Molecular Sciences - Posters B
Szimonetta Tamás1,2,3, Benoit Roux1,3, Boldizsár Vámosi1, Balázs Enyedi1,2,3
1 Department of Physiology, Semmelweis University, Budapest
2 MTA-SE Lendület Tissue Damage Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest
3 HCEMM-SE Inflammatory Signaling Research Group, Department of Physiology, Semmelweis University, Budapest
Introduction: Leukotriene B4 (LTB4) plays a prominent role in launching and propagating inflammatory responses. Acting as a signal relay molecule, LTB4 creates local and long-range gradients to orchestrate leukocyte recruitment and neutrophil swarming during tissue damage. Despite its profound biological relevance, we lack tools to directly measure and visualize these chemoattractant gradients in vivo.
Aims: Create a genetically encoded biosensor to detect and monitor levels of LTB4 in vivo in zebrafish larvae during tissue damage induced neutrophil swarming.
Methods & Results: We developed GEM-LTB4, a GPCR-based fluorescent indicator by inserting a circularly permuted EGFP (cpEGFP) module into the third intracellular loop of the high-affinity LTB4 receptor, BLT1. We optimized and increased the sensitivity of our sensor in HEK293 cells by screening different length linkers flanking the cpEGFP module. The best version, namely GEM-LTB4, shows high affinity and specificity towards LTB4, and a robust shift in fluorescence upon LTB4 exposure. To measure whether GEM-LTB4 could detect physiological concentrations of LTB4, we isolated murine neutrophils and seeded them on GEM-LTB4 expressing cells. When activated with fMLP, we detected an overall increase in LTB4 production and also captured LTB4 signals derived from individual neutrophils.
We then created a transgenic GEM-LTB4 expressing zebrafish line. To determine whether we could detect endogenous LTB4 release in 4 days old zebrafish larvae, we first recruited neutrophils (known to produce LTB4) to an open wound and then activated them using the Ca2+ ionophore A23187. With our sensor, we were able to visualize the generation of endogenous LTB4 gradients, which was completely abolished by adding zileuton, a strong inhibitor of LTB4 production.
Conclusion: In summary, we developed a fluorescent biosensor with high sensitivity and specificity for LTB4. With GEM-LTB4 we could monitor the live release of LTB4 from murine neutrophils and were able to detect and visualize LTB4 gradients likely derived from single cells. When expressed in zebrafish, we could detected neutrophil-induced endogenous LTB4 production and gradients. Since LTB4 plays a central role in inflammatory processes as a main driver of chemotaxis and neutrophil swarming, we believe that GEM-LTB4 will further help to understand this complex response.