Abstract
Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10−/−, TLR5−/− mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5−/− mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10−/− mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.
Highlights
Bacterial flagellins, including flagellin A (flaA) derived from L. monocytogenes, were identified as the ligands for TLR5 [1,2], shown to be proinflammatory at the picomolar range and have strong immune modulatory activities [3,4,5,6,7]
In previous studies we showed that fusion of L. monocytogenes flagellin A and the model allergen Ova resulted in strongly enhanced myeloid dendritic cells (mDC) activation, mDC derived cytokine secretion, a suppression of TH1 and TH2 cytokine secretion from Ova-specific CD4 T cells and prevention of an allergic phenotype upon intraperitoneal application [9]
To further elucidate the mechanisms by which flagellin fusion proteins induce such strong protective immune responses, we characterized the effects of rflaA:Ova were 9.05 fold (rflaA):Ova stimulation on murine mDCs as target cells for a successful vaccination [29]
Summary
Bacterial flagellins, including flaA derived from L. monocytogenes, were identified as the ligands for TLR5 [1,2], shown to be proinflammatory at the picomolar range and have strong immune modulatory activities [3,4,5,6,7]. In vivo Salmonella flagellin C application stimulated strong TLR5 dependent allergic airway responses to inhaled Ova and primed allergic responses to natural indoor allergens present in house dust extracts [8]. Antigens conjugated with flagellin were shown to be effective vaccines promoting antigen-specific TH1 responses in vivo [9,10,11,12,13,14]. Even if a single cell internalizes both proteins, the flagellin:antigen ratios and the type of immune response induced will be different from cell to cell. In a worst case scenario this may result in bystander activation of antigen-specific TH2-responses by flagellin activated cells, as observed for the flagellin induced priming of allergic responses to house dust extracts [8]
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