Neutrophil and natural killer cell imbalances prevent muscle stem cell-mediated regeneration following murine volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell­ and progenitor-mediated myogenic repair. However, how immune cell infiltration and intercellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in the fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observed the heightened infiltration of natural killer (NK) cells as well as the persistence of neutrophils beyond 2 wk postinjury. Functional validation of NK cells revealed an antagonistic role in neutrophil accumulation in part via inducing apoptosis and CCR1-mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFß1). Blocking TGFß signaling reduced neutrophil accumulation and fibrosis and improved muscle-specific force. Collectively, these results enhance our understanding of immune cell­stem cell cross talk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

Engineering nanoparticle therapeutics for food allergy.

Food allergy is a growing public health issue among children and adults that can lead to life-threatening anaphylaxis following allergen exposure. The criterion standard for disease management includes food avoidance and emergency epinephrine administration because current allergen-specific immunotherapy treatments are limited by adverse events and unsustained desensitization. A promising approach to remedy these shortcomings is the use of nanoparticle-based therapies that disrupt disease-driving immune mechanisms and induce more sustained tolerogenic immune pathways. The pathophysiology of food allergy includes multifaceted interactions between effector immune cells, including lymphocytes, antigen-presenting cells, mast cells, and basophils, mainly characterized by a TH2 cell response. Regulatory T cells, TH1 cell responses, and suppression of other major allergic effector cells have been found to be major drivers of beneficial outcomes in these nanoparticle therapies. Engineered nanoparticle formulations that have shown efficacy at reducing allergic responses and revealed new mechanisms of tolerance include polymeric-, lipid-, and emulsion-based nanotherapeutics. This review highlights the recent engineering design of these nanoparticles, the mechanisms induced by them, and their future potential therapeutic targets.

Polymer scaffolds delineate healthy from diseased states at sites distal from the pancreas in two models of type 1 diabetes.

Type 1 diabetes (T1D) prevention is currently limited by the lack of diagnostic tools able to identify disease before autoimmune destruction of the pancreatic ß cells. Autoantibody tests are used to predict risk and, in combination with glucose dysregulation indicative of ß cell loss, to determine administration of immunotherapies. Our objective was to remotely identify immune changes associated with the disease, and we have employed a subcutaneously implanted microporous poly(e-caprolactone) (PCL) scaffold to function as an immunological niche (IN) in two models of T1D. Biopsy and analysis of the IN enables disease monitoring using transcriptomic changes at a distal site from autoimmune destruction of the pancreas, thereby gaining cellular level information about disease without the need for a biopsy of the native organ. Using this approach, we identified gene signatures that stratify healthy and diseased mice in both an adoptive transfer model and a spontaneous onset model of T1D. The gene signatures identified herein demonstrate the ability of the IN to identify immune activation associated with diabetes across models.

Membrane-coated nanoparticles for direct recognition by T cells.

The direct modulation of T cell responses is an emerging therapeutic strategy with the potential to modulate undesired immune responses including, autoimmune disease, and allogeneic cells transplantation. We have previously demonstrated that poly(lactide-co-glycolide) particles were able to modulate T cell responses indirectly through antigen-presenting cells (APCs). In this report, we investigated the design of nanoparticles that can directly interact and modulate T cells by coating the membranes from APCs onto nanoparticles to form membrane-coated nanoparticles (MCNPs). Proteins within the membranes of the APCs, such as Major Histocompatibility Complex class II and co-stimulatory factors, were effectively transferred to the MCNP. Using alloreactive T cell models, MCNP derived from allogeneic dendritic cells were able to stimulate proliferation, which was not observed with membranes from syngeneic dendritic cells and influenced cytokine secretion. Furthermore, we investigated the engineering of the membranes either on the dendritic cells or postfabrication of MCNP. Engineered membranes could be to promote antigen-specific responses, to differentially activate T cells, or to directly induce apoptosis. Collectively, MCNPs represent a tunable platform that can directly interact with and modulate T cell responses.

Nanoparticle dose and antigen loading attenuate antigen-specific T-cell responses.

