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.

Mechanistic contributions of Kupffer cells and liver sinusoidal endothelial cells in nanoparticle-induced antigen-specific immune tolerance.

The intravenous delivery of disease-relevant antigens (Ag) by polymeric nanoparticles (NP-Ags) has demonstrated Ag-specific immune tolerance in autoimmune and allergic disorders as well as allogeneic transplant rejection. NP-Ags are observed to distribute to the spleen, which has an established role in the induction of immune tolerance. However, studies have shown that the spleen is dispensable for NP-Ag-induced tolerance, suggesting significant contributions from other immunological sites. Here, we investigated the tolerogenic contributions of Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) to NP-Ag-induced tolerance in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Intravenously delivered Ag-conjugated poly(lactide-co-glycolide) NPs (PLG-Ag) distributed largely to the liver, where they associated with both KCs and LSECs. This distribution was accompanied by CD4 T cell accumulation, clonal deletion, and PD-L1 expression by KCs and LSECs. Ex vivo co-cultures of PLG-Ag-treated KCs or LSECs with Ag-specific CD4 T cells resulted in PGE2 and IL-10 or PGE2 secretion, respectively. KC depletion and adoptive transfer experiments demonstrated that KCs were sufficient, but not necessary, to mediate PLG-Ag-induced tolerance in EAE. The durability of PLG-Ag-induced tolerance in the absence of KCs may be attributed to the distribution of PLG-Ags to LSECs, which demonstrated similar levels of PD-L1, PGE2, and T cell stimulatory ability. Collectively, these studies provide mechanistic support for the role of liver KCs and LSECs in Ag-specific tolerance for a biomaterial platform that is currently being evaluated in clinical trials.

Cargo-less nanoparticles program innate immune cell responses to toll-like receptor activation.

Developing biomaterials to control the responsiveness of innate immune cells represents a clinically relevant approach to treat diseases with an underlying inflammatory basis, such as sepsis. Sepsis can involve activation of Toll-like receptor (TLR) signaling, which activates numerous inflammatory pathways. The breadth of this inflammation has limited the efficacy of pharmacological interventions that target a single molecular pathway. Here, we developed cargo-less particles as a single-agent, multi-target platform to elicit broad anti-inflammatory action against innate immune cells challenged by multiple TLR agonists. The particles, prepared from poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA), displayed potent molecular weight-, polymer composition-, and charge-dependent immunomodulatory properties, including downregulation of TLR-induced costimulatory molecule expression and cytokine secretion. Particles prepared using the anionic surfactant poly(ethylene-alt-maleic acid) (PEMA) significantly blunted the responses of antigen presenting cells to TLR4 (lipopolysaccharide) and TLR9 (CpG-ODN) agonists, demonstrating broad inhibitory activity to both extracellular and intracellular TLR ligands. Interestingly, particles prepared using poly(vinyl alcohol) (PVA), a neutrally-charged surfactant, only marginally inhibited inflammatory cytokine secretions. The biochemical pathways modulated by particles were investigated using TRanscriptional Activity CEll aRrays (TRACER), which implicated IRF1, STAT1, and AP-1 in the mechanism of action for PLA-PEMA particles. Using an LPS-induced endotoxemia mouse model, administration of PLA-PEMA particles prior to or following a lethal challenge resulted in significantly improved mean survival. Cargo-less particles affect multiple biological pathways involved in the development of inflammatory responses by innate immune cells and represent a potentially promising therapeutic strategy to treat severe inflammation.

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.

Controlled Delivery of Single or Multiple Antigens in Tolerogenic Nanoparticles Using Peptide-Polymer Bioconjugates.

Polymeric nanoparticles (NPs) have demonstrated their potential to induce antigen (Ag)-specific immunological tolerance in multiple immune models and are at various stages of commercial development. Association of Ag with NPs is typically achieved through surface coupling or encapsulation methods. However, these methods have limitations that include high polydispersity, uncontrollable Ag loading and release, and possible immunogenicity. Here, using antigenic peptides conjugated to poly(lactide-co-glycolide), we developed Ag-polymer conjugate NPs (acNPs) with modular loading of single or multiple Ags, negligible burst release, and minimally exposed surface Ag. Tolerogenic responses of acNPs were studied in vitro to decouple the role of NP size, concentration, and Ag loading on regulatory T cell (Treg) induction. CD4+CD25+Foxp3+ Treg induction was dependent on NP size, but CD25 expression of CD4+ T cells was not. NP concentration and Ag loading could be modulated to achieve maximal levels of Treg induction. In relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a murine model of multiple sclerosis, acNPs were effective in inhibiting disease induced by a single peptide or multiple peptides. The acNPs provide a simple, modular, and well-defined platform, and the NP physicochemical properties offer potential to design and answer complex mechanistic questions surrounding NP-induced tolerance.

In vivo reprogramming of immune cells: Technologies for induction of antigen-specific tolerance.

Technologies that induce antigen-specific immune tolerance by mimicking naturally occurring mechanisms have the potential to revolutionize the treatment of many immune-mediated pathologies such as autoimmunity, allograft rejection, and allergy. The immune system intrinsically has central and peripheral tolerance pathways for eliminating or modulating antigen-specific responses, which are being exploited through emerging technologies. Antigen-specific tolerogenic responses have been achieved through the functional reprogramming of antigen-presenting cells or lymphocytes. Alternatively, immune privileged sites have been mimicked using biomaterial scaffolds to locally suppress immune responses and promote long-term allograft survival. This review describes natural mechanisms of peripheral tolerance induction and the various technologies being developed to achieve antigen-specific immune tolerance in vivo. As currently approved therapies are non-specific and carry significant associated risks, these therapies offer significant progress towards replacing systemic immune suppression with antigen-specific therapies to curb aberrant immune responses.

