Characterization of the Immune-Modulating Properties of Different ß-Glucans on Myeloid Dendritic Cells.

In allergen-specific immunotherapy, adjuvants are explored for modulating allergen-specific Th2 immune responses to re-establish clinical tolerance. One promising class of adjuvants are ß-glucans, which are naturally derived sugar structures and components of dietary fibers that activate C-type lectin (CLR)-, "Toll"-like receptors (TLRs), and complement receptors (CRs). We characterized the immune-modulating properties of six commercially available ß-glucans, using immunological (receptor activation, cytokine secretion, and T cell modulating potential) as well as metabolic parameters (metabolic state) in mouse bone marrow-derived myeloid dendritic cells (mDCs). All tested ß-glucans activated the CLR Dectin-1a, whereas TLR2 was predominantly activated by Zymosan. Further, the tested ß-glucans differentially induced mDC-derived cytokine secretion and activation of mDC metabolism. Subsequent analyses focusing on Zymosan, Zymosan depleted, ß-1,3 glucan, and ß-1,3 1,6 glucan revealed robust mDC activation with the upregulation of the cluster of differentiation 40 (CD40), CD80, CD86, and MHCII to different extents. ß-glucan-induced cytokine secretion was shown to be, in part, dependent on the activation of the intracellular Dectin-1 adapter molecule Syk. In co-cultures of mDCs with Th2-biased CD4+ T cells isolated from birch allergen Bet v 1 plus aluminum hydroxide (Alum)-sensitized mice, these four ß-glucans suppressed allergen-induced IL-5 secretion, while only Zymosan and ß-1,3 glucan significantly suppressed allergen-induced interferon gamma (IFNγ) secretion, suggesting the tested ß-glucans to have distinct effects on mDC T cell priming capacity. Our experiments indicate that ß-glucans have distinct immune-modulating properties, making them interesting adjuvants for future allergy treatment.

The Role of IgA in the Manifestation and Prevention of Allergic Immune Responses.

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.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

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.

Evaluation of the in vitro Function of Platelet Concentrates from Pooled Buffy Coats or Apheresis.

BACKGROUND: Platelet concentrates play an important role in transfusion medicine. Their short lifespan and lack of robustness require efforts to ensure adequate product quality. In this study, we compared the in vitro quality of the main concentrate types, pooled platelet concentrate (PPC) from whole blood donations, and platelet concentrate from single-donor apheresis (APC). METHODS: Twenty PPCs and 20 APCs prepared in plasma were analyzed on days 2, 4, and 7 of storage. Variables related to metabolism, degranulation, platelet aggregation, P-selectin expression, and annexin V binding were analyzed. Morphology was assessed by transmission electron microscopy of ultrathin sections. A microfluidic device was applied to test the effects of shear stress on platelet function. RESULTS: The metabolic parameters indicated stable storage conditions throughout the 7-day period. The resting discoid form was the prevailing morphology on days 2 and 4 in the PPCs and APCs. Chemokine release and receptor shedding of soluble P-selectin and soluble CD40L equally increased in PPCs and APCs. Aggregation responses to ADP and collagen were heterogeneous, with marked losses in collagen responsiveness on day 4 in individual concentrates. Baseline expression of P-selectin in PPCs and APCs was low, and inducibility of P-selectin was well preserved until day 4. Under shear stress, equal adhesiveness and stability were found with platelets from PPCs and APCs. CONCLUSIONS: Platelets from PPCs and APCs showed similar in vitro function and stability parameters. However, platelet concentrates presented a high variability and individual concentrates an impaired functional capability. Identifying the factors contributing to this would help increase product reliability.

Critical role of mammalian target of rapamycin for IL-10 dendritic cell induction by a flagellin A conjugate in preventing allergic sensitization.

