stable and field deployable for cost‐effective vaccine delivery.—Pusic, K., Aguilar, Z., McLoughlin, J., Kobuch, S., Xu, H., Tsang, M., Wang, A., Hui, G. Iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood‐stage human malaria vaccine. FASEB J. 27, 1153–1166 (2013). www.fasebj.org

Blood stage merozoite surface protein conjugated to nanoparticles induce potent parasite inhibitory antibodies.

In this proof-of-concept study we report the use of <15 nm, water soluble, inorganic nanoparticles as a vaccine delivery system for a blood stage malaria vaccine. The recombinant malarial antigen, Merozoite Surface Protein 1 (rMSP1) of Plasmodium falciparum served as the model vaccine. The rMSP1 was covalently conjugated to polymer-coated quantum dot CdSe/ZnS nanoparticles (QDs) via surface carboxyl groups, forming rMSP1-QDs. Anti-MSP1 antibody responses induced by rMSP1-QDs were found to have 2-3 log higher titers than those obtained with rMSP1 administered with the conventional adjuvants, Montanide ISA51 and CFA. Moreover, the immune responsiveness and the induction of parasite inhibitory antibodies were significantly superior in mice injected with rMSP1-QDs. The rMSP1-QDs delivered via intra-peritoneal (i.p.), intra-muscular (i.m.), and subcutaneous (s.c.) routes were equally efficacious. The high level of immunogenicity exhibited by the rMSP1-QDs was achieved without further addition of other adjuvant components. Bone marrow derived dendritic cells were shown to efficiently take up the nanoparticles leading to their activation and the expression/secretion of key cytokines, suggesting that this may be a mode of action for the enhanced immunogenicity. This study provides promising results for the use of water soluble, inorganic nanoparticles (<15 nm) as potent vehicles/platforms to enhance the immunogenicity of polypeptide antigens in adjuvant-free immunizations.

What are exosomes and how can they be used in multiple sclerosis therapy

Current treatment options for multiple sclerosis are limited and consist of immunosuppressors or agents to prevent immune infiltration of the brain. These therapies have potentially harmful side effects and do little to promote myelin repair. Instead, we suggest using exosomes, naturally occurring small vesicles that exert influence through the delivery of mRNA, microRNA and protein. Dendritic cells can be cultured from bone marrow and stimulated to release exosomes. When administered to the brain, these exosomes significantly increase myelination and improve remyelination following injury by prompting preoligodendrocytes to differentiate into myelin producing cells. Additionally, they are non-toxic and can easily cross the blood-brain barrier and, thus, have great potential as a therapeutic.

Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium

The NIH Extracellular RNA Communication Programs initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.

Antibody and T cell responses in reciprocal prime-boost studies with full-length and truncated merozoite surface protein 1-42 vaccines.

The P. falciparum Merozoite Surface Protein 1–42 (MSP1-42) is one of the most studied malaria subunit vaccine candidates. The N-terminal fragment of MSP1-42, MSP1-33, is primarily composed of allelic sequences, and has been shown to possess T helper epitopes that influence protective antibody responses toward the C-terminal region, MSP1-19. A truncated MSP1-42 vaccine, Construct 33-I, consisting of exclusively conserved T epitope regions of MSP1-33 expressed in tandem with MSP1-19, was previously shown to be a more effective immunogen than the full-length MSP1-42 vaccine. Here, by way of reciprocal priming/boosting immunization regimens, we studied the immunogenicity of Construct 33-I in the context of recognition by immune responses induced by the full-length native MSP1-42 protein, in order to gauge the effects of pre- and post-exposures to MSP1-42 on vaccine induced responses. Judging by immune responsiveness, antibody and T cell responses, Construct 33-I was effective as the priming antigen followed by full-length MSP1-42 boosting, as well as the boosting antigen following full-length MSP1-42 priming. In particular, Construct 33-I priming elicited the broadest responsiveness in immunized animals subsequently exposed to MSP1-42. Moreover, Construct 33-I, with its conserved MSP1-33 specific T cell epitopes, was equally well recognized by homologous and heterologous allelic forms of MSP1-42. Serum antibodies raised against Construct 33-I efficiently inhibited the growth of parasites carrying the heterologous MSP1-42 allele. These results suggest that Construct 33-I maintains and/or enhances its immunogenicity in an allelic or strain transcending fashion when deployed in populations having prior or subsequent exposures to native MSP1-42s.