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

Abstract

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 1β was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1β, 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-1β). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1β (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.

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

“…Our previous publications showed rFlaA:Betv1 to activate different cell types involved in allergic responses, resulting in the suppression of allergen-induced Th2 inflammation ( 3 , 5 – 7 , 14 , 15 ). In these studies, the rFlaA:Betv-induced IL-1β secretion was shown to be differently regulated by different types of APCs, including mDCs and BMDMs ( 3 , 5 , 14 ).…”

“…In our previous studies, rFlaA:Betv1 efficiently suppressed sensitization towards Bet v 1 in vivo ( 3 ) while also activating mouse bone marrow-derived myeloid dendritic cells (mDCs) ( 3 , 7 , 14 ), bone marrow-derived macrophages (BMDMs) ( 5 ), lung epithelial cells ( 15 ), and naïve mouse and human B cells ( 6 ) more strongly than the equimolar mixture of both proteins either provided alone or as a non-fused mixture. Here, rFlaA:Betv1 induced a pro-inflammatory cytokine production and the subsequent formation of Bet v 1-specific Th1 responses with Th2-suppressing capacity while also mediating a prominent production of the anti-inflammatory cytokine IL-10 in all investigated types of APCs (mDCs ( 3 , 7 , 14 ), BMDMs ( 7 ), and B cells ( 8 )), which was shown to significantly suppress Th2 responses.…”

“…In an effort to test the potential of flagellin as a novel adjuvant, we recently generated a recombinant fusion protein, rFlaA:Betv1, that combines the TLR5-ligand flagellin A from Listeria monocytogenes with the major birch pollen allergen Bet v 1 (3). Such flagellincontaining fusion proteins efficiently target the fused antigen to TLR5-expressing target cells in vivo, which, in case of antigen presenting cells (APCs), were shown to take up, process, and present the fused antigen in the context of the flagellin-mediated immune cell activation, resulting in superior induction of immune responses (4)(5)(6)(7). In line with this, flagellin-containing fusion proteins incorporating influenza antigens were shown to efficiently induce immune responses in elderly patients with a good safety profile (8)(9)(10)(11)(12)(13).…”

When the allergy alarm bells toll: The role of Toll-like receptors in allergic diseases and treatment

“…To modulate allergen-specific immune responses, TLR-ligand allergen fusion proteins combining allergens or allergen peptides with an inherent adjuvant activity were proposed and recently reviewed by Blanco-Pérez et al ( Blanco-Pérez et al, 2019 ). Despite promising preclinical data regarding the induction of immunotolerance by the fusion proteins, i.e., for the birch pollen allergen Bet v 1 fused to flagellin, or T cell peptides of the peach LTP Pru p 3 conjugated to TLR7 and TLR4 ligand, so far none of the candidates were clinically evaluated ( Schülke et al, 2010 ; Losada Méndez et al, 2021 ; Goretzki et al, 2022 ).…”

Editorial of Special Issue “Molecular Mechanisms of Allergy and Asthma 2.0”

“…Considering that NOD2 adjuvants are used in vaccine design to boost immune responses, whereas care needs to be taken in asthmatics, those might be used in non-sensitized individuals. Goretzki et al [5], investigated in their highly ambitious study the metabolic mechanisms behind the activation and T cell-modulating potential of DCs stimulated with a fusion protein combining Toll-like (TLR) 5 ligand flagellin (FlaA) and major birch pollen allergen Bet v 1 into a single molecule (rFlaA:Betv1). They found that the effector function of rFlaA:Betv1-activated myeloid DCs mainly depended on glycolysis, with fatty acid synthesis also substantially participating in rFlaA:Betv1-mediated secretion of cytokines, production of antimicrobial molecules, as well as modulation of T cell responses.…”