BACKGROUND: Fusion proteins incorporating the Toll-like receptor 5 ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. OBJECTIVE: We studied the mechanisms of immune modulation by a flagellin:allergen fusion protein containing the Toll-like receptor 5 ligand flagellin A from Listeria monocytogenes and the birch pollen allergen Bet v 1 (recombinant flagellin A [rFlaA]:Betv1). METHODS: BALB/c mice were vaccinated with rFlaA:Betv1 in an experimental Bet v 1 sensitization model. Myeloid dendritic cells (mDCs) were differentiated from mouse bone marrow, and PBMCs were isolated from subjects with birch pollen allergy. Cells were stimulated with equimolar amounts of rFlaA, rBet v 1, rFlaA plus rBet v 1, or the rFlaA:Betv1 conjugate and analyzed for cell activation, cytokine secretion, and metabolic state. RESULTS: rFlaA:Betv1 displayed strong immune-modulating properties both in vivo and in vitro, as characterized by secretion of both proinflammatory and anti-inflammatory cytokines from murine mDCs and PBMCs from patients with birch allergy. rFlaA:Betv1 suppressed TH2 responses from Bet v 1-specific CD4+ T cells and prevented allergic sensitization in a mouse allergy model. Aggregation of rFlaA:Betv1 resulted in stronger protein uptake accompanied by an increased resistance to microsomal digestion. Remarkably, rFlaA:Betv1 induced activation of mammalian target of rapamycin, which increased the metabolic activity of the stimulated mDCs. rFlaA:Betv1-mediated IL-10 secretion, but not proinflammatory cytokine secretion, was inhibited by rapamycin in mDCs. CONCLUSION: These results provide evidence that mammalian target of rapamycin is a key player involved in prevention of TH2 responses by flagellin A conjugate vaccines.

A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

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.

Anaphylaxis to additives in vaccines.

Anaphylaxis in connection with the administration of vaccines occurs only very rarely. Triggers of immunoglobulin IgE-mediated and non-IgE-mediated anaphylaxis-in addition to the active ingredient itself-may be excipients contained in the vaccine due to their special properties. Some of the excipients in medicinal products are the same compounds used as additives in food. Furthermore, residues from the manufacturing process (e.g., chicken egg white, casein, antibiotics, formaldehyde) or contaminants (e.g., from the primary packaging material) may be potential triggers of anaphylaxis in vaccines. This review article provides an overview of ingredients in vaccines that pose an allergenic risk potential. The components of COVID-19 vaccines approved and marketed in Germany are discussed with regard to their potential for triggering anaphylaxis and possible pathophysiological mechanisms involved.

A Fusion Protein Consisting of the Vaccine Adjuvant Monophosphoryl Lipid A and the Allergen Ovalbumin Boosts Allergen-Specific Th1, Th2, and Th17 Responses In Vitro.

Background. The detoxified TLR4-ligand Monophosphoryl Lipid A (MPLA) is the first approved TLR-agonist used as adjuvant in licensed vaccines but has not yet been explored as part of conjugated vaccines. Objective. To investigate the immune-modulating properties of a fusion protein consisting of MPLA and Ovalbumin (MPLA : Ova). Results. MPLA and Ova were chemically coupled by stable carbamate linkage. MPLA : Ova was highly pure without detectable product-related impurities by either noncoupled MPLA or Ova. Light scattering analysis revealed MPLA : Ova to be aggregated. Stimulation of mDC and mDC : DO11.10 CD4(+) TC cocultures showed a stronger activation of both mDC and Ova-specific DO11.10 CD4(+) TC by MPLA : Ova compared to the mixture of both components. MPLA : Ova induced both strong proinflammatory (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokine responses from mDCs while also boosting allergen-specific Th1, Th2, and Th17 cytokine secretion. Conclusion. Conjugation of MPLA and antigen enhanced the immune response compared to the mixture of both components. Due to the nonbiased boost of Ova-specific Th2 and Th17 responses while also inducing Th1 responses, this fusion protein may not be a suitable vaccine candidate for allergy treatment but may hold potential for the treatment of other diseases that require a strong stimulation of the host's immune system (e.g., cancer).