RESUMEN
In order to ensure valid diagnostics for occupational test allergen solutions despite the ongoing reduction in the availability of commercial test extracts, a plan B was initiated for the possible production of skin prick test (SPT) solutions in public pharmacies. For important occupational allergen sources (wheat and rye, storage mites, animal epithelia, mold material) laboratory extraction methods were analyzed in comparison to pharmacy compatible extraction methods regarding protein quantity and quality in SDS-PAGE combined with silver staining. Subsequently, using the example of bovine epithelia, adapted extraction procedures as well as in-process and final product controls were transferred to a public pharmacy. Allergen sources with a high protein content, such as wheat and rye grains as well as storage mites, showed good comparability of the extractable protein quantity and protein pattern, regardless of the applied extraction method. In contrast, allergen source materials with a low total protein content, such as animal epithelia and molds, can benefit from laboratory extraction conditions such as mechanical disruption and specific buffer additives. In the qualitative protein silver staining, characteristic protein patterns were identified for each allergen source. Depending on the extraction method, only minor differences in total protein patterns were observed in animal epithelia and molds. Using source materials from two suppliers, the resulting allergen extracts displayed clear differences in protein content in storage mites and quantitative and qualitative differences in molds. A practical preparation attempt of SPT solutions in a public pharmacy was successful. SPT solutions prepared with adapted pharmacy extraction methods showed a comparable protein and Bos d 2 allergen content and equivalent qualities in the protein pattern compared to a previously available commercial SPT solution. Accordingly, it can be assumed that standardized SPT solutions with sufficient allergen quality for occupational allergen sources can be prepared in public pharmacies if certified allergen sources with appropriate protein content are available.
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The availability of high-quality skin test allergens is a prerequisite for the reliable diagnosis of occupational type I allergies. Due to the withdrawal of existing marketing authorizations (MAs) by pharmaceutical companies and the lack of new MAs for commercial test allergens, there is an increasing diagnostic gap in Germany and other EU member states, which makes it necessary to investigate alternative ways of providing in vivo diagnostics. The German Medicinal Products Act (Arzneimittelgesetz = AMG) allows for the possibility of preparing medicinal products in pharmacies without the need for an MA or a manufacturing authorization pursuant to Section 13 (2) No. 1 in conjunction with Section 13 (2a) Sentence 2 No. 3 AMG. This also includes test allergens. In addition to the AMG, the requirements of the German Ordinance on the Operation of Pharmacies (Apothekenbetriebsordnung - ApBetrO) and the European Pharmacopoeia apply in particular. Medicolegal and practical challenges, as well as potentials of manufacturing skin prick test solutions in public pharmacies are presented based on examples of different allergen source materials.
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Pectins are dietary fibers that are attributed with several beneficial immunomodulatory effects. Depending on the degree of esterification (DE), pectins can be classified as high methoxyl pectin (HMP) or low methoxyl pectin (LMP). The aim of this study was to investigate the effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice. Supplementation of the diet with LMP or HMP induced changes in the composition of the intestinal microbiota in mice toward Bacteroides, which was mainly promoted by HMP. Metabolome analysis of stool samples from pectin-fed mice showed a different effect of the two types of pectin on the levels of short-chain fatty acids and bile acids, which was consistent with highly efficient in vivo fermentation of LMP. Analysis of serum antibody levels showed a significant increase in IgG and IgA levels by both pectins, while FACS analysis revealed a decrease of infiltrating inflammatory cells in the intestinal lamina propria by HMP. Our study revealed that the structural properties of the investigated pectins determine fermentability, effects on microbial composition, metabolite production, and modulation of immune responses. Consumption of HMP preferentially altered the gut microbiota and suppressed pro-inflammatory immune responses, suggesting a beneficial role in inflammatory diseases.
