The Fusion Protein rFlaA:Betv1 Modulates DC Responses by a p38-MAPK and COX2-Dependent Secretion of PGE2 from Epithelial Cells

Abstract

Developing new adjuvants/vaccines and better understanding their mode-of-action is an important task. To specifically improve birch pollen allergy treatment, we designed a fusion protein consisting of major birch pollen allergen Betv1 conjugated to the TLR5-ligand flagellin (rFlaA:Betv1). This study investigates the immune-modulatory effects of rFlaA:Betv1 on airway epithelial cells. LA-4 mouse lung epithelial cells were stimulated with rFlaA:Betv1 in the presence/absence of various inhibitors with cytokine- and chemokine secretion quantified by ELISA and activation of intracellular signaling cascades demonstrated by Western blot (WB). Either LA-4 cells or LA-4-derived supernatants were co-cultured with BALB/c bone marrow-derived myeloid dendritic cells (mDCs). Compared to equimolar amounts of flagellin and Betv1 provided as a mixture, rFlaA:Betv1 induced higher secretion of IL-6 and the chemokines CCL2 and CCL20 from LA-4 cells and a pronounced MAPK- and NFκB-activation. Mechanistically, rFlaA:Betv1 was taken up more strongly and the induced cytokine production was inhibited by NFκB-inhibitors, while ERK- and p38-MAPK-inhibitors only suppressed IL-6 and CCL2 secretion. In co-cultures of LA-4 cells with mDCs, rFlaA:Betv1-stimulated LA-4 cells p38-MAPK- and COX2-dependently secreted PGE2, which modulated DC responses by suppressing pro-inflammatory IL-12 and TNF-α secretion. Taken together, these results contribute to our understanding of the mechanisms underlying the strong immune-modulatory effects of flagellin-containing fusion proteins.

The Role of Defective Epithelial Barriers in Allergic Lung Disease and Asthma Development

“…117 Classically regarded as a pro-inflammatory mediator, PGE2 (prostaglandin E2) can promote resolution. 118 PGE2 by inhibiting the proliferation, activation, and secretion of cytokines by ILC2. 119 Studies in asthmatic patients reported an inverse correlation between the sputum levels of PGE2 and eosinophil numbers, thus suggesting it can play a role in reducing airway eosinophilia.…”

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

“…As a model for such novel therapeutics in the field of allergies, we 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 into a single molecule [ 3 ]. In previous studies, we were able to show that rFlaA:Betv1 efficiently suppressed allergic sensitization in vivo [ 3 ] while also activating myeloid DCs (mDCs) [ 3 , 4 ], macrophages [ 5 ], epithelial cells [ 6 ], and B cells [ 7 ], leading to pro-inflammatory cytokine production and the subsequent formation of allergen-specific TH1 responses with TH2-suppressing capacity [ 3 , 5 ]. Furthermore, rFlaA:Betv1 induced a prominent production of the anti-inflammatory cytokine IL-10 in all investigated types of antigen presenting cells (APCs) (mDCs, macrophages, and B cells), which was shown to significantly suppress TH2 responses [ 3 , 5 , 7 ].…”

Allergen Immunotherapy: New Insights into an Old Treatment

“…Two articles with original data on the experimental production of novel recombinants of the major birch pollen allergen Betv1 have been published by a German team [ 5 , 6 ]. A recombinant fusion protein of flagellin A (serving as a TLR5-ligand) and Betv1, namely rFlaA:Betv1, has been produced, to target immune cells, in order to create adjuvant-mediated pro- and anti-inflammatory responses and suppress the allergen-induced Th2 responses [ 5 ].…”

“…Using a mouse-model, the stimulation of bone marrow-derived macrophages (BMDMs) by rFlaA:Betv1 and the boosted IL-10 and INF-γ secretion in BMDM and T cell co-cultures, suggest an important role of macrophages as target cells of immunotherapy [ 5 ]. The immune-modulatory effects of rFlaA:Betv1 were confirmed in cultures of murine airway LA-4 epithelial cells with myeloid dendritic cells (mDC) [ 6 ]. It was shown that, upon stimulation with rFlaA:Betv1, epithelial cells can modulate pro-inflammatory mDC responses by p38 MAPK- and COX2-dependent production of PGE2 [ 6 ].…”

“…The immune-modulatory effects of rFlaA:Betv1 were confirmed in cultures of murine airway LA-4 epithelial cells with myeloid dendritic cells (mDC) [ 6 ]. It was shown that, upon stimulation with rFlaA:Betv1, epithelial cells can modulate pro-inflammatory mDC responses by p38 MAPK- and COX2-dependent production of PGE2 [ 6 ].…”