D1.429 - The major birch pollen allergen activates the NLRP3 inflammasome in THP1-ASC-GFP reporter cells

The major birch pollen allergen Bet v 1 is the main cause of birch pollen allergy. Current studies are investigating the link between the NLRP3 inflammasome and allergic diseases. The inflammasome comprises the NLRP3 sensor, the ASC adaptor and pro-caspase-1. While the NLRP3 sensor recognizes PAMPs from various pathogens as well as DAMPs, its main function is sensing cellular damage. It also acts as a transcription factor for IL-33, linking it to allergic diseases. The aim of this project is to investigate the role of the NLRP3 inflammasome formation in sensing the presence of allergens.

THP1-ASC-GFP (Invivogen) cells were primed with LPS to express the ASC-GFP fusion protein. Six hours after priming, natural (n) and recombinant (r) Bet v 1, birch pollen extract (BPE), and nBet v 2 were added. The allergens’ potential to activate inflammasome formation was monitored in 2h intervals for 24h in the Incucyte® S3 (Sartorius). Fluorescence images were analyzed by CellProfiler and Python for the number of ASC-specks formed. The total number of fluorescent cells was calculated based on the number of cells with fluorescent cytosol and those forming specks at the first timepoint after activation. The number of specks was divided by the total number of fluorescent cells, resulting in the relative number of specks formed per cell. Nigericin was used as a technical positive control and in combination with MCC950 as a technical negative control.

Primed cells were detected by their fluorescent cytosol. At a concentration of 25 µg/mL nBet v 1 activated 90% (± 8%), rBet v 1 77% (± 7%) and BPE 73% (± 6%) of the cells at the reaction’s peak. The activation reaction was slightly different for each allergen, resulting in activation peaks 3-5h after addition of the allergen. Natural Bet v 2, a minor allergen, led to a maximum activation of 38% (± 8%) of the cells 5h after its addition. When adding medium alone 41% (± 10%) of cells formed specks. After the reaction had peaked, the inflammasome specks started to degrade resulting in decreasing values. After 13h of activation, most of the specks were degraded.

Our study, which was supported by Danube-ARC projects 4 and 10 funded by the Federal State of Lower Austria, shows that nBet v 1, rBet v 1 and BPE induce NLRP3 inflammasome formation in the primed reporter cell line THP1-ASC-GFP. Our data suggest that Bet v 1 may induce cell damage.