Immune-mediated hypersensitivities such as autoimmunity, allergy, and allogeneic graft rejection are treated with therapeutics that suppress the immune system, and the lack of specificity is associated with significant side effects. The delivery of disease-relevant antigens (Ags) by carrier systems such as poly(lactide-co-glycolide) nanoparticles (PLG-Ag) and carbodiimide (ECDI)-fixed splenocytes (SP-Ag) has demonstrated Ag-specific tolerance induction in model systems of these diseases. Despite therapeutic outcomes by both platforms, tolerance is conferred with different efficacy. This investigation evaluated Ag loading and total particle dose of PLG-Ag on Ag presentation in a coculture system of dendritic cells (DCs) and Ag-restricted T cells, with SP-Ag employed as a control. CD25 expression was observed in nearly all T cells even at low concentrations of PLG-Ag, indicating efficient presentation of Ag by dendritic cells. However, the secretion of IL-2, Th1, and Th2 cytokines (IFNγ and IL-4, respectively) varied depending on PLG-Ag concentration and Ag loading. Concentration escalation of soluble Ag resulted in an increase in IL-2 and IFNγ and a decrease in IL-4. Treatment with PLG-Ag followed a similar trend but with lower levels of IL-2 and IFNγ secreted. Transcriptional Activity CEll ARrays (TRACER) were employed to measure the real-time transcription factor (TF) activity in Ag-presenting DCs. The kinetics and magnitude of TF activity was dependent on the Ag delivery method, concentration, and Ag loading. Ag positively regulated IRF1 activity and, as carriers, NPs and ECDI-treated SP negatively regulated this signaling. The effect of Ag loading and dose on tolerance induction were corroborated in vivo using the delayed-type hypersensitivity (DTH) and experimental autoimmune encephalomyelitis (EAE) mouse models where a threshold of 8 µg/mg Ag loading and 0.5 mg PLG-Ag dose were required for tolerance. Together, the effect of Ag loading and dosing on in vitro and in vivo immune regulation provide useful insights for translating Ag-carrier systems for the clinical treatment of immune disorders.

Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

Intravascular innate immune cells reprogrammed via intravenous nanoparticles to promote functional recovery after spinal cord injury.

Traumatic primary spinal cord injury (SCI) results in paralysis below the level of injury and is associated with infiltration of hematogenous innate immune cells into the injured cord. Methylprednisolone has been applied to reduce inflammation following SCI, yet was discontinued due to an unfavorable risk-benefit ratio associated with off-target effects. In this study, i.v. administered poly(lactide-coglycolide) nanoparticles were internalized by circulating monocytes and neutrophils, reprogramming these cells based on their physicochemical properties and not by an active pharmaceutical ingredient, to exhibit altered biodistribution, gene expression, and function. Approximately 80% of nanoparticle-positive immune cells were observed within the injury, and, additionally, the overall accumulation of innate immune cells at the injury was reduced 4-fold, coinciding with down-regulated expression of proinflammatory factors and increased expression of antiinflammatory and proregenerative genes. Furthermore, nanoparticle administration induced macrophage polarization toward proregenerative phenotypes at the injury and markedly reduced both fibrotic and gliotic scarring 3-fold. Moreover, nanoparticle administration with the implanted multichannel bridge led to increased numbers of regenerating axons, increased myelination with about 40% of axons myelinated, and an enhanced locomotor function (score of 6 versus 3 for control group). These data demonstrate that nanoparticles provide a platform that limits acute inflammation and tissue destruction, at a favorable risk-benefit ratio, leading to a proregenerative microenvironment that supports regeneration and functional recovery. These particles may have applications to trauma and potentially other inflammatory diseases.

Gliadin Nanoparticles Induce Immune Tolerance to Gliadin in Mouse Models of Celiac Disease.

BACKGROUND & AIMS: Celiac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease. METHODS: We performed studies with C57BL/6; RAG1-/- (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG-/- mice were given intraperitoneal injections of CD4+CD62L-CD44hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A-based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed. RESULTS: TIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction. CONCLUSIONS: In mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease.

IL-10 lentivirus-laden hydrogel tubes increase spinal progenitor survival and neuronal differentiation after spinal cord injury.

A complex cellular cascade characterizes the pathophysiological response following spinal cord injury (SCI) limiting regeneration. Biomaterial and stem cell combination therapies together have shown synergistic effects, compared to the independent benefits of each intervention, and represent a promising approach towards regaining function after injury. In this study, we combine our polyethylene glycol (PEG) cell delivery platform with lentiviral-mediated overexpression of the anti-inflammatory cytokine interleukin (IL)-10 to improve mouse embryonic Day 14 (E14) spinal progenitor transplant survival. Immediately following injury in a mouse SCI hemisection model, five PEG tubes were implanted followed by direct injection into the tubes of lentivirus encoding for IL-10. Two weeks after tube implantation, mouse E14 spinal progenitors were injected directly into the integrated tubes, which served as a soft substrate for cell transplantation. Together, the tubes with the IL-10 encoding lentivirus improved E14 spinal progenitor survival, assessed at 2 weeks posttransplantation (4 weeks postinjury). On average, 8.1% of E14 spinal progenitors survived in mice receiving IL-10 lentivirus-laden tubes compared with 0.7% in mice receiving transplants without tubes, an 11.5-fold difference. Surviving E14 spinal progenitors gave rise to neurons when injected into tubes. Axon elongation and remyelination were observed, in addition to a significant increase in functional recovery in mice receiving IL-10 lentivirus-laden tubes with E14 spinal progenitor delivery compared to the injury only control by 4 weeks postinjury. All other conditions did not exhibit increased stepping until 8 or 12 weeks postinjury. This system affords increased control over the transplantation microenvironment, offering the potential to improve stem cell-mediated tissue regeneration.