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Microbioma Gastrointestinal , Pectinas , Ratones , Animales , Pectinas/química , Esterificación , Fibras de la Dieta/farmacología , Fibras de la Dieta/metabolismo , FermentaciónRESUMEN
Type I hypersensitivity, or so-called type I allergy, is caused by Th2-mediated immune responses directed against otherwise harmless environmental antigens. Currently, allergen-specific immunotherapy (AIT) is the only disease-modifying treatment with the potential to re-establish clinical tolerance towards the corresponding allergen(s). However, conventional AIT has certain drawbacks, including long treatment durations, the risk of inducing allergic side effects, and the fact that allergens by themselves have a rather low immunogenicity. To improve AIT, adjuvants can be a powerful tool not only to increase the immunogenicity of co-applied allergens but also to induce the desired immune activation, such as promoting allergen-specific Th1- or regulatory responses. This review summarizes the knowledge on adjuvants currently approved for use in human AIT: aluminum hydroxide, calcium phosphate, microcrystalline tyrosine, and MPLA, as well as novel adjuvants that have been studied in recent years: oil-in-water emulsions, virus-like particles, viral components, carbohydrate-based adjuvants (QS-21, glucans, and mannan) and TLR-ligands (flagellin and CpG-ODN). The investigated adjuvants show distinct properties, such as prolonging allergen release at the injection site, inducing allergen-specific IgG production while also reducing IgE levels, as well as promoting differentiation and activation of different immune cells. In the future, better understanding of the immunological mechanisms underlying the effects of these adjuvants in clinical settings may help us to improve AIT.
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Desensibilización Inmunológica , Hipersensibilidad , Humanos , Adyuvantes Inmunológicos/uso terapéutico , Alérgenos , Hidróxido de Aluminio , Adyuvantes FarmacéuticosRESUMEN
PURPOSE OF REVIEW: Immunoglobulin A (IgA) mediates immune exclusion of antigens in the gut. Notably, IgA plays also a role in the prevention of IgE-mediated allergies and induction of immune tolerance. The present review addresses the role of IgA in the manifestation of IgE-mediated allergies, including allergen-specific immunotherapy (AIT), the regulation of IgA production, and the mechanism of IgA in immune cell activation. RECENT FINDINGS: The majority of studies report an association of IgA with the induction of immune tolerance in IgE-mediated allergies. However, reports on the involvement of humoral and mucosal IgA, IgA subtypes, monomeric and polymeric IgA, and the mechanism of IgA-mediated immune cell activation are confounding. Effects by IgA are likely mediated by alteration of microbiota, IgE-blocking capacity, or activation of inhibitory signaling pathways. However, the precise mechanism of IgA-regulation, the contribution of serum and/or mucosal IgA, and IgA1/2 subtypes, on the manifestation of IgE-mediated allergies, and the underlying immune modulatory mechanism are still elusive.
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Hipersensibilidad Inmediata , Inmunoglobulina A , Humanos , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad Inmediata/prevención & control , Tolerancia Inmunológica , Inmunidad , Inmunoglobulina ERESUMEN
Background: A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1ß secretion and their contribution to the overall immune responses remains elusive. Objective: To investigate the mechanisms underlying the production of IL-1ß from rFlaA:Betv1 stimulated macrophages. Methods: Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1ß to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. Results: rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1ß secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1ß-expression in THP-1 macrophages. Finally, lack of IL-1ß positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1ß, IL-6, and TNF-α from peritoneal macrophages. Conclusion: The mechanisms contributing to rFlaA:Betv1-induced IL-1ß secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.
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Flagelina , Inflamasomas , Animales , Humanos , Ratones , Adyuvantes Inmunológicos/farmacología , Alérgenos , Proteínas de Unión al Calcio/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Inflamasomas/metabolismo , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Recombinantes , Receptor Toll-Like 5/metabolismoRESUMEN
Antigen-presenting cells (APCs) are critical cells bridging innate and adaptive immune responses by taking up, processing, and presenting antigens to naïve T cells. At steady state, APCs thus control both tissue homeostasis and the induction of tolerance. In allergies however, APCs drive a Th2-biased immune response that is directed against otherwise harmless antigens from the environment. The main types of APCs involved in the induction of allergy are dendritic cells, monocytes, and macrophages. However, these cell types can be further divided into local, tissue-specific populations that differ in their phenotype, migratory capacity, T-cell activating potential, and production of effector molecules. Understanding if distinct populations of APCs contribute to either tissue-specific immune tolerance, allergen sensitization, or allergic inflammation will allow us to better understand disease pathology and develop targeted treatment options for different stages of allergic disease. Therefore, this review describes the main characteristics, phenotypes, and effector molecules of the APCs involved in the induction of allergen-specific Th2 responses in affected barrier sites, such as the skin, nose, lung, and gastrointestinal tract. Furthermore, we highlight open questions that remain to be addressed to fully understand the contribution of different APCs to allergic disease.
