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1.
Sci Transl Med ; 15(726): eadf9561, 2023 12 13.
Article in English | MEDLINE | ID: mdl-38091405

ABSTRACT

Immunoglobulin E (IgE) is a key driver of type 1 hypersensitivity reactions and allergic disorders, which are globally increasing in number and severity. Although eliminating pathogenic IgE may be a powerful way to treat allergy, no therapeutic strategy reported to date can fully ablate IgE production. Interleukin-4 receptor α (IL-4Rα) signaling is required for IgE class switching, and IL-4Rα blockade gradually reduces, but does not eliminate, IgE. The persistence of IgE after IL-4Rα blockade may be due to long-lived IgE+ plasma cells that maintain serological memory to allergens and thus may be susceptible to plasma cell-targeted therapeutics. We demonstrate that transient administration of a B cell maturation antigen x CD3 (BCMAxCD3) bispecific antibody markedly depletes IgE, as well as other immunoglobulins, by ablating long-lived plasma cells, although IgE and other immunoglobulins rapidly rebound after treatment. Concomitant IL-4Rα blockade specifically and durably prevents the reemergence of IgE by blocking IgE class switching while allowing the restoration of other immunoglobulins. Moreover, this combination treatment prevented anaphylaxis in mice. Together with additional cynomolgus monkey and human data, our studies demonstrate that allergic memory is primarily maintained by both non-IgE+ memory B cells that require class switching and long-lived IgE+ plasma cells. Our combination approach to durably eliminate pathogenic IgE has potential to benefit allergy in humans while preserving antibody-mediated immunity.


Subject(s)
Anaphylaxis , Immunoglobulin E , Mice , Humans , Animals , Macaca fascicularis , Plasma Cells , Allergens
2.
Sci Adv ; 8(31): eabo0502, 2022 Aug 05.
Article in English | MEDLINE | ID: mdl-35930644

ABSTRACT

Improving the thermal stability of biologics, including vaccines, is critical to reduce the economic costs and health risks associated with the cold chain. Here, we designed a versatile, safe, and easy-to-use reversible PEG-based hydrogel platform formed via dynamic covalent boronic ester cross-linking for the encapsulation, stabilization, and on-demand release of biologics. Using these reversible hydrogels, we thermally stabilized a wide range of biologics up to 65°C, including model enzymes, heat-sensitive clinical diagnostic enzymes (DNA gyrase and topoisomerase I), protein-based vaccines (H5N1 hemagglutinin), and whole viruses (adenovirus type 5). Our data support a generalized protection mechanism for the thermal stabilization of diverse biologics using direct encapsulation in reversible hydrogels. Furthermore, preliminary toxicology data suggest that the components of our hydrogel are safe for in vivo use. Our reversible hydrogel platform offers a simple material solution to mitigate the costs and risks associated with reliance on a continuous cold chain for biologic transport and storage.

3.
Biomacromolecules ; 19(3): 740-747, 2018 03 12.
Article in English | MEDLINE | ID: mdl-29394044

ABSTRACT

Modern medicine, biological research, and clinical diagnostics depend on the reliable supply and storage of complex biomolecules. However, biomolecules are inherently susceptible to thermal stress and the global distribution of value-added biologics, including vaccines, biotherapeutics, and Research Use Only (RUO) proteins, requires an integrated cold chain from point of manufacture to point of use. To mitigate reliance on the cold chain, formulations have been engineered to protect biologics from thermal stress, including materials-based strategies that impart thermal stability via direct encapsulation of the molecule. While direct encapsulation has demonstrated pronounced stabilization of proteins and complex biological fluids, no solution offers thermal stability while enabling facile and on-demand release from the encapsulating material, a critical feature for broad use. Here we show that direct encapsulation within synthetic, photoresponsive hydrogels protected biologics from thermal stress and afforded user-defined release at the point of use. The poly(ethylene glycol) (PEG)-based hydrogel was formed via a bioorthogonal, click reaction in the presence of biologics without impact on biologic activity. Cleavage of the installed photolabile moiety enabled subsequent dissolution of the network with light and release of the encapsulated biologic. Hydrogel encapsulation improved stability for encapsulated enzymes commonly used in molecular biology (ß-galactosidase, alkaline phosphatase, and T4 DNA ligase) following thermal stress. ß-galactosidase and alkaline phosphatase were stabilized for 4 weeks at temperatures up to 60 °C, and for 60 min at 85 °C for alkaline phosphatase. T4 DNA ligase, which loses activity rapidly at moderately elevated temperatures, was protected during thermal stress of 40 °C for 24 h and 60 °C for 30 min. These data demonstrate a general method to employ reversible polymer networks as robust excipients for thermal stability of complex biologics during storage and shipment that additionally enable on-demand release of active molecules at the point of use.


