Biodegradable nanoparticles targeting circulating immune cells reduce central and peripheral sensitization to alleviate neuropathic pain following spinal cord injury.

ABSTRACT: Neuropathic pain is a critical source of comorbidity following spinal cord injury (SCI) that can be exacerbated by immune-mediated pathologies in the central and peripheral nervous systems. In this article, we investigate whether drug-free, biodegradable, poly(lactide- co -glycolide) (PLG) nanoparticle treatment mitigates the development of post-SCI neuropathic pain in female mice. Our results show that acute treatment with PLG nanoparticles following thoracic SCI significantly reduces tactile and cold hypersensitivity scores in a durable fashion. Nanoparticles primarily reduce peripheral immune-mediated mechanisms of neuropathic pain, including neuropathic pain-associated gene transcript frequency, transient receptor potential ankyrin 1 nociceptor expression, and MCP-1 (CCL2) chemokine production in the subacute period after injury. Altered central neuropathic pain mechanisms during this period are limited to reduced innate immune cell cytokine expression. However, in the chronic phase of SCI, nanoparticle treatment induces changes in both central and peripheral neuropathic pain signaling, driving reductions in cytokine production and other immune-relevant markers. This research suggests that drug-free PLG nanoparticles reprogram peripheral proalgesic pathways subacutely after SCI to reduce neuropathic pain outcomes and improve chronic central pain signaling.

Immunotherapy with biodegradable nanoparticles encapsulating the oligosaccharide galactose-alpha-1,3-galactose enhance immune tolerance against alpha-gal sensitization in a murine model of alpha-gal syndrome.

IgE antibodies against the mammalian oligosaccharide allergen galactose-α-1,3-galactose (αGal) can result in a severe allergic disease known as alpha-gal syndrome (AGS). This syndrome, acquired by tick bites that cause αGal sensitization, leads to allergic reactions after ingestion of non-primate mammalian meat and mammalian-derived products that contain αGal. Allergen-specific immunotherapies for this tickborne allergic syndrome are understudied, as are the immune mechanisms of allergic desensitization that induce clinical tolerance to αGal. Here, we reveal that prophylactic administration of αGal glycoprotein-containing nanoparticles to mice prior to tick protein-induced αGal IgE sensitization blunts the production of Th2 cytokines IL-4, IL-5, and IL-13 in an αGal-dependent manner. Furthermore, these effects correlated with suppressed production of αGal-specific IgE and hypersensitivity reactions, as measured by reduced basophil activation and histamine release and the systemic release of mast cell protease-1 (MCPT-1). Therapeutic administration of two doses of αGal-containing nanoparticles to mice sensitized to αGal had partial efficacy by reducing the Th2 cytokine production, αGal-specific IgE production, and MCPT-1 release without reducing basophil activation or histamine release. These data identify nanoparticles carrying encapsulated αGal glycoprotein as a potential strategy for augmenting αGal-specific immune tolerance and reveal diverse mechanisms by which αGal nanoparticles modify immune responses for established αGal-specific IgE-mediated allergic reactions.

Allergen-Encapsulating Nanoparticles Reprogram Pathogenic Allergen-Specific Th2 Cells to Suppress Food Allergy.

Food allergy is a prevalent, potentially deadly disease caused by inadvertent sensitization to benign food antigens. Pathogenic Th2 cells are a major driver for disease, and allergen-specific immunotherapies (AIT) aim to increase the allergen threshold required to elicit severe allergic symptoms. However, the majority of AIT approaches require lengthy treatments and convey transient disease suppression, likely due to insufficient targeting of pathogenic Th2 responses. Here, the ability of allergen-encapsulating nanoparticles to directly suppress pathogenic Th2 responses and reactivity is investigated in a mouse model of food allergy. NPs associate with pro-tolerogenic antigen presenting cells, provoking accumulation of antigen-specific, functionally suppressive regulatory T cells in the small intestine lamina propria. Two intravenous doses of allergen encapsulated in poly(lactide-co-glycolide) nanoparticles (NPs) significantly reduces oral food challenge (OFC)-induced anaphylaxis. Importantly, NP treatment alters the fates of pathogenic allergen-specific Th2 cells, reprogramming these cells toward CD25+FoxP3+ regulatory and CD73+FR4+ anergic phenotypes. NP-mediated reductions in the frequency of effector cells in the gut and mast cell degranulation following OFC are also demonstrated. These studies reveal mechanisms by which an allergen-encapsulating NP therapy and, more broadly, allergen-specific immunotherapies, can rapidly attenuate allergic responses by targeting pathogenic Th2 cells.