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Células Presentadoras de Antígenos , Hipersensibilidad , Humanos , Alérgenos , Linfocitos T , FenotipoRESUMEN
PURPOSE OF REVIEW: Over the last years, we have learned that the metabolic phenotype of immune cells is closely connected to the cell's effector function. Understanding these changes will allow us to better understand allergic disease pathology and improve allergy treatment by modulating immune metabolic pathways. As part two of a two-article series, this review reports on the recent studies investigating the metabolism of the cell types involved in allergies and discusses the initial application of these discoveries in allergy treatment. RECENT FINDINGS: The cell types involved in allergic reactions display pronounced and highly specific metabolic changes (here discussed for epithelial cells, APCs, ILC2s, mast cells, eosinophils, and Th2 cells). Currently, the first drugs targeting metabolic pathways are tested for their potential to improve allergy treatment. Immune-metabolic changes observed in allergy so far are complex and depend on the investigated disease and cell type. However, our increased understanding of the underlying principles has pointed to several promising target molecules that are now being investigated to improve allergy treatment.
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Hipersensibilidad , Inmunidad Innata , Humanos , Linfocitos , Hipersensibilidad/terapia , Células Th2/metabolismo , Mastocitos , Citocinas/metabolismoRESUMEN
BACKGROUND: The experimental fusion protein rFlaA:Betv1 was shown to efficiently suppress allergen-specific sensitization in mice. However, the detailed mechanism of rFlaA:Betv1-mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaA:Betv1 on naïve murine B cells. METHODS: Immune modulating capacity of rFlaA:Betv1 was screened in IL-10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5-/- , or MyD88-/- mice, stimulated with rFlaA:Betv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaA:Betv1-stimulated B cells was tested in mDC:CD4+ T cell:B cell triple cultures. RESULTS: Upon in vivo application of rFlaA:Betv1 into IL-10-GFP reporter mice, CD19+ B cells were shown to produce anti-inflammatory IL-10, suggesting B cells to contribute to the immune-modulatory properties of rFlaA:Betv1. rFlaA:Betv1-induced IL-10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaA:Betv1 resulted in an mTOR- and MyD88-dependent production of IL-10 and rFlaA:Betv1 induced Bet v 1-reactive IgG production, which was not observed for IgA. rFlaA:Betv1-stimulated B cells formed a CD19+ CD24+ CD1d+ IgM+ CD38+ Breg subpopulation capable of suppressing Bet v 1-induced TH2 cytokine secretion in vitro. CONCLUSION: rFlaA:Betv1 can act as a thymus-independent B cell antigen, stimulating the mTOR- and MyD88-dependent differentiation of B cells displaying a regulatory phenotype, IL-10 secretion, antigen-binding antibody production, and a suppressive capacity in vitro.
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Linfocitos B Reguladores , Interleucina-10 , Ratones , Humanos , Animales , Factor 88 de Diferenciación Mieloide/genética , Flagelina/química , Flagelina/genética , Serina-Treonina Quinasas TOR , Inmunoglobulina MRESUMEN
PURPOSE OF REVIEW: Recent high-level publications have shown an intricate connection between immune effector function and the metabolic state of the respective cells. In the last years, studies have begun analyzing the metabolic changes associated with allergies. As the first part of a two-article series, this review will briefly summarize the basics of immune metabolism and then focus on the recently published studies on metabolic changes observed in allergic patients. RECENT FINDINGS: In the last 3 years, immune-metabolic research in allergology had a clear focus on asthma with some studies also reporting findings in food allergy and atopic dermatitis. Current results suggest asthma to be associated with a shift in cellular metabolism towards increased aerobic glycolysis (Warburg metabolism), while also displaying substantial changes in fatty acid- and amino acid metabolism (depending on investigated patient collective, asthma phenotype, and disease severity). Understanding immune-metabolic changes in allergies will allow us to (I) better understand allergic disease pathology and (II) modulate immune-metabolic pathways to improve allergy treatment.
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Asma , Dermatitis Atópica , Hipersensibilidad a los Alimentos , Humanos , Dermatitis Atópica/terapiaRESUMEN
Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.