Mechanical Characterization of Hybrid Vesicles Based on Linear Poly(Dimethylsiloxane-b-Ethylene Oxide) and Poly(Butadiene-b-Ethylene Oxide) Block Copolymers.

Poly(dimethylsiloxane-ethylene oxide) (PDMS-PEO) and poly(butadiene-b-ethylene oxide) (PBd-PEO) are two block copolymers which separately form vesicles with disparate membrane permeabilities and fluidities. Thus, hybrid vesicles formed from both PDMS-PEO and PBd-PEO may ultimately allow for systematic, application-specific tuning of vesicle membrane fluidity and permeability. However, given the relatively low strength previously noted for comb-type PDMS-PEO vesicles, the mechanical robustness of the resulting hybrid vesicles must first be confirmed. Toward this end, we have characterized the mechanical behavior of vesicles formed from mixtures of linear PDMS-PEO and linear PBd-PEO using micropipette aspiration. Tension versus strain plots of pure PDMS12-PEO46 vesicles revealed a non-linear response in the high tension regime, in contrast to the approximately linear response of pure PBd33-PEO20 vesicles. Remarkably, the area expansion modulus, critical tension, and cohesive energy density of PDMS12-PEO46 vesicles were each significantly greater than for PBd33-PEO20 vesicles, although critical strain was not significantly different between these vesicle types. PDMS12-PEO46/PBd33-PEO20 hybrid vesicles generally displayed graded responses in between that of the pure component vesicles. Thus, the PDMS12-PEO46/PBd33-PEO20 hybrid vesicles retained or exceeded the strength and toughness characteristic of pure PBd-PEO vesicles, indicating that future assessment of the membrane permeability and fluidity of these hybrid vesicles may be warranted.

Small molecule disruption of G beta gamma signaling inhibits the progression of heart failure.

RATIONALE: Excess signaling through cardiac Gbetagamma subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein-coupled receptor kinase (GRK)2 to agonist-stimulated beta-adrenergic receptors (beta-ARs) in HF, leading to chronic beta-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gbetagamma signaling and its interaction with GRK2 could be of therapeutic value in HF. OBJECTIVE: We sought to investigate small molecule Gbetagamma inhibition in HF. METHODS AND RESULTS: We recently described novel small molecule Gbetagamma inhibitors that selectively block Gbetagamma-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of Gbetagamma and GRK2 in vitro and in HL60 cells. Here, we show they reduced beta-AR-mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to beta-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and beta-myosin heavy chain). CONCLUSIONS: These data suggest a promising therapeutic role for small molecule inhibition of pathological Gbetagamma signaling in the treatment of HF.

Protease-activated receptor-1 contributes to cardiac remodeling and hypertrophy.

BACKGROUND: Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. METHODS AND RESULTS: We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. CONCLUSIONS: Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Jag2-Notch1 signaling regulates oral epithelial differentiation and palate development.

During mammalian palatogenesis, palatal shelves initially grow vertically from the medial sides of the paired maxillary processes flanking the developing tongue and subsequently elevate and fuse with each other above the tongue to form the intact secondary palate. Pathological palate-mandible or palate-tongue fusions have been reported in humans and other mammals, but the molecular and cellular mechanisms that prevent such aberrant adhesions during normal palate development are unknown. We previously reported that mice deficient in Jag2, which encodes a cell surface ligand for the Notch family receptors, have cleft palate associated with palate-tongue fusions. In this report, we show that Jag2 is expressed throughout the oral epithelium and is required for Notch1 activation during oral epithelial differentiation. We show that Notch1 is normally highly activated in the differentiating oral periderm cells covering the developing tongue and the lateral oral surfaces of the mandibular and maxillary processes during palate development. Oral periderm activation of Notch1 is significantly attenuated during palate development in the Jag2 mutants. Further molecular and ultrastructural analyses indicate that oral epithelial organization and periderm differentiation are disrupted in the Jag2 mutants. Moreover, we show that the Jag2 mutant tongue fused to wild-type palatal shelves in recombinant explant cultures. These data indicate that Jag2-Notch1 signaling is spatiotemporally regulated in the oral epithelia during palate development to prevent premature palatal shelf adhesion to other oral tissues and to facilitate normal adhesion between the elevated palatal shelves.

Muscle-specific microtubule-associated protein 4 is expressed early in myogenesis and is not sufficient to induce microtubule reorganization.

The expression of a muscle-specific variant of microtubule-associated protein 4 (mMAP4) has been analyzed during myogenesis of C(2)C(12) cells using an isoform-specific antibody. MMAP4 localizes to microtubules (MTs) and is expressed prior to a very early morphogenetic event, the formation of mononucleate spindle-shaped cells. MMAP4 protein appears at about the same time as titin and coincident with Golgi reorganization, but antedates myosin expression. Misexpression of EGFP-mMAP4 in non-muscle and proliferating C(2)C(12) cells does not induce dramatic changes in MT organization or stability, nor in Golgi organization. Expression of full-length mMAP4 or of a truncated form lacking the MT-binding domain does not disrupt myotube formation or myofibrillogenesis. While previous antisense studies indicated that mMAP4 is necessary for normal myotube formation [Mangan and Olmsted, 1996: Development 122:771-781], these data indicate mMAP4 is not sufficient to induce the reorganization of MTs or the Golgi into patterns typical of muscle cells. Thus, with respect to MT organizing properties, this tissue-specific variant differs from related neuronal MAPs, MAP2, and tau, which induce neural-like changes in MT organization.