Building-Block Size Mediates Microporous Annealed Particle Hydrogel Tube Microenvironment Following Spinal Cord Injury.

Spinal cord injury (SCI) is a life-altering event, which often results in loss of sensory and motor function below the level of trauma. Biomaterial therapies have been widely investigated in SCI to promote directional regeneration but are often limited by their pre-constructed size and shape. Herein, the design parameters of microporous annealed particles (MAPs) are investigated with tubular geometries that conform to the injury and direct axons across the defect to support functional recovery. MAP tubes prepared from 20-, 40-, and 60-micron polyethylene glycol (PEG) beads are generated and implanted in a T9-10 murine hemisection model of SCI. Tubes attenuate glial and fibrotic scarring, increase innate immune cell density, and reduce inflammatory phenotypes in a bead size-dependent manner. Tubes composed of 60-micron beads increase the cell density of the chronic macrophage response, while neutrophil infiltration and phenotypes do not deviate from those seen in controls. At 8 weeks postinjury, implantation of tubes composed of 60-micron beads results in enhanced locomotor function, robust axonal ingrowth, and remyelination through both lumens and the inter-tube space. Collectively, these studies demonstrate the importance of bead size in MAP construction and highlight PEG tubes as a biomaterial therapy to promote regeneration and functional recovery in SCI.

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.

Masked Delivery of Allergen in Nanoparticles Safely Attenuates Anaphylactic Response in Murine Models of Peanut Allergy.

Food allergy is a growing health concern worldwide. Current allergen-specific immunotherapy (AIT) approaches require frequent dosing over extended periods of time and may induce anaphylaxis due to allergen-effector cell interactions. A critical need remains to develop novel approaches that refine AIT for the treatment of food allergies. Previous studies show that poly(lactide-co-glycolide) (PLG) nanoscale particles (NP) effectively suppress Th1- and Th17-driven immune pathologies. However, their ability to suppress the distinct Th2-polarized immune responses driving food allergy are unknown. Herein, we describe the safety and efficacy of NPs containing encapsulated peanut allergen in desensitizing murine models of peanut allergy. Peanut extract encapsulation allowed for the safe intravenous delivery of allergen relative to non-encapsulated approaches. Application of 2-3 doses, without the need for dose escalation, was sufficient to achieve prophylactic and therapeutic efficacy, which correlated with suppression of Th2-mediated disease and reduced mast cell degranulation. Efficacy was associated with strong reductions in a broad panel of Th1, Th2, and Th17 cytokines. These results demonstrate the ability of PLG NPs to suppress allergen-specific immune responses to induce a more tolerogenic phenotype, conferring protection from intragastric allergen challenge. These promising studies represent a step forward in the development of improved immunotherapies for food allergy.

Different Mixed Astrocyte Populations Derived from Embryonic Stem Cells Have Variable Neuronal Growth Support Capacities.

Central nervous system injury often leads to functional impairment due, in part, to the formation of an inhibitory glial scar following injury that contributes to poor regeneration. Astrocytes are the major cellular components of the glial scar, which has led to the belief that they are primarily inhibitory following injury. Recent work has challenged this by demonstrating that some astrocytes are required for spinal cord regeneration and astrocytic roles in recovery depend on their phenotype. In this work, two mixed populations containing primarily either fibrous or protoplasmic astrocytes were derived from mouse embryonic stem cells (mESCs). Motoneuron and V2a interneuron growth on live cultures, freeze-lysed cultures, or decellularized extracellular matrix (ECM) from astrocytes were assessed. Both neuronal populations were found to extend significantly longer neurites on protoplasmic-derived substrates than fibrous-derived substrates. Interestingly, neurons extended longer neurites on protoplasmic-derived ECM than fibrous-derived ECM. ECM proteins were compared with in vivo astrocyte expression profiles, and it was found that the ESC-derived ECMs were enriched for astrocyte-specific proteins. Further characterization revealed that protoplasmic ECM had significantly higher levels of axon growth promoting proteins, while fibrous ECM had significantly higher levels of proteins that inhibit axon growth. Supporting this observation, knockdown of spondin-1 improved neurite growth on fibrous ECM, while laminin α5 and γ1 knockdown decreased neurite growth on protoplasmic ECM. These methods allow for scalable production of specific astrocyte subtype-containing populations with different neuronal growth support capacities, and can be used for further studies of the functional importance of astrocyte heterogeneity.