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Receptor Toll-Like 5 , Vacunas , Receptor Toll-Like 5/metabolismo , Alérgenos , Interleucina-10/metabolismo , Flagelina/metabolismo , Hexoquinasa/metabolismo , Glutaminasa/metabolismo , Ligandos , Antimicina A/metabolismo , Antimicina A/farmacología , Cerulenina/metabolismo , Cerulenina/farmacología , Células Dendríticas , Proteínas Recombinantes/metabolismo , Citocinas/metabolismo , Adyuvantes Inmunológicos/farmacología , Vacunas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Glucólisis , Serina-Treonina Quinasas TOR/metabolismo , Desoxiglucosa/farmacología , Oligomicinas/farmacología , Ácidos Grasos/metabolismoRESUMEN
Background: Recently, bacterial components were shown to enhance immune responses by shifting immune cell metabolism towards glycolysis and lactic acid production, also known as the Warburg Effect. Currently, the effect of allergen products for immunotherapy (AIT) and commercial vaccines on immune cell metabolism is mostly unknown. Objective: To investigate the effect of AIT products (adjuvanted with either MPLA or Alum) on myeloid dendritic cell (mDC) metabolism and activation. Methods: Bone marrow-derived mDCs were stimulated with five allergoid-based AIT products (one adjuvanted with MPLA, four adjuvanted with Alum) and two MPLA-adjuvanted vaccines and analyzed for their metabolic activation, expression of cell surface markers, and cytokine secretion by ELISA. mDCs were pre-incubated with either immunological or metabolic inhibitors or cultured in glucose- or glutamine-free culture media and subsequently stimulated with the MPLA-containing AIT product (AIT product 1). mDCs were co-cultured with allergen-specific CD4+ T cells to investigate the contribution of metabolic pathways to the T cell priming capacity of mDCs stimulated with AIT product 1. Results: Both the MPLA-containing AIT product 1 and commercial vaccines, but not the Alum-adjuvanted AIT products, activated Warburg metabolism and TNF-α secretion in mDCs. Further experiments focused on AIT product 1. Metabolic analysis showed that AIT product 1 increased glycolytic activity while also inducing the secretion of IL-1ß, IL-10, IL-12, and TNF-α. Both rapamycin (mTOR-inhibitor) and SP600125 (SAP/JNK MAPK-inhibitor) dose-dependently suppressed the AIT product 1-induced Warburg Effect, glucose consumption, IL-10-, and TNF-α secretion. Moreover, both glucose- and glutamine deficiency suppressed secretion of all investigated cytokines (IL-1ß, IL-10, and TNF-α). Glucose metabolism in mDCs was also critical for the (Th1-biased) T cell priming capacity of AIT product 1-stimulated mDCs, as inhibition of mTOR signaling abrogated their ability to induce Th1-responses. Conclusion: The AIT product and commercial vaccines containing the adjuvant MPLA were shown to modulate the induction of immune responses by changing the metabolic state of mDCs. Better understanding the mechanisms underlying the interactions between cell metabolism and immune responses will allow us to further improve vaccine development and AIT.
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Alérgenos , Vacunas , Adyuvantes Inmunológicos/farmacología , Adyuvantes Farmacéuticos/metabolismo , Adyuvantes Farmacéuticos/farmacología , Células Dendríticas , Glucosa/metabolismo , Factores Inmunológicos/farmacología , Inmunoterapia , Interleucina-10 , Serina-Treonina Quinasas TOR/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas/farmacologíaRESUMEN
Over the last decades, the frequency of allergic disorders has steadily increased. Immunologically, allergies are caused by abnormal immune responses directed against otherwise harmless antigens derived from our environment. Two of the main cell types driving allergic sensitization and inflammation are IgE-producing plasma cells and Th2 cells. The acute activation of T and B cells, their differentiation into effector cells, as well as the formation of immunological memory are paralleled by distinct changes in cellular metabolism. Understanding the functional consequences of these metabolic changes is the focus of a new research field termed "immune metabolism". Currently, the contribution of metabolic changes in T and B cells to either the development or maintenance of allergies is not completely understood. Therefore, this mini review will introduce the fundamentals of energy metabolism, its connection to immune metabolism, and subsequently focus on the metabolic phenotypes of IL-4-activated B cells and Th2 cells.
