Abstract
Fusion proteins incorporating the TLR5-ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. We recently reported a flagellin:allergen fusion protein containing the TLR5-ligand flagellin A (FlaA) from Listeria monocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to prevent allergic sensitization in an experimental mouse model. This study analyzes the signaling pathways contributing to rFlaA:Betv1-mediated pro- and anti-inflammatory cytokine secretion and cell metabolism in myeloid dendritic cells (mDCs) in vitro. The influence of mammalian target of rapamycin (mTOR)-, NFκB-, and MAP kinase (MAPK)-signaling on cytokine secretion and metabolic activity of bone marrow (BM)-derived mDCs stimulated with rFlaA:Betv1 were investigated by pre-treatment with either mTOR- (rapamycin), NFκB- (dexamethason, BMS-345541, TPCA-1, triptolide, or BAY-11) or MAPK- (SP600125, U0126, or SB202190) inhibitors, respectively. rFlaA:Betv1-mediated IL-10 secretion as well as activation of mDC metabolism, rather than pro-inflammatory cytokine secretion, were inhibited by rapamycin. Inhibition of NFκB-signaling suppressed rFlaA:Betv1-induced IL-12, while inhibition of MAPK-signaling dose-dependently suppressed rFlaA:Betv1-induced IL-10 as well as pro-inflammatory IL-6 and TNF-α production. Notably, with the exception of a partial JNK-dependency, rFlaA:Betv1-mediated effects on mDC metabolism were mostly NFκB- and MAPK-independent. Therefore, MAPK-mediated activation of both NFκB- and mTOR-signaling likely is a key pathway for the production of pro- and anti-inflammatory cytokines by flagellin fusion protein vaccines.
Highlights
The incidence of allergic diseases has steadily increased over the last 70 years, causing significant decreases in quality of life in affected patients and economic problems [1,2]
Since the rather unspecific NFκB- and Mitogen-activated protein (MAP) kinase-inhibitor dexamethason dose-dependently inhibited all cytokine production induced by stimulation of myeloid dendritic cells (mDCs) with rFlaA:Betv1, we investigated the contribution of MAP kinase-signaling to rFlaA:Betv1-induced activation of mDC metabolism and production of pro- and anti-inflammatory cytokines by applying MAPK-specific inhibitors (Figures 4 and 5)
The aim of the present study was to characterize the signaling pathways contributing to the induction of pro- and anti-inflammatory cytokine secretion in mDCs stimulated with flagellin: antigen fusion proteins and their contribution to the observed activation of mDC metabolism
Summary
The incidence of allergic diseases has steadily increased over the last 70 years, causing significant decreases in quality of life in affected patients and economic problems [1,2]. Besides symptomatic treatment or avoidance of the respective allergens, allergen specific immunotherapy (AIT) with allergen extracts is the only disease altering treatment option available so far. AIT is not convenient for patients due to a multi-year treatment regimen, only partially efficacious for some allergies, and can be hampered by unwanted side effects [3]. To improve AIT, novel vaccine candidates and accompanying adjuvants that increase efficacy while decreasing unwanted adverse-effects are needed [4]. TLR-ligands with an intrinsic ability to induce robust innate immune responses are of special interest for their utilization as adjuvants.
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