ABSTRACT
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.
Subject(s)
Allergens/immunology , Antigens, Bacterial/immunology , Antigens, Plant/immunology , Flagellin/immunology , Recombinant Fusion Proteins/immunology , Animals , Bacterial Proteins/immunology , Cells, Cultured , Glucose/metabolism , Macrophages , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Plant Proteins/immunology , Pollen/immunologyABSTRACT
While acute allergic symptoms can be managed by emergency medication, to date, allergen-specific immunotherapy (SIT) with allergen extracts is the only available curative treatment option. However, the risk of anaphylactic reactions, long treatment duration, varying extract quality, and underrepresentation of certain allergens currently prevent many patients from successfully undergoing SIT. Novel strategies are needed to enhance efficacy, safety, and convenience of allergy treatment. Fusion proteins combining allergen and adjuvant into a single molecule can efficiently induce immune responses by targeting the allergen to the relevant immune cells in vivo. Simultaneous co-delivery of both antigen and adjuvant to the same cell in a fixed molecular ratio triggers the uptake and presentation of the conjugated allergen in the context of the adjuvant-induced immune cell activation. This review summarizes the published strategies to improve the treatment of type I allergies using fusion proteins consisting of allergen (peptides) and either (1) immune-activating bacterial (flagellin, MPLA, S-layer, cholera-, and tetanus toxin), (2) viral (PreS, VP-1, TAT), or (3) fungal (FIP-fve) components, (4) immune-activating DNA motifs, (5) forced delivery of allergens to the MHC-II loading pathway, and (6) killing of immune cells expressing allergen-specific IgE by fusion of the allergen to diphtheria toxin.