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Linfocitos B/inmunología , Hipersensibilidad/inmunología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Células Th2/inmunología , Linfocitos B/metabolismo , Metabolismo Energético , Centro Germinal/metabolismo , Humanos , Hipersensibilidad/metabolismo , Células Th2/metabolismoRESUMEN
Developing new adjuvants/vaccines and better understanding their mode-of-action is an important task. To specifically improve birch pollen allergy treatment, we designed a fusion protein consisting of major birch pollen allergen Betv1 conjugated to the TLR5-ligand flagellin (rFlaA:Betv1). This study investigates the immune-modulatory effects of rFlaA:Betv1 on airway epithelial cells. LA-4 mouse lung epithelial cells were stimulated with rFlaA:Betv1 in the presence/absence of various inhibitors with cytokine- and chemokine secretion quantified by ELISA and activation of intracellular signaling cascades demonstrated by Western blot (WB). Either LA-4 cells or LA-4-derived supernatants were co-cultured with BALB/c bone marrow-derived myeloid dendritic cells (mDCs). Compared to equimolar amounts of flagellin and Betv1 provided as a mixture, rFlaA:Betv1 induced higher secretion of IL-6 and the chemokines CCL2 and CCL20 from LA-4 cells and a pronounced MAPK- and NFκB-activation. Mechanistically, rFlaA:Betv1 was taken up more strongly and the induced cytokine production was inhibited by NFκB-inhibitors, while ERK- and p38-MAPK-inhibitors only suppressed IL-6 and CCL2 secretion. In co-cultures of LA-4 cells with mDCs, rFlaA:Betv1-stimulated LA-4 cells p38-MAPK- and COX2-dependently secreted PGE2, which modulated DC responses by suppressing pro-inflammatory IL-12 and TNF-α secretion. Taken together, these results contribute to our understanding of the mechanisms underlying the strong immune-modulatory effects of flagellin-containing fusion proteins.
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Ciclooxigenasa 2/metabolismo , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Células Epiteliales/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Quimiocina CCL2/metabolismo , Quimiocina CCL20/metabolismo , Quimiocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos BALB C , Modelos Biológicos , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Solubilidad , Receptor Toll-Like 5/metabolismoRESUMEN
TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differentiated from BALB/c, C57BL/6, TLR5-/-, or MyD88-/- mice were pre-treated with inhibitors, stimulated with rFlaA:Betv1 or respective controls, and analyzed for activation, cytokine secretion, metabolic state, RNA transcriptome, and modulation of allergen-specific Th2 responses. Stimulation of BMDMs with rFlaA:Betv1 resulted in MyD88-dependent production of IL-1ß, IL-6, TNF-α, IL-10, CD69 upregulation, and a pronounced shift towards glycolysis paralleled by activation of MAPK, NFκB, and mTOR signaling. Inhibition of either mTOR (rapamycin) or SAP/JNK-MAPK signaling (SP600125) resulted in dose-dependent metabolic suppression. In BMDM and T cell co-cultures, rFlaA:Betv1 stimulation suppressed rBet v 1-induced IL-5 and IL-13 secretion while inducing IFN-γ production. mRNA-Seq analyses showed HIF-1a, JAK, STAT, phagosome, NLR, NFκB, TNF, TLR, and chemokine signaling to participate in the interplay of cell activation, glycolysis, and immune response. rFlaA:Betv1 strongly activated BMDMs, resulting in MyD88-, MAPK-, and mTOR-dependent enhancement of glucose metabolism. Our results suggest macrophages are important target cells to consider during restauration of allergen tolerance during AIT.
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Alérgenos/inmunología , Antígenos Bacterianos/inmunología , Antígenos de Plantas/inmunología , Flagelina/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Proteínas Bacterianas/inmunología , Células Cultivadas , Glucosa/metabolismo , Macrófagos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Plantas/inmunología , Polen/inmunologíaRESUMEN
The COVID-19 pandemic highlights the importance of efficient and safe vaccine development. Vaccine adjuvants are essential to boost and tailor the immune response to the corresponding pathogen. To allow for an educated selection, we assessed the effect of different adjuvants on human monocyte-derived dendritic cells (DCs) and their ability to polarize innate and adaptive immune responses. In contrast to commonly used adjuvants, such as aluminum hydroxide, Toll-like receptor (TLR) agonists induced robust phenotypic and functional DC maturation. In a DC-lymphocyte coculture system, we investigated the ensuing immune reactions. While monophosphoryl lipid A synthetic, a TLR4 ligand, induced checkpoint inhibitors indicative for immune exhaustion, the TLR7/8 agonist Resiquimod (R848) induced prominent type-1 interferon and interleukin 6 responses and robust CTL, B-cell, and NK-cell proliferation, which is particularly suited for antiviral immune responses. The recently licensed COVID-19 vaccines, BNT162b and mRNA-1273, are both based on single-stranded RNA. Indeed, we could confirm that the cytokine profile induced by lipid-complexed RNA was almost identical to the pattern induced by R848. Although this awaits further investigation, our results suggest that their efficacy involves the highly efficient antiviral response pattern stimulated by the RNAs' TLR7/8 activation.
