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

Yen-Ju Lin,Stefan Schülke,Stefan Vieths,Adam Flaczyk,Stephan Scheurer,Andrea Wangorsch,Annette Jamin,Alexandra Goretzki,Sonja Wolfheimer

doi:10.3390/cells10123415

Yen-Ju Lin, Stefan Schülke + Show 7 more

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https://doi.org/10.3390/cells10123415

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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.

Highlights

The current SARS-CoV-2 pandemic has clearly demonstrated the need to develop both safe and efficacious vaccines
In this experimental setting no secretion of either GM-CSF, G-CSF, IL-33, TSLP, IL-10, TNF-α, IL-12, or IL-1β was detected from stimulated LA-4 cells
While the effects of flagellin on epithelial cells are well-known [43,44,45,46,47,48,49,50,51] and the interaction of flagellin with epithelial cells has often been described as a key factor for its adjuvant activity [52], our study is the first to characterize the response of epithelial cells to a flagellin:antigen fusion protein

Summary

Introduction

The current SARS-CoV-2 pandemic has clearly demonstrated the need to develop both safe and efficacious vaccines. Protein-antigen-based vaccines that typically have a low immunogenicity often need to be adjuvanted in order to induce robust immune responses. In this context, some “Toll”-like receptor (TLR) ligands with intrinsic immune-activating properties are attractive adjuvant candidates. Several vaccines containing MPLA as a component of more complex adjuvant systems have been licensed or are undergoing phase III clinical trials. These include Fendrix® (for the prevention of hepatitis B), Cervarix®

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