Subject(s)
Bacteriophage T4/enzymology , DNA Ligases/chemistry , Hot Temperature , Hydrogels/chemistry , Photochemical Processes , Polyethylene Glycols/chemistry , Viral Proteins/chemistry , Enzyme Stability
4.
PLoS One ; 11(3): e0151252, 2016.
Article in English | MEDLINE | ID: mdl-26978520

ABSTRACT

The precise context in which the innate immune system is activated plays a pivotal role in the subsequent instruction of CD4+ T helper (Th) cell responses. Th1 responses are downregulated when antigen is encountered in the presence of antigen-IgG immune complexes. To assess if Th17 responses to antigen are subject to similar influences in the presence of immune complexes we utilized an inflammatory airway disease model in which immunization of mice with Complete Freund's Adjuvant (CFA) and ovalbumin (Ova) induces a powerful Ova-specific Th1 and Th17 response. Here we show that modification of that immunization with CFA to include IgG-Ova immune complexes results in the suppression of CFA-induced Th17 responses and a concurrent enhancement of Ova-specific Th2 responses. Furthermore, we show the mechanism by which these immune complexes suppress Th17 responses is through the enhancement of IL-10 production. In addition, the generation of Th17 responses following immunization with CFA and Ova were dependent on IL-1α but independent of NLRP3 inflammasome activation. Together these data represent a novel mechanism by which the generation of Th17 responses is regulated.


Subject(s)
Antigen-Antibody Complex/immunology , Inflammasomes/immunology , Th17 Cells/immunology , Animals , Antigen-Antibody Complex/metabolism , Freund's Adjuvant , Immunization , Inflammasomes/metabolism , Interleukin-1alpha/metabolism , Mice , Ovalbumin , Th17 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism
5.
J Immunol ; 193(10): 5190-8, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25320279

ABSTRACT

IgG immune complexes have been shown to modify immune responses driven by APCs in either a pro- or anti-inflammatory direction depending upon the context of stimulation. However, the ability of immune complexes to modulate the inflammasome-dependent innate immune response is unknown. In this study, we show that IgG immune complexes suppress IL-1α and IL-1ß secretion through inhibition of inflammasome activation. The mechanism by which this inhibition occurs is via immune complex ligation of activating FcγRs, resulting in prevention of both activation and assembly of the inflammasome complex in response to nucleotide-binding domain leucine-rich repeat (NLR) P3, NLRC4, or AIM2 agonists. In vivo, administration of Ag in the form of an immune complex during priming of the immune response inhibited resultant adaptive immune responses in an NLRP3-dependent model of allergic airway disease. Our data reveal an unexpected mechanism regulating CD4(+) T cell differentiation, by which immune complexes suppress inflammasome activation and the generation of IL-1α and IL-1ß from APCs, which are critical for the Ag-driven differentiation of CD4(+) T cells.


