Antigen specificity is a primary goal in developing curative therapies for autoimmune disease. Dendritic cells (DCs), as the most effective antigen presenting cells in the body, represent a key target to mediate restoration of antigen-specific immune regulation. Here, we describe an injectable, dual-sized microparticle (MP) approach that employs phagocytosable ∼1 μm and nonphagocytosable ∼30 μm MPs to deliver tolerance-promoting factors both intracellularly and extracellularly, as well as the type 1 diabetes autoantigen, insulin, to DCs for reprogramming of immune responses and remediation of autoimmunity. This poly(lactic-co-glycolic acid) (PLGA) MP system prevented diabetes onset in 60% of nonobese diabetic (NOD) mice when administered subcutaneously in 8 week old mice. Prevention of disease was dependent upon antigen inclusion and required encapsulation of factors in MPs. Moreover, administration of this “suppressive-vaccine” boosted pancreatic lymph node and splenic regulatory T cells (Tregs), upregulated PD-1 on CD4+ and CD8+ T cells, and reversed hyperglycemia for up to 100 days in recent-onset NOD mice. Our results demonstrate that a MP-based platform can reeducate the immune system in an antigen-specific manner, augment immunomodulation compared to soluble administration of drugs, and provide a promising alternative to systemic immunosuppression for autoimmunity.
Targeted delivery of self-antigens to the immune system in a mode that stimulates a tolerance-inducing pathway has proven difficult. To address this hurdle, we developed a vaccine based-approach comprised of two synthetic controlled-release biomaterials, poly(lactide-co-glycolide; PLGA) microparticles (MPs) encapsulating denatured insulin (key self-antigen in type 1 diabetes; T1D), and PuraMatrixTM peptide hydrogel containing granulocyte macrophage colony-stimulating factor (GM-CSF) and CpG ODN1826 (CpG), which were included as vaccine adjuvants to recruit and activate immune cells. Although CpG is normally considered pro-inflammatory, it also has anti-inflammatory effects, including enhancing IL-10 production. Three subcutaneous administrations of this hydrogel (GM-CSF/CpG)/insulin-MP vaccine protected 40% of NOD mice from T1D. In contrast, all control mice became diabetic. In vitro studies indicate CpG stimulation increased IL-10 production, as a potential mechanism. Multiple subcutaneous injections of the insulin containing formulation resulted in formation of granulomas, which resolved by 28 weeks. Histological analysis of these granulomas indicated infiltration of a diverse cadre of immune cells, with characteristics reminiscent of a tertiary lymphoid organ, suggesting the creation of a microenvironment to recruit and educate immune cells. These results demonstrate the feasibility of this injectable hydrogel/MP based vaccine system to prevent T1D.
Certain commercial carbohydrate replacement products include slowly absorbed carbohydrates such as isomaltulose. Few studies have investigated the metabolic effects of ingesting isomaltulose during exercise and none have evaluated exercise performance and gastrointestinal comfort. Nine male cyclists participated postprandially during three trials of 2-h steady-state (S-S) exercise (60%Wmax) followed by a 16 km time trial (TT) while ingesting 63 g·h-1 of either, 0.8:1 fructose: maltodextrin (F:M) or isomaltulose (ISO) or placebo- flavored water (PL). Data were analyzed by magnitude-based inferences. During S-S exercise, ISO and PL similarly increased plasma nonesterified fatty acid (NEFA) concentration (mean change ISO versus F:M: 0.18, 90%CI ±0.21 mmol·L-1, 88% likelihood) and fat oxidation (10, 90%CI ±9 g, 89% likelihood) while decreasing carbohydrate oxidation (-36, 90%CI ±30.2 g, 91% likelihood) compared with F:M, despite equal elevations in blood glucose concentration with ISO and F:M. Rating of stomach cramps and bloating increased progressively with ISO (rating: 0-90 min S-S, weak; 120 min S-S, moderate; TT, strong) compared with F:M and PL (0-120 min S-S and TT, very weak). TT performance was substantially slower with ISO (mean change: 1.5, 90%CI ±1.4 min, 94% likely harmful) compared with F:M. The metabolic response of ISO ingestion during moderate exercise to increase NEFA availability and fat oxidation despite elevating blood glucose concentration is anomalous for a carbohydrate supplement. However, ingesting isomaltulose at a continuous high frequency to meet the recommended carbohydrate replacement dose, results in severe gastrointestinal symptoms during prolonged or high intensity exercise and negatively affects exercise performance compared with fructose-maltodextrin supplementation.
The protein type most suitable for ingestion during endurance exercise is undefined. This study compared co-ingestion of either 15 g/h whey or casein hydrolysate with 63 g/h fructose: maltodextrin (0.8:1) on exogenous carbohydrate oxidation, exercise metabolism and performance. 2 h postprandial, 8 male cyclists ingested either: carbohydrate-only, carbohydrate-whey hydrolysate, carbohydrate-casein hydrolysate or placebo-water in a crossover, double-blind design during 2 h of exercise at 60%W max followed by a 16-km time trial. Data were evaluated by magnitude-based inferential statistics. Exogenous carbohydrate oxidation, measured from (13)CO2 breath enrichment, was not substantially influenced by co-ingestion of either protein hydrolysate. However, only co-ingestion of carbohydrate-casein hydrolysate substantially decreased (98% very likely decrease) total carbohydrate oxidation (mean±SD, 242±44; 258±47; 277±33 g for carbohydrate-casein, carbohydrate-whey and carbohydrate-only, respectively) and substantially increased (93% likely increase) total fat oxidation (92±14; 83±27; 73±19 g) compared with carbohydrate-only. Furthermore, only carbohydrate-casein hydrolysate ingestion resulted in a faster time trial (-3.6%; 90% CI: ±3.2%) compared with placebo-water (95% likely benefit). However, neither protein hydrolysate enhanced time trial performance when compared with carbohydrate-only. Under the conditions of this study, ingesting carbohydrate-casein, but not carbohydrate-whey hydrolysate, favourably alters metabolism during prolonged moderate-strenuous cycling without substantially altering cycling performance compared with carbohydrate-only.
Experimental vaccine adjuvants are being designed to target specific toll-like receptors (TLRs) alone or in combination, expressed by antigen presenting cells, notably dendritic cells (DCs). There is a need for high-content screening (HCS) platforms to explore how DC activation is affected by adjuvant combinations. Presented is a cell-based microarray approach, “immunoarray”, exposing DCs to a large number of adjuvant combinations. Microparticles encapsulating TLR ligands are printed onto arrays in a range of doses for each ligand, in all possible dose combinations. Dendritic cells are then co-localized with physisorbed microparticles on the immunoarray, adherent to isolated islands surrounded by a non-fouling background, and DC activation is quantified. Delivery of individual TLR ligands was capable of eliciting high levels of specific DC activation markers. For example, either TLR9 ligand, CpG, or TLR3 ligand, poly I:C, was capable of inducing among the highest 10% expression levels of CD86. In contrast, MHC-II expression in response to TLR4 agonist MPLA was among the highest, whereas either MPLA or poly I:C, was capable of producing among the highest levels of CCR7 expression, as well as inflammatory cytokine IL-12. However, in order to produce robust responses across all activation markers, adjuvant combinations were required, and combinations were more represented among the high responders. The immunoarray also enables investigation of interactions between adjuvants, and each TLR ligand suggested antagonism to other ligands, for various markers. Altogether, this work demonstrates feasibility of the immunoarray platform to screen microparticle-encapsulated adjuvant combinations for the development of improved and personalized vaccines.
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