Porous Silicon Nanoparticles Embedded in Poly(lactic-co-glycolic acid) Nanofiber Scaffolds Deliver Neurotrophic Payloads to Enhance Neuronal Growth.

Scaffolds made from biocompatible polymers provide physical cues to direct the extension of neurites and to encourage repair of damaged nerves. The inclusion of neurotrophic payloads in these scaffolds can substantially enhance regrowth and repair processes. However, many promising neurotrophic candidates are excluded from this approach due to incompatibilities with the polymer or with the polymer processing conditions. This work provides one solution to this problem by incorporating porous silicon nanoparticles (pSiNPs) that are pre-loaded with the therapeutic into a polymer scaffold during fabrication. The nanoparticle-drug-polymer hybrids are prepared in the form of oriented poly(lactic-co-glycolic acid) nanofiber scaffolds. We test three different therapeutic payloads: bpV(HOpic), a small molecule inhibitor of phosphatase and tensin homolog (PTEN); an RNA aptamer specific to tropomyosin-related kinase receptor type B (TrkB); and the protein nerve growth factor (NGF). Each therapeutic is loaded using a loading chemistry that is optimized to slow the rate of release of these water-soluble payloads. The drug-loaded pSiNP-nanofiber hybrids release approximately half of their TrkB aptamer, bpV(HOpic), or NGF payload in 2, 10, and >40 days, respectively. The nanofiber hybrids increase neurite extension relative to drug-free control nanofibers in a dorsal root ganglion explant assay.

Microporous scaffolds loaded with immunomodulatory lentivirus to study the contribution of immune cell populations to tumor cell recruitment in vivo.

Metastases are preceded by stochastic formation of a hospitable microenvironment known as the premetastatic niche, which has been difficult to study. Herein, we employ implantable polycaprolactone scaffolds as an engineered premetastatic niche to independently investigate the role of interleukin-10 (IL10), CXCL12, and CCL2 in recruiting immune and tumor cells and impacting breast cancer cell phenotype via lentiviral overexpression. Lentivirus delivered from scaffolds in vivo achieved sustained transgene expression for 56 days. IL10 lentiviral expression, but not CXCL12 or CCL2, significantly decreased tumor cell recruitment to scaffolds in vivo. Delivery of CXCL12 enhanced CD45+ immune cell recruitment to scaffolds while delivery of IL10 reduced immune cell recruitment. CCL2 did not alter immune cell recruitment. Tumor cell phenotype was investigated using conditioned media from immunomodulated scaffolds, with CXCL12 microenvironments reducing proliferation, and IL10 microenvironments enhancing proliferation. Migration was enhanced with CCL2 and reduced with IL10-driven microenvironments. Multiple linear regression identified populations of immune cells associated with tumor cell abundance. CD45+ immune and CD8+ T cells were associated with reduced tumor cell abundance, while CD11b+Gr1+ neutrophils and CD4+ T cells were associated with enhanced tumor cell abundance. Collectively, biomaterial scaffolds provide a tool to probe the formation and function of the premetastatic niche.

Regulation of adipose tissue inflammation and systemic metabolism in murine obesity by polymer implants loaded with lentiviral vectors encoding human interleukin-4.