Subject(s)
Antigen-Antibody Complex/genetics , CD4-Positive T-Lymphocytes/immunology , Inflammasomes/immunology , Interleukin-1alpha/metabolism , Interleukin-1beta/metabolism , Lung/immunology , Respiratory Hypersensitivity/immunology , Adjuvants, Immunologic/administration & dosage , Alum Compounds/administration & dosage , Animals , CD4-Positive T-Lymphocytes/pathology , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/pathology , Gene Expression Regulation , Immunity, Innate , Inflammasomes/genetics , Interleukin-1alpha/biosynthesis , Interleukin-1beta/biosynthesis , Lung/pathology , Lymph Nodes/immunology , Lymph Nodes/pathology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin , Receptors, IgG/genetics , Receptors, IgG/immunology , Respiratory Hypersensitivity/chemically induced , Respiratory Hypersensitivity/genetics , Respiratory Hypersensitivity/pathology , Signal Transduction
6.
Proc Natl Acad Sci U S A ; 111(3): 1072-7, 2014 Jan 21.
Article in English | MEDLINE | ID: mdl-24395802

ABSTRACT

Chronic recurrent multifocal osteomyelitis (CRMO) is a human autoinflammatory disorder that primarily affects bone. Missense mutation (L98P) of proline-serine-threonine phosphatase-interacting protein 2 (Pstpip2) in mice leads to a disease that is phenotypically similar to CRMO called chronic multifocal osteomyelitis (cmo). Here we show that deficiency of IL-1RI in cmo mice resulted in a significant reduction in the time to onset of disease as well as the degree of bone pathology. Additionally, the proinflammatory cytokine IL-1ß, but not IL-1α, played a critical role in the pathology observed in cmo mice. In contrast, disease in cmo mice was found to be independent of the nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome as well as caspase-1. Neutrophils, but not bone marrow-derived macrophages, from cmo mice secreted increased IL-1ß in response to ATP, silica, and Pseudomonas aeruginosa compared with neutrophils from WT mice. This aberrant neutrophil response was sensitive to inhibition by serine protease inhibitors. These results demonstrate an inflammasome-independent role for IL-1ß in disease progression of cmo and implicate neutrophils and neutrophil serine proteases in disease pathogenesis. These data provide a rationale for directly targeting IL-1RI or IL-1ß as a therapeutic strategy in CRMO.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cytoskeletal Proteins/genetics , Gene Expression Regulation , Interleukin-1beta/metabolism , Osteomyelitis/immunology , Animals , Bone Marrow Cells/cytology , Cytokines/metabolism , Disease Models, Animal , Disease Progression , Inflammasomes/metabolism , Macrophages/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Mutation, Missense , Neutrophils/cytology , Neutrophils/metabolism , Osteomyelitis/genetics , Protein Structure, Tertiary , Receptors, Interleukin-1/genetics
7.
Immunity ; 39(2): 311-323, 2013 Aug 22.
Article in English | MEDLINE | ID: mdl-23954133

ABSTRACT

Nlrp3 inflammasome activation occurs in response to numerous agonists but the specific mechanism by which this takes place remains unclear. All previously evaluated activators of the Nlrp3 inflammasome induce the generation of mitochondrial reactive oxygen species (ROS), suggesting a model in which ROS is a required upstream mediator of Nlrp3 inflammasome activation. Here we have identified the oxazolidinone antibiotic linezolid as a Nlrp3 agonist that activates the Nlrp3 inflammasome independently of ROS. The pathways for ROS-dependent and ROS-independent Nlrp3 activation converged upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. Cardiolipin bound to Nlrp3 directly and interference with cardiolipin synthesis specifically inhibited Nlrp3 inflammasome activation. Together these data suggest that mitochondria play a critical role in the activation of the Nlrp3 inflammasome through the direct binding of Nlrp3 to cardiolipin.