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Adyuvantes Inmunológicos/farmacología , COVID-19/inmunología , Células Dendríticas/inmunología , Inmunidad Celular/efectos de los fármacos , SARS-CoV-2/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Anciano , Femenino , Humanos , Imidazoles/farmacología , Lípido A/análogos & derivados , Lípido A/farmacología , Masculino , Persona de Mediana Edad , Receptores Toll-Like/inmunologíaRESUMEN
PURPOSE OF REVIEW: The incidence of allergies is increasing and has been associated with several environmental factors including westernized diets. Changes in environment and nutrition can result in dysbiosis of the skin, gut, and lung microbiota altering the production of microbial metabolites, which may in turn generate epigenetic modifications. The present review addresses studies on pectin-mediated effects on allergies, including the immune modulating mechanisms by bacterial metabolites. RECENT FINDINGS: Recently, microbiota have gained attention as target for allergy intervention, especially with prebiotics, that are able to stimulate the growth and activity of certain microorganisms. Dietary fibers, which cannot be digested in the gastrointestinal tract, can alter the gut microbiota and lead to increased local and systemic concentrations of gut microbiota-derived short chain fatty acids (SCFAs). These can promote the generation of peripheral regulatory T cells (Treg) by epigenetic modulation and suppress the inflammatory function of dendritic cells (DCs) by transcriptional modulation. The dietary fiber pectin (a plant-derived polysaccharide commonly used as gelling agent and dietary supplement) can alter the ratio of Firmicutes to Bacteroidetes in gut and lung microbiota, increasing the concentrations of SCFAs in feces and sera, and reducing the development of airway inflammation by suppressing DC function. Pectin has shown immunomodulatory effects on allergies, although the underlying mechanisms still need to be elucidated. It has been suggested that the different types of pectin may exert direct and/or indirect immunomodulatory effects through different mechanisms. However, little is known about the relation of certain pectin structures to allergies.
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Microbioma Gastrointestinal , Hipersensibilidad , Fibras de la Dieta , Ácidos Grasos Volátiles , Humanos , Hipersensibilidad/tratamiento farmacológico , PectinasRESUMEN
Some ß-mannans, including those in coffee bean and soy, contain a mannose backbone with ß-(1â4) bonds. Such mannooligosaccharides could have immunological functions involving direct interaction with immune cells, in addition to acting as prebiotics. This study aimed at assessing the immunological function of mannooligosaccharides with ß-(1â4) bond, and elucidating their mechanism of action using bone marrow-derived murine dendritic cells (BMDCs). When BMDCs were stimulated with the mannooligosaccharides, only ß-Man-(1â4)-Man significantly induced production of cytokines that included IL-6, IL-10, TNF-α, and IFN-ß, and enhanced CD4+ T-cell stimulatory capacity. Use of putative receptor inhibitors revealed the binding of ß-Man-(1â4)-Man to TLR4/MD2 complex and involvement with the complement C3a receptor (C3aR) for BMDC activation. Interestingly, ß-Man-(1â4)-Man prolonged the production of pro-inflammatory cytokines (IL-6 and TNF-α), but not of the IL-10 anti-inflammatory cytokine during extended culture of BMDCs, associated with high glucose consumption. The results suggest that ß-Man-(1â4)-Man is an immunostimulatory molecule, and that the promotion of glycolysis could be involved in the production of pro-inflammatory cytokine in ß-Man-(1â4)-Man-stimulated BMDCs. This study could contribute to development of immune-boosting functional foods and a novel vaccine adjuvant.