Dysfunctional adipose tissue plays a central role in the pathogenesis of the obesity-related metabolic disease, including type 2 diabetes. Targeting adipose tissue using biopolymer implants is a novel therapeutic approach for metabolic disease. We transplanted porous poly(lactide-co-glycolide) (PLG) implants coated with human interleukin-4 (hIL-4)-expressing lentivirus into epididymal white adipose tissue (eWAT) of mice fed a high-fat diet. Tissue and systemic inflammation and metabolism were studied with flow cytometry, immunohistochemistry, quantitative real-time polymerase chain reaction, adipose tissue histology, and in vivo glucose tolerance testing at 2 and 10 weeks of a high-fat diet. PLG implants carrying hIL-4-expressing lentivirus implanted into epididymal white adipose tissue of mice-regulated adipose tissue inflammation, including increased CD3+ CD4+ T-cell frequency, increased eWAT adipocyte hypertrophy, and decreased FASN and ATGL expression, along with reduced fasting blood glucose levels. These effects were observed in early obesity but were not maintained in established obesity. Local delivery of bioimplants loaded with cytokine-expressing lentivirus vectors to adipose tissue influences tissue inflammation and systemic metabolism in early obesity. Further study will be required to show more durable metabolic effects. These data demonstrate that polymer biomaterials implanted into adipose tissue have the potential to modulate local tissue and systemic inflammation and metabolism.

Dynamic genome-scale cell-specific metabolic models reveal novel inter-cellular and intra-cellular metabolic communications during ovarian follicle development.

BACKGROUND: The maturation of the female germ cell, the oocyte, requires the synthesis and storing of all the necessary metabolites to support multiple divisions after fertilization. Oocyte maturation is only possible in the presence of surrounding, diverse, and changing layers of somatic cells. Our understanding of metabolic interactions between the oocyte and somatic cells has been limited due to dynamic nature of ovarian follicle development, thus warranting a systems approach. RESULTS: Here, we developed a genome-scale metabolic model of the mouse ovarian follicle. This model was constructed using an updated mouse general metabolic model (Mouse Recon 2) and contains several key ovarian follicle development metabolic pathways. We used this model to characterize the changes in the metabolism of each follicular cell type (i.e., oocyte, granulosa cells, including cumulus and mural cells), during ovarian follicle development in vivo. Using this model, we predicted major metabolic pathways that are differentially active across multiple follicle stages. We identified a set of possible secreted and consumed metabolites that could potentially serve as biomarkers for monitoring follicle development, as well as metabolites for addition to in vitro culture media that support the growth and maturation of primordial follicles. CONCLUSIONS: Our systems approach to model follicle metabolism can guide future experimental studies to validate the model results and improve oocyte maturation approaches and support growth of primordial follicles in vitro.

Combinatorial lentiviral gene delivery of pro-oligodendrogenic factors for improving myelination of regenerating axons after spinal cord injury.

Spinal cord injury (SCI) results in paralysis below the injury and strategies are being developed that support axonal regrowth, yet recovery lags, in part, because many axons are not remyelinated. Herein, we investigated strategies to increase myelination of regenerating axons by overexpression of platelet-derived growth factor (PDGF)-AA and noggin either alone or in combination in a mouse SCI model. Noggin and PDGF-AA have been identified as factors that enhance recruitment and differentiation of endogenous progenitors to promote myelination. Lentivirus encoding for these factors was delivered from a multichannel bridge, which we have previously shown creates a permissive environment and supports robust axonal growth through channels. The combination of noggin+PDGF enhanced total myelination of regenerating axons relative to either factor alone, and importantly, enhanced functional recovery relative to the control condition. The increase in myelination was consistent with an increase in oligodendrocyte-derived myelin, which was also associated with a greater density of cells of an oligodendroglial lineage relative to each factor individually and control conditions. These results suggest enhanced myelination of regenerating axons by noggin+PDGF that act on oligodendrocyte-lineage cells post-SCI, which ultimately led to improved functional outcomes.

It's All in the Delivery: Designing Hydrogels for Cell and Non-viral Gene Therapies.

Hydrogels provide a regenerative medicine platform with their ability to create an environment that supports transplanted or endogenous infiltrating cells and enables these cells to restore or replace the function of tissues lost to disease or trauma. Furthermore, these systems have been employed as delivery vehicles for therapeutic genes, which can direct and/or enhance the function of the transplanted or endogenous cells. Herein, we review recent advances in the development of hydrogels for cell and non-viral gene delivery through understanding the design parameters, including both physical and biological components, on promoting transgene expression, cell engraftment, and ultimately cell function. Furthermore, this review identifies emerging opportunities for combining cell and gene delivery approaches to overcome challenges to the field.

Local Immunomodulation with Anti-inflammatory Cytokine-Encoding Lentivirus Enhances Functional Recovery after Spinal Cord Injury.

