The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88- and MAPK-Dependent Activation of Glucose Metabolism in Macrophages.

Yen-Ju Lin,Jennifer Zimmermann,Zoltán Ivics,Sonja Wolfheimer,Stephan Scheurer,Stefan Vieths,Garibald Papp,Alexandra Goretzki,Csaba Miskey,Ger Van Zandbergen,Anna Fiedler,Peter Crauwels,Stefan Schülke

doi:10.3390/cells10102614

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-1β, 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.

Highlights

In the future, novel adjuvants and vaccines may be valuable tools to improve the efficacy, safety, and convenience of allergen immunotherapy
While differences between the rFlaA:Betv1- and either the rFlaA− or rFlaA + rBet v 1induced Warburg effect were significant, there was no difference in glucose consumption between rFlaA:Betv1− and rFlaA + Bet v 1-stimulated bone marrow-derived macrophages (BMDMs) (Figure 1B)
Activation of BMDM glucose metabolism was paralleled by a highly significantly increased production of both pro- (IL-1β, IL-6, TNF-α) and anti-inflammatory (IL-10) cytokines from rFlaA:Betv1-stimulated BMDMs compared to cells stimulated with either both proteins alone or provided as a non-fused mixture

Summary

Introduction

Novel adjuvants and vaccines may be valuable tools to improve the efficacy, safety, and convenience of allergen immunotherapy. For their safe and efficient application, basic knowledge describing their immune modulating capacity, activated cell types, as well as a characterization of the underlying immunological mechanisms is needed. Since flagellin is the only proteinaceous TLR ligand, its application for the generation of recombinant flagellin:antigen fusion proteins is of special interest in vaccine development. Fusion proteins combining flagellin with different antigens have been investigated for their potential to generate immune protection against different diseases including, among others: influenza [7,8,9], poxvirus [10], West

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Conclusion