Subject(s)
Cardiolipins/metabolism , Carrier Proteins/metabolism , Inflammasomes/metabolism , Mitochondria/metabolism , Acetamides/metabolism , Acetamides/pharmacology , Animals , Cardiolipins/immunology , Cell Line , Cyclosporine/metabolism , Enzyme Activation , Humans , Inflammation/chemically induced , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Linezolid , Macrophages/immunology , Macrophages/metabolism , Mice , Mitochondria/immunology , NLR Family, Pyrin Domain-Containing 3 Protein , Oxazolidinones/metabolism , Oxazolidinones/pharmacology , Potassium/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/metabolism
8.
Microbes Infect ; 14(14): 1263-70, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22841804

ABSTRACT

The importance of innate immunity lies not only in directly confronting pathogenic and non-pathogenic insults but also in instructing the development of an efficient adaptive immune response. The Nlrp3 inflammasome provides a platform for the activation of caspase-1 with the subsequent processing and secretion of IL-1 family members. Given the importance of IL-1 in a variety of inflammatory diseases, understanding the role of the Nlrp3 inflammasome in the initiation of innate and adaptive immune responses cannot be overstated. This review examines recent advances in inflammasome biology with an emphasis on its roles in sterile inflammation and triggering of adaptive immune responses.


Subject(s)
Adaptive Immunity/physiology , Immunity, Innate/physiology , Inflammasomes/immunology , Animals , Humans
9.
Anesthesiology ; 116(5): 1104-15, 2012 May.
Article in English | MEDLINE | ID: mdl-22531249

ABSTRACT

BACKGROUND: The innate immune response is important in ventilator-induced lung injury (VILI) but the exact pathways involved are not elucidated. The authors studied the role of the intracellular danger sensor NLRP3 inflammasome. METHODS: NLRP3 inflammasome gene expression was analyzed in respiratory epithelial cells and alveolar macrophages obtained from ventilated patients (n = 40). In addition, wild-type and NLRP3 inflammasome deficient mice were randomized to low tidal volume (approximately 7.5 ml/kg) and high tidal volume (approximately 15 ml/kg) ventilation. The presence of uric acid in lung lavage, activation of caspase-1, and NLRP3 inflammasome gene expression in lung tissue were investigated. Moreover, mice were pretreated with interleukin-1 receptor antagonist, glibenclamide, or vehicle before start of mechanical ventilation. VILI endpoints were relative lung weights, total protein in lavage fluid, neutrophil influx, and pulmonary and systemic cytokine and chemokine concentrations. Data represent mean ± SD. RESULTS: Mechanical ventilation up-regulated messenger RNA expression levels of NLRP3 in alveolar macrophages (1.0 ± 0 vs. 1.70 ± 1.65, P less than 0.05). In mice, mechanical ventilation increased both NLRP3 and apoptosis-associated speck-like protein messenger RNA levels, respectively (1.08 ± 0.55 vs. 3.98 ± 2.89; P less than 0.001 and 0.95 ± 0.53 vs. 6.0 ± 3.55; P less than 0.001), activated caspase-1, and increased uric acid levels (6.36 ± 1.85 vs. 41.9 ± 32.0, P less than 0.001). NLRP3 inflammasome deficient mice displayed less VILI due to high tidal volume mechanical ventilation compared with wild-type mice. Furthermore, treatment with interleukin-1 receptor antagonist or glibenclamide reduced VILI. CONCLUSIONS: Mechanical ventilation induced a NLRP3 inflammasome dependent pulmonary inflammatory response. NLRP3 inflammasome deficiency partially protected mice from VILI.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/physiology , Inflammasomes/genetics , Ventilator-Induced Lung Injury/genetics , Ventilator-Induced Lung Injury/pathology , Animals , Bronchoalveolar Lavage Fluid/chemistry , Caspase 1/metabolism , Cytokines/blood , Cytokines/metabolism , Enzyme Activation/physiology , Epithelial Cells/metabolism , Glyburide/pharmacology , Humans , Macrophages, Alveolar/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Neutrophil Infiltration , Organ Size/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Interleukin-1/antagonists & inhibitors , Respiration, Artificial , Tidal Volume/physiology , Up-Regulation/physiology , Uric Acid/metabolism
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