Trauma to the spinal cord and associated secondary inflammation can lead to permanent loss of sensory and motor function below the injury level, with the resulting environment serving as a barrier that limits regeneration. In this study, we investigate the localized expression of anti-inflammatory cytokines IL-10 and IL-4 via lentiviral transduction in multichannel bridges. Porous multichannel bridges provide physical guidance for axonal outgrowth with the cytokines hypothesized to modulate the neuroinflammatory microenvironment and enhance axonal regeneration. Gene expression analyses indicated that induced IL-10 and IL-4 expression decreased expression of pro-inflammatory genes and increased pro-regenerative genes relative to control. Moreover, these factors led to increased numbers of axons and myelination, with approximately 45% of axons myelinated and the number of oligodendrocyte myelinated axons significantly increased by 3- to 4-fold. Furthermore, the combination of a bridge with IL-10 and IL-4 expression improved locomotor function after injury to an average score of 6 relative to an average score of 3 for injury alone. Collectively, these studies highlight the potential for localized immunomodulation to decrease secondary inflammation and enhance regeneration that may have numerous applications.

Overcoming challenges in treating autoimmuntity: Development of tolerogenic immune-modifying nanoparticles.

Autoimmune diseases, such as celiac disease, multiple sclerosis, and type 1 diabetes, are leading causes of morbidity and mortality in the United States. In these disease states, immune regulatory mechanisms fail that result in T and B cell-mediated destruction of self-tissues. The known role of T cells in mediating autoimmune diseases has led to the emergence of numerous therapies aimed at inactivating T cells, however successful 'tolerance-inducing' strategies have not yet emerged for approved standard-of-care clinical use. In this review, we describe relevant examples of antigen-specific tolerance approaches that have been applied in clinical trials for human diseases. Furthermore, we describe the evolution of biomaterial approaches from cell-based therapies to induce immune tolerance with a focus on the Tolerogenic Immune-Modifying nanoParticle (TIMP) platform. The TIMP platform can be designed to treat various autoimmune conditions and is currently in clinical trials testing its ability to reverse celiac disease.

Biodegradable antigen-associated PLG nanoparticles tolerize Th2-mediated allergic airway inflammation pre- and postsensitization.

Specific immunotherapy (SIT) is the most widely used treatment for allergic diseases that directly targets the T helper 2 (Th2) bias underlying allergy. However, the most widespread clinical applications of SIT require a long period of dose escalation with soluble antigen (Ag) and carry a significant risk of adverse reactions, particularly in highly sensitized patients who stand to benefit most from a curative treatment. Thus, the development of safer, more efficient methods to induce Ag-specific immune tolerance is critical to advancing allergy treatment. We hypothesized that antigen-associated nanoparticles (Ag-NPs), which we have used to prevent and treat Th1/Th17-mediated autoimmune disease, would also be effective for the induction of tolerance in a murine model of Th2-mediated ovalbumin/alum-induced allergic airway inflammation. We demonstrate here that antigen-conjugated polystyrene (Ag-PS) NPs, although effective for the prophylactic induction of tolerance, induce anaphylaxis in presensitized mice. Antigen-conjugated NPs made of biodegradable poly(lactide-co-glycolide) (Ag-PLG) are similarly effective prophylactically, are well tolerated by sensitized animals, but only partially inhibit Th2 responses when administered therapeutically. PLG NPs containing encapsulated antigen [PLG(Ag)], however, were well tolerated and effectively inhibited Th2 responses and airway inflammation both prophylactically and therapeutically. Thus, we illustrate progression toward PLG(Ag) as a biodegradable Ag carrier platform for the safe and effective inhibition of allergic airway inflammation without the need for nonspecific immunosuppression in animals with established Th2 sensitization.

Tolerogenic Ag-PLG nanoparticles induce tregs to suppress activated diabetogenic CD4 and CD8 T cells.

Type 1 diabetes (T1D) is mediated by destruction of pancreatic ß cells by autoantigen-specific CD4+ and CD8+ T cells, thus the ideal solution for T1D is the restoration of immune tolerance to ß cell antigens. We demonstrate the ability of carboxylated 500 nm biodegradable poly(lactide-co-glycolide) (PLG) nanoparticles PLG nanoparticles (either surface coupled with or encapsulating the cognate diabetogenic peptides) to rapidly and efficiently restore tolerance in NOD.SCID recipients of both activated diabetogenic CD4+ BDC2.5 chromagranin A-specific and CD8+ NY8.3 islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific TCR transgenic T cells in an antigen-specific manner. Further, initiation and maintenance of Ag-PLG tolerance operates via several overlapping, but independent, pathways including regulation via negative-co-stimulatory molecules (CTLA-4 and PD-1) and the systemic induction of peptide-specific Tregs which were critical for long-term maintenance of tolerance by controlling both trafficking of effector T cells to, and their release of pro-inflammatory cytokines within the pancreas, concomitant with selective retention of effector cells in the spleens of recipient mice.

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.