Conjugation of Transforming Growth Factor Beta to Antigen-Loaded Poly(lactide- co-glycolide) Nanoparticles Enhances Efficiency of Antigen-Specific Tolerance.

Current strategies for treating autoimmunity involve the administration of broad-acting immunosuppressive agents that impair healthy immunity. Intravenous (i.v.) administration of poly(lactide- co-glycolide) nanoparticles (NPs) containing disease-relevant antigens (Ag-NPs) have demonstrated antigen (Ag)-specific immune tolerance in models of autoimmunity. However, subcutaneous (s.c.) delivery of Ag-NPs has not been effective. This investigation tested the hypothesis that codelivery of the immunomodulatory cytokine, transforming growth factor beta 1 (TGF-ß), on Ag-NPs would modulate the immune response to Ag-NPs and improve the efficiency of tolerance induction. TGF-ß was coupled to the surface of Ag-NPs such that the loadings of Ag and TGF-ß were independently tunable. The particles demonstrated bioactive delivery of Ag and TGF-ß in vitro by reducing the inflammatory phenotype of bone marrow-derived dendritic cells and inducing regulatory T cells in a coculture system. Using an in vivo mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, TGF-ß codelivery on Ag-NPs resulted in improved efficacy at lower doses by i.v. administration and significantly reduced disease severity by s.c. administration. This study demonstrates that the codelivery of immunomodulatory cytokines on Ag-NPs may enhance the efficacy of Ag-specific tolerance therapies by programming Ag presenting cells for more efficient tolerance induction.

Evaluation of biomaterial scaffold delivery of IL-33 as a localized immunomodulatory agent to support cell transplantation in adipose tissue.

INTRODUCTION: The development of novel immunomodulatory strategies that might decrease the need for systemic immune suppression would greatly enable the utility of cell-based therapies. Cell transplantation on biomaterial scaffolds offers a unique opportunity to engineer a site to locally polarize immunogenic antigen generation. Herein, we investigated the localized delivery of IL-33, which is a novel cytokine that has been shown to have beneficial immunomodulatory effects in certain transplant models as mediating anti-inflammatory properties in the adipose tissue, to determine its feasibility for use as an immunomodulatory agent. RESULTS: Localized IL-33 delivery from poly(lactide-co-glycolide) (PLG) scaffolds implanted into the epididymal fat specifically increased the Foxp3+ population of CD4+ T cells in both blank scaffold implants and scaffolds seeded with allogeneic islets. In allogeneic islet transplantation, we found IL-33 delivery results in a local upregulation of graft-protective T cells where 80% of the local CD4+ population is Foxp3+ and overall numbers of graft destructive CD8+ T cells are decreased, resulting in a prolonged graft survival. Interestingly, local IL-33 also delayed islet engraftment by primarily inducing a local upregulation of Th2 cytokines, including IL-4 and IL-5, leading to increased populations of ST2+ Type 2 innate lymphoid cells (ILC2s) and Siglec F+ eosinophils. CONCLUSIONS: These results suggest that local IL-33 delivery from biomaterial scaffolds can be used to increase Tregs enriched in adipose tissue and reduce graft-destructive T cell populations but may also promote innate cell populations that can delay cell engraftment.

TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae.

Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.

Transforming growth factor-beta 1 delivery from microporous scaffolds decreases inflammation post-implant and enhances function of transplanted islets.

Biomaterial scaffolds are central to many regenerative strategies as they create a space for infiltration of host tissue and provide a platform to deliver growth factors and progenitor cells. However, biomaterial implantation results in an unavoidable inflammatory response, which can impair tissue regeneration and promote loss or dysfunction of transplanted cells. We investigated localized TGF-ß1 delivery to modulate this immunological environment around scaffolds and transplanted cells. TGF-ß1 was delivered from layered scaffolds, with protein entrapped within an inner layer and outer layers designed for cell seeding and host tissue integration. Scaffolds were implanted into the epididymal fat pad, a site frequently used for cell transplantation. Expression of cytokines TNF-α, IL-12, and MCP-1 were decreased by at least 40% for scaffolds releasing TGF-ß1 relative to control scaffolds. This decrease in inflammatory cytokine production corresponded to a 60% decrease in leukocyte infiltration. Transplantation of islets into diabetic mice on TGF-ß1 scaffolds significantly improved the ability of syngeneic islets to control blood glucose levels within the first week of transplant and delayed rejection of allogeneic islets. Together, these studies emphasize the ability of localized TGF-ß1 delivery to modulate the immune response to biomaterial implants and enhance cell function in cell-based therapies.